RECENT ADVANCES IN VACCINE AND SERUM THERAPY THE RECENT ADVANCES SERIES ALLERGY (Asthma, Yay foyer: Eczema, Migraine, etc.). By G. W. Bray, M.B., Ch.N ANESTHESIA AND CSA. By C. LANGTON HEWER, ANATOMY. By H. WooLLArD, M.D. BACTERIOLOGY. By J. Henry DisLE, F.R.C.P. 2nd Edition. BIOCHEMISTRY. By J. Prypg, M.Sc. 3rd Edition. CARDIOLOGY. By Terence East, M.D., and C. W. C. BAIN, M.B. 2nd Edition. CHEMOTHERAPY. By G. M. FixprLay, M.D. CHILDREN. By W. J. Pearson, F.R.C.P., and W. G. WYLLIE, M.D. 2nd Edition. ENDOCRINOLOGY. By A. T. Cameron, D.Sc, F.R.S.C. FORENSIC MEDICINE. By Sypney Smith, M.D, and Joun GLAISTER, M.D HAEMATOLOGY. By A. Piney, M.D. 3rd Edition. MATERIA MEDICA (Sera and Vaccines, Hormones and Vitamins). By J. H. Burn, M.D. MEDICINE. By G. E. Beaumont, F.R.C.P.,, and E. C. Dobbs, M.V.0., F.R.C.P. 7th Edition. NEUROLOGY. By W. RusseLL Bray, D.M., and E. B. Strauss, B.M. 2nd Edition. OBSTETRICS AND GYNACOLOGY. By A. W. BOURNE, F.R.C.S,, and LL. WiLLiams, F.R.C.S. 3rd Edition. OPHTHALMOLOGY. By Sir W. STEWART DUKE-ELDER, M.D. 2nd Edition. PATHOLOGY. By G. HaprieLp, M.D., and L. P. GArRrOD, M.B. PHYSIOLOGY. By C. Lovarr Evans, F.R.C.P., F.R.S. 4th Edition. PREVENTIVE MEDICINE. By J. F. C. HasLam, M.D. PSYCHIATRY. By H. DeviNg, M.D. 2nd Edition. PSYCHONEUROSES. By Mitrais CureiN, M.D. PULMONARY TUBERCULOSIS. By L. S. T. BURRELL, F.R.C.P. 2nd Edition. RADIOLOGY. By Peter KerLEy, M.D., D.M.R.E. RADIUM. By W. Roy Warp, M.B.,, and A. J. DURDEN Smita, M.B. RAEUMATISM, By F. J. Poy~NToN, M.D., and B. SCHLESINGER, SURGERY. By W. H. Ocirvig, F R.C.S. 2nd Edition. TROPICAL MEDICINE. By Sik L. Rocers, F.R.S.,, M.D. 2nd Edition. VACCINE AND SERUM THERAPY. By ALEXANDER FLeming, F.R.C.S,, and G. F. PeTriE, M.D. RECENT ADVANCES IN VACCINE AND SERUM THERAPY By ALEXANDER FLEMING F.R.C.S.(Eng.) Professor of Bacteriology in the University of London ; Assistant Director of the Inoculation Department, St. Mary's Hospital G. F. PETRIE M.D.(Aberd.) Bacteriologist-in-charge, Serum Department, Lister Institute, Elstree WITH 5 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON’S SON & CO. INC. 1012 WALNUT STREET 1934 CAT. FOR PUBLIC HEALTH Printed in Great Britain [SM 741 Fo PUBLIC HEALTH LIBRARY PREFACE Our purpose in writing this book has been to survey the present state of knowledge regarding the employment of vaccines and sera in the prevention and treatment of disease. Care has been taken to refer to the methods of their preparation in order that the clinical worker may obtain some idea of the processes of manipula- tion and control through which they pass before they are released for issue. We hope, too, that the specialist whose work is concerned with the technique of their production will find interest in learning something of the successes and the failures that have attended their use. We wish to state that each author is solely responsible for his own part of the work. Thus Part I., which deals with the preven- tion and treatment of disease by means of sera and with the specific prophylaxis and treatment of virus diseases of man and of animals, has been written by Dr. Petrie. Part IL. gives an account of the prevention and treatment of disease by means of vaccines, and is the work of Professor Fleming. Progress in serum therapy during the past decade has been made in several directions. Thus improvements in the technical methods for the production of therapeutic sera have resulted from a fuller’ knowledge of the conditions that are necessary for obtaining with constancy effective preparations of the requisite antigens. Again, research workers in different countries are extending the application of precise methods for assessing the potency of therapeutic sera in terms of standard units, and are co-operating to secure International authority for the new stan- “dards. Credit for the work that has been accomplished should be given to the Commission on Biological Standardization which was appointed some years ago by the League of Nations, and, in this country, to the Biological Standards Department of the National Institute for Medical Research, London. An important group of v .619 vi PREFACE researches which is showing rapid expansion concerns the produe- tion of specific sera against virus infections. It may safely be predicted that as our knowledge of the nature of viruses increases, antiviral sera will be more extensively employed. A difficult problem which requires further investigation is the improvement of serum preparations with the object of removing from them the elements that may contribute to those unpleasant and, on occasion, alarming symptoms which are apt to appear in individuals who are serum-sensitive. The author of the section on vaccine therapy has based his opinions on the experience he has gained at St. Mary's Hospital in close co-operation with Sir Almroth Wright, who is recognized as a pioneer in this field. He has not touched upon every aspect of the subject and some diseases in which vaccines have been found to be of considerable value are not mentioned ; it should not, therefore, be assumed that he has no belief in the efficacy of vaccines for the treatment of such diseases but only that there have been no recent advances to record or that the time has not yet arrived when knowledge has sufficiently crystallized to form a profitable subject for discussion. A few years ago there was no control over the production of vaccines in England, but this has now been established by means of the provisions of the Therapeutic Substances Act. Under the Act all laboratories that prepare vaccines must be licenced, but this condition does not imply that vaccines from all sources can be standardized so that they are equally efficient. The Regulations ensure that vaccines shall be composed of the bacteria they are stated to contain and that they shall be sterile, but they do not control the potency of the product. Our knowledge is too scanty for standards of potency to be laid down, and, indeed, one of the greatest advances in vaccine therapy will be the discovery of potency tests so that vaccines may be issued for use in terms of antigenic units without reference to numerical estimates of their bacterial content. A section on the employment of vaccines and sera in veterinary practice has been included. There is clearly no reason why this part of the subject should be omitted, since an artificial division PREFACE vii between the specific prophylaxis and treatment of man and of animals cannot be justified on logical grounds : the barriers that have separated the domains of human and animal pathology in the past have delayed progress. The reader will find that frequent reference is made in the following chapters to ‘“ antigens ”* and *“ antibodies,” terms that, unfortunately, ignore the rules of word-construction, however defensible they may be on the score of convenience. It seems ¢ expedient to define these substances, because their operations and interactions form the subject-matter of this book. When we speak of antigens, we mean a group of substances of bacterial or viral origin which, as the result of their introduction into the living tissues of animals give rise to antibodies that are capable of reacting either in vitro or in vivo with the corresponding antigens. For example, a dose of an appropriate antibody will protect a susceptible animal from illness and death when many multiples of the lethal dose of a toxin or a highly virulent bacterium are administered to it. The efficacy of vaccine preparations depends upon their antigenic components, and sera owe their therapeutic activity to the antibodies—whether antitoxic or antibacterial’ or antiviral-—that they contain. We desire to express our grateful thanks to colleagues at the Lister Institute and St. Mary’s Hospital who have assisted us during the preparation of this volume. The author of Part I. is deeply indebted to Mr. H. W. Parker, of the Zoological Depart- ment, British Museum (Natural History), for valuable help in revising Chapter IX. ; to Dr. J. T. Edwards and Mr. W. A. Pool, Deputy Director, Imperial Bureau of Animal Health, Ministry of Agriculture and Fisheries, for similar services in respect of Chap- ter XIX. : and to Dr. J. M. Petrie for critical revision of the text. The author of Part II. has pleasure in acknowledging the help of Dr. R. A. O’Brien and Dr. V. D. Allison in the preparation of certain chapters in the section on vaccine therapy. Thanks are also due to Professors Topley and Wilson, and to their publishers, Messrs. Edward Arnold & Co., for permission to reproduce five charts from the * Principles of Bacteriology and Immunity.” of these authors. vii PREFACE We desire, in conclusion, to express our gratitude to Messrs. J. & A. Churchill for help and advice extended to us during the preparation and publication of this volume. A. FLEMING. G. F. PETRIE, CHAP. 11. 111. Vv. V. V1. VII. VIII. IX. XI. X11. XIII. XIV. XV. XVI. XVII. XVIII. XIX. CONTENTS PART 1 SERUM THERAPY (G. F. PETRIE) GENERAL CONSIDERATIONS ON THE PRODUCTION AND THE USE OF THERAPEUTIC SERA . J y . Tue SERUM TREATMENT OF DIPHTHERIA . , " Tue SERUM TREATMENT OF TETANUS . : : THE SERUM TREATMENT OF GAS GANGRENE 5 : THE SERUM TREATMENT OF SCARLET FEVER AND OTHER STREPTOCOCCAL INFECTIONS . . . ’ . ANTI-STAPHYLOCOCCUS SERUM . . . . . THE SERUM TREATMENT OF DYSENTERY . . . THE SERUM TREATMENT OF BoTULISM . . . THE SPECIFIC SERUM TREATMENT OF SNAKE-BITE, THE STING OF SCORPIONS AND THE BITE OF SPIDERS ‘ THE SERUM TREATMENT OF LOBAR PNEUMONIA ’ THE SERUM TREATMENT OF CEREBROSPINAL FEVER . ANTI-ANTHRAX SERUM . " ’ . . ’ ANTI-TYPHOID SERUM : . . . ’ . THE SERUM TREATMENT OF PLAGUE, ’ ’ . ANTI-CHOLERA SERUM . . . ‘ ’ ‘ Tie PROPHYLAXIS OF MEASLES BY MEANS OF SERUM . THE SERUM TREATMENT OF ACUTE ANTERIOR PoOLIO- MYELITIS : . g s . t 5 y Tue VACCINE AND SERUM TREATMENT OF CERTAIN Diseases IN MAN CAUSED BY FILTER-PASSING VIRUSES Tine EMPLOYMENT OF VACCINES AND SERA IN VETERI- NARY PRracTICE > v . . ’ . R.A. VACCINES, ix PAGE 18 38 51 65 83 88 99 102 113 130 152 157 160 162 165 CHAP. II. 111. Iv, VT. VII. VIL. IX. XI. XII. XIII. Xv. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII. XXIII. XXIV. XXV. CONTENTS PART II VACCINE THERAPY (A. FLEMING) THE PREPARATION OF A VACCINE . v BACTERIAL VARIATION IN REGARD TO IMMUNITY . LocArn IMMUNIZATION ANTI-TYPHOID INOCULATION IMMUNIZATION AGAINST PARATYPHOID INFECTIONS BAcCiLLARY DYSENTERY ANTI-CHOLERA IMMUNIZATION . ANTI-PLAGUE INOCULATION ” r 3 ’ . LoBAR PNEUMONIA PrEVENTION OF THE CoMMON COLD AND OTHER CATARRHAL RESPIRATORY INFECTIONS INFLUENZA Wuooring CoucH Acute Rueumartic FEVER CuaroNic RHEUMATIC CONDITIONS ACNE VULGARIS FuruNcULOSIS B. CoLi INFECTIONS . ULCERATIVE COLITIS GONOCOCCAL INFECTIONS . HAY FEVER AND OTHER IDIOSYNCRASIES . ASTHMA . Non-specIFi¢c VACCINE THERAPY ACTIVE IMMUNIZATION AGAINST DIPHTHERIA AcTIVE IMMUNIZATION AGAINST TETANUS . PREVENTION OF CERTAIN ANIMAL DISEASES BY THE ADMINISTRATION OF ForMOL CULTURES y v INDEX OF SUBJECTS INDEX OF AUTHORS PAGE 243 258 268 ‘273 280 283 290 293 208 314 327 334 343 350 365 370 376 380 383 393 402 408 420 439 442 RECENT ADVANCES IN VACCINE AND SERUM THERAPY PART 1 SERUM THERAPY CHAPTER I GENERAL CONSIDERATIONS ON THE PRODUCTION AND THE USE OF THERAPEUTIC SERA PAGE THE PREPARATION OF THERAPEUTIC SERA . 2 Antitoxic Sera 2 Antibacterial Sera 4 Antiviral Sera. 5 THE CONCENTRATION OF THERAPEUTIC SEF RA 5 THE TITRATION OF THERAPEUTIC SERA 6 TuE OrricIAL CONTROL OF THERAPEUTIC SERA . 6 STATISTICAL EVIDENCE OF THE CLINICAL VALUE OF THE RAPEUTIC SERA . v SERUM SICKNESS, SE RUM " ACCIDENTS, AND y OTHER COMPLICATIONS OF SERUM TREATMENT . ’ A : . . 8 Serum Sickness : > ’ ’ : : : a 8 Serum Accidents . . . . » 10 The Mechanism of Serum Sensitiveness . : g 13 Febrile Reactions after Intravenous Injections of Se rum . . 13 Neurological Complications of Serum Treatment ‘ . 14 The practical work of producing therapeutic sera is based on researches into the processes of immunity that are being pursued in laboratories in many countries, and thus the methods of production are constantly undergoing improvement. Thera- peutic sera may be arranged in three groups: (1) antitoxins, such as diphtheria and tetanus antitoxin; (2) antibacterial sera, as, for instance, anti-pneumococcus serum ; and (3) antiviral sera, R.A. VACCINE, l 1 2 PRODUCTION AND USE OF THERAPEUTIC SERA of which anti-poliomyelitis serum is an example. They are obtainable from two chief sources of supply : the horse, an animal with a natural capacity for the production of antibodies in response to many diverse antigens when it is subjected to a succession of immunizing doses ; and man. The use of human immune-sera has greatly extended in recent years; the serum is provided by persons who have acquired an immunity as a result of having previously been infected with a particular virus or bacterium. The Preparation of Therapeutic Sera Antitoxic Sera. Formolized toxins are now generally employed for immunizing horses intended to be used for the production of antitoxic sera. Formalin is added to the crude toxic filtrate in amounts varying from 0-2 to 0-4 per cent. ; the filtrate is then incubated at 837° C. for from 4 to 6 weeks, and at the end of this period it is found that the toxin has been rendered harmless to susceptible animals when it is administered to them in considerable doses. The formalin converts, in a way which is not yet under- stood, the toxic molecules into a non-toxic modification or “toxoid the ‘“anatoxine ’’ of Ramon; this, nevertheless, acts as an effective immunizing agent. The toxins of the diphtheria bacillus, the tetanus bacillus, the dysentery bacillus (Shiga), the streptococcus associated with scarlet fever, and the plague bacillus can all be treated by the formalin method for the purpose of preparing antitoxic sera. The employment of modified antigens has the advantage that the reactions in the horse are less severe than those caused by the unmodified toxin, and also that the first immunizing course can be proceeded with more rapidly. Some workers, however, still prefer the original method of giving care- fully graduated doses of unmodified toxin to fresh horses (Wernicke and Schmidt). Ramon noted an increase in the amount of antitoxin in the blood of horses when sterile abscesses happened to form at the site of inoculation ; and from this observation he was led to produce artificial sterile abscesses by mixing the toxin with a tapioca preparation. The average titre of the sera of horses so treated PREPARATION OF THERAPEUTIC SERA 3 was higher than that in a control group. Walbum found that a solution of manganese chloride acted as a stimulus to antitoxin production, but others have not confirmed this result. Glenny and his colleagues (1930) have shown that the addition of a small amount of potash alum solution to diphtheria and tetanus toxoids brings about a considerable increase in their antigenic efficiency. The average antitoxin value in groups of 50 horses was nearly 700 units of diphtheria antitoxin for the group to which alum was not given, and 1,100 units for the group that received alum ; the addition of alum to tetanus toxoid has doubled the resulting yield of antitoxin. Ramon has found that a similar effect may be obtained by the use of calcium chloride. When these salts are added to the toxoid solutions they cause a considerable precipitate of some of the constituents of the broth, and it is probable that the toxoid molecules become adsorbed on the particles of the precipitate. This physical reaction may explain the enhanced production of antitoxin, because, as Glenny has suggested, the absorption of the toxoid molecules after their deposition in the tissues of the horse is delayed, and thus the antigenic stimulus is maintained. Glenny’s method is undoubtedly a valuable one in the preparation of antitoxic sera. When immunizing horses against diphtheria toxin most workers are careful to choose animals whose blood normally contains small amounts of diphtheria antitoxin, because they respond more rapidly to immunization with diphtheria toxin or toxoid ; more- over, the resulting antitoxin content in the blood is, on the average, higher than in horses whose blood contains no natural antitoxin (Glenny ; Celarek and Porebski). It is well known that horses not infrequently harbour the diphtheria bacillus in wounds or skin lesions of various kinds, and that the older the horse the more likely it is to possess a naturally acquired immunity. Nevertheless, it appears that the lack of immunity in a fresh horse can be compensated for by the use of a powerful antigen, although the response may be slower in it than in an animal which has already some degree of immunity. In the early days of the manufacture of diphtheria antitoxin difficulties were encountered in obtaining a regular supply of a 1-—2 4 PRODUCTION AND USE OF THERAPEUTIC SERA uniformly strong toxin from successive lots of broth cultures. For unaccountable reasons the lethal dose varied from batch to batch, and the average level of toxicity was low. The improved results obtained during recent years are due to the recognition of two important factors in the preparation of the culture medium : (1) the reduction of the temperature of sterilization to the lowest point that is consistent with sterility, and (2) the suitability of the peptone that is added to the medium (P. Hartley and O. Hartley). When it is desired to obtain highly toxic and antigenic prepara- tions, sterilization of the medium by means of filtration is prefer- able to sterilization by heat. Investigations into the conditions for the successful production of diphtheria toxin have been made by Pope (1933). Antibacterial Sera. Experience has shown that sera of this kind are best prepared by injecting the appropriate antigen intra- venously. The technique of the immunization, that is, the amount and spacing of the doses, is on a much less satisfactory basis than that for obtaining antitoxic sera, because there are no easily applied methods for the titration of antibacterial sera, which would serve as a guide. The immunizing material consists of a suspension in physiological saline of the bacterium in question, either in the living state or killed by heat. The fact that some bacterial species, such as the pneumococcus and the meningo- coccus, are divisible into serological types or groups has a bearing on the preparation of therapeutic sera, since the type or group antigens and the corresponding antibodies are highly specific. The simultaneous injection of a variety of antigens intravenously into the horse is believed to result in a low titre for each antibody that is formed. An alternative procedure is to use separate groups of horses for each type, and to prepare a multivalent serum by mixing the type-specific sera when these have attained the requisite titre; this method has the disadvantage that the potency of each serum is weakened by the resulting dilution. The antigens that are used in the preparation of antibacterial sera give rise to a number of antibodies, for example, agglutinins, tropins and complement-fixing antibodies, which are titratable in vitro ; protective antibodies, whose relation to those just men- CONCENTRATION OF THERAPEUTIC SERA 5 tioned is stil obscure, are also formed and may be titrated by means of animal experiments. Our knowledge of the precise mode of action of antibacterial sera is incomplete, and accordingly a discussion of it would be out of place here. With some infections, such as plague and gas gangrene, it is likely that a combined serum which contains both antibacterial and antitoxic components will be found to be more effective than a simple serum that has been prepared with the object either of neutralizing the toxin or of limiting the invasive power of the infecting bacterium, Antiviral Sera. This is a branch of the subject which is only now beginning to receive attention. Ample evidence exists that immune-bodies make their appearance in the blood in the course of certain virus infections, such as measles and poliomyelitis, and that these immune-bodies can exert a prophylactic and perhaps even a curative action. It has proved to be possible to immunize animals with this kind of infective agent, and thereby to obtain a serum which is capable of modifying the symptoms of a natural or experimental infection in the same species, provided that an adequate dose of the serum is administered at an early stage of the illness. Reference to work on the subject of virus diseases and their specific prophylaxis and treatment will be found in Chapters XVI. (p. 165), XVII. (p. 177), XVIII. (p. 200), and XIX. (p. 215) The Concentration of Therapeutic Sera The purification and concentration of antitoxic sera are effected by adding appropriate concentrations of ammonium or sodium sulphate to the plasma or serum in order to separate the three serum proteins—the euglobulin, the pseudoglobulin and the albumin. The method depends upon the different solubilities of the protein fractions in varying concentrations of neutral salts. The bulk of the antitoxin is associated with the pseudoglobulin fraction, whereas the euglobulin and the albumin contain little or no antitoxin. In practice, it is found that by means of a process of this kind the number of antitoxin units in the final concentrated product may be from three to five times as many as those in an 6 PRODUCTION AND USE OF THERAPEUTIC SERA equal volume of the original plasma or serum. Other technical methods, such as electrodialysis, have been employed for separating the least soluble globulins, but difficulties inherent in these methods have prevented their general adoption. The aim of the maker of therapeutic sera is to obtain a product which combines a high concentration of antitoxin with a minimal amount of protein. The volume of fluid injected is thus lessened, and therefore also the degree of discomfort to the patient from the injection ; the risk, too, of symptoms referable to the introduction of a foreign protein is diminished. The ultimate aim of biochemical research on antitoxins is to isolate the antitoxin in as pure a state as possible, and free from all non-specific substances. The protective substance in antibacterial and antiviral sera is associated for the most part with the euglobulin fraction. Thus the specific antibody in anti-pneumococcus serum has been found by Felton to be associated with a somewhat narrow fraction of the globulins, and the purified product which is used for the treatment of pneumonia is obtained by separating this fraction from the remaining inactive proteins of the serum. The Titration of Therapeutic Sera When a serum has been prepared, it is necessary to estimate its strength by means of a method of titration which has been pre- scribed by the official control authorities. During the past ten years valuable service has been rendered by a Permanent Com- mission of the Health Organization of the League of Nations, which has co-ordinated the work of serologists in many countries with a view to the improvement of existing methods of standardiza- tion. A summary of the work accomplished by the Commission has been prepared by Professor Prausnitz (1929); the reader who desires detailed information on the subject should consult this account. The Official Control of Therapeutic Sera In most countries a Government control department exists which regulates the standards of purity and potency to which therapeutic STATISTICAL EVIDENCE OF VALUE OF SERA 7 sera must conform before they are released for issue. In Great Britain the Ministry of Health, with the co-operation of the Medical Research Council, exercises control in accordance with The Therapeutic Substances Regulations, 1931. The supervising authority in the United States has its headquarters in the National Institute of Health in Washington ; and its Director, Dr. G. W. McCoy, has described the work of this department in a recent article. Statistical Evidence of the Clinical Value of Therapeutic Sera When a serum passes into the hands of the clinician, evidence of its value, whether it be positive or negative, accumulates, and the collected experience of different observers, after it has been subjected to analysis, generally permits a conclusion to be drawn regarding the efficacy of the serum. This is not, however, always a simple matter, for the results may be difficult to interpret when they are expressed as a percentage fatality-rate in a serum-treated and in a control group. Consideration should be given to the number of cases in each group, since sampling errors are more likely to arise in small groups. The accuracy of a series of obser- vations is in proportion to the square of their number, so that, in order to double the accuracy, it is necessary to multiply the observations fourfold. When a comparison is sought between the fatality-rates of a treated and an untreated group of cases, the statistical test for significance may be applied by determining the standard error of the difference. The reader who desires to acquaint himself with the ideas which underlie statistical methods may consult the list of references that will be found at the end of this chapter; he may also read with profit a discussion by Greenwood (1913) on the methods by which a therapeutic problem ought to be investigated. The hypothetical question discussed by this writer is whether a certain treatment is efficacious in acute lobar pneumonia ; and the example is an apt one at the present time in connection with the serum treatment of this disease. The value of statistical methods for the analysis of experimental observations in animals is now gaining general 8 PRODUCTION AND USE OF THERAPEUTIC SERA recognition ; and although strictly controlled conditions in the treatment of human infections are not always easy to arrange, a knowledge of biometric methods will help the clinician to appre- ciate more fully the true significance of his observations. Serum Sickness, Serum Accidents, and other Complications of Serum Treatment Serum sickness or ¢ serum disease ” is the name that is given to the group of symptoms which follow the parenteral administration of a foreign serum, that is, its administration by routes other than the alimentary canal : the reactions are of moderate severity and never constitute any danger to life; it may be noted that the introduction of human serum, even by the intravenous route, is rarely followed by unpleasant symptoms. Serum accidents take the form of sudden and severe reactions in persons who are hyper- sensitive to serum; they are rarely fatal. Atopy (aromia, an unnatural state) is the term given by Coca to indicate certain heritable forms of hypersensitiveness such as asthma and hay fever that occur. as far as is known, only in human beings ; the group includes those persons who are highly sensitive to horse emanations and horse dander and who are prone to serum accidents. The terms * anaphylaxis ”* and * allergy ” are often applied to the complex of symptoms which are apt to appear in this type of person. The factors that bring about these undesirable symptoms —whether they be mild or severe—are bound up in part with the serum, irrespective of the specific antibody it may contain, and in part with the degree of sensitiveness of the recipient of the serum to the alien protein. Serum Sickness. The incidence of serum sickness has been lowered in consequence of the more extended use of concentrated sera. Rolleston (1929) states that in his hospital the introduction of concentrated diphtheria antitoxin has caused a considerable diminution in the incidence of skin rashes and other unpleasant sequelae ; urticaria is now almost the only symptom noted : it is often transient, limited to the site of injection, and not accom- panied by any general reaction, Ramon (1926) and Janvier (1926) COMPLICATIONS OF SERUM TREATMENT 9 have supplied data that lead to a similar conclusion. In contrast with this experience, Mackenzie and Hanger (1930), in a useful summary of the subject, mention that out of 100 consecutive patients with lobar pneumonia who were treated with uncon- centrated serum at the Presbyterian Hospital, New York, 93 had symptoms of serum sickness. In the treatment of pneumonia the doses are often large and are given, in the United States at least, by the intravenous route. Von Pirquet and Schick (cited by Kleinschmidt) used 100-200 c.cm. of Moser’s serum —presumably whole serum-—for the treatment of scarlet fever in Vienna, with the result that more than 85 per cent. of their patients had symptoms due to the serum. In the Blegdams Fever Hospital in Copenhagen 55:6 per cent. of the diphtheria patients showed signs of serum sickness ; apparently a natural serum was used and large doses—200,000-300,000 units—were given to about 15 per cent. of the patients, namely, those who were suffering from a severe form of diphtheria (Hecksher, 1926). The experience of the writer just cited is that the incidence and severity of serum sickness increase with the age of the patient, and that its frequency is less after intravenous than after intramuscular injections. The serum of some horses is especially liable to produce reactions, but the reason for this has not vet been discovered ; it is not practicable to test each horse before immunization with a view to its dismissal if it should prove to possess a * toxic > serum. Dale and Hartley (1916) found that a distinction can be made by means of anaphylactic experiments between the three proteins that are separable from natural horse serum. Thus a guinea-pig which had received a preparatory injection of the pure euglobulin became sensitive to it but remained unaffected by the pure albumin, and conversely. When a small preliminary injection of the whole serum was given to the animal, the symp- toms of sensitiveness to euglobulin appeared first—in 8 to 10 days —and to albumin only after an interval of 16 to 20 days. In the same way the euglobulin and the pseudoglobulin could be clearly distinguished. Clinical experience has afforded similar observa- tions, for, as Dale has pointed out, there are numerous records of rashes which have recurred at intervals of a few days after a 10 PRODUCTION AND USE OF THERAPEUTIC SERA single injection of horse serum had been given, but there is no record of more than three such rashes in one patient ; the explana- tion is obvious in the light of the experimental work on the three serum fractions. Recurring rashes are not seen when purified antitoxin is used. Serum Accidents. Among those who are liable to serum acci- dents there is a group of patients who, when serum is administered to them for the first time, react almost at once with severe symptoms, even when the dose is small and given subcutaneously. Some of them are ‘ horse-asthmatics,” who are highly sensitive to horses or horse dander, and who are probably carriers of an hereditary allergic factor. Another group is composed of those who have been sensitized by a previous injection of serum. In 1924 Lamson brought together from the literature 41 reports of sudden death after an injection of serum: in only eight of the cases was there a history of a previous injection. From about 34 per cent. of the patients a history of asthma or hay fever was obtained, and it was ascertained that in several of them the allergic symptoms were made worse by proximity to horses. In practice, the risk of death from allergic shock is a slight one. Thus Park (1928) has estimated that alarming symptoms appear in only one out of 20,000 patients, and that a fatal result occurs only once in 50,000 times. Gaffky searched the world literature of ten years and found a reference to severe illness after the first injection in seven cases; these included three deaths. In an analysis by Pfaundler (cited by Schick) there were only three deaths among one million persons who had received serum treat- ment. There is general agreement that, when serum is injected intravenously, the risk is very great if the patient has a history of asthma or sensitiveness to horses ; and that it is considerable if he has had serum given to him, by whatever route, on a previous occasion. In Coca’s opinion the physician should not administer serum or even attempt to desensitize a patient who is known to be a ** horse-asthmatic ”’ subject, but, on the other hand, he may give an intravenous injection to patients who are already sensitized, but in whom there is no history of allergy, if their condition urgently demands it. Park (1924) affirms that an immediate COMPLICATIONS OF SERUM TREATMENT 11 intravenous injection is justified in every case of severe diphtheria, without waiting for the result of an intracutaneous test, unless there are signs of “status lymphaticus or a history of asthma has been obtained ; and he emphasizes the fact that almost all of the few deaths that have resulted from the administration of antitoxin followed the first injection. The article of Mackenzie and Hanger, already referred to, gives an admirable account of the subject from the point of view of the clinician, and describes the precautionary measures which should be taken when there is reason to suspect danger. Thus an intracutaneous injection of 0-05 c.cm. of a tenfold dilution of serum in saline may be given. If, after 10 to 20 minutes, the resulting wheal has increased in size and shows a zone of erythema, the reaction should be regarded as positive. The extent of the reaction is a rough measure of the hypersensitiveness, so that a wheal with pseudopodial projections is significant of the condition in an extreme degree. A conjunctival test may be done, but it is not considered advisable, on account of the risk of damage to the cornea if the patient should chance to be hypersensitive. Nevertheless, the usual practice in the United States at the present time is to use the conjunctival test in preference to the intra- cutaneous test, which is regarded as being too sensitive. A drop of a tenfold dilution of normal horse serum in saline is introduced into one eye ; if after 15 to 20 minutes there is no conjunctival redness as compared with the other eye, the serum may be given intravenously, whereas a positive reaction is held to contra- indicate the intravenous procedure. If the conjunctival reaction should be excessive, it can be controlled by the instillation of adrenaline solution. A comparison between the conjunctival and the intracutaneous test has been made by Claiborn (1932) in 465 persons, who afterwards received a prophylactic injection of 1,500 U S.A. units of tetanus antitoxin intramuscularly ; 5-6 per cent. of these gave positive reactions in both tests, and 21-3 per cent. gave a positive reaction only in the skin test. The symp- toms of the ordinary delayed serum sickness were noted in approximately the same proportion (14-18 per cent.) of patients with both tests positive; with the conjunctival test negative 12 PRODUCTION AND USE OF THERAPEUTIC SERA and the skin test positive ; and with both tests negative. Even when the skin reaction is absent precautions should still be taken ; on the other hand, according to Park, in a patient with a moderate skin reaction only slight allergic symptoms may follow. Banks (1933) in an article on the treatment of scarlet fever by the intravenous administration of the specific antitoxin gives an account of the reactions that may occur and the means he adopts to prevent or at least minimize them. His experience includes the intravenous treatment of 1,204 patients with scarlet fever and of many cases of diphtheria. When the indications enjoin caution, an attempt should be made to desensitize the patient before giving the greater part of the dose. The method of desensitization is based on: (1) partition of the dose, and (2) slow administration of the serum. If, then, the skin test is positive, it is advisable to give not more than 0-01 c.cm. of the serum subcutancously and to double this amount every 30 minutes till 1 c.em. in all is given. After a lapse of 30 minutes, 0-1 c.cm. should be given intravenously and then the amount is doubled every 20 minutes until the full dose is administered. If allergic symptoms appear during the intravenous administration, the previous dose should be repeated. The intravenous doses may be diluted with warm saline and should be given very slowly. A hypodermic injection of tlgth—rfsth grain (0-4-0-6 mgm.) of atropine may be administered beforehand, and a solution (1 in 1,000) of adrenaline chloride (epinephrine) should be kept ready for use if symptoms should appear, when 5-10 minims (0-3-0-6 c.cm.) may be given, and repeated if it is thought to be necessary. In the event of the onset of grave symptoms, it is worth remember- ing the stimulant action of 1 c.em. of adrenaline solution when it is injected directly into the heart. After iodine has been applied to the skin the needle should be inserted at the upper margin of the fifth rib just to the left of the sternum ; when the posterior edge of the sternum is reached the point is turned toward the midline and pushed into the heart. If artificial respiration is being practised, the needle should be inserted during the expiratory phase (Fantus, 1926). Experimental work by Sharpey-Schafer and Bain (1932) supports the clinical evidence, and confirms the MECHANISM OF SERUM SENSITIVENESS 13 value of intracardiac injections of adrenaline in restoring the cardiac contractions after they have been arrested by the induction of asphyxia. A number of physicians have reported an increase in the incidence and severity of serum reactions in patients who had previously received toxin-antitoxin immunization (Hooker ; Stewart; Gate- wood and Baldridge, 1927 ; Gordon and Creswell, 1929). Park, however, is not disposed to attach undue weight to the possibility that this method of prophylaxis has a sensitizing influence, and Spicer (1928) has given an account of observations, which she made in the Willard Parker Hospital, New York, that support his belief. The trend of immunizing procedures at the present time is towards the use of a simple formolized toxoid solution with- out the addition of diphtheria antitoxin, so that it seems likely that this complication of serum therapy will disappear. The Mechanism of Serum Sensitiveness. Research that has been carried out within the past decade has thrown light upon the nature of the anaphylactic reaction. Excellent summaries of this work are available (Dale; Scott; Bray ; Coca; Wells; Karsner; and Doerr), and therefore only the briefest statement need be added. According to the modern view, anaphylactic shock is the result of cellular injury brought about by the intracellular reaction of the antigen with a precipitating antibody" which is contained in the cell protoplasm (Dale, 1920 and 1929). As an immediate consequence histamine, which is a normal constituent of the tissues, is released, and its effects are added to those of other constituents which are liberated at the same time from the injured cells. This theory assumes a close relation, amounting indeed to identity, between the anaphylactic antibody and the precipitins of immune-sera. In man precipitins appear in the blood between the ninth and the twenty-first day after the injection of horse serum. Insusceptible persons do not form precipitins, and in them the serum continues to circulate in the tissues for many weeks and perhaps months, whereas susceptible persons rapidly excrete the serum (Mackenzie and Hanger). Febrile Reactions after Intravenous Injections of Serum. The so-called thermal reaction” is frequently mentioned in con- 14 PRODUCTION AND USE OF THERAPEUTIC SERA nection with the administration of anti-pneumococcus serum. This type of reaction generally occurs about one hour after an intravenous dose has been given, and it is characterized by a rigor and an increase in the cyanosis ; profuse sweating and a fall in the temperature follow : these symptoms are not likely to be serious. The question of the nature of the rigor-producing substance in concentrated preparations of anti-pneumococcus serum has been investigated by Felton and Kauffmann (1931) and by Sabin and Wallace (1931). There is agreement among these workers that the phospholipin content of the serum preparation is probably not a factor in the production of febrile reactions ; that rigor-producing preparations contain a protein which is precipitable at a pH value of about 5:0 in N/20 salt solution ; and that good preparations contain but little of this protein. Felton and Kauffmann have observed that preparations derived from grossly contaminated serum always cause febrile reactions, and they express the opinion that the unavoidable introduction of bacterial protein during the processes of concentration increases the difficulty of obtaining a satisfactory product. Here it may be mentioned that, whenever it is considered advisable to dilute a serum preparation with normal saline before administering it intravenously, care should be taken that the saline solution is made with sterile, freshly- prepared distilled water, in order to ensure its freedom from rigor- producing substances of bacterial origin. Neurological Complications of Serum Treatment. During the past few years increasing attention has been paid to certain neurological complications of serum treatment which, however, seem fortunately to be rare, for J. D. Rolleston states that he has never seen a case, and probably less than 100 cases have been reported. A good review of the subject has been given by Allen (1931), who had the opportunity of making observations on a patient whose symptoms appeared after he had received a dose of 10 c.cm. of scarlet fever antitoxin. This author classifies the complications into four groups: (1) a radicular type, resembl- ing an Erb-Duchenne paralysis of acute onset; (2) a neuritic type, in which single nerve trunks are affected ; (3) a polyneuritic type, in which the clinical condition resembles that of a toxic NEUROLOGICAL COMPLICATIONS 15 polyneuritis ; and (4) a cerebral type, in which the prominent features are perhaps referable to intracranial cedema. The condition has most often followed the use of tetanus antitoxin, and in the majority of the cases the symptoms have appeared a few days after an attack of serum sickness. The patient complains of intermittent stabbing pains in one or more segmental areas on one or both sides, or in the limbs and joints, or in the distribution of a peripheral nerve. Muscular weakness and wasting follow and may persist for many months. With regard to the pathogenesis of the condition, Allen points out that its appearance from two to five days after the onset of serum sickness, at a time when the urticaria and the concomitant meningeal reaction are most pronounced, strongly suggests that the origin of the condition is an cedema of the nerve roots or of the meninges. The prognosis is, in general, good. Observations on this complication have been made by physicians in the United States, and an account of them will be found in the articles by Wilson and Hadden (1932), Young (1932), and Gordon (1932). The intrathecal injection of a therapeutic serum derived from the horse may cause a localised aseptic meningitis, which is accom- panied by a rise of temperature and sometimes by signs of meningeal irritation ; the symptoms that are already present are aggravated, but improvement sets in after a short time. Stone refers to a few cases of serous meningitis that followed intravenous injections of tetanus antitoxin ; the spinal symptoms were inter- preted as an allergic reaction caused by the serum, for the cerebro- spinal fluid was found to be under pressure, and relief, as a rule, quickly followed lumbar puncture. REFERENCES ALLEN, I. M. 1931. Lancet, 2, 1128. Banks, H. S. 1933. J. of Hyg., 33, 282. Bray, G. W. 1931. “ Recent Advances in Allergy.” London. CELAREK, J., and Poresskr, W. 1928. C. R. Soc. Biol., 99, 1017. CraiBorN, L. N. 1932. J. Amer. Med. Ass., 98, 1718. Coca, A. F. 1928. * The Newer Knowledge of Bacteriology,” p. 1011. " 1931. ¢ Asthma and Hay Fever.” London. Pp. 3-114. 16 PRODUCTION AND USE OF THERAPEUTIC SERA Dare, H. H. 1920. Proc. Roy. Soc., B.91, 126. 1922. Brit. Med. J.,1, 45. ss 1929. Lancet, 1, 1179, 1233, 1285. Dave, H. H., and Harrrey, P. 1916. Biochem. J., 10, 408. Doerr, R. 1929. * Handb. der path. Mikroorg.” (Kolle, Kraus u. Uhlenhuth), 1, 918. Fanrus, B. 1926. J. Amer. Med. Ass., 87, 564. Fevron, L. D., and KAurrMaNN, G. 1931. J. Inf. Dis., 49, 337. Garewoop, W. E., and BaLpripee, C. W. 1927. J. Amer. Med. Ass., 88, 1068. GLENNY, A. T. 1925. J. Hygiene, 24, 301. % 1930. Brit. Med. J., 2, 244. GorpoN, A. 1932. J. Amer. Med. Ass., 98, 1625. GorpoN, J. E., and CRESWELL, S. M. 1929. J. Prev. Med., 3, 21. GREENWOOD, M. 1913. Lancet, 1, Jan. 18th. Harwrrrey, P., and Hartoey, O. 1922. J. Path. and Bact., 25, 458. Heckscurr, H. 1926. Ugeskrift f. Laeger, 88, 1167, 1169. "5 1926. Acta Med. Scand., 64, 505. Hooker, S. B. 1924. J. Immunol., 9, 7. JANVIER, LI. 1926. Thése de Paris, Ref. Brit. Med. J. Epit., Oct. 2nd, 1926. Karsner, H. T. 1928. “The Newer Knowledge of Bacteriology,” p. 966. Kreinscumor, I. H. 1926. * Hand. der Exp. Ther. Serum u. Chemother.,” ed. Wolff-Eisner, p. 209. Lamson, R. W. 1924. J. Amer. Med. Ass., 82, 1091. Mackenzie, G. M., and Hanger, F. M. 1930. J. Amer. Med. Ass., 94, 260. McCoy, G. W. 1928. “The Newer Knowledge of Bacteriology,” p. 947. Park, W. H. 1921. J. Amer. Med. Ass., 76, 109. 1924. J. Immunol., 9, 17. ’ 1928. Amer. J. Pub. Hlth., 18, 354. Praunprer. Cited by Schick, B., 1930, Kinderarzt. Praxis, Sept., p- 12. Por, C. G., and Hearry, M. 1933. Brit. J. Exp. Path., 14, 77. iy ”" 1933. Brit. J. Exp. Path., 14, 87. Prauvs~irz, C. 1929. Mem. on the Internat. Standardization of Therapeutic Sera and Bacterial Products, Geneva. Ramon, G. 1926. Presse Méd., 1, 323. Rorreston, J. D. 1929. ** Acute Infectious Diseases.” London. SaBIN, A. B., and Warrace, G. B. 1931. J. Exp. Med., 53, 339. Scorr, W. M. 1931. “A System of Bacteriology.” London, H.M. Stationery Office, 6, 457. SHARPEY-SCHAFER, E., and Bain, W. A. 1981-1932. Proc. Roy. Soc. Edin., 52, 139. SorpELLI, A. 1920. Cited by Glenny, A. T., 1925. J. Hyg., 24, 301. SPICER, S. 1928. J. Amer. Med. Ass., 90, 1778. STEWART, C. A. 1926. J. Amer. Med. Ass., 86, 113. Stone, W. J. 1922. J. Amer. Med. Ass., 78, 1939. ’ ’ REFERENCES 17 WerLs, H. Gipeon. 1929. * The Chemical Aspects of Immunity,” New York. WerNICKE, E., and Scamipr, H. 1928. * Handb. der path. Mikroorg.” (Kolle, Kraus u. Uhlenhuth), 5, 525. WiLson, G., and HApDDEN, S. B. 1932. J. Amer. Med. Ass., 98, 123. Youna, F. 1932. J. Amer. Med. Ass., 98, 1139. Statistical Methods DunN, HaLBerT L. 1929. Physiological Reviews, 9, 275. Prarr, R. 1930. °° Biometry and Vital Statistics in relation to the Science of Medicine.” Philadelphia and London. Woobs, H. M., and Russerr, W. T. ‘ An Introduction to Medical Statistics.” London. 1931. Yure, G. Ubp~xy. “An Introduction to the Theory of Statistics.” London. 1929. CHAPTER 1I THE SERUM TREATMENT OF DIPHTHERIA PAGE THE SERUM TREATMENT OF MALIGNANT DIPHTHERIA . 18 The Intensive Treatment of Malignant Diphtheria by the Intravenous Administration of Antitoxin. " . 24 Tae PROBLEM OF AVIDITY OF DIPHTHERIA ANTITOXIN : . 28 Tue MODES OF ADMINISTRATION OF THE ANTITOXIN . 30 THE IMPORTANCE OF THE KARLY ADMINISTRATION OF THE "ANTI- TOXIN ’ 31 THe EMPLOYMENT OF AN TITOXIN IN DIPHTHERITIC PAR ALYSIS ’ 32 THE THERAPEUTIC EMPLOYMENT OF AN ANTIBACTERIAL SERUM . 33 Tue Proruvracric Use or DIPHTHERIA ANTITOXIN. ’ ’ 34 The Serum Treatment of Malignant Diphtheria THE occurrence of an unusually high incidence of the severe type of diphtheria has been reported in recent years from various countries, and especially from Germany. The increased incidence of this type was observed about the same time, namely, in the latter half of the year 1926, both in Europe and in the United States. The reports on the subject are mainly concerned with large cities—for example, Berlin, Vienna, Budapest, Paris, Marseilles, Padua, Barcelona and Detroit ; the available informa- tion indicates that the rural districts of Germany and France have a normal death-rate. When the disease assumes a septic or highly toxic form, it is characterized by swelling of the glands in the neck, at times of such a degree as to cause respiratory embarrassment ; by damage to the kidneys in varying amount ; and, in the worst cases, by haemorrhagic manifestations. Faucial diphtheria, associated frequently with extension of the disease to the nasal mucous membrane, has been the rule; the incidence of the laryngeal type is low. In Berlin the mortality has been particu- larly high in school children, whereas formerly it was highest in infants. Deicher and Agulnik (1927) state that the mortality from the disease in the Rudolf Virchow hospital in Berlin was 17-4 per cent. in 1926 as compared with 6:1 per cent. in 1923. 18 MALIGNANT DIPHTHERIA 19 In a critical article on the enhanced lethality of diphtheria in recent years, Wolff (1928) utters a warning against drawing conclusions from hospital statistics without first submitting the numerical data to the usual statistical methods for testing the significance of differences in mortality-rates (the standard error ratio) ; he points out that hospital statistics are vitiated to some extent by the circumstance that they do not represent an unselected group, since mild cases of the disease are not always sent to hospital : statistical methods are applicable only to wholly random groups. The apparently fortuitous increase of malignant diphtheria is not easy to explain, but its simultaneous and widespread occurrence is in favour of a common factor which affects the diphtheria bacillus rather than its host. Some seek for an explanation of periodic outbreaks of the severe form of the disease in cyclical variations in its infectivity ; this hypothesis may be true, but it is not based on indisputable evidence. Opinions differ both with regard to the cause of the malignant form and the efficacy of serum treatment in patients who suffer from it. A conjecture which has been much discussed, especially in Germany, is that the association of streptococci with the diphtheria bacillus in the throats of these patients accounts for the severity of the symptoms. Canon (1927) obtained evidence of participation by the streptococcus while he was in charge of the diphtheria wards in the Moabit Hospital in Berlin in 1892-1893 during an epidemic of grave diphtheria; he isolated a streptococcus from the blood of 12 out of 18 cases of the septic form. Again, Finkelstein and Kénigsberger (1928) and F. Meyer (1928) found in 50 per cent. of their cases a hemolytic strepto- coccus in the throat, which by means of its toxin caused local inflammatory and necrotic changes, anaemia, and myocarditis. They therefore treated every case of severe diphtheria at the earliest possible moment with a streptococcus antitoxin, in addition to diphtheria antitoxin. They assert that the results from the double serum treatment were better, but at the same time admit that the haemorrhagic type of case was uninfluenced by the treat- ment, and that diphtheritic paralysis, especially of the diaphragm, 20 THE SERUM TREATMENT OF DIPHTHERIA occurred in spite of it. Von Bormann (1931) also advocates the use of a combined diphtheria and streptococcus antitoxin as a means of preventing the onset of septic symptoms; the dose should be administered as early as possible because, if invasion by the streptococcus has begun, serum treatment has only a slight effect. The theory of a streptococcal association is without doubt a plausible one, yet the bulk of the evidence, which includes the experience of some highly trained clinical observers, does not confirm belief in the value of the combined serum treat- ment. Moreover, it is certain that there are grave cases which owe nothing of their malignant character to a combined infection. Thus, out of 20 cases under Deicher’s care that were examined post mortem, although a streptococcus was found in the organs in nine of them, the clinical signs that preceded death were those of diphtheria and not of streptococcal sepsis ; indeed, in the remain- ing 11 cases in which there was no evidence of streptococcal invasion, treatment with diphtheria antitoxin failed to prevent a fatal issue (Schmidt, 1927). H. Schmidt (1927), in reviewing the problem of the serum treatment of malignant diphtheria, gives the results of his own experiments. He isolated strains of the diphtheria bacillus and a variety of associated bacteria from the throat swabs taken from nine cases of the disease in hospitals in Berlin. The freshly isolated diphtheria strains proved to be highly virulent when they were inoculated into guinea-pigs, but the virulence was not raised by simultaneous inoculation with any of the associated bacteria. The diphtheria strains were not specially toxigenic, and symbiosis in culture with the associated bacteria did not bring about any increase in toxin production. Some workers think that a symbiosis in artificial culture of the diphtheria bacillus with a streptococcal strain is favourable to the formation of diphtheria toxin, but others, including Schmidt, have not been able to confirm this belief. A conceivable reason for the alleged failure of serum treatment in malignant diphtheria is that the toxin formed in these cases is not neutralized by the ordinary commercial antitoxins, which in most laboratories are produced by employing the toxin derived from the No. 8 strain of the bacillus isolated many years ago by MALIGNANT DIPHTHERIA 21 Park and Williams. Recent work has shown that strains of the diphtheria bacillus are divisible into a number of serological groups, and the idea has been entertained that, corresponding to these groups, there are a number of closely related toxins which differ from each other sufficiently to cause incomplete neutraliza- tion of any one toxin by an antitoxin prepared from any of the other toxins (Havens, 1920). But the available evidence is altogether against the view that diphtheria toxin derived from different strains has variable antigenic properties. Hartley (1923) investigated the toxins produced by eight strains that were representative of the three serological types into which Major Bell had separated 130 cultures of C. diphtherie, mostly obtained in the London area. He could not find that any of those strains produced a toxin which required more than the usual amount of antitoxin for its neutralization. His conclusion is in agreement with that reached by other workers, for example, Park, Williams, and Mann (1922). As already mentioned, Schmidt found that the strains of C. diphtherice which he isolated from the Berlin cases possessed considerable virulence for the guinea-pig. Feierabend and Schubert (1930), working in Prague, examined strains from malignant cases and especially from those in which serum treat- ment appeared to fail. They were unable to discover any qualita- tive differences from the normal type, but thought that there was a quantitative difference, not in regard to the toxigenicity in vitro of the cultures, but in regard to their virulence, as judged by experimental results in susceptible animals. Concordant evidence from pathological studies of the human disease is contained in the statement by Deicher and Agulnik, and also by Simic (1931), that C. diphtherie is present in the organs, and especially the lungs, in a large proportion of the cases. Since the virulence of cultures, as estimated by the method of inoculation into guinea-pigs, measures the sum of two qualities : (1) the invasive power of the bacillus, and (2) its ability to produce toxin in vivo, it is likely that an influential factor in the etiology of malignant diphtheria is the aptitude of the infecting strain for invading the tissues of the host, with the consequent liberation of exceptionally large amounts of 22 THE SERUM TREATMENT OF DIPHTHERIA toxin. This represents the view adopted by Kolle (1930), who considers that it explains the apparent failure of the antitoxin, even when it has been given on the first day of the disease ; a concomitant streptococcal infection is regarded by him as an occasional etiological fa-tor. Lignieres (1930) has suggested that in the refractory type of case the diminished efficacy of diphtheria antitoxin, in spite of the administration of larger doses than were formerly given, may be due to the almost universal use of the highly toxigenic Park- Williams No. 8 strain in the preparation of the serum. He thinks that the loss of virulence which this strain has sustained owing to prolonged cultivation in artificial media has rendered it less suitable for use as an antigen than a freshly isolated strain with respect to the production of an antibacterial serum. In his view the bactericidal property of the serum should be kept in mind when treating the severe form of the disease. An interesting paper by Anderson, Happold, McLeod, and Thomson contains a description of two main types of the diphtheria bacillus, to which the authors have applied the terms gravis »’ and ““ mitis 7 ; their work is the outcome of observations on the disease in Leeds. They believe that the occurrence of the * gravis” strain is correlated with severe and intractable cases of diphtheria, and that the mitis > strain is associated with the milder cases, which respond to serum treatment. C. diphtheric gravis grows with a pellicle and a granular deposit in broth ; forms a flattened, lustreless colony of irregular outline; and actively ferments polysaccharides. In contrast with these characters, C. diphtherie mitis grows with uniform turbidity in broth; forms a convex, partly translucent and light-reflecting colony; and does not ferment polysaccharides. Intermediate forms, of which Park-Williams No. 8 is an example, were also found. The experience of Parish, Whatley and O’Brien has been that the * mitis ”* strains are as pathogenic to the guinea-pig as the “ gravis strains, and that the former are the better toxin producers. These workers have not observed any close relation between the distribution of *“ gravis 7 strains and the incidence of severe and fatal diphtheria, and they suggest that the correlation which was noted by Anderson MALIGNANT DIPHTHERIA 23 and his colleagues in Leeds may be of local significance. Wright and Rankin (1932) examined 50 cases of diphtheria in Edinburgh, but were unable to place the strains they isolated from the patients in any rigid scheme of classification. They could find no grounds for believing that the type of the disease is correlated with the type of colony of the infecting strain, and that a particular strain is associated with an apparently poor response to serum treatment. If the original thesis receives support from further experience, especially in communities that show a high incidence of the malignant form of diphtheria, it will afford a simple explanation of a difficult problem. Even if the theory of an enhanced invasive power of the bacillus should be accepted, contributory factors may play a part in the causation of severe diphtheria. French observers ascribe an important rdle to concomitant diseases, especially measles, chicken-pox, and influenza, as well as to associated streptococcal and pneumococcal infections. Schmidt, too, refers to the harmful effects of repeated influenzal attacks upon the nervous system of under-fed children who are reared in unhygienic homes. A share in the alleged failure of the serum treatment of diphtheria in Germany has been attributed to the high cost of the antitoxin, and to the prejudice of the relatives against the injection of serum. The apparent failure of diphtheria antitoxin in a particular case may be due, as Dold (1927) has pointed out, to the circumstance that a mistaken interpretation may be attached to the presence of diphtheria bacilli in febrile anginas when staphylococci and streptococci are also present. In such an event the practitioner naturally makes a diagnosis of diphtheria, and yet the disease may really be a coccal angina in which C. diphtherie is deprived of its pathogenic action owing to the patient possessing a natural or acquired antitoxic immunity. It is obvious that diphtheria antitoxin will exert a partial influence upon a mixed coccal and diphtheritic infection ; only in a pure diphtheritic infection can it be expected to exert its full effect. In the experience of some physicians the early administration of even large doses of serum has not always been successful in 24 THE SERUM TREATMENT OF DIPHTHERIA mitigating the symptoms of highly toxic diphtheria, or in saving the life of the patient. For example, Friedberger went so far as to discredit the value of diphtheria antitoxin, and mentioned that in August, 1927, the mortality in the Rudolf Virchow Hospital in Berlin was 86-3 per cent., although early and apparently adequate serum treatment was given. This point of view is opposed by such a mass of evidence that we regard it as unduly pessimistic, and prefer to lay stress upon the general belief that the prognosis becomes worse in proportion to the delay in administering the serum. Recent work has shown that in the febrile stage of diphtheria the carbohydrate metabolism is profoundly disturbed as the result of the toxwmia, and that the injection of insulin, as first suggested by Hector, when controlled by blood-sugar estimations, is a valuable supplementary method of treatment (Schwentker and Noel ; Hector). Benn, Hughes, and Alstead (1932) have published their observations on the method when it is employed in combina- tion with antitoxin. The fatality-rate of 89 consecutive toxic cases which were treated by the combined method was 22-5 per cent., whereas 35-9 per cent. of 131 patients who were treated with antitoxin alone, died ; the difference is probably significant and not due merely to chance. Woodcock (1932) treated 25 cases of malignant diphtheria with insulin, and gained the impression that the patients experienced relief from the physical distress which is a feature of this type of the disease. The reader who is interested in the subject of malignant diphtheria should read the report (1931) of the discussion at the Berlin Medical Society, in which Friedberger and others took part. He will find, too, a considerable body of evidence in the collection of papers by various workers in European countries which appeared in the May issue of Annales de Médecine (1931). The Intensive Treatment of Malignant Diphtheria by the Intra- venous Administration of Antitoxin. Clinicians are not yet in agreement upon a scheme of dosage, as estimated in antitoxic units, for the different types of the disease. The reason, doubt- less, is that a number of factors have to be considered ; for example, the age of the patient, the degree of severity of the INTENSIVE SERUM TREATMENT 25 illness, and the interval elapsing between the onset of the disease and the time of administration of the serum. Useful guides to dosage are those provided by Park in his Table of Dosage and the table drawn up by F. Thomson. The scale of doses recommended by Rolleston (1928) is similar. Since the year 1920 at the Blegdams Hospital in Copenhagen, Bie has treated the severe type of case with doses of serum that are considerably in excess of those usually employed, and has chosen the intravenous route for its administration. In his first paper (1922) he concluded that the use of very large doses of antitoxin removed altogether the risk of death from respiratory paralysis, and reduced the mortality of the severe cases to about one-half. An article by Hecksher (1926) shows that of 4,819 patients who were treated in the Blegdams Hospital for faucial and nasal diphtheria in the period from 1921 to 1925, there were only 57 deaths, giving a death-rate of 1-2 per cent., although this material included more than 10 per cent. of the malignant type of case ; in these figures 11 deaths that occurred within 24 hours after admission to hospital are not included. A later paper by Bie analyses the results he obtained during the five months from September 1st, 1928, to January 31st, 1929. In this period there were 1,113 cases, of which 62 per cent. were mild, 28 per cent. moderately severe, and 10 per cent. were of the severe type; cases of croup, nasal diphtheria, and carriers are not included in the series. Of the 1,113 cases 23 died, giving a death-rate of 2:07 per cent., and if five patients who died within the first 24 hours after admission are excluded, the rate becomes 1:62 per cent. There were no deaths among the 687 mild cases in this series ; 0-96 per cent. of the moderately severe cases died ; and 14:15 per cent. of the severe cases died. In a group comprising 1,159 cases of faucial and laryngeal diphtheria the death-rate was 2-8 per cent. In order to lessen the risk from protein shock when the serum is given intravenously, Bie limits the maximum amount of the serum for adults to 50 c.em.; and for children to 1 c.cm. per kilogramme of body-weight. If the antitoxin content of the serum 26 THE SERUM TREATMENT OF DIPHTHERIA is such that the requisite number of units are not provided by this scale of dosage, the remainder is given by the intramuscular route. The serum is an unconcentrated one, which is supplied by the Danish State Serum Institute, and the scheme of dosage is as follows :— (1) Mild cases : 0-16,000—60,000 units. (2) Moderate cases: 32,000-48,000-96,000 units ; if thought necessary, the dose is repeated on the next day. (3) Severe cases : 80,000—-100,000—200,000 units ; 100,000 units are sometimes given on the next day. (4) The very severe cases in older children and adults receive a dose of between 300,000 and 500,000 units. Banks and McCracken (1928) have published the results of similar treatment carried out by them in the City of Leicester Isolation Hospital during the twelve months April 1st, 1927, to March 31st, 1928. The death-rate during the eleven years 1916-1926 was 9-3 per cent., the lowest in any year being 7-3 per cent. ; their analysis shows that in contrast with these ficures only 2-6 per cent. of 300 patients treated by the intensive method died, and this rate is reduced to 1-7 per cent. if laryngeal cases and patients who died within 12 hours of admission are excluded. In this series the disease was of the highly toxic type in 9 per cent. of the patients. Every patient who was admitted to hospital in a grave condition received 70,000 to 200,000 units, administered both intravenously and intramuscularly—generally in doses of 50,000 units intravenously and 20,000 units intramuscularly. The dose was repeated at discretion after about 12 hours ; thus, if the mental condition of the patient remained stuporous, a second intravenous dose was given. In the group of moderately toxic cases 20,000 units were given intravenously and 20,000 units intramuscularly ; and in that of the mild and moderately severe cases 8,000-16,000 units were administered intramuscularly. These authors now aim at giving by the intravenous route the whole of the dose that they judge to be adequate for each case, thus saving time by reducing the number of the injections and obviating the discomfort caused by large intramuscular injections. They believe that this method lessens the risk of complications and INTENSIVE SERUM TREATMENT 27 particularly that of the more serious forms of paralysis ; moreover, the average stay in hospital of the patients who recover is shor- tened, a circumstance which helps to offset the additional cost of the serum. They suggest, too, that the treatment diminishes the incidence of carriers. A disadvantage of the method is the risk to life from the allergic reactions which may follow the intravenous administration of the serum. This risk should not be accepted lightly nor, on the other hand, should it be exaggerated, because the benefits that have been conferred by the intravenous method probably more than compensate for the few serious accidents that have been imputed to it. Certainly the experience with it in Copenhagen and in Leicester must be regarded as an encouraging one in this respect, since only one death can be directly attributed to the serum (Hecksher, 1926). In spite of these favourable results, there seems to be little disposition on the part of other physicians to adopt the method, and, indeed, some authorities do not agree that very large doses are necessary. Park (1921) mentions the amount of 50,000 units as the maximum needed in any circumstances, and Goodall (1928) regards an amount of antitoxin exceeding 30,000 units as wasted, even when it is spread over two or three days. A comparison of the case-mortality in two hospitals in Hamburg was made by Reiche and Reye (1930). In one of the hospitals from 42,000 to 72,000 units of antitoxin were given, and in the other the dosage was 9,000 to 27,000 units; the death-rate was not significantly different. Evidence of a similar kind has been adduced by Lichtenstein in an analysis of the records from 1900 to 1929 of the Hospital for Infectious Diseases in Stockholm. During the early years of this period the average dosage was 5,000 to 10,000 units, as compared with 100,000 to 200,000 units in the period 1924-1929. The death-rate was not appreciably lessened by the increased dosage. The only additional evidence of which the present writer is aware that supports the value of the inten- sive method of serum treatment in diphtheria by the intra- venous route is that given by Harries and MacFarlane (1928), as the result of their experience with it in the City Hospital, Birmingham. 28 THE SERUM TREATMENT OF DIPHTHERIA The Problem of Avidity of Diphtheria Antitoxin Roux (1900) was the first to suggest that the antitoxin content of a serum, as measured by Ehrlich’s method, does not always correspond to its curative value when tested in a guinea-pig against a virulent culture of C. diphtherice. ¥rom the year 1908 onwards Kraus and his colleagues have from time to time advanced considerations in support of this view. But it is the flocculation method introduced some years ago by Ramon, whereby antitoxic sera can be titrated by noting the time of formation of toxin- antitoxin floccules in a series of tubes containing varying amounts of serum with a constant quantity of toxin, that has given a fresh impulse to research into the avidity problem. Ramon demon- strated that the tube which first shows flocculation contains toxin and antitoxin in a proportion which corresponds to a neutralized mixture for the guinea-pig. He observed also that sera taken from different horses flocculate at different rates, some in less than one hour and others only after several hours. Madsen and his co-workers have published a number of papers on the subject, in which they give experimental evidence for the existence of a relation between the rate of flocculation of different sera and their power of protecting rabbits against a lethal dose of diphtheria toxin. They believe that the therapeutic effect of sera of equal antitoxic value depends upon the speed of flocculation which is characteristic of each serum. Thus a quickly flocculating serum, that is, one with a high avidity for toxin, may save the test animal from a fatal toxamia, whereas a slowly flocculating serum may fail to protect it in conditions that are otherwise comparable. With these observations in view, Madsen and his colleagues suggest that therapeutic sera should possess not only the requisite amount of antitoxin, but also an energetic avidity for toxin. They point out that the methods employed for the concentration and purification of diphtheria antitoxin entail a risk of diminishing this quality, and it is certainly true that concentrated sera are sometimes difficult to titrate by the Ramon method, because they flocculate poorly ; it is necessary to add to them a proportion of a natural serum in order to bring about flocculation. Biicher and AVIDITY OF DIPHTHERIA ANTITOXIN 29 Kraus (1929) have hinted that some of the failures of serum treatment in the course of recent epidemics of diphtheria may be thus explained ; and Madsen and Schmidt (1930) believe that the extended use of sera which possess a high avidity for toxin would help to combat the malignant type of diphtheria that has been prevalent in Europe and elsewhere. An important investigation which throws light upon the problem was conducted in two of the hospitals of the Metropolitan Asylums Board (now administered by the London County Council) from November 29th, 1927, to June 23rd, 1928. The observations were made with the object of determining: (1) the relative incidence and severity of serum sickness, and (2) the therapeutic efficacy of a concentrated and an unconcentrated antitoxin. Both batches of serum contained about 900 units per c.cm. and also approximately the same amount of total protein. The results of the investiga- tion bear on the point under discussion, for it is stated that there was no evidence of any appreciable difference in the efficacy of the two kinds of serum. From the data published in this Report the present writer has calculated by the usual method the statistical significance of the difference in the mortality-rates in the two groups. The ratio of the difference to its standard error is equal to 0-49, a value from which it may be concluded that the difference is negligible and is due merely to errors of sampling. White and Mair (1927-1928) carried out laboratory tests with the batches of natural and of concentrated serum which were used in this investigation. They compared the influence of each kind of serum in modifying or preventing the skin lesions produced in guinea-pigs by the intracutaneous injection of virulent diphtheria cultures, and found that they seemed to be equally effective. A comparison of the results of antitoxin treatment obtained by Bie and by Banks and McCracken affords corroborative evidence, since the serum used by Bie is a natural serum, and for the severe cases preferably one with a high avidity for toxin (that is, one with a flocculating time of less than one hour), whereas the English physicians use concentrated serum; vet in both series the clinical results must be considered to be equally satisfactory. It appears then that the clinical evidence which we have adduced does not 30 THE SERUM TREATMENT OF DIPHTHERIA support the view that the avidity of anti-diphtheritic sera is an essential factor in the successful treatment of the disease. More- over, the exact significance of the experimental work on which the hypothesis is based is still in dispute. The Modes of Administration of the Antitoxin The choice of the route by which a therapeutic serum is to be administered to a patient is an important consideration, because upon it depends the rapidity of absorption of the antitoxin or other antibodies. When the serum is given intramuscularly, it is absorbed three to six times more quickly than when it is injected into the subcutaneous tissues. A convenient place for an intra- muscular injection is the middle of the outer side of the thigh, the needle being pushed into the vastus externus muscle. When antitoxin is injected directly into a vein, it is immediately available for neutralizing the toxin. The occasional difficulty of finding suitable veins for intravenous injection in infants suffering from laryngeal or severe diphtheria led Platou (1923) to use the intraperitoneal method ; he believed that absorption by this route is about five times more rapid than after intramuscular injection. The best site for insertion of the needle is stated to be through the linea alba, just below the umbilicus ; the serum is given by gravity from a container. The method was used as early as 1917 in a case of malignant diphtheria ; and in 1925 Toomey, Goehle, and Dauer compared the results of treatment in three groups, each comprising 50 chil- dren, in which the serum was administered by the intraperitoneal, the intravenous, and the intramuscular route. They found clinical evidence of rapid absorption of the antitoxin from the peritoneal cavity, and noted that the rate of disappearance of the membrane in the throat was about the same as when the serum had been given by the intravenous route. They used the intra- peritoneal method in a further series of 168 cases, and were con- firmed in their belief that it is safe and well tolerated by the patients. They recommend its use in severe diphtheria associated with toxic myocarditis, and in infants and children with incon- MODES AND TIME OF ADMINISTRATION 31 spicuous veins. Musser (1927) advises the administration of 10,000 units intramuscularly and 10,000 units intraperitoneally in children under three years of age who are suffering from highly toxic diphtheria. For reasons that are not obvious, the intra- peritoneal method, in spite of its apparent advantages, does not seem to have been employed to any extent ; it is believed, perhaps, that the risk of trauma or sepsis cannot be disregarded. The Importance of the Early Administration of the Antitoxin The risk of death in the severe type is determined to a great extent by the time of administration of the serum in relation to the onset of the disease. Many observations have been made that confirm this statement. A recent inquiry is that of Stevens (1931), who has analysed the clinical history of 3,122 cases of diphtheria ; the work was done at the Johns Hopkins University and with the co-operation of the California State Board of Health. She obtained abundant evidence in favour of the early administration of antitoxin. Place (1923) estimated that in the United States alone about 15,000 deaths were caused every year by delayed treatment with antitoxin or by the lack of it. F. H. Thomson (1927-1928) emphasizes the importance of prompt administration of antitoxin without waiting for a report from the laboratory, and states that it is still common for severe cases, untreated by antitoxin, to be admitted to hospital so late in the course of the disease that treatment is of no avail. He notes also that in nearly 23 per cent. of those cases which were admitted to hospital with a previous diagnosis of diphtheria the diagnosis was not confirmed. Similar statements have been made by Dr. E. H. R. Harries ; in the year 1929, of 69 patients who died in the Birmingham City Hospital, only five had received antitoxin before admission, and of these the antitoxin had been given to three after receipt of the bacterio- logical report. Inquiry showed that in 26 of the 69 cases the practitioner was called in after the fourth day—a delay which made the need for prompt administration of the serum still more urgent. Of 2,099 patients admitted for diphtheria to the 32 THE SERUM TREATMENT OF DIPHTHERIA Birmingham City Hospital, 34 per cent. were found to have no clinical signs of the disease. From these figures the conclusion must be drawn that the value of antitoxin treatment is seriously reduced, either in consequence of difficulties in the way of accurate diagnosis or of undue delay, from whatever cause, in commencing the specific treatment; and they indicate lines along which progress may be expected in the future. The Employment of Antitoxin in Diphtheritic Paralysis Paralysis is nearly always an avoidable complication of diphtheria, for it is an indication of delayed or inadequate treat- ment with antitoxin. Opinions differ among clinicians with regard to the effect of antitoxin on the condition. Some deny that it is of any value and cite experimental evidence in support of their view. The natural tendency of post-diphtheritic paralysis to spontaneous recovery makes it difficult, as J. D. Rolleston has remarked, to be certain of the therapeutic action of the serum ; he suggests that any apparent benefit is possibly due to a psycho- therapeutic rather than to a specific action. A recent paper by Friedemann and Elkeles (1930) reviews the subject and summarizes the results of experimental work which they undertook in order to ascertain whether the antitoxin treatment of diphtheritic paralysis has a rational basis. They found that in rabbits even large doses of antitoxin, when given by the intravenous route, did not pass through the barrier of the cerebral blood-vessels into the cerebrospinal fluid ; and thus confirmed previous results, which showed that, when the meninges are healthy, they do not allow antibodies to penetrate to the central nervous system. Nor could they find any evidence of the presence of antitoxin in the cerebrospinal fluid of patients who had received large doses of serum, for example, 20,000 units intra- venously and even 50,000-100,000 units; the only exception was a patient who died a few hours after the specimen of cerebro- spinal fluid had been obtained. Nevertheless, they consider that their experimental evidence points to some increase of permeability in the cerebral vessels as a consequence of cellular USE OF AN ANTIBACTERIAL SERUM 33 damage by the toxin, when this was injected directly into the brain. These authors admit that their experiments do not com- pletely solve the problem of the therapeutic action of antitoxin on post-diphtheritic paralysis in children. Kleinschmidt (1926) estimated the antitoxin content in the serum of paralysed children, and found that their recovery or death was quite independent of the presence or absence of antitoxin in the blood. Ramon, Debré and Uhry (1982) point out that diph- theritic paralysis which has been induced experimentally in guinea-pigs coexists with an antitoxic immunity, and conclude that the administration of antitoxin to patients, after the signs of paralysis have appeared, is without effect. Those who advocate serum treatment for this complication agree that, to be efficacious, large doses must be given. The Therapeutic Employment of an Antibacterial Serum The recognition of an antimicrobic property of diphtheria antitoxin, in addition to a purely antitoxic action, dates back to Behring’s original papers. G. Dean (1908) reviewed the scanty experimental work on the subject, and referred to the production by L. Martin (1903) of an antibacterial serum in the horse by means of intravenous or intraperitoneal injections of the bacillary bodies. The serum contained agglutinins, and it is stated that its local application in the form of pastilles brought about a rapid disappearance of the false membrane in the throat of patients. Dean prepared a serum of this kind and arranged for a clinical test of its efficacy in the local treatment of diphtheria ; he found no evidence, however, that the period of persistence of the bacilli in the throat was shortened. An antibacterial serum has not won any general application as a method of treatment, and a systematic investigation of its clinical worth does not seem to have been carried out during the past twenty years. The diphtheria bacillus is said to contain an endotoxin, which is thermostable and is not neutralized by anti- toxin, but there is little evidence that it plays an appreciable part in the pathology of the disease, Topley and Wilson (1929) R.A. VACCINE, 2 34 THE SERUM TREATMENT OF DIPHTHERIA speculate whether an antibacterial immune-body would influence the natural or an experimental infection, and they suggest that if this component should prove to be of value the different serological types of the bacillus should be used in preparing a serum. Barlow and Burnell (1925) sprayed the throats of seven carriers with an antibacterial serum prepared from a human volunteer who had received a course of injections of a virulent culture of the diphtheria bacillus ; the bacilli in the throat quickly disappeared in six of the seven carriers. Grenet and Delarue (1927) compared the thera- peutic effect of a natural serum of 300 units per c.cm. with that of a serum containing 150 units per c.cm., but possessing in addition a high antibacterial potency, and they obtained a lower death-rate with the weaker antitoxic serum ; the groups of patients under test are, however, too small for accurate comparison. In the opinion of the present writer, it would be worth re- investigating the subject by testing on an adequate scale, under controlled conditions, the therapeutic value of a combined anti- toxic and antibacterial serum as compared with the ordinary antitoxic serum ; in this way it should be possible to ascertain whether the composite serum is superior to the antitoxic serum (1) in reducing the fatality-rate of the malignant type of case, and (2) in lessening the carrier-rate. In an inquiry of this kind bias should be avoided as far as possible in order that the results may be suitable for examination by the usual tests for statistical significance. The Prophylactic Use of Diphtheria Antitoxin The tendency among practitioners at the present day is to restrict the prophylactic use of antitoxin, partly because the resulting immunity lasts for only two or three weeks, and partly because of the circumstance that the recipient may be rendered serum-sensitive. The method of active immunization is to be preferred when possible, but whereas immediate protection is obtained by the administration of antitoxin, the protection that follows active immunization with toxoid is due to a gradual process which takes weeks and even months for its full development, PROPHYLACTIC USE 35 The need for a prophylactic dose of antitoxin is less in older children and adults, since many of them possess an immunity to the infection ; nor is it necessary that younger children should receive it, provided that they are under constant medical super- vision such as exists in hospitals, or are carefully looked after at home. But a dose of 500-1,000 units of antitoxin given subcu- taneously has proved to be of undoubted value in lessening the incidence of the disease among contacts in the home. Doull and Sandidge (1924) quote Chapin’s figures for Providence City, which show an attack-rate of 16-3 per cent. in 2,213 such contacts of six years of age or under ; Seligmann’s figures are very similar (17-6 per cent.). This risk can be reduced to from 1 to 4 per cent, by the prophylactic use of antitoxin. A good instance of its preventive effect is to be found in the experience of Braun (cited by Kleinschmidt). He gave 6,869 prophylactic injections in children who were exposed to the risk of infection; 73 of the children were attacked by diphtheria (1-1 per cent.); and of this number 20 came under observation within the first three days of exposure. He believed that the danger to the children of unprotected families is at least ten times as great as that in the protected homes. Groups of children who have been exposed to the risk of infec- tion may be tested first by the Schick method, and those who prove to be susceptible can then be given a prophylactic dose of anti- toxin. Children in hospital wards who are suffering from measles or scarlet fever and who may have incurred the risk of a diphtheritic infection should receive 1,000 units of diphtheria antitoxin in order to protect them from the possibility of a dangerous double infection. In Germany the ox, the sheep and the goat have been utilized for the production of antitoxin intended for prophylactic use, with the object of eliminating the risk of serum sickness which may follow a subsequent dose of a therapeutic serum prepared from horses. REFERENCES ANDERSON, J. S., Harrop, F. C., McLEoD, J. W., and THOMSON, J. G. 1931. J. Path. and Bact., 34, 667. Bicner and Kraus. 1929. Centralbl. f. Bakt., 110, 142. 36 THE SERUM TREATMENT OF DIPHTHERIA Banks, H. S., and McCrACKEN, G. 1928. Lancet, 2, 4. Barrow, D. L., and BurNELL, G. H. 1924. Med. J. Australia, Supp. 1, 290. BENN, E. C., HucHes, E., and ALsTEAD, S. 1932. Lancet, 1, 281. Berlin Medical Society. Discussion on Malignant Diphtheria. 1931. Med. Welt., 5, 241. ’ Bie, V. 1922. Acta med. Skand., 56, 537. = 1929. Deutsche med. Woch., 55, 563. BorMmaNN, F. von. 1931. Arch. f. Kinderheilk., 94, 241. Canon. 1927. Deutsche med. Woch., 53, 1171. Crooks, T. T. 1925. Amer. J. Dis. Child., 29, 269. Dean, G. 1908. * The Bacteriology of Diphtheria,” p. 571. Cam- bridge. DeicHER, H., and AcuLNik, F. 1927. Deutsch. Med. Woch., 53, 825. Dorp, H. 1926. Miinch. med. Woch., 73, 358. - 1927. Deutsche med. Woch., 53, 1760. Dour, J. A., and SANDIDGE, R. P. 1924. Reprint No. 901, Pub. Hith. Rep., p. 283. U.S. Pub. Hlth. Service, Washington. Dovury; J. A., and SANDIDGE, R. P. 1924. Pub. Hlth. Reps., Feb. 15th. U.S. Pub. Hlth. Service, Washington. Fanrtus, B. 1926. J. Amer. Med. Ass., 87, 667. FrIErABEND, B., and ScHUBERT, O. 1930. Les Travaux de U'Inst. d’Hyg. Pub. de IEtat Tcheco-slovaque, 1, 48. FINKELSTEIN, H., and KONIGSBERGER, E. 1928. Deutsche med. Woch., 54, 218. . FRIEDBERGER, E. 1928. Med. Klin., 24, 767. FRIEDBERGER, KONIGSBERGER, et al. 1931. Med. Welt., Feb. 14th, p. 241. Friepemann, U., and EvLkeLes, A. 1930. Deutsche med. Woch., 56, 1725. GreNET, H., and DELARUE, J. 1927. Bull. et Mém. Soc. Méd. Hépit. de Paris, 51, 164. GooparL, F. W. 1928. “A Text-book of Infectious Diseases,” London. . Harries, E. H. R. 1930. Brit. Med. J., 2, 587. Harries, E. H. R., and MacrarLaNe, W. M. 1928. Lancet, 2, 146. HarTLEY, P. 1923. Lancet, 1, 17. Havens, L. C. 1920. J. Inf. Diseases, 26, 388. HecksHER, H. 1926. Deutsche med. Woch., 52, 58. 5 1926. Acta med. Skand., 63, 522. Hector, F. J. 1926. Lancet, 2, 642. KreinscamipT. 1926. °° Handb. der exp. Ther. Serum. u. Chemother.,” Munich, p. 159. KorLe, W. 1930. Med. Klin., 26, 1809, 1848. Kraus, R. 1920. Deutsche med. Woch., 46, 687. LICHTENSTEIN, A. 1931. Zeitschr. f. Kinderheilk., 51, 755. LieNiires, J. 1930. Bull. de I’ Acad. de Méd., 104, 698, 720. MabseN, TH., and Scamipr, S. 1930. Zeitschr. f. Immunititsf., 85, 357. REFERENCES 37 MarTIN, L. 1903. Compt. Rend. Soc. de Biol., 4, 624. METROPOLITAN AsYLUMS BoArD. 1928-1929. Annual Report (The relative value of concentrated and unconcentrated diphtheria anti- toxin), p. 240. MEYER, F. 1928. Deutsche med. Woch., 54, 215. Musser, J. H. 1927. J. Amer. Med. Ass., 88, 1125. Parisu, H. J., Wat Ey, E. E., and O’Brien, R. A. 1932. J. Path. and Bact., 35, 653. Park, W. H. 1921. J. Amer. Med. Ass., 76, 109. Park, W. H., Wirriams, A. W., and MANN, A. G. 1922. J. Immunol. 7, 243. Prace, E. H. 1923. Boston Med. and Surg. J., 188, 32. Prarou, KE. S. 1923. Arch. of Pediatrics, 40, 3. Ramon, G. 1922. C. R. Soc. Biol., 86, 661. Ramon, G., Derg, R., and Unry, P. 1932. C. R. Soc. Biol., May 13th, p. 42. ReicHE, F., and Reve, E. 1930. Deutsch. med. Woch., 56, 1162. RoLLEsTON, J. D. 1928. Brit. Med. J., 2, 337. Rosexow, M. J., and ANDERSON, J. F. 1907. U.S. Pub. Hlth. and Marine Hosp. Serv. Bull., 38. Roux, E. 1900. Congress Internat. d’Hyg. et Démographie, 5. Scumipr, H. 1927. Deutsche med. Woch., 53, 1810. ScHWENTKER, F. F., and NoerL, W. W. 1929. Bull. Johns Hopkins Hosp., 45, 259 ; ibid., 46, 359. SELIGMANN, E., cited by KueinscumipT, H. 1926. * Handb. d. Exp. Ther. Serum u. Chemotherap.,”” Munich, p. 159. Siig, T. V. 1931. Centralbl. f. Bakt., 1 Abt., Orig. 120, 385. Stevens, I. M. 1931. Amer. J. Hyg., 13, 392. Toomson, F. H. 1924. ‘ Diagnosis and Treatment of Infectious Diseases,” London, p. 137. Tuoomson, F. H. 1928. ‘Ann. Rep. Metropol. Asylums Bd.,” 1927-1928. TooMmEY, J. A., GOEHLE, O. L., and DAUER, C. C. 1925. Amer. J. Dis. Childr., 29, 214. Torrey, W. W. C., and WiLsoN, G. S. 1929. * The Principles of Bacteriology and Immunology,” 2, 866. Wire, R. G., and Mair, W. 1928-1929. ** Metropol. Asylums Bd. Ann. Rep.,” p. 260. Wouvrrr, G. 1928. Klin. Woch., 7, 1477. ‘Woobcock, H. E. pe C. 1932. Lancet, 2, 894. WricHT, H. A., and Rankin, A. L. K. 1932. Lancet, 2, 884. CHAPTER III THE SERUM TREATMENT OF TETANUS PAGE Tae PROPHYLACTIC VALUE OF TETANUS ANTITOXIN . : . 38 Tue DIAGNOSTIC IMPORTANCE OF THE EARLY SYMPTOMS OF TETANUS . : ’ 2 ‘ . . 40 Tue SpeciFic TREATMENT OF TE TANUS " . ' . 42 THE TREATMENT OF SPECIAL FORMS OF TETANUS ; . . 47 Local Tetanus . ’ . ‘ . . . . 47 Delayed Tetanus ‘ . . . . : i Recurrent Tetanus . . . : v : . 48 Cephalic Tetanus : : . . 48 THE PREPARATION OF TETANUS ANTITOXIN . . . . 48 The Prophylactic Value of Tetanus Antitoxin Tur experience that was gained during the Great War has firmly established the value of tetanus antitoxin as a prophylactic agent. An analysis of 1,458 cases of tetanus, which were treated in Home Military Hospitals in Great Britain during the years 1914-1918, was made by Bruce (1920). To this figure there should be added 1,071 cases that were reported from British hospitals on the Western Front, making a total of 2,529 cases among 1,710,369 wounded men or 1-47 per 1,000 (Cummins). The number of recorded cases for the British Army in all the theatres of war is 2,549 : a figure which affords a striking example of the danger of tetanus from the contamination of wounds with heavily manured soil, for only 20 cases were reported elsewhere than in France and Belgium. Antitoxin first came into use as a prophylactic for the British troops about the middle of October, 1914. The incidence of tetanus in the early stages of the war in France is not accurately known, but Bruce gives the rate as nine per 1,000 in September, 1914 ; it fell to 1-4 per 1,000 in December, 1914, as the result of serum prophylaxis. In the United States Expeditionary Force among 224,000 wounded men only 36 were attacked by the disease, 38 PROPHYLACTIC USE 39 that is, a proportion of one to 6,224 wounds, as compared with one to 487 wounds in the American Civil War. The administration of a prophylactic dose influenced the character and the progress of the disease in those patients who subsequently contracted it. Bruce asserts that the clinical picture of tetanus was thereby changed from an acute disease which was almost invariably fatal to a subacute disease having a case- mortality of only some 20 per cent. Again, its period of incubation was lengthened, so that in 31-7 per cent. of the cases the symptoms appeared five weeks or more after the date of infliction of the wound. The average incubation period of a protected group was 455 days; of an unprotected group it was 10-9 days. The importance of this result is enhanced by the fact that the longer the period of incubation the lower is the rate of mortality ; the death-rate in a group of 429 cases with an incubation period of more than five weeks was only 15-4 per cent. (Bruce). Further, the number of cases of local tetanus, when compared with the cases of general tetanus, tended to increase in each year that the war lasted ; thus in the year 1914 there were 1-1 per cent. of cases of local tetanus and in the period 1917-1918 about 20 per cent. The difference is mainly attributable to the effect of the prophy- lactic injections, and perhaps also to greater experience in the diagnosis of the mild localized forms of the disease. Both in Great Britain and in France the procedure was adopted of giving a course of four prophylactic injections, each of 1,000 International units (500 U.S.A. units) ; they were administered subcutaneously at intervals of seven days. The effect was to lower the attack- rate, and to lengthen the incubation period in those cases in which tetanus appeared later. In civil practice the prompt surgical treatment of wounds that have been contaminated with soil is an important prophylactic measure. During the later stages of the war it was shown that thorough excision of the damaged tissues around the track of a wound favoured aseptic healing. The removal by this procedure of necrotic tissue, which provides the condition of reduced oxygen tension that favours the germination of tetanus spores, greatly decreases the risk of the onset of the disease. Operative treatment, 40 THE SERUM TREATMENT OF TETANUS however, should not be undertaken until an adequate prophylactic dose of antitoxin has been given. The amount will depend upon the severity and the degree of contamination of the wound as well as the interval that has elapsed since its infliction : 1,000-3,000 International units (500-1,500 U.S.A. units) should, if possible, be given intramuscularly from three to six hours before any attempt is made to clean the wound by surgical methods. If a subsequent operation should be needed, for example, to remove a foreign body (a sequestrum of bone or a fragment of metal or clothing), a prophylactic injection of antitoxin should always be given, even although the original wound is completely healed, if more than seven days have elapsed since the administration of the last dose of antitoxin. Ramon and Zoeller (1927) have recommended a method of prophylaxis which combines active with passive immunization. Ten c.cm. of antitoxin and 1 c.cm. of ‘‘anatoxine ”—an atoxic formolized preparation—are given at different sites; ten days later the dose of antitoxin is repeated ; after another ten days 2 c.cm. of ‘ anatoxine” is given. The combined method is specially indicated for the protection of those patients who have incurred a risk of tetanus and whose wounds are likely to be long in healing. The Diagnostic Importance of the Early Symptoms of Tetanus Success in the treatment of tetanus depends largely upon the institution of specific treatment at the earliest possible moment. For this reason the early diagnosis of the disease assumes great importance, especially as commencing tetanus may be mistaken for such diverse illnesses as sore throat, mumps, and rheumatism. MacConkey (1914) laid stress upon the necessity for the careful study of suspicious symptoms in the wounded, and in a Memorandum (3rd Edit., 1917) the Committee for the Study of Tetanus invited Army Medical Officers to furnish information of this kind which would promote the early diagnosis of the disease : yet in the medical literature of the war the question has not apparently received consideration. The probable explanation is EARLY SYMPTOMS OF TETANUS 41 to be found in the difficulty of recognizing the import of the group of symptoms that have been observed in early tetanus and of which a brief account is given below ; for these are not in them- selves really characteristic, although they possess some collective significance. Moreover, many of the patients suffered from multiple and septic wounds; in these men the symptoms that were due to sepsis and to nervous shock must often have masked those of an incipient tetanus toxemia. In the generally simpler injuries of civil practice the premonitory symptoms of tetanus deserve special attention, since their recognition may lead to the prompt institution of serum treatment; and the chances of success, other factors being equal, are in proportion to the shortness of the interval between the onset of the symptoms and the administration of antitoxin. Tetanus toxin is formed at the site of the injury and is trans- ported to the motor nerve cells of the central nervous system by way of the axis cylinders of the nerves that supply the muscles in the neighbourhood of the wound ; and, therefore, these muscles will give the first indication of the presence of the toxin : rigidity, twitchings, or an increased reflex response to tapping or pressure may be noted. As the toxin continues to accumulate in the wound it is absorbed by the lymphatics and the blood-vessels and, passing along nerves remote from the wound, it may cause wide- spread damage to the motor nerve cells; this diffusion of the toxin is manifested by tetanic spasms of groups of muscles else- where than in the neighbourhood of the wound. Thus there may be a feeling of soreness in the throat or reflex yawning and difficulty in swallowing due to spasm of the pharyngeal muscles ; sensations of stiffness in the neck or jaws; deviation of the tongue ; facial spasm or paralysis ; paralysis or spasm of the eye muscles producing strabismus ; changes in the facial expression varying in degree from an anxious look to the classical risus sardonicus ; an increased flow of saliva; a spasmodic cough with pain in the side; and difficulty in micturition caused by spasm of the sphincter vesicz. Evler gives a list of premonitory symptoms in 13 cases which he personally observed ; his interest in the subject was increased by 42 THE SERUM TREATMENT OF TETANUS the circumstance that he himself had passed through an attack. The symptoms include a feeling of restlessness, alternating suddenly with a desire for rest ; increasing nervousness, accom- panied by unreasonable outbursts of temper; sleeplessness, distressing dreams, nocturnal delirium; violent headache and attacks of giddiness ; diffuse backache ; darting pains in various parts; profuse sweating; and pain persisting after the muscular contraction induced by effort. In Evler’s opinion the appearance of such symptoms following a wound which is likely to be infected by the tetanus bacillus should lead the physician to consider the advisability of administering the specific serum forthwith. The Specific Treatment of Tetanus The therapeutic use of tetanus antitoxin rests on a much less solid basis than its prophylactic application, for the clinical and experimental results obtained by different workers are conflicting. There is, however, general agreement that as soon as any manifes- tations of tetanus are observed energetic specific treatment should be commenced, and that the aim of the practitioner should be to flood the system with antitoxin as quickly as possible. Differences of opinion exist regarding the best way of achieving this result. Most physicians, influenced possibly by the view of the War Office Committee, prefer the intrathecal route. The con- sidered opinion of this Committee was that in acute tetanus large doses should be administered without delay by the in- trathecal route ; the doses to be repeated on two, three or four days in succession, and combined, if thought desirable, with intramuscular injections. The risk of fatal anaphylactic shock, which may follow intravenous injections, deterred them from recommending the intravenous route, although it is admittedly the best of all for neutralizing any toxin that may be circulating in the blood-stream. The scheme of dosage which they give as an example is shown in the accompanying table ; it will be seen that the initial dose is 82,000 International units intrathecally and 16,000 units intra- muscularly ; the total number is 156,000 International units. SPECIFIC TREATMENT OF TETANUS 43 This scheme may be modified according to the necessities of the case, and in modern practice the total amount administered is often as much as 500,000 International units (250,000 U.S.A. units). TABLE I Scheme of Dosage for the Curative Treatment of Tetanus as recommended by the War Office Committee (1917) No. of International Units to be given by Dag-op Disease, Subcutaneous Intramuscular Intrathecal Route. Route. Route. 1st day ’ . _ 16,000 32,000 2nd ,, . v som 16,000 32,000 3rd ,, . # —_ 8,000 — 4th ,, . x _ 8,000 _ 5th ,, . . 4,000 ee — 7th ,, . 4,000 — os Sh ,, . 4,000 — en In the United States the opinion of most physicians is in favour of the intrathecal route as the method of choice, supplemented by intravenous, intramuscular or subcutaneous injections ; it is hoped by this means to maintain the concentration of antitoxin in the blood and tissues (Nicoll; Ashhurst; Ferris and Fuerth; Calvin and Goldberg). On the other hand, Wainwright holds the opinion that intrathecal injections are harmful, that they increase the mortality-rate, and that they should be abolished; in his experience intravenous doses of from 60,000 to 100,000 Inter- national units have reduced the deaths by one-half or one-third. Ashhurst questions the validity of Wainwright’s data, and expresses his firm belief in the efficacy of early intrathecal injec- tions. Freedlander’s analysis of 25 consecutive cases that were treated by him with large and frequently repeated intravenous injections of antitoxin gave a death-rate of 36 per cent.; and this figure is reduced to 12 per cent. by excluding six fatal cases 44 THE SERUM TREATMENT OF TETANUS in which death took place before an effective dose of serum could be given. H. R. Dean (1917) gave to 14 patients with severe symptoms, including trismus, a dose of 60,000 International units intravenously ; 13 of them recovered, but apparently all had received a prophylactic injection : the incubation periods were long. Recently Paterson has published the results of the intensive treatment by the intravenous method of 26 children, of whom 19 recovered ; his scale of dosage considerably exceeds any hitherto recorded. Thus, as a routine method, 200,000 International units were given intravenously and 80,000 International units intra- muscularly. The intravenous dose was repeated in 12 hours and afterwards at intervals of 24 hours, the frequency depending upon the condition of the patient; if his progress was satisfactory, smaller doses were given at longer intervals. The doses administered to the patients who recovered ranged from 240,000 to 340,000 International units. Paterson admits that the number of his patients is small, and that for this reason the results cannot be regarded as conclusive. Obvious difficulties attached to the method are its cost and the dangers attendant on the adminis- tration of massive intravenous doses of antitoxin, even in the form of a highly purified preparation. A scrutiny of the clinical journals for the past five years has shown that most of the articles on tetanus give a description of a single case or a quite small group of cases, and that the patients were treated by antitoxin administered in various ways. The present writer has collected 82 cases from this source ; of these 11 died (34 per cent.). The comparatively high recovery-rate in this group may in some measure be accounted for by the natural tendency to report the successes rather than the failures of treatment. The disease in four of the fatal cases originated from wounds that were inflicted by a toy pistol ; it is well known from experience in America that tetanus, when it is due to this type of injury, causes a high mortality. Yodh (1932) has summarized the experience he gained during five years in the male tetanus wards of the Sir Jamshet;ji Jeejeebhoy Hospital, Bombay. His analysis is based on 278 consecutive cases without selection, of which 229 came under his personal SPECIFIC TREATMENT OF TETANUS 45 observation. A group of 72 patients were treated in the usual way by intravenous, intramuscular, and intrathecal injections, and of these 85 died (50 per cent.). In another group of 77 cases there were only 18 deaths (23-4 per cent.) ; these patients were treated by serum in much the same way as those of the first group, except that the serum was given by cisternal and not by lumbar puncture. It should be noted that these figures do not include a number of cases that were fatal within 24 hours of admission to hospital. Yodh concludes that the combined intrathecal (by cisternal puncture), intravenous and intramuscular method is the best of all, since it relieves the symptoms far more than intravenous and intramuscular administration and, moreover, definitely lowers the fatality-rate. This author avoids the use of chloroform in performing cisternal puncture, and also as a means of treatment according to the methods recommended by French authors in late years, because he thinks that the employment of this drug increases the risk of a fatal pulmonary cedema. He regards the evidence for the utility of magnesium sulphate and carbolic ‘acid as insufficient ; and he states that when they have been tried in a large series of con- secutive cases the results were not satisfactory. : Suvansa (1931) has reported the results he has obtained in Bangkok in the treatment of tetanus by the intrathecal injection of a 1/400 solution of carbolic acid in saline ; he gives from 12 to 40 c.cm. of the solution, the amount varying with the age of the patient and the severity of the symptoms. There were four deaths among 14 patients who received the treatment. The elimination of the carbolic acid by the kidneys caused an attack of acute nephritis in three of the patients, and the author utters a warning against employing the method in patients with chronic renal disease and in old persons. In addition to the usual modes of administration, tetanus patients have been treated by intracranial and intraneural injections of antitoxin. Clinical experience of these methods appears to be small; their value to be doubtful; and their application to be not without danger. It is not easy to form an estimate of the curative effects of 46 THE SERUM TREATMENT OF TETANUS tetanus antitoxin, and in attempting to do so a distinction needs to be drawn between patients whose attack has been mitigated by a prophylactic injection and those who have not been so protected. Bruce (1920), in his analysis of 1,458 cases that were treated in Home Military Hospitals, concludes that the data yield no trust- worthy information on the value of serum therapy. In a recent review Fildes states his belief that the curative treatment of tetanus by antitoxin is not as ineffective as individual authors have concluded from a small experience of it, and he cites in support of his opinion the fact that at the London Hospital the mortality in 71 non-serum cases in the period from 1878 to 1923 was 88-7 per cent., and 71-8 per cent. in 103 treated cases. Permin (1914) makes a similar comparison ; 79 per cent. of 199 patients died in pre-serum days and 57-7 per cent. of 189 patients died who had received serum treatment. The present writer finds that, if the value of three times the standard error is accepted as the test for significance, the difference between the London Hospital mortality rates is on the verge of significance (d/od = 2-7), and that the corre- sponding difference in the Danish statistics analysed by Permin is certainly significant (d/od = 4-5). There appears to be a lack of correlation between clinical experience of the effects of treatment and experimental observa- tions made on animals. Thus evidence from the experimental side does not support the idea that antitoxin can reach the damaged nerve cells. Fildes has pointed out that cerebral tetanus —an experimental form of tetanus which follows the inoculation of the toxin directly into the substance of the brain—can be produced in an animal whose blood contains a high concentration of antitoxin. He adds that there is little reason to suppose that substances injected into the cerebrospinal fluid are in a favourable position for combining with toxin in the tissue spaces of the spinal cord, since the flow of cerebrospinal fluid is probably not directed to those areas which are believed to be the site of intoxication. Nevertheless, the experiments of Sherrington (1917) on monkeys indicated—although he refrained from drawing any conclusions— that intrathecal injections of antitoxin had a definite curative effect, and that the fatality-rate was considerably lower when the SPECIAL FORMS OF TETANUS 47 serum was given by this route than when it was given intra- venously, intramuscularly or subcutaneously. Topley and Wilson suggest that the favourable results in these experiments were partly due to the use of toxin alone and not of culture, and partly to the relatively enormous dose of serum, in proportion to the body weight of the animals, which was given to them. Park and Nicoll (1914) had previously carried out similar experiments with guinea-pigs, and the results seemed to confirm the superiority of the intrathecal route over the intravenous route. Florey and Fildes (1927), however, remark that the number of guinea-pigs used by these workers was so small as to be subject to sampling errors. They repeated the same type of experiment in rabbits, and could find no advantage in intrathecal as compared with intravenous administration. Fildes sums up the matter in the statement that if the intrathecal route offers any advantage for the introduction of antitoxin, it is unimportant as far as the cure of experimental tetanus in animals is concerned ; and that the failure to cure tetanus in man or animals depends upon factors that are unrelated to the site of injection of the antitoxin. The Treatment of Special Forms of Tetanus Local Tetanus. This form of tetanus may appear in patients who have received one or more prophylactic doses of antitoxin. The signs are limited to the site of the injury and take the form of intermittent local cramps, which are usually painful, and persistent tonic rigidity, which is often painless. The two kinds of spasm may occur singly or together (Andrewes). In the experience of Cummins uncomplicated local tetanus is never fatal. A less drastic course of treatment than that required for generalized tetanus is sufficient, and may consist of a series of intramuscular doses, but, if signs of extension should appear, active treatment must be begun at once. Delayed Tetanus. Tetanus sometimes appears long after the original wound has healed, as, for example, after a local injury or some slight operative procedure, or it may follow the recrudescence of a septic infection in the wound. Worster-Drought refers to a 48 THE SERUM TREATMENT OF TETANUS case of subacute, generalized tetanus which came on six months after a gunshot wound of the knee ; the patient had received three prophylactic doses of antitoxin at weekly intervals after the infliction of the wound ; he made a good recovery. In cases of this kind the dosage and mode of administration of the antitoxin should correspond to the degree of severity of the toxwmia. Recurrent Tetanus. This term indicates a reappearance of the symptoms after serum treatment has effected an apparent cure. Andrewes has reported two such cases ; in one the recurrence took place 43 days after the last dose of serum had been given, and it assumed the form chiefly of local tetanus; in the other the symptoms recurred and led to a fatal issue 57 days after the last dose of serum, although the patient had recovered from a severe attack of general tetanus. In this group also the thoroughness of the serum treatment should be proportionate to the extent and the severity of the symptoms. : Cephalic Tetanus. This is a comparatively rare form of tetanus, which follows injuries of the face, and is of interest because it belongs to the local ascending type (Fildes). The prognosis is similar to that of the more common generalized form. A case has been described recently by Jenkins and Barnett (1929), who append sources of information on the condition. These authors treated their patient, who made a good recovery, in a very thorough fashion. They gave a total of 510,000 International units of antitoxin distributed as follows : cisternal route, 70,000 units ; spinal route, 20,000 units; intravenous route, 260,000 units ; subcutaneous or intramuscular route, 110,000 units ; and facial nerve and wound, 50,000 units. They attribute the recovery of the patient mainly to the massive doses of serum which were administered, and particularly to the cisternal injections. The Preparation of Tetanus Antitoxin In this country and in France formolized tetanus toxin is used for the immunization of the horses. The effect of the formalin is to render the lethal toxin non-toxic, without at the same time destroying its antigenic power (Lowenstein, 1921). The addition PREPARATION OF TETANUS ANTITOXIN 49 of potash alum to the formolized toxin, as recommended by Glenny (1930), has a powerfully stimulating action on the production of antitoxin in the horse. Moreover, this author’s method of giving an initial dose of the alum antigen to a fresh horse, and waiting for one month before proceeding with the immunization, yields a much better response than was formerly obtained by a sequence of doses at short intervals. Tulloch, as a result of work during the war, divided strains of B. tetani into a number of sero- logical types, but it is agreed that the toxins produced by the various types are the same and that the antitoxins produced by them are likewise identical. The strain in use in serum laboratories appears to belong to the Type I. group. Until recently four or five different methods of titration were used in Europe and in the United States. The basis of the present method is that which was worked out in the United States. A new International unit has lately been accepted by the European members of the Standardization Commission of the League of Nations ; this unit is exactly one-half the value of the U.S.A. unit. It is important that authors of papers on tetanus should mention the particular unit in terms of which they state their dosage. A flocculation method similar to that which is so useful for titrating diphtheria antitoxin has been alleged by French workers (Abt and Erber) to give fair agreement with the results of animal tests, but the general view is that the method possesses inherent difficulties that make uncertain the interpretation of the results. Tetanus antitoxin doubtless owes its efficacy in great part to its antitoxic component, since the tetanus bacillus has little or no power of invading the tissues of its host. The suggestion is occasionally met with in the literature that in experimental infections the curative value of a tetanus serum is not wholly related to its antitoxin content (Tulloch, 1919; Tenbroeck and Bauer, 1926 ; Weinberg and Ginsbourg, 1927 ; and Reymann, 1930). But at the present time the evidence in favour of this view is by no means complete, and for this reason further reference to it need not be made. 50 THE SERUM TREATMENT OF TETANUS REFERENCES ABT, G., and ErRBER, B. 1926. Ann. de I'Inst. Past., 40, 659. ANDREWES, F. W. 1923. History of the Great War, Med. Services, Pathology,” p. 188. ASHHURST, A. P. C. 1920. Arch. Surgery, 1, 407. ” 1926. J. Amer. Med. Ass., 87, 2089. Bruce, D. 1920. J. Hygiene, 19, 1. Carvin, J. K., and GOLDBERG, A. H. 1930. J. Amer. Med. Ass., 94, 1977. Cummins, S. L. 1923. History of the Great War, Med. Services, Pathology,” p. 164. Dean, H. R. 1917, Lancet, 1, 673. Evier, K. 1910. Deutsche med. Woch., p. 961. FERRis, J. L., and Fuerth, A. L. 1925. Missouri State Med. Ass. i 22, 253 ; ref. in J. Amer. Med. Ass., 85, 704. 1925. Fibres, P. 1929. “A System of Bacteriology, London, H.M. Stationery Office,” 8, 358. FREEDLANDER, S. O. 1927. Ann. Surgery, 85, 405. Frorey, H., and Firpes, P. 1927. Brit. J. Exp. Path., 8, 393. GLENNY, A. T. 1930. Brit. Med. J., 2, 244. JENKINS, J. A., and BARNETT, G. M. F. 1929. Brit. Med. J., 1, 547. LoewEeNsTEIN, E. 1921. Deutsche med. Woch., 47, 833. MacCoNkEY, A. T. 1914. Brit. Med. J., 2, 609. 3 1916. J. Roy. Army Med. Corps, 26, 291. Nicorr, M. 1921. J. Amer. Med. Ass., 76, 112, Park, W. H., and Nicorr, M. 1914. J. Amer. Med. Ass., 63, 235. PATERSON, A. E. 1930. Med. J. Australia, 1, 832. Permin, C. 1914. Mitt. Grenzgeb. Med. Chir., 27, 1. Ramon, G., and ZokLLER, C. 1927. Ann. de I'Inst. Past., 41, 803. ReymanN, C. C. 1930. Communic. de UInst. Sérothérap. de UEtat Danois, 20, 335. SHERRINGTON, C. S. 1917. Lancet, 2, 964. Suvansa, S. 1931. Lancet, 1, 1075. TENBROECK, C., and Baver, J. H. 1926. J. Exp. Med., 43, 361. Torrey, W. W. C., and WiLsoN, G. S. 1929. * The Principles of Bacteriology and Immunity,” 2, 1154. TurLrocu, W. J. 1919. Spec. Rep. Series Med. Res. Council, No. 39. WaiNwricHT, J. M. 1926. Arch. Surgery, 12, 1062. ” 1926. J. Amer. Med. Ass., 86, 1642. War Office Committee. 1917. Mem. on Tetanus, 3rd Edit. London. - ss 1919. Mem. on Tetanus, 4th Edit., London. WEINBERG, M., and GINSBOURG, B. 1927. Bull. Past. Inst., 25, 97. WorsTeER-DrouGHT, C. 1926. Lancet, 1, 726. Yoon, B. B. 1932. Brit. Med. J., 2, 589. CHAPTER IV THE SERUM TREATMENT OF GAS GANGRENE A NOTE ON THE BACTERIOLOGY OF GAS GANGRENE . 3 . 51 THE SERUM TREATMENT OF GAS GANGRENE. DURING THE War 52 TE SERUM TREATMENT OF GAS GANGRENE IN CIviL PRACTICE 54 THE SERUM TREATMENT OF ACUTE ABDOMINAL CONDITIONS ’ 55 The treatment of intestinal obstruction and of peritonitis . 55 The use of an anti-gangrene serum in typhoid fever ‘ . 58 THE SERUM TREATMENT OF PUERPERAL INFECTIONS CAUSED BY PATHOGENIC ANAEROBES : > . ’ ’ : 58 ANAEROBIC INFECTIONS THAT MAY BE BENEFITED BY THE USE OF GAS-GANGRENE ANTITOXIN : . . . . . 60 THE PREPARATION AND TITRATION OF GAS-GANGRENE ANTI- TOXIN . . ’ . . ‘ ' . 5 v 61 A Note on the Bacteriology of Gas Gangrene TrE pathogenic organisms that are associated with gas gangrene belong to a well-defined group of bacilli, which are characterized by growth under anaerobic conditions, the production of an exotoxin, and the formation of resistant spores. These characters furnish the clue to the pathology of the disease. In the first place, the bacilli will proliferate freely in tissues that have suffered a severe trauma, particularly if their blood supply is deficient owing to an accompanying vascular injury. The trauma and the nutritional disturbances caused by interference with the circulation in the injured part bring about necrosis of the tissues ; moreover, in the infected tissues the toxins attack the cellular elements of the epidermis, muscles, nerves, and blood-vessels, and produce characteristic necrotic lesions. The diminished oxygenation of the surviving tissues and the well-known property which dead tissue possesses of reducing the oxygen tension in its neighbour- hood provide an environment which is suitable for the multiplica- tion of the invading anaerobes. Secondly, the characteristic symptoms of the disease are mainly due to the specific bacterial 51 52 THE SERUM TREATMENT OF GAS GANGRENE toxins, which are formed either at the site of the injury or in secondary foci of the infection : in fatal cases the toxemia is the cause of death. Lastly, the resistance that the spores offer to unfavourable environmental conditions accounts for the universal distribution of this group of bacilli in soil, dust, and the intestinal tract of man and animals ; this in its turn explains why manured soil is such a dangerous material when it gains entrance to wounds. The chief members of the group that are pathogenic to man are in the order of their importance : (1) B. welchii (syn. B. aerogenes capsulatus, B. perfringens, and Fraenkel’s bacillus); (2) B. adematis maligni (Vibrion septique); and (8) B. novyi (B. wde- matiens). Wounds that manifest the signs of gas gangrene have usually been grossly contaminated with soil, and a mixture of anaerobes with various kinds of aerobes is often present in them. Each of the three types is capable of invading the blood-stream. On the Western Front during the war, B. welchii was the predominant organism in wounds that were infected with gas-gangrene bacilli, for it occurred in about 70 per cent. of the cases investigated ; the frequency of the other two species was about equal (15 per cent.). In Great Britain at the present time B. welchii is, of the three organisms, by far the commonest and most important infecting agent, and it is therefore questionable whether any account need be taken of its associates when considering the preparation of a specific serum for treatment. The bacteriology of anaerobic infections has been discussed at length in monographs by well-known authorities (M. Robertson, 1929 ; Weinberg and Ginsbourg, 1927 ; and Zeissler, 1928). The Serum Treatment of Gas Gangrene during the War The incidence of gas gangrene among the British troops in France was estimated by Wallace to be from 1 to 5 per cent. of all wounds, with a death-rate of about 22 per cent. The medical departments of the belligerent armies on the Western Front tried a specific serum only at a late period in the war, so that data on which a trustworthy opinion of its worth can be based are not easily USE OF SERUM DURING THE WAR 58 found. Rumpel in 1917 used * Hochst ” serum with apparently good results when it was given soon after the infliction of the wound ; at first he injected the serum in the neighbourhood of the wound, and later gave considerable amounts of it intravenously. Aschoff (1917) observed a series which comprised 223 serum- treated patients ; among these there were 44 per cent. deaths, as compared with 68-7 per cent. deaths among 2,183 wounded men who did not receive the serum. In 1918 Weinberg and Séguin gave a prophylactic injection of a serum to seriously wounded patients within 5 to 18 hours after they had received the wound, with the result that, although the mortality from other causes was high, none of them showed the signs of gas gangrene. In the early months of the year 1918 antitoxic sera were available for use in the British Army ; the supplies were insufficient until later in the same year, and the titre of the serum was not high ; the sera used were prepared by immunizing horses with the toxins of B. welchii and B. wdematis maligni. Although in a few cases the results were so striking that they could reasonably be attributed to the employ- ment of the serum, in general they were disappointing ; this may have been due to ignorance of the proper dosage and of the best route for administering it (Stokes and Wallace). Moreover, as Stokes pointed out, to decide on its efficacy from clinical observa- tion is not easy, for the disease may take a surprising change for the better, apart from any special treatment, even when the patient appears to be moribund. In the summer of 1918 observations on a considerable scale were made to determine, if possible, the prophylactic value of an anti- toxin for B. welchii when it was given soon after the wound was in- flicted. The serum was injected together with a prophylactic dose of tetanus antitoxin, and the combined dose was repeated in the base hospitals. There does not seem to have been any significant diminution of the incidence of gas gangrene in the group to which the specific serum was given, but the mortality in this group was definitely lower than that in the control group (11-6 per cent. : 22-8 per cent.); these figures are based on observations that were made on nearly 40,000 men in the base hospitals. Stokes concluded his account of the specific therapy of gas gangrene in 34 THE SERUM TREATMENT OF GAS GANGRENE war wounds by adding a number of recommendations which deserve the consideration of those who may be confronted by a similar problem. The Committee who were appointed by the Medical Research Council in 1919 to collect information on the infections caused by anaerobic bacteria, summarized in an admirable manner the experience of the treatment that was gained during the war. They thought that the results obtained in France surpassed ex- pectations, and considered it probable that prophylactic injections of the serum would prove in the future to be more effective than curative treatment by its means. They mention, too, the possi- bility of protecting troops that are exposed to the risk of wound gangrene by immunizing them with a properly balanced toxin- antitoxin mixture ; improved methods of producing active immunity against the toxins of the chief anaerobes may yet supplant to a large extent the prophylactic and curative use of a specific serum. The Serum Treatment of Gas Gangrene in Civil Practice Gas gangrene is not a common infection from injuries in times of peace. Thus Siebner (1929) states that there were only six cases among 4,500 patients in the surgical department of a hospital in Stuttgart during a period of three years; four of them died, and serum treatment probably aided the recovery of one of the two patients who survived. Siebner recommends that an immediate dose of a multivalent serum which is combined with tetanus anti- toxin should always be given when it is likely that a wound has been infected with anaerobic bacilli. Larson and Pulford (1930) used a serum which was capable of neutralizing the toxins of the three chief types ; seven patients received the treatment, and six of them recovered. The serum was given intravenously and was also injected into the deep tissues near the wound; no patient received more than 1,000 c.em. of it. These authors insist on the necessity for treating the wound by radical surgical methods in addition to the use of serum. Timely and thorough cleansing of the injured part, together with the removal of foreign material TREATMENT OF ABDOMINAL CONDITIONS 55 and devitalized tissues, should be practised, and afterwards the wound should be frequently irrigated with antiseptic solutions such as one per cent. chloramine. The recognition of the early symptoms of the infection is important ; these include pain in the wound, which is free from any inflammatory reaction and, indeed, may show pallor of the surrounding skin ; a feeling of malaise and restlessness ; and a rise in the temperature and pulse-rate. Vomiting may be an early symptom, and later in the illness it may cause much distress to the patient (Wallace). The Serum Treatment of Acute Abdominal Conditions The Treatment of Intestinal Obstruction and of Peritonitis. Williams (1926, 1927) has pointed out that the tox@mic symptoms of acute intestinal obstruction resemble those of acute peritonitis when this is accompanied by a secondary paralytic obstruction of the intestine ; and that the features which are common to both of these conditions recall the clinical picture of the toxsemia of gas gangrene. He formed the theory that the toxemia in primary intestinal obstruction and in peritonitis with obstruction is attributable to absorption of the toxin of one of the gas-gangrene group of bacilli, and in particular B. welchii. He believes that the lower portion of the small bowel, especially when it is obstructed or paralysed, is the only part of the intestinal tract that is suitable for the proliferation of B. welchii, because the contents of this part of the bowel, when they become stagnant, are alkaline and provide the requisite anaerobic conditions. In pyloric obstruction the acidity of the stomach contents is likely to destroy any toxin that may be formed by B. welchit, and, similarly, acute toxaemia is never seen when stagnation is confined to the large intestine, owing to the acid character of its contents. Williams obtained some evidence of the presence of this toxin in the small intestine in cases of intestinal obstruction and peritonitis in man and in dogs. Other workers, however, are not convinced that B. welchit gives rise to the toxic symptoms, so that there is at present no agreement regarding the pathogeny of the toxsemia which is associated with intestinal obstruction. 56 THE SERUM TREATMENT OF GAS GANGRENE “Acting on his belief that the toxin of B. welchii plays an important part in acute abdominal emergencies, Williams gave a trial to a specific antitoxic serum for patients who were suffering from peritonitis with paralytic obstruction ; he found that their condition showed a noticeable improvement. Similar results were obtained in cases of organic obstruction, but it was not always possible to distinguish between the effects of the serum treatment and of the removal of the obstruction by operation. The clinical effects of the serum treatment were often immediate. Restless- ness was greatly diminished or wholly disappeared, and the patients slept better and required less morphia ; cyanosis, when it had been present, passed off ; the pulse-rate became slower soon after the serum was given; the signs of abdominal distension lessened, and the bowels sometimes acted spontaneously within a few hours. The mortality-rate in 256 serum-treated cases of acute appendicitis was 1-2 per cent. as compared with a rate of 6-3 per cent. in a control group ; in 54 cases of acute intestinal obstruction the rate was 9-3 per cent., as compared with a rate of 24-8 per cent. in control patients. The initial dose of serum which Williams recommends is at least 80 c.cm. intramuscularly, and in serious cases an additional 40 c.cm. intravenously. On subsequent days 40-80 c.cm. should be given intramuscularly until the distension has disappeared and the bowels are moving spontaneously and regularly. A patient who is suffering from acute intestinal obstruction may die of toxaemia soon after the condition has been relieved by operation. Williams advises that, in order to forestall this risk, a dose of antitoxin should be administered intravenously before operation. It is true that this procedure tends to create a false sense of security in the mind of the surgeon, so that he may be tempted to minimize the gravity of the patient’s condition ; yet it cannot be denied that, as with any therapeutic serum, a dose given early is much more likely to be effective than one that is given at a later stage of the illness. Bower and Clark (1928) have reported on a series of 25 cases in which the treatment followed the lines laid down by Williams. The serum was administered to 11 patients with acute suppurative TREATMENT OF ABDOMINAL CONDITIONS 57 peritonitis; in 9 of them the primary focus was a perforated appendix. Five of the patients were operated on at once and antitoxin was given after the operation ; two of them died. Anti- toxin was given to the remaining six patients and operation was postponed : when the patients were operated upon later the appen- dix was not removed ; there were no deaths. The serum was given intravenously and intramuscularly in doses of 40 c.cm. of natural serum and 20 c.cm. of concentrated serum. Bower and Clark noted that the tox@mic symptoms were mitigated in much the same way as that described by Williams. They give a guarded opinion upon the value of the serum, but think that the results were favourable. Fagge (1981) from his experience at Guy’s Hospital believes that the use of a gas-gangrene serum in the treatment of patients with advanced peritonitis or of those who are suffering from acute ob- struction that has lasted for some days is at times of the greatest value. His view is shared by Corry (1931), who has given an account of five cases of strangulated femoral hernia, in which the obstruction had persisted for several days and was accompanied by regurgitant or feeculent vomiting. The treatment consisted in the relief of the obstruction by operation under a local anaesthetic, and the introduction intravenously during the operation of from 2} to 8} pints of saline, and of from 40 to 50 c.cm. of B. welchii anti- toxin. After the operation saline and glucose were given continu- ously by the rectum in order to combat the dehydration of the tissues caused by the vomiting. Corry remarks that the general condition of the patients on the day following the operation agreeably surprised him ; all of them made a good recovery. The treatment of appendicitis with an anti-gangrene serum as an adjuvant to operation has been carried out on a considerable scale in Paris since the war by Weinberg and his colleagues (Weinberg, 1929). They have given a description of 52 acute non-gangrenous cases of appendicitis that were successfully treated with the serum without a death, and of 44 cases of gan- grenous appendicitis in two of which no benefit resulted ; the failures were attributed to the lack in the serum of specific anti- bodies against the anaerobes which were multiplying in the gangrenous tissues. 58 THE SERUM TREATMENT OF GAS GANGRENE This group of workers have noted that B. welchii and B. coli are the chief pathogenic bacteria in acute appendicitis, the former being present in about one-third of the cases ; B. welchii is invariably found when the appendix is gangrenous. They there- fore use a multivalent anti-gangrene serum to which has been added one that has been prepared by immunizing horses with cultures of B. coli; they give the mixed serum according to circumstances, either prophylactically or curatively, with bene- ficial results. The Use of an Anti-gangrene Serum in Typhoid Fever. Weinberg and Thibault (1927) have described cases of typhoid fever associated with numerous B. welchii in the stools. They believe that this bacillus is able to pass through the intestinal wall at the site of a typhoidal lesion and invade the blood-stream. The treatment consists of an anti-typhoid serum administered subcu- taneously and an anti-gangrene serum administered per rectum. Other observers (Tapia and Aznar) were unable to obtain favourable results with this method of treatment. The Serum Treatment of Puerperal Infections caused by Pathogenic Anaerobes Infections of the uterus with pathogenic anaerobes (B. welchit) frequently follow attempts at criminal abortion and often prove fatal, but puerperal infections of this nature are comparatively rare ; Wrigley (1930), in a search of the literature, was able to discover only 20 cases. An examination of the records of the Obstetrical Department of St. Thomas's Hospital showed that between the years 1922-1927 there were 16 deaths from puerperal sepsis, and that in six of them post-mortem examination revealed the presence of generalized gas gangrene. Wrigley made observa- tions on the frequency of occurrence of B. welchii during pregnancy and the puerperium. He concluded that this organism is not present in the cervix before delivery nor in the lochia after delivery, when pregnancy, labour and the puerperium are normal ; on the other hand, in 69 cases in which these were abnormal, B. welchii was isolated in 13 cases from the lochia. The puerperium SERUM IN PUERPERAL INFECTIONS 59 in four of these patients was complicated by a generalized gas- gangrene infection, a condition which is always associated with previous intrauterine manipulations and which is nearly always fatal. In Wrigley’s view the generalized type of infection is the terminal stage of a sequence of events which may be summed up briefly as follows : (1) the introduction of the bacteria as the result of intrauterine manipulations ; (2) the circumstance that the child is dead when the infection is introduced, thereby pro- viding material for the growth of the bacteria ; (3) the retention of the dead child for some hours in utero, so that time is allowed for the proliferation of the bacteria; and (4) the access of the infective material to the tissues of the uterus at the site of trauma caused by the manipulations. When the traumatic factor is absent the patient may recover in spite of the existence of puerperal infection ; this is indicated by the finding of B. welchii in the lochial discharge for many days after delivery. Wrigley empha- sizes the grave risk of leaving a dead foetus undelivered, or of performing any but the most gentle intrauterine manipulations when removing a child which has been dead for some hours in utero. He strongly urges that when the combination of circumstances related above is encountered a prophylactic dose of at least 40 c.cm. of a concentrated specific antitoxin should be administered at the earliest possible moment and without waiting for bacteriological evidence of anaerobic infection; a general infection may proceed with such rapidity that no time should be lost in giving the serum. When the infection is already estab- lished the serum should be given freely either by local injection or by the intravenous route. Wrigley is doubtful whether hysterectomy is advisable in any circumstances when the uterus is attacked by gas-gangrene infection; he believes that this operation is perhaps justifiable at an early stage in a patient in whom no demonstrable extrauterine source of infection has been discovered ; if operative treatment is undertaken, it should be reinforced by vigorous treatment with antitoxin. A case of puerperal infection due to B. welchii which was treated by the use of an anti-gangrene serum has been described by 60 THE SERUM TREATMENT OF GAS GANGRENE Ivens (1929). Labour was protracted and delivery difficult owing to the large size of the child, which had been dead for some time. Thirty c.cm. of anti-streptococcal serum were given, and next morning 20 c.cm. of anti-gangrene serum and 20 c.em. of an anti-coli serum were administered subcutaneously ; the patient recovered. Adams and Adams (1931) have given details of the illness of a woman, aged 25 years, who died, during parturition, of a generalized gas-gangrene infection accompanied by extensive subcutaneous emphysema. The symptoms began during the first stage of labour, and the patient died before the third stage was completed. The foetus was macerated and emphysematous, and had probably been dead for some time; its condition was almost certainly attributable to syphilis in the mother. An intramuscular injection of 30 c.cm. of gas-gangrene serum was given to the patient, although the prognosis seemed to be hopeless. Anaerobic Infections that may be benefited by the use of Gas-gangrene Antitoxin For the sake of completeness, it may be worth enumerating a number of conditions which are associated with the gas-gangrene group of bacilli and in which specific serum therapy may be given a trial. Pearce Gould and Whitby (1926-1927) relate the case of a woman, 43 years of age, whose gall-bladder, when removed at operation, showed gangrenous patches in the mucous membrane. A smear of the fluid contents revealed numerous bacilli resembling B. welchii. The gall-bladder contained 20-25 pale facetted stones, and from six of them B. welchii was cultivated. The patient made a complete recovery. The authors cite two similar cases, one of which was fatal. Gordon-Taylor and Whitby (1930) demonstrated the presence of B. welchii in nearly 9 per cent. of gall-bladders removed by operation, and in 13 per cent. of gall-stones obtained from the post-mortem room. In two gangrenous cases this anaerobe was isolated in pure culture from the wall of the gall-bladder, the bile, PREPARATION OF ANTITOXIN 61 and the central portion of the gall-stones. One of the patients, although desperately ill at the time of operation, quickly recovered, and the authors attribute the rapid subsidence of the toxemia, in part at least, to the administration of gas-gangrene antitoxin. In Bucharest, since the war, scarlet fever has been characterized by an increase in the severity of the symptoms, a necrotic angina having been present in 7 per cent. of the cases ; this complication had proved to be refractory to the action of scarlet fever antitoxin. A multivalent serum prepared against the chief types of anaerobe was given early and in large doses, with the result that of 70 patients, 39 of whom were gravely ill, only 9 died (15 per cent.). The death-rate (23 per cent.) in the group of severe cases thus treated contrasts favourably with the usual mortality of 80 per cent. in severe necrotic angina (Stro¢, Hortopan, and Bazgan, 1931). Lesions that become infected from the intestinal tract, for example, perirectal abscess, are likely to contain sporing anaerobes. Again, pulmonary gangrene has been treated by Flaum (1926) in Sweden with anti-gangrene serum; doses of from 20 to 80 c.cm. were injected daily into a muscle, and, of 8 patients, 4 recovered. Within eleven years 41 cases were treated by him with the serum, and in 23 per cent. recovery or improvement followed; the temperature fell and the amount of expectoration and its feetor diminished. The serum is also worthy of trial in cases of diabetic gangrene. Lastly, the resistance of the spores to heat and to the action of the ordinary disinfectants explains the occasional occurrence of local gas gangrene after the hypodermic injection of therapeutic substances, for example, morphine, adrenaline, caffeine and camphor. The Preparation and Titration of Gas-gangrene Antitoxin The principles that are followed in the preparation of a gas- gangrene serum are those which have already been mentioned in connection with the ordinary antitoxic or antibacterial sera. A multivalent serum is often prepared, that is to say, one against the more common infective types of anaerobe. The serum is always 62 THE SERUM TREATMENT OF GAS GANGRENE an antitoxic one, but in the opinion of some authorities (Robertson and Felix ; and Weinberg and Barotte) it should also possess an antibacterial component. Robertson and Felix (1930), in their prophylactic experiments on mice, showed that a serum which was prepared in a horse by means of a particular strain of Vibrion septique and which contained no antitoxin, but only an anti- bacterial immune-body, was more effective in saving life than two sera that were purely antitoxic; this immune-serum was found to be type-specific. They recommend, therefore, that a thera- peutic serum should be made by employing a mixture of all the four types of V. septique, or, if this is not practicable, that the hyperimmunization should be pushed to such a point as to ensure that the serum will be effective against the common antigen of all the four types; in their view, a therapeutic serum should be both antitoxic and antibacterial. Most serologists agree that multi- valent sera should be prepared by mixing the appropriate mono- valent sera, since otherwise it is very difficult to titrate the various antibodies which they may contain. Experimental work by Craddock and Parish (1931) shows that a serum prepared against the toxin of B. wdematis maligni (Vibrion septique), when injected intravenously or intraperitoneally in divided doses, protected 75 to 100 per cent. of mice against 100 fatal doses of washed spores, with calcium chloride as the activator. Mice that were nearly moribund five hours after the infecting dose had been given recovered after the intravenous administration of antitoxin, They conclude that when this antitoxin is used in the treatment of anaerobic infections in man, large doses should be given without delay by the intravenous route. Much has been written in late years on the titration of antitoxins for B. welchii toxin and the toxins of other members of the group, but the only method that has received official recognition in Great Britain and the United States is the method of testing B. welchii antitoxin. In the United States the titration is carried out by the intramuscular injection of serum-toxin mixtures into pigeons in accordance with the standard unit of antitoxin pre- scribed by Bengston. Recently the British and the Washington control authorities have agreed upon a unit which represents TITRATION OF ANTITOXIN 63 exactly 1/100 part of the original ‘ Bengston’ unit. In Great Britain the method of carrying out the titration consists in giving the serum-toxin mixtures intravenously to mice ; it is an excellent one and permits of the accurate testing of the toxins and anti- toxins. REFERENCES Apawms, J., and Apams, P. 1931. Brit. Med. J., 2, 1179. Ascuorr, L. 1923. Klin. Woch., p. 2224. Ref. in Handb. der path. Mikroorg., 1928, 4, 1177. BenGstoN, I. A. 1920. Bull. U.S. Hyg. Lab., No. 122, 20. Bower, J. O., and CrArk, J. 1928. Amer. J. Med. Sci., 176, 97. Corry, D. C. 1931. Brit. Med. J., 1, 219. CRADDOCK, S., and Parisu, H. J. 1931. Brit. J. Exp. Path., 12, 389. Facer, C. H. 1931. Brit. Med. J., 1, 50. Fraum, A. 1926. C. R. Soc. de Biol., 95, 1518. Ref. in Bull. Inst. Past., 1927, 25, 324. GORDON-TAYLOR, G., and Wuirsy, L. E. H. 1930. Brit. J. Surg., 18, 78. GouLp PEARCE, E.,and Wuitsy, L. E.H. 1926-1927. Brit. J. Surg., 14, 646. Ivens, F. 1929. Lancet, 1, 606. Larson, E. E., and Puvrrorp, D. S. 1930. J. Amer. Med. Ass., 94, 612. Reports of the Committee upon Anaerobic Bacteria and Infections. Special Report Series, No. 39, Medical Research Council, 1919. ROBERTSON, M. 1929. “A System of Bacteriology, London, H.M. Stationery Office,” 3, 225. ROBERTSON, M., and Ferix, A. 1930. Brit. J. Exp. Path., 11, 14. RumpeL, F. 1918. Verdff. Milit. Sanitdtsw., 1918, H. 68, S. 35. Ref. in Zeissler, J., 1928, Handb. der path. Mikroorg., 4, 1177. SIEBNER, M. 1929. Deut. Z. f. Chir., April, p. 59. Ref. Brit. M. J., Epit., 1929, June 22nd. Stokes, A. 1923. ¢ History of the Great War, Medical Services Pathology,” p. 78. StTrO#, A., HOorRTOPAN, D., and BazcanN, J. 1931. Rev. frang. de pédiat., vy 23, Tapia, M., and AzNAR, P. 1929. La med. Ibera, October 26th, p. 405. Ref. Brit. M. J., Epit., January 11th, 1930. WALLACE, C. S. 1922. ‘ History of the Great War, Medical Services, Surgery of the War,” 1, 134. WEINBERG, M. 1929. Bull. Inst. Past., 27, 529 and 577. WEINBERG, M., and BarorrTE, J. 1929. Ann. Inst. Past., 43, 453. WEINBERG, M., and GINSBOURG, B. 1927. Données récentes sur les microbes anaerobies. (Paris.) 64 THE SERUM TREATMENT OF GAS GANGRENE WEINBERG, M., and StcuiN. 1918. La gangréne gazeuse. (Paris.) WEINBERG, M., and THiBAULT, G. 1927. C. R. Soc. de Biol., 97, 1476. Ref. Bull Hyg., 1928, 3, 615. Wirniams, B. W. 1926. Brit. J. Surg., 14, 54; Lancet, 1927, 1, 907. WRIGLEY, A. J. 1930. Proc. Roy. Soc. Med., 23, 1645. ZE1ssLER, J. 1928. Handb. der path. Mikroorg. (Kolle, Kraus, Uhlenhuth), 4, 1177. CHAPTER V THE SERUM TREATMENT OF SCARLET FEVER AND OTHER STREPTOCOCCAL INFECTIONS PAGE AN Historicarn NOTE . " 65 THE INFLUENCE OF THE SPECIFIC ANTIT OXIN uU PON THE Sy MPTOMS OF SCARLET FEVER 67 THE TREATMENT OF SCARLET FEVER R BY ME ANS OF INTRAVENOUS INJECTIONS OF ANTITOXIN ’ ’ . 69 SCARLET FEVER ANTITOXIN AS A PROPHYLACTIC AGENT . y 71 THE PREPARATION AND TITRATION OF SCARLET FEVER ANTI- TOXIN ” ’ . ” 72 Tue Use or ** CONVAL ESCE NT SERUM 1 IN THE TREATMENT OF SCARLET FEVER ” ; . 73 The USE OF SCARLET FEVER "ANTITOXIN IN V. ARIOUS KINDS OF STREPTOCOCCAL INFECTION 5 5 . " 73 THE SERUM TREATMENT OF ERYSIPELAS . . . . 75 THE SERUM TREATMENT OF PUERPERAL FEVER 1 . v kid MULTIVALENT ANTI-STREPTOCOCCUS SERUM 3 . ” ; 78 An Historical Note In the year 1887 Klein directed attention to the association of streptococci with scarlet fever; from the throat and blood of patients he isolated streptococci which he regarded as belonging to a specific type, and which he therefore named S. scarlatine. Marmorek (1895) and Aronson (1902) prepared an anti-strepto- coccus serum for the treatment of scarlet fever, but the results of their work were less striking than those obtained by Moser (1902) in Vienna. Moser’s paper makes interesting reading in the light of present knowledge, first, because his mode of attack upon the problem was a logical one, and, secondly, because the description of the effects produced by his serum agrees remarkably well with recent accounts of the therapeutic action of scarlet fever antitoxin. There is every reason to believe that Moser’s serum owed its success in treatment to a specific antitoxic property. Unfortu- nately, it was prone to cause severe serum reactions, as might be R.A. VACCINES, 65 3 66 THE SERUM TREATMENT OF SCARLET FEVER expected from the circumstance that it was a natural serum and that, owing to the lack of any method of titration, it was necessary to give large doses of it (100-200 c.cm.). KEscherich (1903) used the serum in the University Clinic for Children in Vienna, and, like Moser, was impressed by its action in quickly relieving the toxsemic symptoms ; an improvement was sometimes evident in as short a time as four hours after its administration. The Russian observers Sawchenko (1905) and Gabritschewsky (1906) correctly appreciated the true direction in which progress would be made, for they worked at the problem of producing a specific toxin and antitoxin. In the year 1918 Schultz and Charlton described the blanching of the skin of a patient with a scarlet fever rash that followed the injection into it of serum from a convalescent patient. This extinction effect was attributed by Mair (1923) to the neutralizing action of a specific antitoxin in the blood of the convalescent patient. In 1923 G. F. Dick and G. H. Dick (1923, 1924! and 19242) brought forward evidence which indicated that the scarlet fever streptococcus with its toxin is the chief agent in the etiology of the disease. Dochez in 1924 prepared a therapeutic serum from horses by means of a special technique which had for its twofold object the growth of the streptococcus and the liberation of its toxin within the tissues of the animal; cultures of the streptococcus were introduced into agar which had previously been inserted subcutaneously into the horse. Dick and Dick succeeded later in producing an antitoxic serum by the simpler method of inoculation of toxic filtrates into the horse. Further details of the early work on the scarlet fever streptococcus will be found in an article by Park (1925). The investigations into the causation of scarlet fever that have been made during the past ten years have undoubtedly put the treatment of the disease upon a rational basis by providing a specific remedy for it. Some writers believe a filter-passing virus to be its essential cause, and regard the streptococcus and its toxin as of secondary importance. But we have found little support for this view in the literature of the subject. On the contrary, it is evident that the symptoms of the disease are toxamic in their nature, that they can be modified by giving the specific strepto- THERAPEUTIC EFFECTS 67 coccal antitoxin, and that the septic complications of scarlet fever are adequately explained by the growth of the streptococcus in the focal lesions. The hypothesis of a primary filter-passing virus is thus rendered unnecessary. The Influence of the Specific Antitoxin upon the Symptoms ot Scarlet Fever Numerous observers in many countries have published the results of their experience with scarlet fever antitoxin, and only a few disagree with the opinion that the serum, if it is given early in the course of the disease, definitely modifies the severity of the symptoms and hastens their disappearance. The temperature subsides to the normal level in from 12 to 48 hours, the time depending upon the method of administering the serum ; an intravenous injection will often bring about a critical drop in the temperature. Park, in his paper (1925), presents a chart which gives the composite temperature curves of 25 severe, 25 moderate and 25 mild cases, which were treated with the serum during the first three days of the disease, together with an equal number of comparable cases which did not receive it. The curves of the serum-treated groups are consistently lower than those of the control groups. According to von Bormann (1929), the temperature curve is the best indication of the action of the serum ; this author noted a distinct difference between the temperature curves of the treated and the untreated cases, but only when the serum was given before the fourth day of the disease. The effect on the pulse-rate and the respiration-rate is observed about the same time as the fall in temperature, and is equally remarkable. The character and course of the eruption are influenced by the serum ; if it is given within the first 24 hours of the onset of the illness the rash will disappear within 12 hours, and will be followed by very slight desquamation. The rapid disappearance of the rash and the scanty desquamation may lead to doubt being cast upon the diagnosis (Cushing, 1926). 3—2 68 THE SERUM TREATMENT OF SCARLET FEVER Symptoms that are characteristic of the more acute form of the disease are favourably influenced. The pain in the throat is greatly relieved. In twelve of the serum-treated cases observed by Scott (1928) a profuse rhinorrheea ceased in a striking manner within 48 hours. Vomiting, cardiac palpitation and convulsions are relieved in a remarkable way by giving the serum (Toyoda et al, 1930). Delirium and joint pains, when present, disappear, and the subjective sensations of the patient are so much improved that he may assert that he feels quite well and is able to get up (Cushing). The benefit conferred by the serum is sometimes so rapid and striking that the physician may be led to doubt whether he has not made a mistaken diagnosis (Dick and Dick, 1929). In most countries the disease is generally so mild that it is not always easy to be certain whether serum treatment has been beneficial or not. Again, lack of success in severe cases may be due in some instances to the use of a serum which is deficient in antitoxic units : scarlet fever antitoxin is not easily titratable. Gabriel (1929), from his observation of more than 400 patients, was unable to satisfy himself that the serum was definitely efficacious. But, as has already been stated, most clinicians affirm that the early administration of the antitoxin is able to ameliorate the symptoms in the severe type of scarlet fever; indeed, as Park declares, the more toxic the case the more manifest will be the improvement in the symptoms. A good critical review of the literature is given by Toomey (1928), whose conclusion is that the value of the serum will be finally determined during an epidemic of scarlet fever that is associated with a high rate of mortality. There is lack of agreement regarding the propriety of giving the serum to every case of scarlet fever, whether it be mild or severe. Dick and Dick insist that it should not be withheld until it is apparent that the attack will be a severe one. Cushing and other clinicians share this view, but Rolleston (1930) and his colleagues in the London Fever Hospitals reserve the use of the antitoxin for those patients whose symptoms presage a severe attack ; Rolleston employs it in not more than about 10 per cent. of his cases. The question has often been asked whether the serum, when it is given early, prevents complications and mitigates the severity TREATMENT BY INTRAVENOUS INJECTIONS 69 of those which make their appearance after its administration, or whether it possesses any curative action on existing septic com- plications. The evidence goes to show that the antitoxin reduces the incidence of complications or modifies them, but that it is powerless to exert a healing action on septic complications when they are already well established. We may note, however, that Comber (1931) administered scarlet fever antitoxin to five patients with scarlet fever complicated by septicemia; four of them recovered. A specific preventive or curative action on scarlatinal nephritis does not appear to have been definitely proved, although Dick and Dick (1929) assert that the incidence in the serum- treated cases is diminished by one-quarter. The mild character of scarlet fever at the present time with its fatality-rate of only a few per cent. makes a reduction in the rate due to the serum less apparent than a proportionate decrease in a lethal type of the infection. Dick and Dick state that antitoxin treatment has halved the number of deaths, and this figure agrees with the results obtained by others. The serum is usually given by the intramuscular route, and a suitable dose is from 10 to 50 c.cm. of a concentrated preparation, the amount varying according to the age of the patient and the severity of the symptoms. Most observers hold the view that the fourth day from the onset of the disease marks the limit for effective action of the serum. Serum reactions are not, as some think, especially liable to follow the administration of scarlet fever antitoxin. They were frequent when the natural serum was used, but their incidence decreased when concentrated pre- parations were obtainable. The Treatment of Scarlet Fever by means of Intravenous Injections of Antitoxin Banks and Mackenzie (1929) used this method for the routine treatment of all cases admitted to hospital in the acute stage, even up to the fifth or sixth day of the disease, with the exception of children under two years of age and patients whose history or physical condition gave an indication of danger from protein 70 THE SERUM TREATMENT OF SCARLET FEVER shock. They have treated 404 patients in this way, and obtained results which resembled those described above, but which differed from them in two respects; first, the response to the treatment was even more rapid, for the acute process was arrested within a few hours, and secondly, the incidence of complications was negligible, because in this series there was not a single instance of acute otitis media, arthritis, or nephritis. Desquamation was usually trifling or absent, and recovery was so rapid and un- eventful that the average stay of the patient in hospital was reduced to a period of just over a fortnight, and yet the rate of return cases was only 1-7 per cent. Unfortunately, one death resulted from protein shock, and serum reactions occurred in fully 60 per cent. of the cases ; they appeared soon after its administra- tion. In a later paper Banks (1931) states that in three years he treated 832 cases by this method with a complication-rate of just above 1 per cent.; the complications were: albuminuria (2), acute suppurative otitis media (5), and mild arthritis (3). The series included nearly all the undoubted cases that were admitted up to the fifth day, as well as ‘septic ”’ cases up to the eighth day of the disease. Further confirmatory experience has been described by this author (Banks, 1933). Similar results are recorded by Lichtenstein (1931) from the Hospital for Infectious Fevers at Stockholm. He gave the antitoxin intravenously to 512 patients with moderate and severe scarlet fever. In uncomplicated cases the benefit was quickly apparent, and there were no deaths among the patients who received the serum before the onset of complications. When the serum was given within the first three days of the illness the frequency of these was lessened. The treatment was well tolerated ; a few patients suffered from symptoms of collapse and only one from serum sickness. The risks accompanying the intravenous injection of the serum are likely to deter most physicians from adopting the method in mild cases, although it is certain that the intravenous route is the most effective one. Rolleston and also Harries have recorded their opinion that the routine practice of this method is not justifiable. ‘ PROPHYLACTIC USE 71 Scarlet Fever Antitoxin as a Prophylactic Agent An outbreak of scarlet fever in a hospital or institution for children seriously disturbs its administrative routine by necessi- tating quarantine of the units in which the infection has appeared. The success of the specific antitoxin as a curative agent naturally suggested its use in circumstances of this kind and also for the protection of family ‘ contacts” of children with scarlet fever who are treated at home (Kirkhope, 1926). Joe and Swyer (1929) made observations on forty separate outbreaks of scarlet fever in the non-scarlet fever wards of a hospital for infectious diseases during a period of two years ; they conferred a passive immunity upon more than 250 persons by giving them the antitoxin. They regard their results as encouraging and think that the case for passive immunization is a strong one. Apart from warding off illness and even death, hospital accommodation is saved by reducing the period of quarantine from 10 to 3 days, the shorter period representing the time occupied in testing the contacts for susceptibility and immunizing them. The immunizing dose of the serum varied from 3 c.cm. to 10 c.cm., and the degree of immunity in those who received it did not appear to differ much, according to the results obtained afterwards by applying the Dick test. Joe and Swyer recommend that susceptible contacts up to the age of 10 years who give a Dick reaction of less than 30 mm. in diameter should receive 5 c.em. of antitoxin intra- muscularly and should be examined again by the Dick test after 48 hours ; when the reaction is positive for the second time the dose should be repeated. Contacts above 10 years of age and those of any age whose reaction is 30 mm. or more in diameter should receive an initial dose of 10 c.em. of the serum. McClean (1927) carried out a similar procedure in a General Hospital for children ; the spread of the disease was apparently prevented, for all the protected patients escaped the infection. The plan was so successful that it was decided to give up quarantine and to proceed with surgical operations and the admission and discharge of patients in accordance with the normal routine of the hospital. 72 THE SERUM TREATMENT OF SCARLET FEVER Other observers who have obtained similar results are Harries, Hervey and Fellowes (1926), James (1928), Cowie (1930), and Schottmiiller (1930). The last-mentioned author tried a combina- tion of active and passive immunization ; he gave first 10 c.cm. of a specific serum and 1 c.em. of a toxin preparation, and after- wards two successive doses of toxin. The combined method is a rational one, for it is well known that only a transient immunity is conferred by the antitoxin, since it disappears after two or three weeks ; on the other hand, an active immunity gives lasting results although it develops slowly. The Preparation and Titration of Scarlet Fever Antitoxin The usual method of immunizing horses closely follows the scheme for producing diphtheria antitoxin, namely, the inoculation, subcutaneously or intramuscularly, of increasing doses of filtered broth cultures. The antitoxin is associated with the pseudo- globulin fraction of the serum proteins, and it can be concentrated by the ordinary processes. At the present time there is no agreed method of titration, although several have been suggested, as, for example, the skin test in goats, used by Wadsworth, Kirkbride and Wheeler (1926) ; the rabbit test of Parish and Okell (1927) ; and the skin test in susceptible human beings which is used by the Dicks and at the National Institute of Health in Washington. None of them is wholly satisfactory. The interpretation of skin tests in man is rendered difficult by : (1) variations in the specific reactions given by members of the susceptible group; (2) the appearance of reactions due to hypersensitiveness to the horse serum which is the medium for the antitoxin ; and (3) the variable reaction of an allergic nature which is caused by some non-specific substance in the test toxin : this is perhaps the most important disturbing factor (O’Brien, Okell and Parish, 1929 ; O’Brien, 1930; James, Joe and Swyer, 1930). Improvements in the methods of standardizing and titrating scarlet fever antitoxin will probably follow the lines of the experimental work on the concentration of the toxin that has been carried out by Hartley (1928) and by Pulvertaft (1928). TREATMENT OF STREPTOCOCCAL INFECTIONS 73 The Use of ‘ Convalescent ’’ Serum in the Treatment of Scarlet Fever Weisbecker, in the year 1897, was, perhaps, the first to use the serum from convalescent patients in the treatment of scarlet fever ; and since then the value of the method has often been confirmed. Nevertheless, its action is apt to be capricious, the uncertainty depending upon the dose, the varying antitoxin content of the serum preparation used, and the time of its adminis- tration in relation to the onset of the illness. Balteano and his colleagues treated 72 cases of malignant scarlet fever at Jassy in Roumania by means of intramuscular or intravenous injections of the serum, and observed an improvement in the symptoms of most of the patients. The fatality-rate was only 23 per cent. as com- pared with the usual rate of 90 to 100 per cent. in this type of case. Lichtenstein (1931) obtained satisfactory results in more than 700 severe and moderately severe cases. Look (1932) from a small experience recommends the transfusion of blood from convalescent patients to patients who are seriously ill and who have failed to benefit from the use of scarlet fever antitoxin derived from the horse. The Use of Scarlet Fever Antitoxin in Various Kinds of Streptococcal Infection The streptococcus that is associated with scarlet fever is a member of the group of streptococci which secrete a filterable hemolysin. This group includes not only the scarlet fever streptococcus, but also those which are associated with erysipelas, puerperal fever, and a variety of acute and chronic inflammatory and suppurative conditions. Many of these hwmolytic strains secrete in cultures a soluble toxin, which produces a reaction in the human skin indistinguishable from that given by the toxin of the scarlet fever streptococcus. In the United States most investigators favour the view that the various streptococcal toxins are strictly specific (Birkhaug, 1925; Lash and Kaplan, 1925; and Dick and Dick, 1929). On the other hand, research on the subject which has been carried out in Great Britain indicates that 74 THE SERUM TREATMENT OF SCARLET FEVER the toxins are closely related to each other, and that they are, perhaps, identical, the only difference, if it exists, being a quanti- tative and not a qualitative one ; the scarlet fever streptococcus is believed to be the most actively toxigenic member of the group (Eagles, 1926 ; McLachlan, 1927 ; Parish and Okell, 1928; Mackie and McLachlan, 1929 ; Okell, 1932). If the latter observa- tions prove to be well founded, they will furnish a scientific basis for the treatment by means of scarlet fever antitoxin of strepto- coccal infections due to other haemolytic strains. The clinical evidence that follows gives some support to the theory of a common toxin or, it may be, of a group of toxins which are produced by haemolytic strains and which are characterized by a considerable degree of antigenic overlapping. Thus Sanderson, Capon and MacWilliam (1927) successfully treated a patient who was suffering from puerperal septicemia and another patient who was ill with septicemia complicating otitis media by injecting concentrated scarlet fever antitoxin intramuscularly ; repeated doses of multivalent anti-streptococcus serum had previously been administered without effect. An account has been given by Rosher (1930) of similar treatment in four severe cases of septicaemia ; all the patients recovered. Burt- White (1930) treated 27 cases of puerperal fever with intravenous injections of concentrated scarlet fever serum in doses varying from 20 to 100 c.cm. ; he believed that improvement in the condi- tion of the patients resulted from the treatment, but was doubtful whether the serum acted specifically or not. A favourable account of the treatment with scarlet fever antitoxin of patients suffering from puerperal and post-abortal sepsis due to hemolytic streptococci has been given by Defoe (1930) from his experience with it in the Toronto General Hospital. References to the use of scarlet fever antitoxin as a prophylactic remedy against other types of streptococcal infection are scanty. A dose of the serum may be given immediately after the infliction of a wound received while performing a post-mortem examination or a surgical operation on a septic case (Okell and Parish, 1928). Baird and Cruickshank (1930) have suggested that a dose of 5-10 c.em, may be given to women at their confinement if for any SERUM TREATMENT OF ERYSIPELAS 75 reason they are considered likely to incur the risk of puerperal sepsis. A similar recommendation has been made by Burt-White, Colebrook et al (1930); they advise that the Dick test should be performed on women who give a history of complicated labours, and that the positive reactors among them should be protected by the injection of streptococcal antitoxic serum. To sum up, it will be seen that there is scarcely enough material upon which to form a judgement on the question at issue, but it can at least be said that the use of scarlet fever antitoxin or an antitoxin obtained by immunizing with any toxin-producing strain of streptococcus is justified in acute inflammatory or septic conditions when they are known to be associated with a hemolytic streptococcus. The Serum Treatment of Erysipelas Experimental work carried out by Birkhaug (1925) has led him to the conclusion that the streptococcus associated with erysipelas is a specific strain which is distinguishable from the streptococcus of scarlet fever and from other pyogenic streptococci. In 1926 he reported on the treatment by means of a specific antitoxin of 60 cases of erysipelas of moderate severity. The most notable effect of the serum was the prompt amelioration of the toxic symptoms, and the rapid and almost critical drop in the tempera- ture and in the pulse- and respiration-rate. The symptoms were relieved within 12 to 18 hours after the injection of the serum. The erysipelatous redness quickly faded, and the fluid in the blebs and cedematous tissues was rapidly absorbed. The dose adminis- tered was 100 c.cm. of an unconcentrated serum or 15-20 c.cm. of a concentrated product. Symmers (1928) has given the results of a year’s experience of the treatment at Bellevue Hospital, New York ; during this period 705 patients were treated with a serum which was appar- ently both antitoxic and antibacterial ; strains of streptococci derived from erysipelas were used for immunizing the horses. The serum was given intramuscularly in 10-25-c.cm. doses, the amount depending upon whether a concentrated or unconcen- trated product was used. Symmers tried the intravenous route 76 THE SERUM TREATMENT OF SCARLET FEVER for its administration, but gave it up after encountering two cases of anaphylaxis, one of which was fatal. The intramuscular injection was repeated every 24 hours until there was a noticeable improvement in the condition of the patient. In some patients the serum had no effect even after repeated doses. He regards the results as marking an advance in the treatment of the disease, and he thinks that the serum, as a therapeutic agent, will bear com- parison with diphtheria antitoxin. The data given in his paper indicate a shortening of the duration of the illness and a lowering of the death-rate as compared with the data of a control group. Symmers and Lewis state that the serum does not confer a lasting immunity, so that it does not prevent recurrences, nor does it lessen the incidence of complications. They mention, too, that erysipelas affecting the face responds better to the treatment than when it is situated on the limbs or trunk. In a later paper (1932) they give additional observations which, in their opinion, confirm the previous results, and they make a comparison between two large groups of cases that were treated with and without antitoxin. The fatality-rate was 7-1 per cent. of 3,311 patients who were given antitoxin in the period May, 1927, to May, 1932, and 10-1 per cent. of 15,277 patients who were treated without anti- toxin prior to the year 1927. Other observers who have reported favourably on the specific treatment of erysipelas are Anderson and Leonard (1927), Huntoon and Hutchison (1928), Gordon and Young (1929), and Eley (1930). In a critical article by McCann (1928) a different view is expressed. His results were not as good as those obtained by the authors just mentioned ; and he regards the evidence in favour of the serum as being inconclusive. He points out the importance of giving due weight to the factors that influence the death-rate in erysipelas, such as the age of the patient, the season of the year, and fluctuations in the severity of the disease from year to year. Benson's experience of the serum treatment of 200 cases of erysipelas in the City Hospital, Edinburgh, has not been altogether favourable, for in spite of liberal doses of erysipelas antitoxin and scarlet fever antitoxin, the spread of the local lesion was not checked. There was, however, in some instances a definite ANTITOXIN IN PUERPERAL FEVER m7 lowering of the temperature, and alleviation of the headache, malaise, and delirium. Wadsworth (1932) distributes for issue a monovalent serum which is prepared by means of Dochez’s “N.Y.5” strain of scarlet fever streptococcus, and he has reported on its use in the treatment of many forms of streptococcal infection. He states that the effect which is produced by the serum on the toxemia of erysipelas is similar to that which may be observed in scarlet fever. The serum was beneficial in 67 per cent. of 106 severe cases ; the mortality in this group was 11 per cent. ; there were 16 deaths in a total of 194 cases (8:2 per cent.). Platou, Schlutz and Collins (1927) compare the results of the antitoxin treatment with those obtained by the use of other methods; a combination of X-ray and antitoxin treatment proved in their hands to be particularly successful. Ude and Platou (1930) found that a single application of ultra-violet rays arrested the disease in 92 per cent. of their cases ; this result was slightly more favour- able than that given by the antitoxin treatment, but they admit that their figures are rather small for comparison. The Serum Treatment of Puerperal Fever Lash (1929), who upholds the specificity of the strain of strepto- coccus that seems to be the chief pathogenic micro-organism in puerperal fever, has treated 57 women in various stages of the disease with a specific concentrated antitoxin. The serum, when given within 36 to 48 hours from the onset of the illness, was comparable in its efficacy to diphtheria antitoxin, but was almost without value when given at a later stage; by its use the fatality-rate was reduced from 61 per cent. to 32 per cent. A memorandum by Cameron and Thomson (1931) on the prevention of puerperal pyrexia by the use of an anti-streptococcus (puerperal) serum includes a table which furnishes evidence of a reduced incidence of pyrexia after confinements and abortions when large doses (50-70 c.cm.) were given as a prophylactic in all complicated labour cases and in normal cases with perineal lacerations. If trouble was anticipated, the serum was given either during labour or a few days before its onset. They record that in the year 78 THE SERUM TREATMENT OF SCARLET FEVER 1929-1930 there was only one case of puerperal pyrexia, as defined by the British Medical Association Standard, among 1,243 confinements. Reference has already been made to the use of scarlet fever antitoxin in the prophylaxis and treatment of puerperal fever (p. 74). An additional example of its use will be found in a paper by Gaessler (1928); he treated by its means 400 febrile puerperal cases ; and in 39 per cent. of them the temperature and pulse became normal within a short time. He believed that the use of the serum was a measure that saved life. Ordinary multi- valent anti-streptococcus serum has also been tried, but the evidence in its favour appears to be slender, although Theobald (1926) thinks that its prophylactic value is undeniable when it is given soon enough and in large enough doses ; he considers that as a curative agent it is quite useless. Benson’s experience in the City Hospital, Edinburgh, has made him feel uncertain whether an effective therapeutic serum is available ; he admits, however, that most of his patients did not receive the serum before the third or fourth day of their illness. Multivalent Anti-streptococcus Serum This preparation has been in use for many years, so that there have been abundant opportunities for testing it and coming to a conclusion upon its value as a remedial agent in streptococcal infections. Doubtless it is occasionally efficacious, but most clinicians who have employed it have obtained disappointing results (Weaver, 1921; Novak, 1926; Lingelsheim, 1928; McLeod, 1929 ; Kinsella, 1929). The Council on Pharmacy and Chemistry in the United States, which advises upon the use of new and non- official remedies, requested Dr. Emil Novak to collect information on its value ; he addressed a questionnaire to a number of well- known surgeons, gynwcologists and obstetricians. The replies yielded no proof of any strong belief in its worth as a thera- peutic agent, and, indeed, of twenty-five opinions, sixteen were explicitly unfavourable. Since January, 1930, the Council has omitted the serum from its list of approved remedies, MULTIVALENT SERUM 79 This experience contrasts strongly with the general belief in the value of streptococcal antitoxin, and it must be supposed that there are difficulties in the way of obtaining a highly potent antibacterial serum against haemolytic streptococci. The sug- gestion has often been made that antitoxic sera for scarlet fever, erysipelas and puerperal fever might be improved by combining the antitoxin with an antibacterial component. In theory, this is a sound procedure, but it is questionable whether a potent antibacterial serum can be easily prepared ; moreover, there are good grounds for believing that the titration of such a serum, whether by animal test or in vitro, would be beset by many difficulties. Cotoni and Césari (1927) studied 136 strains of streptococcus that were associated with various diseased condi- tions. The great majority of the cultures were found to be non- pathogenic for laboratory animals (mice and rabbits), and the virulence could not be exalted to any extent by passage. Their attempts to immunize mice, rabbits and horses by means of a variety of antigenic preparations yielded insignificant and irregular results. The streptococcal group as a whole contains a variety of races or types whose antigenic nature and relationships are still obscure. It is probable, therefore, that until our knowledge of the group has increased, the preparation of a serum which will contain the antibodies needed for effective therapeutic use is likely to prove a difficult matter. A full and authoritative discussion on the subject of the serum treatment of streptococcal infections will be found in the Milroy Lectures dealing with the 7éle of the hemolytic streptococci in infective disease (Okell, 1932). The author notes the diversity of types that are being discovered in the streptococcal group, and observes that in consequence of this diversity nearly every strain of streptococcus will need to be countered by its own antibacterial serum. He believes that an antibody which is suitable as a prophylactic and therapeutic agent in all types of streptococcal infection has not yet been prepared against the group of hemolytic streptococci, with the exception of the antitoxin which neutralizes the erythrogenic toxin ; in his view there is no difference between the toxins that are produced by various 80 THE SERUM TREATMENT OF SCARLET FEVER members of the group. Okell points out that attempts to prepare a serum which will neutralize the pyogenic effects of the strepto- coceus in experimental conditions have not been successful. If we may judge from the arguments that have been used in clinical discussions on the efficacy of the serum treatment of streptococcal septicemia, it would seem that the complexity of the problem from the immunological point of view is not always appreciated. Observations that the present writer had the opportunity of making during the war on streptococcal septicaemia as a complica- tion of wounds led him to realize the importance of thorough surgical treatment of the primary septic focus which was acting as the nidus of the streptococci and from which they overflowed into the blood-stream. It is obvious that as long as the local conditions are favourable for maintaining a generalized infection serum treatment can be of no avail. REFERENCES ANDERSON, J. F., and LEoNarD, G. F. 1926. Amer. J. Med. Sei., 172, 334. A ARONSON, H. 1902. Berl. klin. Wschr., p. 979. Barb, D., and Cruicksuank, R. 1930. Lancet, 2, 1009. BavrreaNO, I., ALEXA, I., and ALExa, E. 1930. Arch. Roumaines Path. Expér. et Microbiol., 3, 231. Banks, H. S. 1929. Lancet, 1, 1248. »8 1931. Brit. Med. J., 2, 34. ge 1933. J. Hyg., 33, 282. Banks, H. S., and Mackenzie, J. C. H. 1929. Lancet, 1, 381. Benson, T. W. 1932. Brit. Med. J., 1, 797. Birkuave, K. E. 1925. Johns Hopkins Hospital Bull., 37, 85 ; Ref. J. Amer. Med. Ass., 1925, 85, 1002. > 1926. J. Amer. Med. Ass., 86, 1411. Boente, J. 1927. Z. f. Kinderheilk., 45, 135. Ref. Bull. Hyg., 1928, 8 527. BormaNN, voN KF. 1929. Jahrb. f. Kinderheilk., December, p. 1. Ref. Brit. Med. J., Epit., March 15th, 1930. Burr-Waite, H. 1930. Lancet, 1, 16. BurT-Wuite, H., and COLEBROOK, L., ef al. 1930. Brit. Med. J, 1, 240. CAMERON, S. J., and Tuomso~N, H. 1931. Brit. Med. J., 1, 350. CoMmBER, V. H. 1931. Lancet, 1, 698. Coron, L., and Cesari, E. 1927. Ann. Inst. Pasteur, 41, 1270. Cowie, J. M. 1930. Brit. Med. J., 1, 588. REFERENCES 81 CusHING, H. B. 1926. Canadian Med. Ass. J., 16, 936. Deror, W. A. 1930. Amer. J. Obst. and Gyn., 20, 174. Ref. in J. Amer. Med. Ass., 1930, 95, 1203. Dick, G. F., and Dick, G. H. 1923. J. Amer. Med. Ass., 81, 1166. ” ’s 19241 Ibid., 82, 265. . . 19242. Ibid., 82, 301. 9 0 1925. J. Amer. Med. Ass., 85, 1693. . . 1929. Ibid., 93, 1784. o 1929. Amer. J. Dis. Child., 38, 905. Docuez, A. R. 1924. Proc. Soc. Exper. Biol. and Med., 4, 184. Eacres, G. H. 1926. Brit. J. Exp. Path., 7, 286. ELey, R. C. 1928. Amer. J. Dis. Child., 35, 14. EscuericH, T. 1903. Wien. klin. Wschr., p. 663. GABRIEL, E. 1929. Jahrb. f. Kinderheilk., 125, 1 ; in Lancet, 1930, 1, 530. GABRITSCHEWSKY, G. 1906. Centralb. f. Bakt. I. Orig., 41, 848. GAESSLER, E. 1928. Miinch. med. Wschr., 75, 164. Ref. J. Amer. Med. Ass., 1928, 90, 1087. GorDON, J. E. 1927. J. Amer. Med. Ass., 88, 382. GorpON, J. E., and Younc, D. C. 1929. Mich. State Med. Soc. J ., 28, 353. Ref. J. Amer. Med. Ass., 1929, 93, 414. Harries, E. H. R. 1929. Brit. Med. J., 1, 997 Harries, E. H. R., HErvVEY, D., and FELLowEs, V. 1926. Brit. Med. J., 1, 864. HArTLEY, P. 1928. Brit. J. Exp. Path., 9, 259. Hun~Ttoon, F. M., and Hurcuison, R. H. 1928. ‘ The Newer Know- ledge of Bacteriology and Immunology,” Chicago, p. 921. James, E. 1928. Lancet, 1, 227. James, G. R., JOE, A., and SwyEr, R. 1930. J. Hyg., 29, 347. JoE, A., and SWyERr, R. 1929. Lancet, 1, 177. KiNsELLA, R. A. 1929. J. Amer. Med. Ass., 93, 1524. Kirknope, D. C. 1926. Brit. Med. J., 1, 214. KLEIN, E. 1887. Proc. Roy. Soc., 42, 158. LasH, A. F. 1929. Amer. J. Obst. and Gyn., p. 297. Ref. Brit. Med. J., Epit., August 3rd, 1929. LasH, A. F., and Karran, B. 1925. J. Amer. Med. Ass., 84, 1991. 5 PT) 1926. Ibid., 86, 1197. LICHTENSTEIN, A. 1931. Abstract in Brit. Med. J., Epit., May 16th. 1981. Acta Pediatrica, June 30th, p. 549. LiNnceLsaeEM, W. von. 1928. Handbuch der path. Mikroorg. (Kolle, Kraus, Uhlenhuth), 4, 841. Look, W. 1932. Arch. f. Kinderheilk., May 6th, p. 129. Mackie, T. J., and McLacuran, D. G. S. 1929. “A System of Bacteriology,” London, H.M. Stationery Office, 2, 71. Mair, W. 1923. 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Zeitschr. f. Klin. Med., 32, 188. CHAPTER VI ANTI-STAPHYLOCOCCUS SERUM Tue pathogenic staphylococci, and particularly the aureus varieties, are characterized by biochemical activities which have been known to bacteriologists for many years and which give a clue to the nature of the suppurative lesions in man. The type species, Staphylococcus aureus, produces a hzemolysin to which the red cells of the rabbit are the most sensitive ; a leucocidin, which is toxic to leucocytes ; and a toxin which causes necrotic lesions in the skin of the rabbit and which, when it is administered intravenously to this animal, may prove fatal within a very short time (Van de Velde, 1894; wv. Lingelsheim, 1900 ; Neisser and Wechsberg, 1901). There is every reason to believe that these toxic substances play an important part in human staphylococcal infections. Neisser and Wechsberg showed that a serum which they had prepared as an anti-hzmolysin was able to protect against the experimental skin lesions which followed the injection of toxic culture-filtrates. When we reflect that this fundamental knowledge has been available for thirty years, it is remarkable that comparatively little work has been done with the object of producing a specific serum for clinical use. The disaster at Bundaberg in Queensland in 1928, when twelve children died within 24 hours after being inoculated with a diph- theria prophylactic consisting of a toxin-antitoxin mixture, which had become contaminated with staphylococci, led to re- newed interest in the subject. Although the rapidly lethal effect was said to have been a surprise to bacteriologists and clinicians, it was generally known that a bacterizmia has a graver prognostic import when it is associated with the staphylococcus than when it is caused by a hmmolytic strain of the streptococcus, for recovery from a staphylococcal septicemia rarely occurs, except 83 84 ANTI-STAPHYLOCOCCUS SERUM when the primary focus of the infection is in the bone marrow. This was the experience of the present writer during the war, and he is not likely to forget one instance, in particular, of a fulminating infection which gave the impression of overwhelming virulence of the invading organism. A young officer was seen one morning apparently perfectly well except for a scarcely visible abrasion on one of his toes ; he had struck his toe on a duckboard in the trenches, and the question was raised whether, in view of the trivial nature of the injury, he should receive a dose of tetanus antitoxin in accordance with the regulations then in force. A lymphatic infection began during the course of the day and pro- ceeded with such rapidity that in the evening he underwent a high amputation through the thigh ; a few hours later he was dead. A blood culture was taken at the operation and it yielded a growth of staphylococcus. In 1903 Proscher immunized goats and horses and titrated the sera in rabbits against an intravenous dose of the living culture ; the serum was given subcutaneously 24 hours before the test-dose of culture ; 1 c.cm. of the horse serum protected a rabbit against a lethal dose. Thomas in 1913 described the preparation and employ- ment of a multivalent anti-staphylococcus serum. He used 18 strains of SS. aureus, which were derived from various sources, and immunized a ram with heat-killed and, later, with living suspen- sions in normal saline given intraperitoneally. The course of the immunity process was examined by means of the opsonic technique. Thomas concluded that the serum possessed undoubted therapeutic value, both general and local, in staphy- lococcal infections. Russ (1916), working in the Serum Institute at Vienna, made a number of interesting observations on the effects of the specific toxin in experimental animals. He believed that the toxin exerted its chief action on the capillary circulation and that, contrary to the opinion which was then held and is still current, the toxin is contained in the bacterial bodies, from which it is set free by their disintegration, and that it is not, therefore, a true exotoxin ; Gross (1929) also has put forward this view. Russ obtained sera that were both antitoxic and anti- infectious. Contributions to the experimental aspects of the PRODUCTION AND TITRATION 85 subject and, in particular, the interrelations of the toxic effects and their neutralization by specific sera have been made by Burnet (1929, 1930, 1931, 1932), Panton and Valentine (1929, 1932), Parish and Clark (1932), Gengou (1931, 1932) and Bigger, (1933). Parker (1924) and Parker, Hopkins and Gunther (1926) investi- gated the conditions that are necessary for the production of the specific toxin in culture media. They recommended growth in an atmosphere containing 10 per cent. carbon dioxide and the use of Difco proteose peptone instead of Witte peptone. In 1927 Parker and Banzhaf gave an account of their method of producing a specific antitoxin in horses, but so far there has been no published report on the therapeutic use of this preparation. Gross (1929) has carried out work in the Behring Institute in Marburg on the production of anti-staphylococcus serum in horses and its titration. He selects as toxigenic strains only those which possess active fermentative properties, and which have a strongly marked necrotizing action on the tissues of experimental animals. He injects not only the fresh toxic filtrates sub- cutaneously so as to stimulate antitoxin production, but he also gives intravenous doses of heat-killed coccal suspensions in order to obtain an antibacterial serum. He estimates the strength of his antitoxic sera by titrating them in the skin of rabbits against a dry preparation of a standard toxin. The test-dose is chosen as the amount of the standard toxin which, when it is injected intracutaneously in rabbits, causes a well-marked necrosis of the skin within 24 hours. The sera contain agglutinins, but the presence of complement-binding antibodies has not been demon- strated. According to Bieling and Meyer (1932) a considerable proportion — nearly 30 per cent.—of puerperal infections in Germany are caused by a staphylococcus. For the treatment of these cases they recommend large daily doses (100 c.cm.) of the serum combined with the administration of collargol. In their view the proper sphere of action for the serum is the forestalling, by means of an early dose, of the tragic results that may follow furuncles near the lip, severe carbuncles, and the type of puerperal fever just men- 86 ANTI-STAPHYLOCOCCUS SERUM tioned. They suggest, too, that blood transfusion should be tried with the aid, if possible, of actively immunized donors or of con- valescent patients. For some years a combined antitoxic and ‘antibacterial’ serum was prepared at the Lister Institute, Elstree, but it has not been possible to obtain reports on its clinical value. Recently, Dr. Panton and Dr. Valentine of the London Hospital, who were investigating the toxic products of the staphylococcus, arranged with the writer for the immunization of a horse by means of a toxin prepared by them. The natural serum taken after the immunization and a concentrated preparation, which consists of the pseudo globulin fraction of the serum, have been employed by Dr. Panton and his colleagues in the treatment of various staphylococcal infections. They state that the antitoxin appeared to be chiefly efficacious in the pywxmic cases; the complete cessation of metastatic abscesses in all the serum-treated patients was also noteworthy. Panton and Valentine believe that the anti-leucocidin is the important antibody in the serum. Our knowledge of the methods of preparation and of the clinical use of staphylococcus antitoxin will be greatly advanced when a unit of antitoxin which is based on a dry preparation of a standard serum has been determined. In the meantime it is to be hoped that surgeons will give the remedy a trial, so that the limits of its usefulness may be better defined. REFERENCES Brering, R., and Meyer, F. * Heilsera und der Impfstoffe in der Praxis,” Leipzig, 1932. Biceer, J. W. 1933. J. Path. and Bact., 36, 87. Burner, F. M. 1929. J. Path. and Bact., 32. 711. " 1930. J. Path. and Bact., 33, 1. % 1931. J. Path. and Bact., 34, 471. 1932. J. Path. and Bact., 35, 477. FreminG, A. © A System of Bacteriology,” London, H.M. Stationery Office, 2, 12. GENGou, O. 1931. Arch. Internat. Méd. Exp. 6, 211. ' 1932. Ann. Inst. Past., 48, 135. Gross, H. 1928. Zeitschr. f. Immunititsforsch., 59, 510. > 1929. Zeitschr. f. Hyg., 109, 479. ” 1929. Klin. Woch., 8, 1079, REFERENCES 87 LINGELSHEIM, V. “ Atiologie und Therapie der Staphylokokkeninfek- tion.” Berlin-Wien, 1900. NEIsser, M. 1927. Handb. der path. Mikroorg., Ed. Kolle, Kraus, Uhlenhuth, 4, 498. NEISSER, M., and WECHSBERG, F. 1901. Zeitschr. f. Hyg., 36, 299. PantON, P. N. 1932. Lancet, 2, 1019. Panton, P. N., and VaLenTINE, F. C. O. 1929. Brit. J. Exp. Path., 10, 257. ”» 3 ”" 1932. Lancet, 1, 506. PaNTON, P. N., VALENTINE, F. C. O., and Dix, V. W. 1931. Lancet, 2, 1180. Parisi, H. J., and Crark, W. H. M. 1932. J. Path. Bact., 35, 251. PARKER, J.T. 1924. J. Exp. Med., 40, 761. PARKER, J. T., and Banzuar, E. J. 1927. J. Immunol., 13, 25. PARKER, J. T., Hopkins, J. G., and GUNTHER, ANNE. 1926. Proc. Soc. Exp. Biol. and Med., 23, 344. Proscuer. 1903. Centralbl. f. Bakt., Orig. I. Abt., 34, 437. Russ, V. K. 1916. Zeitschr. f. Exp. Path. w. Ther., 18, 220. TroMas, B. A. 1913. J. Amer. Med. Ass., 60, 1070. Van DE VELDE, H. 1894. Cellule, 10, Fasc. 2. CHAPTER VII THE SERUM TREATMENT OF DYSENTERY PAGE THE IMPORTANCE OF EARLY DIAGNOSIS IN THE TREATMENT OF DYSENTERY . 3 ’ ? . . . 88 GENERAL CONSIDE RATIONS ON THE USE OF ANTI-DYSENTERY SERUM . . . . . : . ' . . 91 THE SERUM TREATMENT OF INFECTIONS CAUSED BY BACILLI OF THE *° FLEXNER ”’ GROUP . : : : ’ 5 92 THE SERUM TREATMENT OF INFECTIONS CAUSED BY B. DysENTERLE (Shiga) : ; : : 94 THE SERUM TREATMENT OF INFECTIONS "CAUSED BY B. DvysENTERIZE (Sonne) 3 : . . 96 THE SERUM TREATMENT OF ULCERATIVE Corrs . ’ ’ 97 The Importance of Early Diagnosis in the Treatment of Dysentery THE early recognition of the infective agent in a patient with an attack of dysentery furnishes the key to the success of his treat- ment. The first step, after a provisional diagnosis of dysentery has been formed, should be to determine whether the attack is due to one of the dysentery group of bacilli or to Entamewba histolytica, or to a mixed bacillary and amaebic infection. Within recent years an increasing number of bacterial species have been described as causative agents in dysentery ; mention may be made of B. dysenterice (Sonne), and B. dysentericw (Schmitz). B. dysenterice (Shiga) and B. dysenteric (Flexner group) still remain the chief representatives of the dysentery group of bacilli, and the illness they cause is often so serious as to call for serum treatment ; the Sonne and Schmitz types of the disease are comparatively mild infections, and, therefore, for the most part, hardly require specific serum treatment. The reader who wishes to obtain information on the bacteriological aspects of the subject should consult the excellent review by Gardner (1929). 88 EARLY DIAGNOSIS OF DYSENTERY 89 The variety of agents that are concerned in the etiology of dysentery may seem at first sight to make it a difficult task to form an exact diagnosis which will serve as a guide to treatment, especially as little help can be derived from the ordinary methods of clinical observation. Fortunately, however, in practice, as will presently appear, the difficulty can be overcome by the employ- ment of a simple technique. The statement is sometimes made that the primary diagnosis of dysentery is solely the concern of the bacteriologist. But it is more correct to say that the responsibility for the diagnosis devolves first of all, not upon the bacteriologist, but upon the medical attendant, who should personally examine the stools of the patient for the presence of blood and mucus as soon as the symptoms suggest to his mind the possibility of a dysenteric infection. The scheme of examination should comprise three procedures. The first is macroscopic observation of the stools ; careful inspection of the stools of an acute case may give an indication of the nature of the infection ; in bacillary dysentery the typical stool consists of blood-streaked mucus, whereas in ameebic dysentery it is com- posed of faeces mixed with blood. The second procedure that is required for an accurate diagnosis is the discrimination, by means of a microscopical examination of the stools, between bacillary and amaebic dysentery. Lastly, after the amaebic type has been excluded, an attempt should be made to isolate the specific bacillus by employing the usual bacteriological methods. Bacteriological methods of diagnosis often fail, especially if the number of specimens sent for examination to the laboratory is such as to put a strain upon its resources, a common experience in outbreaks of dysentery during the war; moreover, the chances of successful isolation of the causative bacillus diminish progres- sively with the duration of the illness. Willmore and Shearman made observations on the cell-exudate in smears from the stools of ameebic and of bacillary dysentery, and showed that the microscopical appearances are characteristic for each type, and, therefore, provide a ready means of distinguishing between them. The essential feature of the exudate in bacillary dysentery is the abundance of polymorphonuclear cells, which show degenerative 90 THE SERUM TREATMENT OF DYSENTERY changes of toxic origin; these vary in degree, and in their extreme form they may reduce the cell to the periplast of the cytoplasm —the so-called *“ ghost-cell ” ; this is an almost certain sign of a bacillary infection. If a cytological examination reveals the typical picture of a bacillary infection, a multivalent anti-dysentery serum should be given without waiting for the bacteriological report, and its administration should be continued on clinical grounds alone, even when attempts to isolate a specific dysentery bacillus are unsuccessful. In the amcebic infection there are, apart from red blood cor- puscles, comparatively few cells in smears of the exudate, and of the white cells present mononuclear cells are more numerous than polynuclear leucocytes. The discovery of motile ameebae possessing the features of E. histolytica, particularly the inclusion of red blood corpuscles, is proof of an amcebic infection. In mixed infections the amoebz may be found in an exudate which exhibits the characters of the bacillary variety. The value of the cytological method of diagnosis is undoubted, and has been attested by a number of authors (Manson-Bahr ; Wenyon and O’Connor; C. J. Martin; and Haughwout and Callender). Martin, from his experience of dysentery among soldiers in France, concluded that bacteriological methods are unsatisfactory for the routine diagnosis of the disease, and that clinical observations, when supplemented by a microscopical examination of the stools, will lead to a diagnosis in 95 per cent. of the cases. He pointed out that the exact identification of the strain of the infecting bacillus does not aid in deciding upon the treatment of the patient or in making arrangements for his dis- position under war service conditions. The present writer had many opportunities for testing the cytological method in an epidemic of bacillary dysentery (Shiga ”/* Flexner-group,” 60/40) among the troops in Northern Italy during the war and was impressed by its utility. The value of the method was evident, not only in the typical acute case but in patients who were recovering and in those who were suffering from diarrhcea of dysenteric origin and whose stools, on careful examination, showed shreds of mucus or muco-pus. GENERAL CONSIDERATIONS 91 The proportion of cases in an epidemic which is attributable to two or more members of the dysentery group of bacilli can be ascertained with sufficient accuracy by the daily examination of a reasonable number of samples of stools. General Considerations on the Use of Anti-dysentery Serum Some observers have professed themselves doubtful of the value of anti-dysentery serum. There is, however, a considerable body of evidence of a general nature which supports a belief in its efficacy, although, for reasons that are not difficult to find, an exact analysis cannot always be applied to the data in the records. In the first place, the prevalent use of the term * anti-dysentery serum’ without further qualification is a source of confusion, since it is often not expressly stated whether the serum that was em- pioyed contained antibodies against both ** Shiga” and * Flexner 2 strains or only against one of these types. When, in comments on the results of the serum treatment of dysentery, we find the phrase * bacillary dysentery ” used without reference to the specific bacillus, the confusion becomes still greater. Thus, in a discussion introduced by Manson-Bahr (1920), little or no attempt was made by the speakers to distinguish between the influence of the serum upon Shiga” and * Flexner-group ” infections. Moreover, the titration of antibodies in a serum, however it may have been prepared, is not an easy matter. At the present time the only antibody which is titratable in terms of a standard unit is the antitoxin that neutralizes the pathogenic action of the specific toxin of B. dysenterie (Shiga) ; the method of titration has been in use during the past few years. There is no satisfactory method of titrating the antibodies that are present in sera prepared with the antigens of the « Flexner” group of bacilli. Until it is possible to titrate the essential components of a multi- valent anti-dysentery serum in terms of standard units, sera from different sources are likely to vary widely with respect to their antibody content. The usual practice of expressing the dosage of the serum in cubic centimetres conveys little real information to the reader and is, therefore, unsatisfactory, but it must be admitted 92 THE SERUM TREATMENT OF DYSENTERY that hitherto there has been no alternative. The number of units of «Shiga ” antitoxin given to a patient should always be included in the records of treatment. Clinical experience has shown that the serum should be adminis- tered as early as possible if the maximum therapeutic effects are to be secured. The time of administration in relation to the onset of the disease should also be recorded when assessing the efficacy of the serum in any particular case or series of cases. The value of reports on the utility of serum treatment is much diminished if these details are omitted. The Serum Treatment of Infections caused by Bacilli of the ‘‘ Flexner ’’ Group The work of Andrewes and Inman showed that strains of the “ Flexner ” group contain in varying proportions at least four or five different antigens, known as V, W, X (Y ?) and Z, with a preponderance in each strain of one of these components, so that the group may be regarded as a single species which is separable by agglutination tests into a ‘ spectrum ” of varieties (Gardner, 1929). On account of this diversity, representatives of all of these types should be used for immunizing horses, and, as Gardner has suggested, a multivalent ¢ Shiga-Flexner ”’ serum is to be preferred for the treatment of a patient while waiting for bacteriological identification of the infective agent. This author supposes that the mode of action of the serum is anti-endotoxic, and certainly, when suspensions of Flexner” cultures are given intravenously to horses, the symptoms that may follow in them are suggestive of a toxic action. If we may judge by experience with other bacterial antigens, sera prepared in this way possess in addition antibacterial properties. Clinical accounts of the potency of the serum are not altogether concordant, as the following examples will show. Huntoon and Hutchison believe that it has proved to be of little value, and they attribute its lack of efficacy to the fact that the bacilli are confined to the intestine and are therefore unlikely to be influenced by antibodies circulating in the blood. Fletcher and Jepps (1924) “ FLEXNER GROUP» INFECTIONS 93 gave the serum to 246 native hospital patients at Kuala Lumpur in the Malay Peninsula, and concluded that it had no effect in relieving the symptoms or lowering the mortality. They regarded the serum, however, as potent, and attributed its seemingly weakened action to the debilitated state of the patients, who were often suffering from a complicating disease, such as malaria ; the serum, when given to Europeans and well-to-do Asiatics, speedily relieved the distressing symptoms. The incidence of the bacillary types ¢ Flexner-group ”’/< Shiga” was 92/8 among the serum- treated hospital patients. This investigation was continued later by Kingsbury and Kanagarayar (1927); they employed a serum which had been specially prepared by MacConkey at the Lister Institute, a number of local * Flexner ” strains having been used as antigens. Their figures for the serum-treated cases and the control cases are small (12 and 16), but they believe that the serum had a beneficial effect in lessening tenesmus and abdominal pain and in making the patients more comfortable. Most of the bacillary infections in Manila and its environs are caused by the ¢ Flexner ” group. They run a comparatively mild clinical course, and respond quickly to relatively small doses of a serum which is prepared in the Serum Laboratory of the Bureau of Science in Manila. When it is given from 12 to 24 hours after the onset of the symptoms the results are said to compare in efficacy with those obtained by the use of the specific antitoxin in diphtheria ; the temperature falls, the stools are reduced in number, and abdominal pain ceases (personal communication from Drs. Schébl and Haughwout, 1925). Gardner (1929) concludes his review of the serum treatment of the * Flexner-group infections with the statement that a specific serum is a useful remedy in the more severe forms, particularly if it is given in the early stage of the illness. He suggests that a « Shiga-Flexner ”’ serum should be employed, but that to avoid weakening the anti-Shiga properties of the serum it should be made by injecting the requisite antigens into the same horse, and not by mixing sera from separately immunized animals. But since a method of titrating dysentery antitoxin (Shiga) is now available, 94 THE SERUM TREATMENT OF DYSENTERY there need be no risk of issuing a multivalent serum which is deficient in antitoxic units. In this connexion it should be noted that clinicians have not yet formulated the number of ‘ Shiga units that are necessary for the treatment of a case of average severity. The Serum Treatment of Infections caused by B. dysenteriae (Shiga) From the serological point of view strains of the dysentery bacillus of Shiga possess a uniformly simple antigenic structure, and thus are not separable into diverse types as they are in the “Flexner” group. The specific toxin is the principal antigen, and at the present time the preparation of a specific serum is concerned solely with the production of an antitoxin of adequate strength. There is reason to believe that the symptoms in human dysentery are caused by the action of the specific toxin ; characteristic toxic effects can be produced in experimental animals by the injection of filtrates of old broth cultures, and the toxin can be extracted from the bacillus by suitable procedures. Accordingly, the serum treatment of “Shiga” infections rests on a rational basis. In the opinion of some workers the efficacy of the serum can be increased by ensuring that it contains a sufficient amount of an antibacterial component. This aspect of the immunizing process is usually considered to be of secondary importance; and it is probably best to postpone a decision upon its significance until clinical experience has ascertained the therapeutic value of sera that have been titrated in terms of the standard unit of antitoxin. This unit was determined by the International Standardization Commission of the League of Nations, and it was adopted in the year 1927 by the Control Authority in Great Britain and Northern Ireland. The method of titration consists in the intravenous injection into groups of mice of graduated amounts of the serum under test to each of which an appropriate and constant test-dose of the ““ Shiga ” toxin has been added. The results are compared with those which are obtained in the same test by injecting a group of mice with a mixture of the same dose of toxin and one unit of the standard serum. INFECTIONS WITH B. DYSENTERIE (SHIGA) 95 The proportion of * Shiga” to ‘ Flexner-group ” infections in the British Salonika Force during the war appears to have been similar to that which was found in Northern Italy ; Graham gives the ratio 75/25 for the group of the severe and moderately severe cases. The death-rate was only about one per cent. The best therapeutic results were obtained by the intravenous injection of from 60 to 80 c.cm. of serum followed by from 150 to 300 c.cm. of normal saline, and administered twice daily for the first two days and once daily for the next two days. In very toxic cases 5 per cent. glucose in distilled water was substituted for the saline. ‘Shiga > infections are said to have responded better to serum treatment than ‘ Flexner-group > infections. The most striking effect produced by the serum was the relief of the tenesmus and abdominal pain ; there was also in many cases a marked reduction in the number of the stools. Corroborative evidence of the value of anti-dysentery serum (Shiga) is given by Phear (1924) and by Acton and Knowles (1924). A paper by Lantin (1930) contains an analysis of 2,259 cases of dysentery that were admitted to the Philippine General Hospital during a period of more than seventeen years (1910-1927). Of these, 1,341 patients did not receive serum treatment; 855 patients were treated by means of intramuscular injections of serum ; and to 83 patients it was given per rectum in the form of a retained enema. It was prepared by immunizing horses with heat-killed ¢ Shiga’ and ¢ Flexner-group * strains. The fatality- rate among the control group was 36-3 per cent., and among the serum-treated group it was 19-7 per cent. The author gives a table which clearly shows the advantages of early administration. He points out that the serum is incapable of dislodging toxin which has become anchored to the cells, although it is able to neutralize the toxin molecules which are still free in the tissues. He therefore endeavours to saturate the tissues of the patient with it in the early days of the illness, and has found that the larger the dose the higher is the recovery-rate. The best results were obtained by administering 50-150 c.cm. of the serum in 20 c.cm. doses three times daily for two or three days, without taking into account the age of the patient. The rectal method of administra- 96 THE SERUM TREATMENT OF DYSENTERY tion gave encouraging results, but Lantin refrains from committing himself to a definite opinion upon its value, because the group of cases in which the method was tried did not include severe cases and the number of patients in the group was small. We may add that there is no proof of the absorption of the antibodies in serum through the normal intestinal mucous membrane; the serum may act, therefore, solely by neutralizing any free toxin that may be present in the lumen of the intestine. The experience of Bieling and Meyer (1932) is based on the treatment of several hundred cases among the German troops during the war, and was so convincing that they have no doubt of the outstanding value of the treatment when the serum is given early and in sufficient amounts. They remark that the statistical data which were obtained during the period of the war show a definite correlation between the curative effects and the degree of thoroughness with which the treatment was carried out. When the serum was administered, the temperature became normal before the seventh day in 53 per cent. of the patients, and blood disappeared from the stools in 76 per cent. ; the corresponding figures for the men who were treated without serum are 5 per cent. and 19 per cent. And when it was given by the combined intra- venous and intramuscular routes the results were even better, for the bleeding ceased after one day in 56 per cent. of the patients, and after five days this symptom had disappeared from all the serum-treated patients. The Serum Treatment of Infections caused by B. dysenteriae (Sonne) Evidence has been accumulating of late years which shows that the generally mild character of *“ Sonne infections, as in healthy young adults, may take the form in very young children of a serious and even fatal illness. Hissler (1931) gives an account of 235 cases that were observed in a children’s hospital in Leipzig. The sex incidence was about equal. The mortality was greatest in the first year of life ; and 50 per cent. of the infants died in the fifth month of life. The experience of Nabarro has been similar, TREATMENT OF ULCERATIVE COLITIS 97 and he proposes, with the co-operation of the Lister Institute, to give a trial to a serum which has been prepared from strains he has isolated in the course of his observations on this infection. The Serum Treatment of Ulcerative Colitis The suggestion has been made by some clinical observers that sporadic ulcerative colitis is caused primarily by one of the types of B. dysenterice. The appearance of the colon as seen with the sigmoidoscope simulates the lesions of dysentery (Hurst). In the belief that ulcerative colitis is really a form of dysentery, Hurst has tried the effect of giving large doses of a multivalent anti-dysentery serum intravenously. He gives 40, 60, 80 and 100 c.cm. on successive days, followed by 100 c.cm. for a few additional days. The results have often been excellent, although occasionally the treatment fails to bring about improvement. Hurst realizes that the beneficial action of the serum may be a non-specific one, and due to a form of protein shock, although in one patient the treatment succeeded after a series of doses of normal horse serum had proved to be of no avail. Ryle has obtained equally good results by this method. On the other hand, Tidy (1930) has failed to obtain any improvement ; he notes that in acute bacillary dysentery a specific serum appears to be ineffec- tive when it is given after the first few days of the illness. Dudgeon has likewise expressed scepticism of the value of the treatment, and Alessandrini (1927) has found that specific serum therapy does not seem to give better results than non-specific protein therapy. The reader will find a discussion on the treatment of ulcera- tive colitis, which includes references to the employment of serum, in the Proceedings of the Royal Society of Medicine (see Hurst, 1931). Some of the speakers mentioned an occasional brilliant cure from the use of anti-dysentery serum, but thought there was no assurance that a satisfactory result would follow. Another form of serum treatment may be briefly referred to here. Bargen has prepared a serum in horses by inoculating them with a diplo-streptococcus of the viridans type which he thinks is of R.A. VACCINES, 4 98 THE SERUM TREATMENT OF DYSENTERY etiological significance in ulcerative colitis. Intramuscular doses of a solution of the euglobulin fraction of the serum are given daily in minimal amounts ; the treatment begins with 0-1 c.cm. and this is followed by increments of 0-1 c.cm. at intervals of 10 hours up to an average maximal amount of 3 c.cm. (Rankin, Bargen and Buie, 1932). The present writer has been unable to find in the literature any clear evidence of its therapeutic effects. The treatment of ulcerative colitis by means of a vaccine prepared from the diplo-streptococcus of Bargen is dealt with on p. 380. REFERENCES Acton, H. W., and KnowLEs, R. 1924. Ind. Med. Gaz., 59, 325. ALESSANDRINI, P. 1927. Il Policlinico, May 22nd, p. 743; ref. epitome Brit. Med. J., 13th Aug., 1927. BIELING, R., and MEYER, F. 1932. * Heilsera und Impfstoffe in der Praxis.” Leipzig. Dubpcron, L. S. 1926-1927. Discussion, Proc. Roy. Soc. Med., 20, 367. Frercuer, W., and Jepps, MARGARET W. 1924. °° Dysentery in the Federated Malay States.” London. GARDNER, A. D. 1929. “A System of Bacteriology,” 4, 161, 220. GrauaM, Duncan, 1918. Lancet, 1, 51. HissLer, BE. 1931. Jahrb. f. Kinderheilk., May, p. 284. Havenwour, F. G., and CALLENDER, G. R. 1925. Internat. Clinics, 2, 104. Hu~toon, F. M., and Hurcuison, R. H. 1928. * The Newer Know- ledge of Bacteriology,” p. 924. Hurst, A. F. 1926-1927. Proc. Roy. Soc. Med., 20, 367. > 1931. Ibid. 24, 785. KinGsBURY, A. N., and KANAGARAYAR, K. 1927. Malayan Med. J., 2, No. 1, March. LantiN, P. T. 1930. Amer. J. Med. Sci., 180, 635. Manson-Banr, P. 1920. Trans. Roy. Soc. Trop. Med. and Hyg., 13, 64. Martin, C.J. 1918. Med. Bull., No. 7, Paris, p. 481. PHEAR, A. G. 1924. Lancet, 2, 232. RANKIN, F. W., BArGEN, J. A., and Buig,L. A. 1932. * The Colon, Rectum and Anus,” Philadelphia and London. Rvyre, J. A. 1927. Discussion, Proc. Roy. Soc. Med., 20, 367. Tiny, H. LErnesy. 1930. Brit. Med. J., 1, 135. WenvoN, C. M., and O’ConNor, F. W. “ Human Intestinal Protozoa in the Near East,” London, 1917, p. 60. WILLMORE, J. G., and Snearman, C. H, 1918, Lancet, 2, Aug. 17th, CHAPTER VIII THE SERUM TREATMENT OF BOTULISM Borurism is fortunately an uncommon disease. Only one outbreak has been recorded from Great Britain—at Loch Maree in Scotland in the year 1922; eight persons were attacked and all died (Monro and Knox, 1923). We owe most of the recent knowledge of the subject to workers in the United States, and particularly to K. F. Meyer and his associates. From 1909 to 1926, 146 outbreaks were reported in the United States and Canada, with a total of about 500 cases and with 330 deaths (Hewlett). For the eight-year period 1918-1925 there was a yearly average of 13 cases in the United States. Bucillus botulinus (Clostridium botulinum of American authors) is a sporing anaerobe whose habitat is the soil; the spores are highly resistant to heat. Jordan (1928) points out that, since the spores are present in the soil in many parts of the world, and must therefore frequently contaminate vegetables and preserved foodstuffs that have been inadequately sterilized in the home, botulism might have been expected to occur more often than the records show. The reason for the apparent immunity appears to be that the factors which favour the production and the persistence of the toxin of the bacillus seldom concur. The specific exotoxin is solely responsible for the symptoms of the disease in man: a true infection does not take place. It is of interest to note that this toxin is the only example of a bacterial poison which produces its effects in consequence of its ingestion. There are three well-recognized types of the bacillus, A, B and C; these differ from each other in some of their biological characters and in their toxigenic ability ; the toxicity diminishes in the order named. In the United States, type A is the most frequent cause of botulism, whereas in Europe only type B is found ; type A, 99 4—2 100 THE SERUM TREATMENT OF BOTULISM nevertheless, caused the outbreak in Scotland. Type C has been isolated from samples of soil in the United States; it has not proved, so far, to be an important member of the group. Authorities on botulism are agreed that a specific antitoxic serum alone offers some hope in its treatment and that, to be effective, it must be given at the earliest possible moment after the appearance of the symptoms ; actually, it often happens that the serum is not available when needed, and this, doubtless, accounts for the scanty observations that have been made on the specific treatment of the disease. It may be useful to add here a brief account of the symptoms as they are described in the monograph by Meyer (1928) and in the clinical account of the Scottish cases; the details apply equally whether the illness is caused by type A, B or C. The early symptoms are gastro-intestinal and are said to be due to irritation from the contaminated foodstuff which has been ingested, and not to the specific toxin. The first to be noted are nausea or vomiting, a feeling of weakness, headache, and giddiness ; these are in part due to the constipation which may be troublesome in the early stages of the illness. Visual disturbances appear early in the form of diplopia and loss of accommodation ; then follow loss of the reflex to light, mydriasis, and blepharoptosis ; the pupils may be unequal and irregular, and nystagmus is noticeable. There is difficulty in swallowing and speaking. Progressive muscular weakness of the neck and limbs is a striking symptom. The patient does not complain of pain, but much distress may be caused by difficulty of breathing due to paresis of the muscles of respiration. The serum should contain an antitoxin for types A and B, because their toxins and the derived antitoxins are not identical ; it should be given by the intravenous route and large doses are required. Intrathecal injections are not likely to be of service, since the lesions are probably peripheral and not central, but the method may be given a trial (Hewlett). Bronfenbrenner and Weiss (1924) inferred from their experimental work that the administration of morphine may delay the progress of the intoxi- cation, and thus give time for the antitoxin to exert its effect. PREPARATION AND TITRATION OF THE SERUM 101 Persons who are exposed to risk from contaminated food may be given a prophylactic dose of antitoxin intramuscularly. The preparation of the specific antitoxin is very similar to that of tetanus antitoxin and does not offer any special difficulty. The toxins for immunization are produced in much the same way as for tetanus toxin ; and the titration of the serum closely follows the method for tetanus antitoxin. The flocculation method of titration has not proved its value (Weinberg and Ginsbourg, 1926). Horses should be immunized separately against the “A” and the “ B” toxins, and the sera, when they attain the requisite titre, should be mixed in order to form the final product for issue. REFERENCES Nore. A full list of references to recent work on botulism need not be given here, since the articles cited below contain excellent biblio- graphies. BRONFENBRENNER, J., and Weiss, H. 1924. J. Exp. Med., 39, 517. Hewrert, R. T. 1929. * A System of Bacteriology,” London, H.M. Stationery Office, 8, 374. Jorpan, E. O. 1928. * The Newer Knowledge of Bacteriology, and Immunology,” p. 447. Chicago. Leicaron, G. 1923. * Botulism and Food Preservation.” London. Meyer, K. F. 1928. * Handbuch der pathogenen Mikroorganismen,’ Bd. IV., p. 1343. Edit. by Kolle, Kraus, and Uhlenhuth, Jena. Monro, T. K., and Knox, W. W. N. 1923. Brit. Med. J., 1, 279. O’Brien, R. A. 1929. “A System of Bacteriology,” London, H.M. Stationery Office, 3, 395. Savage, W. G. 1929. * A System of Bacteriology,” London, H.M. Stationery Office, 3, 407. WEINBERG, M., and GINSBOURG, B. 1926. Bull. Inst. Past., 24, 969. CHAPTER IX THE SPECIFIC SERUM TREATMENT OF SNAKE-BITE, THE STING OF SCORPIONS AND THE BITE OF SPIDERS PAGE ANTIVENINS FOR SNAKE-BITE ‘ . . v . « 302 Introductory . 3 : . 3 7 : . 102 The European Vipers g : ' . 103 The Chief Poisonous Snakes of ‘North America 3 y . 105 The Snakes of Central America . 3 . . . 105 The Venomous Snakes of Australia v . ’ . 105 The Poisonous Snakes of Africa 4 . , : . 106 The Mode of Preparation of Antivenins 2 ‘ : + 206 The Titration of Antivenins ‘ v . ‘ . 106 The Mode of Administration of the Serum. . x : L107 The General Treatment of Snake-bite . ‘ : v . 108 Statistical Evidence of the Value of Antivenins : . 109 SPECIFIC SERA FOR THE STING OF SCORPIONS . 3 . 109 SPECIFIC SERA FOR THE BITE OF SPIDERS ’ v . . 110 Antivenins for Snake-bite Introductory. Researches on the toxic constituents of snake venoms and on the preparation of specific sera for the treatment of snake-bite are being pursued in those countries in which poisonous snakes abound. In the United States there are more than 1,000 cases of snake-bite annually, with a fatality-rate varying from 10 to 35 per cent. according to the district ; most of the cases are reported from the south-western regions (Amaral, 1927). Exact statistics from other countries are unobtainable, but it is probable that in India at least 20,000 persons die each year, the fatality- rate being 35-40 per cent. The technique of production of antivenins is a complicated one owing to the now generally accepted fact that the antigenic substances contained in snake venoms are on the whole highly specific, and also because the infested areas of the world harbour a variety of genera and species which are characteristic for each 102 ANTIVENINS FOR SNAKE-BITE 103 country. Thus, in Europe, small vipers or adders (Vipera aspis, V. berus, V. ammodytes) cause death in 10-14 per cent. of the cases; V. berus is the species that is found in England. In India and Southern Asia the important snakes are the Indian cobra (Naia naia), the hamadryad (Naia hannah), the ‘kraits (Bungarus spp.) and the daboia (Vipera russelli); for an account of the Indian species the reader is referred to the monograph by Wall (1928). An analysis of a group of 451 cases of snake-bite within the United States in the year 1928 showed that ten species were incriminated, and that 60 per cent. of the bites were caused by the American copperhead snake (Agkistrodon mokasen) and the Texas rattler (Crotalus atrox) (Hutchison, 1929). Among the venomous snakes of South America we may mention the rattle- snake (Crotalus terrificus), several species of Bothrops—for example, the Fer de lance (Bothrops atrox)—and species of the genus Lachesis. In Australia the dangerous species include the tiger snake, the death adder, the Australian copperhead, the Australian black snake, and the Australian brown snake. The Pasteur Institute in France prepares a serum against the venom of the cobra and that of the European vipers ; the Pasteur Institute at Saigon manufactures Calmette’s cobra antivenin ; the Butantan Institute in Sao Paulo, Brazil, issues a univalent serum against the venom of the rattlesnake and a multivalent serum which is employed for bites by the Central American snakes ; at Kasauli in India a bivalent serum is prepared against the venom of the cobra and that of the daboia ; in the Antivenin Institute of America, at Glenolden, two kinds of antivenin are made, one for the bite of the North American rattler (Crotalus spp.), the American copperhead, and the moccasin or the cotton-mouth water snake (Agkistrodon piscivorus), and the other for the bites of the Central American species of Bothrops ; the Bacteriological Institutes in Tokyo and Buenos Aires prepare antivenins that neutralize the venom of the dangerous snakes in their respective countries ; and lastly, FitzSimons at Port Elizabeth manufactures an antivenin which he asserts is powerfully polyvalent and is able to cure the bites of all venomous snakes. The European Vipers. The symptoms that follow the bite of 104 THE SPECIFIC TREATMENT OF SNAKE-BITE these vipers resemble those resulting from a small dose of rattle- snake venom (Martin and Lamb, 1909). There is an immediate burning pain locally with a rapidly spreading cedema and discolora- tion of the affected part, and within a few hours of the bite the patient is in a state of great prostration, which is accompanied by vomiting, diarrhcea, and a cold perspiration ; the heart action is feeble and he may become comatose. Calmette has prepared an antivenin which is labelled * E.R.” The difficulty and cost of preparing this serum led Kraus to try to ascertain whether the anti-viperine serum for the South American species of Crotalide is effective against the venom of the European vipers, and he discovered that a serum prepared by Vital Brazil at the Butantan Institute, Brazil, with the venom of Bothrops jararaca, was capable of neutralizing the venom of V. aspis as effectively as Calmette’s serum. Moritsch found that the multi- valent anti-bothrops serum prepared by Brazil was equally potent against this venom. The work has been confirmed and extended by Otto, who has given his results in a series of papers published during recent years. He has ascertained that a multivalent serum from Java was without effect upon the venom of European vipers ; nevertheless, he was able to neutralize the venom of the European viper by means of the multivalent anti-viperine serum from the Bandoeng Institute in Java when this was administered subcutaneously, but not when it was given by the intravenous route ; the test animal used was the guinea-pig. He found that the venoms of the European vipers differ in their lethal power, their resistance to heat, and their reaction to alien anti-venoms. Otto’s work indicates the complexity of the subject, and this is due in part to the circumstance that crude snake venoms contain a variety of toxic substances, such as neurotoxins and cytolysins, and enzymes which are present in variable amounts, perhaps even in samples from the same species, and which, although they may exert a similar toxic effect upon susceptible cells and tissues, are not necessarily antigenically identical, even when they are derived from related species. These crude mixtures serve not only as the material for producing the antivenins, but as the test material for THE SNAKES OF AMERICA AND AUSTRALIA 105 estimating the neutralizing value of various antivenins. More- over, at the present time, there are no universally recognized methods of standardizing and titrating venoms and antivenins in terms of standard units. The Chief Poisonous Snakes of North America. Useful informa- tion on this group is given in the Bulletin of the Antivenin Institute of America (1927-1932). In this journal there will be found a good general description of the important species, with excellent illustrations. As has already been stated, a composite serum is obtainable for the treatment of the bites of the Nearctic Crotalidee (rattlesnake, copperhead and moccasin). The problem of snake- bite in the southern territory of the United States is becoming increasingly important, owing to the extension of agriculture there and the modern tendency to lead an outdoor life. The Snakes of Central America. The Butantan Institute at Sdo Paulo, Brazil, issues a serum for the rattlesnake (C. terrificus), and a multivalent serum for some half-dozen species of Bothrops ; the Bacteriological Institute at Buenos Aires makes a bivalent serum for C. terrificus and Bothrops alternata. Lindsay (1926) states that in Paraguay there are very few estancias which are not provided with a supply of antivenin accompanied by clear instruc- tions for its use, together with a suitable syringe and with potas- sium permanganate for the local treatment of the bite. He cites a few cases in which the polyvalent serum was remarkably efficacious. Twenty years ago about 5,000 persons died of snake- bite poisoning in Brazil every year, but this figure has been materially reduced by the growing use of the serum. The Venomous Snakes of Australia. The notable work of C. J. Martin (1892-1905) on the venoms of these snakes and the nature of their interaction with specific sera was not continued, but fresh observations on the natural history of the Australian species and on the toxicological and immunological relations of their venoms have been made by Hamilton Fairley and by Kellaway (1929, 1931). The important snakes are the death-adder (Acanthophis antarcticus), the mortality from whose bite is very high—50 per cent. ; the tiger snake (Notechis scutatus), with a fatal result in 45 per cent. of the cases; the Australian copper-head (Denisonia 106 THE SPECIFIC TREATMENT OF SNAKE-BITE superba) ; the Australian brown snake (Demansia textilis) ; and the Australian black snake (Pseudechis porphyriacus). A help- ful article on the specific and general treatment of snake-bite in Australia has been written by Kellaway and Morgan (1931). An antivenin of high potency against the venom of the tiger snake is prepared in the Commonwealth serum laboratories, and has been used with excellent results. Dr. F. G. Morgan, Director of the Laboratories, has informed the writer that he has succeeded in obtaining a considerable degree of active immunity against the venom of the death adder in the horse but that, even after a pro- longed immunization, the serum fails to protect susceptible experimental animals. The Poisonous Snakes of Africa. In Africa the species that are responsible for most of the cases of snake-bite are the various cobras (Naia spp.), the puff-adder (Bitis arietans), and the mamba (Deudraspis angusticeps). A comprehensive account of the occurrence of snake-bite in Africa has not yet been published. The Mode of Preparation of Antivenins. Horses are used for the production of the antivenin. The immunizing process must be cautiously carried out owing to the sensitiveness of this animal to snake venoms, and therefore it takes a considerable time— according to Kraus, 16 months. The details of the technique vary with different workers; for example, Calmette endeavours to lessen the reactions by employing venom the toxicity of which has been destroyed by the addition of a solution of calcium hypochlorite ; Brazil prefers to use the unaltered venom ; and other workers shorten the period of immunization by combining antivenin with the doses of venom, at least in the early stages. Kraus, Bicher and Lowenstein treated the venom of Bothrops jararaca with 0-5-1 per cent. formalin in an attempt to destroy its toxic action while sparing its antigenic efficacy, but they believe that the results are not as good as those obtained with bacterial toxins when these are treated in the same way. The Titration of Antivenins. Various methods have been described for the titration of antivenins. Some workers prefer to use a prophylactic test and others a simultaneous test, such as that which is employed for the titration of the ordinary therapeutic MODE OF ADMINISTRATION 107 sera ; others, again, carry out curative tests with the serum. In the first method the serum is given an hour or two before the test dose of venom, whereas the second method allows for the neutrali- zation of serum-venom mixtures to take place in vitro before their injection into the test animals. The curative test is the method prescribed by the American authorities for the control of thera- peutic sera ; the venom is injected intramuscularly into rabbits and about 20 minutes later the antivenin is given by the subcutaneous route (McCoy, 1928). Amaral believes that for accurate titration more than one method should be employed ; the best combination in his experience is the neutralization test in pigeons and the estimation of both the preventive and the curative potency of the serum in rabbits and white mice. Kraus has pointed out the desirability of international action with the object of establishing standard units. The Mode of Administration of the Serum. The serum should be issued in syringe-containers, so that no time may be lost in preparing for its injection. For the treatment of bites by the North American snakes 10 c.cm. should be injected without delay into a muscle, or, if the condition of the patient is grave and a considerable time has elapsed since the bite, by the intravenous route. In young children an intraperitoneal injection is likely to be easier to give than an intravenous one. If the local swelling continues to increase, the dose should be repeated every hour or two. Two or three c.em. may be injected into the tissues at the site of the wound if the patient comes under observation soon after he is bitten. The dose of the serum for a child is two or three times greater than that for an adult, because the toxicity of the venom is inversely proportional to the body-weight of the patient. Hutchison believes that it is never too late to give anti- venin, and that there is a chance of recovery, however desperate the state of the patient may be. It is certain that rapid retrogres- sion of the symptoms often follows the injection of the serum and particularly the intravenous doses. The relief of pain is, too, a noticeable feature. The antivenin is associated with the pseudo- globulin fraction of the serum, so that it is easy to produce a concentrated preparation with the advantages of small volume 108 THE SPECIFIC TREATMENT OF SNAKE-BITE and elimination of the useless proteins. Kraus thought that an avidity factor plays a part in the curative action of antivenins. The General Treatment of Snake-bite. Apart from the adminis- tration of a specific serum, the lo:al and general treatment should be thoroughly understood by phy:icians who are called upon to act in this emergency. An excellent summary of the procedure recommended for the treatment of snake-bite poisoning in the United States has been given by ITutchison (1929); the chief recommendations may be noted here. 1. Apply a ligature or tourniquet a few inches above the bite, if possible ; this delays absorption of the poison into the general circulation. Release the tourniquet every 10 or 15 minutes for about a minute at a time. Remove it after the serum has been given. 2. If the bite is a severe one, after washing the skin make a cross-cut incision at each fang-mark } inch deep and } inch long. Apply suction with a breast pump or a Bier’s suction glass, and at the same time irrigate the wound with 1 or 2 per cent. salt solution. If the condition of the patient demands energetic measures, continue this treatment for 20 minutes in each hour for a period of 15 hours. Jackson and Githens (1931) have shown, by means of experiments on dogs, that this method of treatment can save animals which have received several lethal doses of the venom of the Texas rattler (Crotalus atrox). 3. Movement and alcoholic stimulants should be avoided, since they seem to aid the diffusion of the venom throughout the body. The American authorities do not recommend the local application of potassium permanganate, because they consider that it is toxic to the tissues and possesses no antidotal value. A serious difficulty frequently met with in the treatment of snake-bite is the remote- ness of the patient from medical aid. It is interesting to learn that in Texas army aeroplanes have carried skilled assistance to patients who were living in distant and inaccessible localities. Major R. E. Scott, from his personal experience, has testified to the satisfactory results obtained by the use of antivenin in such circumstances. In the State of Queensland, Australia, only five serum depdts serve an area of 670,000 square miles, but the SERUM TREATMENT OF SCORPION-STING 109 problem of the rapid distribution of the serum is likely to be solved by the highly developed airway communications in that country. Statistical Evidence of the Value of Antivenins. Vital Brazil is of opinion that the death-rate of persons in Brazil who have not received serum is 25 per cent., and of serum-treated persons 2:5 per cent. Useful data for the United States are given by Hutchison ; of 832 serum-treated cases in the years 1928 and 1929, 28 died (3-4 per cent.), and of 258 patients who received no serum 31 died (12 per cent.), a difference which the present writer finds to be definitely significant (d/od = 5-4). Specific Sera for the Sting of Scorpions An antivenin against scorpion sting is prepared at the Butantan Institute in Brazil ; the Pasteur Institute in Algiers; the Lister Institute, Elstree, in England ; and the Behring Institute in Germany. Charles Todd (1909) was the first to obtain an effective serum from horses. His article and the monograph by Wilson (1901) contain much interesting information on the venom of Egyptian scorpions and its toxic and lethal action in experimental animals. At the time these authors made their observations there were many deaths every year in Egypt from scorpion sting ; almost all were in children under twelve years of age. Cairo and Upper Egypt, that is, the strip of the Nile Valley stretching between Cairo and Assouan, accounted for the bulk of the fatal cases; in the town of Assouan the deaths represented 1:6 per cent. of the total death-rate. Apart from its lethal action, the sting causes very severe pain and often leads to collapse. Buthus quinque- striatus is the common variety of scorpion in Egypt. Todd found that the anti-venom from immunized horses neutralized the venom when it was mixed with it in vitro, and that it also acted prophylactically and curatively in experimental animals. He was unable to discover any neutralizing action on the scorpion venom of Calmette’s antivenin for snake-bite. The serum prepared by Todd seemed to diminish the intense pain which follows the sting. He arranged for the preparation of the serum at the Lister Institute, Elstree, where for many years a concentrated serum has been issued for use in Egypt. The 110 THE SPECIFIC TREATMENT OF SNAKE-BITE antivenin, like that for snake venom, is associated with the pseudoglobulin fraction of the serum and is capable of being concentrated tenfold by volume. In Brazil numerous deaths are said to occur from scorpion stings, and especially from that of T'ityus bahiensis. The Egyptian serum has no beneficial action on the effects of the venom of this species. A specific concentrated serum is available and has given excellent results ; the risk of death has, indeed, been completely removed by its use. Magalhies has studied the venoms of 7". bahiensis and of three other South American species, and has been able to make effective specific sera against them. The horse is very susceptible to the action of the venom of the Egyptian scorpions. Todd mixed the venom with Gram’s solution of iodine and believed that this expedient was of value in reducing the severity of the symptoms in his serum-producing horses, while leaving unaffected its antigenic property; at the Lister Institute the venom solution is formolized. At this Institute the titration of the serum is carried out in white rats, the serum-venom mixtures being injected subcuta- neously after standing at room temperature for three-quarters of an hour to give time for neutralization to take place. The statement of Arthus that immediate combination occurs in vitro is not true for every serum, although it may be for some sera that possess an exceptional avidity for the venom. The test allows of quite regular titrations, and it is rendered easier by the fact that the toxicity of the venom solution in saline remains constant over long periods without any special care, such as cold storage, being taken for its preservation. The principles that guide the treatment of snake-bite apply in general to that of scorpion sting. The concentrated serum is put into syringe-phials, each containing a dose which is capable of neutralizing the total amount of venom in one poison gland ; it should be injected at the site of the sting, if this is possible. Specific Sera for the bite of Spiders In various parts of the world, for example, in California, Brazil, South Russia and Australia, toxic and even lethal effects in man SERUM TREATMENT OF SPIDER-BITE 111 and animals from the bites of poisonous spiders have been reported. In California the bite of the species Latrodectus mactans, the so-called * black widow ” spider, can give rise to exceedingly unpleasant symptoms (Bogen, 1926). The pain caused by the bite is excruciating, although there are few, if any, visible local signs, and complete relief is not felt in some cases even after a week. Bogen gave a trial to the serum of convalescent patients and obtained encouraging results. Troise (1928) found that 0-5 mgm. of the venom, when given intravenously, caused death in guinea-pigs of 600 gms. weight in less than two hours; he succeeded in immunizing rabbits by the administration of graduated doses of the venom. Kellaway (1930) has studied the action of the venom of L. hasseltii, an Australian species, in experimental animals ; the symptoms are characteristic of those produced by a neurotoxin, and bronchial constriction plays an important part in determining the fatal issue. The Brazilian species have been investigated by V. Brazil and Vellard, and by Troise (1929). Brazil found that Lycosa raptoria and Ctenus nigriventer are responsible for most of the cases observed in Sao Paulo (cited by Amaral, 1928). The venom of the former species produces only local signs, namely, necrosis of the skin and swelling of the tissues, whereas Ctenus gives rise to generalized symptoms indicative of the action of a neurotoxin. Brazil and Vellard have prepared a bivalent antivenin by immunizing sheep, and have demonstrated its therapeutic efficacy in man; rapid improvement follows its use. For titrating the potency of the anti-Lycosa serum they determine its anti-necrotizing power in rabbits ; the Ctenus antivenin is tested by inoculating rabbits intravenously with appropriate serum-venom mixtures. Spider antivenins, in common with those derived from snakes and scorpions, are highly specific. REFERENCES Snake-bite AMARAL, A. Do. 1927. Bull. Antivenin Inst. America, 1, 31, 61. ”" o 1928. “ The Newer Knowledge of Bacteriology and Immunology,” p. 1066. Ed. Jordan and Falk, Chicago. 112 THE SPECIFIC TREATMENT OF SNAKE-BITE Braz, V. 1927. Cited by Kraus, R., ¢“ Handb. der path. Mikroorg,” Sy. Farry, N. Hamiuron. 1929. Med. J. Australia, 1, pp. 1, 19, 34, 70, 296. FrrzSivmons, F. W. 1929. “ Snakes and the Treatment of Snake- bite,” Capetown. Hurcnaison, R. H. 1929. Bull. Antivenin Inst. America, 3, 43. 1980. Loc. cit., 4, 41. JACKSON, D., and GrruENs, T. S. 1931. Bull. Antivenin Inst., 5, 1. KELLAWAY, C. H. 1929. ‘Med. J. Australia, 1, pp. 46, 56, 64, 87, 97, 336, 372. or 1929. Brit. J. Exp. Path., 10, 281. 1931. Med. J. Australia, 2, 1, 85. KELLAWAY, C. H., and Eapes, T. 1929. Med. J. ’ Australia, 2, p. 249. KELLAWAY, Cc. H, and MorcGaN, F. G. 1931. Med. J. Australia, 2, 482. Kraus, R. 1927. < Handb. der path. Mikroorg.” (Kolle, Kraus and Uhlenhuth), Bd. 3, 1. Kraus, BACHER, and LOWENSTEIN : cited in Kraus, R., 1927. ‘ Handb. der path. Mikroorg.” .(Kolle, Kraus and Uhlenhuth), 3, 1. Linpsay, J. W. 1926. Trans. Roy. Soc. Trop. Med. Hyg., 20, 275. MARTIN, C. J., and Lams, G. 1909. ¢ A System of Medicine,” Vol. II., Pt. IL, p. 783. Ed. Allbutt and Rolleston. McCoy, G. W. 1928. * The Newer Knowledge of Bacteriology and Immunology,” p. 947. Ed. Jordan and Falk, Chicago. Morirscu, P. 1926. Wien. klin. Woch., 39, 1514. Morirsch, P., and Pirxer, H. 1928. Wien. klin. Woch., 41, 1255. Otro, R. 1930. Arch. f. Hyg. uw. Bakt., 103, 165. Warr, F. 1928. “The Poisonous Terrestrial Snakes of our British Indian Dominions,” 4th Ed., Bombay. Scorpion Sting MacGarHAES, O. DE. 1928. Mem. Inst. Oswaldo Cruz., 21, 5. Topp, C. 1909. J. Hyg., 9, 69. WiLson, W. H. 1904. Rec. Egypt. Gov. School of Medicine, 2, 11. Spider Bite AMARAL, A. Do. 1928. Bull. Antivenin Inst. America, 2, 69. Bogen, E. 1926. J. Amer. Med. Ass., 86, 1894. Brazin, V., and VErLARD, J. 1926. Mem. Inst. de Butantan, 8, 243. Ref. in Trop. Dis. Bull., 1927, 24, 402. Kerraway, C. H. 1930. Med. J. Australia, 1, 41. Troise, E. 1928. C. R. Soc. Biol., 99, 1431, 1433. iH 1929. C. R. Soc. Biol., 102, 1097, 1098. CHAPTER X THE SERUM TREATMENT OF LOBAR PNEUMONIA PAGE INTRODUCTORY . v ‘ v ‘ . ‘ L113 TaE SERUM TREATMENT OF TYPE I. PNEUMONIA . . 114 Frequency and age-distribution of Type I. infections . . 114 Factors, apart from serum treatment, that influence the mortality from the disease ’ ‘ 9 . . 115 Serum preparations used in the treatment of pneumonia . 115 The therapeutic effects produced by the serum . . 116 The influence of the serum upon the fatality-rate . 2 . 118 The mode of action of the serum 3 % . 3 . 119 The mode of administration of the serum . 3 : . 120 Dosage of the serum . : . . y ‘ 121 The technique for determining the type of the infecting pneumococcus . . . : ’ . 122 Tae Serum TREATMENT OF TYPE II. PNEUMONIA : . 124 TaE PREPARATION OF ANTI-PNEUMOCOCCUS SERUM . # . 125 The production of the serum. : v . . 125 The concentration of the serum > ’ . 3 . 125 The standardization and titration of the serum . ’ . 126 Introductory Trae work of American investigators during the past ten years, both in the laboratory and in hospital wards, has greatly advanced our knowledge of the specific serum therapy of lobar pneumonia. The strict specificity of the various races of the pneumococcus, which now include at least thirty types, has made it necessary to prepare either separate sera or, on the other hand, multivalent sera that contain immune-bodies against the chief types ; and also to take steps to ascertain with the least possible delay the type that has caused the patient’s illness. The nature of the type- specificity was made clear by the work of Heidelberger and Avery (1923 and 1924), who isolated from each of the chief pneumococcal races a substance which possesses the chemical properties of a polysaccharide ; the specificity of each type of pneumococcus is related to the chemical constitution of the corresponding poly- 113 114 SERUM TREATMENT OF LOBAR PNEUMONIA saccharide. The type-specific carbohydrate, in combination with the species-specific nucleoprotein in the bacterial cell, forms the specific antigen for each type. A good review of recent work on the significance of the pneumococcal types has been given by F. Griffith (1929), who has made valuable contributions to the subject. In Great Britain and America Types I. and II. are found in 55 or 65 per cent. of cases of lobar pneumonia in adults. Type I. is chiefly associated with the pneumonia of young adults, and has a comparatively low mortality ; infections caused by Type II. are more severe ; Type IIL is prevalent among old people, and for this reason shows a high death-rate : it is not an especially fatal infection when it attacks younger persons (Blake, 1932). The distribution of the types has been less thoroughly worked out in children than in adults; the collection of strains derived from children which were examined by Raia, Plummer, and Shultz (1931), included a considerable number that did not conform to the recognized types. This circumstance restricts the opportunities for applying serum treatment in the disease in children, although fortunately the mortality is least among them. It follows that most of the available information on the treatment of pneumonia by serum has been obtained by noting its effects in adults. The Serum Treatment of Type I. Pneumonia Frequency and Age-distribution of Type I. Infections. As already stated, Type I. pneumococcus is prone to attack the younger members of a community. Thus, of 1,127 cases of lobar pneu- monia of this type which were investigated by Cecil and Plummer (1930), nearly 62 per cent. were in persons between 10 and 40 years of age. These authors found Type I. to be the commonest type of pneumococcus in patients with lobar pneumonia, and state that it was present in almost one-third of all the cases under their care in Bellevue Hospital, New York. The reader is advised to consult the summary (1930) of their observations on 1,161 patients with Type I. pneumonia whom they treated in the previous ten years ; their review devotes special attention to the results that followed SERUM PREPARATIONS USED IN PNEUMONIA 115 serum therapy. They note that the illness of the Type 1. patient has a typical onset, and runs a typical course which terminates as a rule by crisis. Factors, apart from Serum Treatment, that influence the Mortality from the Disease. The death-rate in pneumonia is a variable one, as is shown when the experience of different hospitals in the same city is compared, as, for instance, Bellevue Hospital and the Rockefeller Hospital, New York. The death-rate is consistently higher in Bellevue Hospital, and this is probably due to the circumstance that the patients come from a lower social stratum, and that their general health and habits are inferior to those of the patients who are admitted to the Rockefeller Hospital. The mortality-rate may vary from year to year in the same hospital ; in Bellevue Hospital during the period from 1921-1929, it increased from 20 per cent. to 43 per cent. for no known reason ; Cecil and Plummer suggest that the influenza epidemic of the year 1918, and the increasing incidence of alcoholism in New York may have been important contributory causes. In general, the mor- tality shows a progressive increase from childhood to old age. The prognosis in all types of pneumonia depends to a great extent upon the presence or absence of a bacterizmia, and the frequency of this complication in Type I. cases is about 30 per cent. A clinical and bacteriological study of 2,000 cases of lobar pneumonia, which was carried out by Cecil, Baldwin and Larsen (1927), showed a death-rate, when the blood culture was positive, of 83-1 per cent. as compared with only 18-7 per cent. in patients with a negative blood culture. In view of the foregoing considerations, it would be necessary in a clinical test of the value of serum treatment to arrange for a control series of comparable cases, and to allocate the patients alternately to each group, if it were desired to obtain results that would satisfy the conditions for statistical analysis. Serum Preparations used in the Treatment of Pneumonia. Huntoon’s antibody solution contains immune-bodies against Types 1, II. and III. The method of preparation is first to absorb the antibody from a specific serum by adding to it living pneumococci, and afterwards to effect a separation of the antibody by washing the coccal deposit in a weak alkaline solution. The 116 SERUM TREATMENT OF LOBAR PNEUMONIA clinical results from its use appear to have been satisfactory ; on the other hand, intravenous injections of it are often followed by severe febrile reactions, although, owing to its freedom from horse-serum protein, it never gives rise to serum sickness or anaphylactic symptoms. The * thermal >’ reactions are sometimes alarming, and in three patients the intravenous administration of the solution appeared to have been the immediate cause of death (Cecil and Plummer). Park, Bullowa and Rosenbliith (1928) have given up the use of this preparation, and Kessel and Hyman (1927) are unable to recommend it as suitable for routine use in hospitals. A concentrated serum prepared according to the methods described by Felton is now preferred by most physicians. This serum is obtained by immunizing horses against Type I. or IL, and is concentrated by separating the protein fraction which contains the protective antibody. It is titrated in terms of a unit adopted by Felton (1928) on experimental grounds, the unit being defined as the smallest amount of serum which, on the average, will neutralize one million lethal doses of a virulent pneumococcus culture, when the test is carried out in mice under certain prescribed conditions. Sera can be prepared that contain 1,000 units of each antibody per cubic centimetre. A concentrated preparation has been tried on a considerable scale by Cecil and Plummer, and by Park and his colleagues, and their testimony agrees in attribut- ing to it valuable therapeutic properties. Cole (1929), however, remains sceptical and prefers the unconcentrated serum, because he is uncertain whether the potency of concentrated sera can be estimated by the particular mouse test which he himself employs for estimating the potency of the natural serum. Nevertheless, there is no reason to doubt that the protective substance can be concentrated by appropriate methods, and that the resulting product is capable of titration by tests in mice. Cole does not think it right to withhold the serum from patients, so convinced is he of its value, and therefore he does not now provide for a simul- taneous control series. The Therapeutic Effects produced by the Serum. The American observers agree that the serum often diminishes the severity of the THERAPEUTIC EFFECTS 117 symptoms, so that even within 24 hours the temperature, the pulse-rate and the respiratory-rate are reduced or have become normal. According to Cecil and Plummer, there is no more striking clinical effect in the whole field of specific therapy than that which frequently follows its early administration. The fall of temperature is so rapid that it simulates the natural crisis, and the toxemic symptoms often disappear within 24 hours. More- over, if pneumococci are present in moderate numbers in the blood stream, they quickly vanish after one or two doses of the serum have been given. It has no apparent influence upon the incidence of complications nor upon the mortality resulting from them (Cecil and Plummer). The inflammatory changes in the lung are checked, but the rate of resolution is not hastened by serum treatment, since this process seems to be independent of the immunity mechanism (Griffith, 1929). Bullowa (1928) believes that the serum not only decreases the number of deaths, but also delays death and shortens the acute stage of the illness in those who recover. A careful clinical study has been made by Sutliff and Finland (1931) with the object of ascertaining how the serum influences the course of the disease. The criteria they kept specially in view as denoting improvement were: (1) the length of the illness ; (2) the course taken by the bacterizzmia ; and (3) the limitation of the spread of inflammation in the lung. They conclude that the group of patients treated with serum showed definite improvement within 80 hours after its administration, and that the illness was shorter on the average than in the control group ; the beneficial action of the serum was proportionately greater the earlier it was given. They thought that the results as they affected the bacterizemia were striking, for no serum-treated patient whose first blood culture was negative showed signs of a bacterizzmia at a later stage of his illness. Lastly, extension of the inflammation to fresh portions of the pulmonary tissues was observed among the untreated patients, but not among those who received the serum. The physicians to the Royal Infirmary, Edinburgh, have reported on the results of a small series of cases. A graph illus- trating the frequency distribution of the day of the crisis in serum- 118 SERUM TREATMENT OF LOBAR PNEUMONIA treated and control groups shows that the critical fall of tempera- ture occurred at least one day earlier in the serum-treated group than in the control group. Even when the illness was not shortened, rapid improvement set in soon after the serum had been given. Speaking on behalf of his colleagues in Edinburgh in the discussion that followed the address given by Dr. Russell Cecil at the British Medical Association meeting (1932), Murray Lyon stated their conviction that an improvement of a kind which was not indicated in the numerical records was evident in many of their serum- treated cases. Cowan and his co-workers, from their experience at the Glasgow Royal Infirmary, are inclined to believe that the serum may shorten the febrile period in some of the Type I. cases, but they could not find that by its use the toxemia was lessened, complications prevented, or resolution of the lung hastened. They consider that the serum should be available for the treatment of pneumonia in every hospital. In the experience of Armstrong and Johnson (1932), the average day of the crisis was accelerated by two and a half days, and the number of cases that ended by lysis was reduced. Moreover, the treatment appeared to lessen the liability to the spread of inflam- mation in the lungs. They admit, however, that there is a group which, perhaps owing to a naturally feeble immunity response, appears to be unaffected by early and vigorous serum treatment. A leucopenia and a positive blood culture were generally present in this type of patient. At the British Medical Association meeting referred to above, the present author was interested to note the concordance of opinion among the speakers to the effect that the serum consti- tutes a specific and efficacious remedy in the treatment of lobar pneumonia (Cruickshank, Davidson, Murray Lyon and Armstrong). The Influence of the Serum upon the Fatality-Rate. During the four years from 1924-1925 to 1928-1929, Cecil and Plummer treated in all 239 cases of Type I. pneumonia with serum : the death-rate was 20-1 per cent. ; 234 alternate control cases had a death-rate of 31-2 per cent., a difference which can be regarded as statistically significant (d/od =2-8). These authors have compared MODE OF ACTION OF THE SERUM 119 the mortality-rates in patients admitted to hospital within 72 hours of the onset of symptoms, and they remark that the contrast between the two groups is even more striking ; of 103 serum- treated patients only 11-7 per cent. died, and of 97 controls 26-8 per cent. died; but according to the calculation of the present writer these figures have approximately the same degree of significance as those in the first-mentioned set of data (d/ od == 27). Park, Bullowa and Rosenbliith (1928) have analyzed the combined experience in three hospitals in New York for the year 1927-1928. Of 266 serum-treated Type I. cases, 19 per cent. died, and of 249 controls 33 per cent. died, a difference of 14 per cent. ; they give the ratio of the difference to its standard error as 3-7, and remark that, since statisticians regard a value of 2-0 as significant, this result is impressive. Griffith (1929) has pointed out that the best single criterion of the value of the serum is its effect upon a bacterizemia. Although the figures that are available are not large, they indicate that the serum has a real value in combating this grave complication. Thus Rosenbliith found that 81 per cent. of twenty-two Type I. cases which were untreated by serum died, as compared with 39 per cent. of twenty-eight serum-treated cases. According to this observer, a late invasion of the blood-stream by pneumococci is a very unfavourable prognostic sign. Concentrated serum is quite as effective as unconcentrated serum and, unless the bacterizmia is extreme, the pneumococci disappear from the circu- lating blood after the administration of one or two doses of the serum (Cecil and Plummer). The Mode of Action of the Serum. Protective antibodies and agglutinins can be demonstrated in the blood of patients who recover from an attack ; they appear about the time of the crisis, and their presence indicates that serum treatment need not be continued. The type-specific carbohydrate is also present in the blood during the illness and has the property of neutralizing the specific antibody, whether it be the natural one formed by the patient or the artificial one in the therapeutic serum he has received. Pneumococci and the protective antibodies are rarely found together in the blood (Baldwin and Cecil, 1926). Thus it is clear 120 SERUM TREATMENT OF LOBAR PNEUMONIA that a bacterizemia is correlated with an inadequate production of natural antibody, and doubtless also with an excessive liberation of the specific soluble substance. Cecil and Blake (1920) found that Type I. anti-pneumococcus serum quickly brought about the disappearance of pneumococci from the blood of experimentally infected monkeys ; and Petrie and Morgan (1982) showed that a single intravenous dose of a natural or a concentrated Type I. serum was able to cure mice—even although a bacterizemia was present—when it was administered up to 12 hours after an infecting dose of a highly virulent culture. The clinical evidence indicates a similar clearing process after the administration to the patient of a specific serum. When the homologous serum is introduced into the vein of a patient it neutralizes the soluble substance, promotes phagocytosis of the pneumococci in the blood and tissues, and inhibits their multiplication. The administration of the serum at an early stage of the illness, by increasing the specific antibody in the blood, has the effect of forestalling a bacterizemia ; it may be necessary to give repeated doses in order to keep up the concentra- tion of antibody in the blood ; and even at a late stage of the illness the antibodies in the therapeutic serum may reinforce the natural defences of the patient, and turn the balance in favour of his recovery. The effects of anti-pneumococcus serum are usually attributed to its antibacterial properties; so far as is known, it has no antitoxic action. The Mode of Administration of the Serum. There is no difference of opinion regarding the best route for giving the serum. The immediate and widespread distribution of the antibody through- out the blood of the patient which occurs when the serum is given intravenously leaves the practitioner no choice, apart from his scruples concerning the risk of serum reactions that may follow the injection. The risk of a severe reaction should not be exaggerated. Cecil and his colleagues have treated more than 500 cases with intravenous injections of the concentrated serum without a single fatal accident, and a like experience has been recorded from Harlem Hospital, New York. In practice, alarming reactions are seldom encountered, and they can, more- over, be readily controlled by the intramuscular or intravenous DOSAGE OF THE SERUM 121 injection of 0-5-1:0 c.cm. of one in 1,000 adrenaline solution. A syringe containing the solution should always be at hand for immediate use if required. Before administering the serum, inquiries should be made regarding previous injections of horse serum, and also regarding the occurrence of attacks of asthma or hay fever ; a positive history denotes the need for caution. Many physicians test for undue sensitiveness to horse serum by introducing a drop of a tenfold dilution in saline of normal horse serum into one eye; if after 15 to 20 minutes there is no con- junctival redness as compared with the other eye, the serum may be given intravenously. A positive reaction is held to contra- indicate the intravenous procedure. An excessive conjunctival reaction can be controlled by the instillation into the eye of a drop of adrenaline solution. The risk to the patient from an allergic reaction that may follow the first dose may be minimized by slowly injecting into the vein 1 c.em. of the serum, either pure or diluted with sterile normal saline, and allowing an interval of 20 or 30 minutes to elapse before the remaining portion of the dose is given. The immediate reac- tions that are met with are of two kinds: (1) the allergic type, which may occur almost at once with pallor, tachycardia and res- piratory embarrassment ; and (2) febrile reactions, which generally appear in about an hour, are characterized by a rigor and an increase in the cyanosis, and are followed by profuse sweating and a fall of temperature (see p. 13). Dosage of the Serum. There is no uniform scheme of dosage ; in the United States larger doses are given than in this country, doubtless because the disease there runs a stormier course with a higher proportion of bacterizemic cases and a consequently greater mortality. Thus Sutliff and Finland (1931) gave 90,000 units on the first day, and on succeeding days 225,000, 360,000 and 495,000 units. Cecil (1932) advises an initial dose of 5 c.cm., followed in one or two hours by 15-20 c.cm., and a dose every two or three hours thereafter until 100,000 units (usually about 100 c.cm. of serum) have been given. The scale of dosage may be halved next day if the patient shows improvement, and a precautionary dose of 10 or 20 c.cm. may be given on the following day. The Glasgow 122 SERUM TREATMENT OF LOBAR PNEUMONIA physicians are content with a dose of 10,000 units every eight hours until the temperature falls below 102-0° F.; their average dose per patient was 86,000 units. Armstrong and Johnson give 20,000 units as a first dose and 40,000 units thereafter, morning and evening, with a total, on the average, of 75,000 units. In view of the expense incurred in giving repeated doses of anti- pneumococcus serum it is important to be able to know whether further doses should be given to a patient or whether, on the other hand, it is safe to stop the treatment. Francis (1933) has described a test which promises to serve as a useful guide. He finds that if a small amount of the homologous polysaccharide solution is injected into the patient’s skin an immediate positive reaction signifies that he is in the stage of recovery and does not need more serum, whereas a negative result indicates that the serum treatment should be continued. The Technique for Determining the Type of the Infecting Pneumo- coccus. A number of methods have been described, but the one that appears to fulfil best the primary object of the technique, namely, rapid determination of the type, is that which was published by Armstrong (1932) and simultaneously by Logan and Smeall. This test is based on the observation made many years ago by Neufeld that virulent pneumococci, when brought into contact with undiluted homologous immune-serum, undergo a characteristic swelling of the capsule so that the diplococei are easily seen under a comparatively low power of the microscope. The technique consists in mixing a Type I. and a Type II. aggluti- nating serum separately with droplets of the sputum, and examining the wet preparations under the microscope ; when a positive result is obtained with one or other serum the difference between the two preparations is striking. Valentine (1933) has described a method of staining the swollen capsules and thus rendering them more easily recognizable. The modification of Sabin (1933) consists in examining the sputum, as soon as possible after it has been expectorated, in a hanging-drop under the oil immersion lens with the addition of alkaline methylene blue and of type sera from the rabbit. The test, however it may be carried DETERMINATION OF THE TYPE 123 out, is applicable to any material containing virulent pneumococci, such as a pure or mixed culture, or the peritoneal effusion of an infected mouse. The capsule phenomenon should always be looked for, since it possesses a diagnostic significance which is at least equal to that of a specific agglutination. It may be noted here that even at an early stage of the disease, for example, within the first three days, a successful preparation may be obtained from a swab of the pharynx. If the preparation of the pharyngeal swab gives an inconclusive result owing to the small number of pneu- mococel present, the swab should be rubbed into a broth tube containing 1 per cent. glucose and 10 per cent. rabbit blood and incubated at 87° C. for two or three hours, when the capsule test may again be applied. The direct method of typing can be confirmed by inoculating a mouse intraperitoneally with a portion of the sputum, taking a sample of the peritoneal fluid after four or six hours, and carrying out agglutination tests and the capsule test with the fluid. When the mouse dies, cultures from its heart blood may be made and diagnostic tests carried out with saline washings of the peritoneal exudate. Cecil (1982) combines the mouse method with the rapid methods of Krumwiede and of Sabin, and is able to determine the type within five hours in a large proportion of the cases. The test devised by Krumwiede consists in boiling 5-10 c.cm. of the sputum in order to coagulate the albuminous material and to set free the specific soluble substance (polysaccharide). The clear fluid is then pipetted carefully into small test tubes containing the undiluted diagnostic sera so as to form a supernatant layer; a precipitation ring appears when the test is positive. In the method described by Sabin a little of the peritoneal fluid is removed by puncture with a glass capillary or a fine syringe needle from a mouse which has been inoculated with the sputum three to six hours previously. Droplets of the exudate are placed on a slide, and a droplet of a 1/10 dilution of each diagnostic serum is mixed with them. The smears are made thin, allowed to dry, fixed in the flame, and stained. The slide that shows agglutination gives the clue to the type. 124 SERUM TREATMENT OF LOBAR PNEUMONIA The Serum Treatment of Type II. Pneumonia In the experience of the American workers a bacterizmia occurs in this type in from 83 to 48 per cent. of the cases, and is associated with a high mortality-rate ; recovery naturally occurs more often among those patients whose blood remains sterile. Baldwin and Rhoades (1927) conclude that a highly potent Type II. serum, if it is administered early, may lessen the chance of occurrence of a bacterizemia, and may sometimes sterilize the blood when a generalized infection already exists. This belief is supported by data supplied by Rosenbliith ; in his series of cases 75 per cent. of the control patients (17) with positive blood cultures died, as compared with 38 per cent. of those (18) who had cocci in the blood-stream and had received serum ; the numbers, however, are perhaps hardly large enough for exact comparison. Park and Cooper (cited by Bullowa et al, 1928) have reported the results of tests which indicate that in patients with a Type II. infection there is a relatively large amount of soluble specific substance in the blood, a finding which points to the importance of adopting early and intensive serum treatment in this type of pneumonia. The difference between the results in the control and the serum- treated cases in Bellevue Hospital is significant (dod = 2-0), but the data for Harlem Hospital are inconclusive, owing appar- ently to the disproportionate number of bacterizemic cases in the serum-treated group (Park et al, 1928). The conclusion may be drawn that specific serum therapy is less efficacious in Type II. than in Type I. pneumonia. A recent article by Cecil and Plummer (1932) provides much useful information on Type II. pneumonia and the results of its treatment by a specific concentrated serum. The clinical features are similar to those of Type I. pneumonia except that the patients tend to be more toxic, and certainly the mortality among them is greater; in Bellevue Hospital nearly half of the untreated patients died, a result which was correlated with the high incidence of bacterizemia (about 50 per cent.). Cecil and Plummer treated 252 cases of Type II. pneumonia with serum, and observed 253 control patients ; the fatality-rates were respectively 40-5 per cent. TYPE II. PNEUMONIA 125 and 45-8 per cent. During the last year of their work the serum was given only to cases treated within 72 hours of the onset, with the result that of 21 serum-treated cases the death-rate was only 14-8 per cent. as compared with 65 per cent. of 20 control cases. Although the figures are small, they indicate progress. It is significant that some of the speakers in the discussion at the British Medical Association meeting felt constrained, from the clinical impressions they had received, to admit the value of serum treatment in Type II. pneumonia. Let it be repeated that the severity of the attack renders it an urgent matter to commence serum treatment at the earliest possible moment. The Preparation of Anti-pneumococcus Serum Only sera prepared against Types I. and IIL need be considered here, since it is not yet practicable to make a potent serum against Type III. pneumococcus, and since at the present time there is no efficacious multivalent serum for the treatment of pneumonia caused by types of pneumococci which were formerly included in the miscellaneous Group IV. A potent Type IIL. serum is more difficult to prepare than a Type I. serum. The Production of the Serum. The immunization of horses is effected by the intravenous injection of serial doses of cocci obtained by centrifuging heat-killed broth cultures of fully virulent strains. The cultures must be young, and the suspension of cocci should be injected without undue delay in order to prevent the dissociation of the antigen, that is, the specific carbohydrate- nucleoprotein complex. A final series of doses of living cocci may be given, but this method entails a risk, even in highly immunized horses, of the occurrence of fatal pneumococcal infections and especially of vegetative endocarditis. After about nine months’ immunization the serum has attained a potency which renders it suitable for concentration. The Concentration of the Serum. Most of the work on this part of the subject has been done by Felton, who has given a general account of it in a recent paper (1930). This author has shown that the protective antibody is associated with a relatively water- 126 SERUM TREATMENT OF LOBAR PNEUMONIA insoluble protein which can be separated from the euglobulin by adjusting the hydrogen-ion concentration of the solution in dilute saline to a pH of 4-8 to 5:0. When this has been done, the inert euglobulin falls out of solution and can be filtered off; the protective protein remains in solution. The fraction that contains the protective protein represents from 10 to 15 per cent. of the total serum proteins, and the final concentration is equivalent to a volume which is one-fourth to one-tenth of that of the original serum. There is a heavy loss of antibody during the process of concentration, and this, together with the difficulties that are inherent in the immunizing processes, adds considerably to the cost of production. The Standardization and Titration of the Serum. At the present time there is no method of testing the potency of anti-pneumo- coccus serum that has won universal acceptance; its titration is indeed still in the experimental stage. The tests in current use seek to determine either: (1) a minimum standard of potency to which the serum must attain; or (2) the value of the serum in terms of an arbitrary unit. Thus the conditions laid down by the National Institute of Health in Washington require that 0-2 c.cm. of the serum when mixed with 0-1 c.em. of a young virulent culture and injected intraperitoneally into mice shall ensure the survival of at least 50 per cent. of the test animals. This test makes no attempt to measure the limit of the protective power of the serum, and for this reason experimental work is being done in some laboratories in order to discover a method of titration by which it may be possible to estimate the protective value of anti- pneumococcus serum in terms of a standard unit. Goodner (1929) has described a method of titrating the curative value of anti- pneumococcus serum by observing the effect of graduated intra- venous doses on lesions of the skin in rabbits that are produced by an appropriate dose of a living virulent culture; exact details of the method do not appear to have been worked out. One method of titration is to choose a constant test-dose of a virulent culture, and to estimate the protective value for mice of varying amounts of the serum under test. Felton has defined the unit he uses as the amount of serum which will protect mice against one TITRATION OF THE SERUM 127 million lethal doses of a culture, when the serum and culture are injected simultaneously. An essential requisite for any method of titration of this kind is the provision of a standard serum, issued preferably by a Control Authority and kept under proper conditions of storage, of which an arbitrary but definite amount shall represent a unit. The need for such a standard serum is evident in view of the apparently uncontrollable factors that cause irregularities in the tests and so render difficult the interpretation of the results. Progress is being made in this direction for, although an official unit for anti-pneumococcus serum (Type I.) has not yet been prescribed for Great Britain and Northern Ireland, the National Institute for Medical Research in London has prepared and arranged to issue a stable standard serum, the unit value of which has been provisionally defined in terms of a particular serum supplied by Dr. Felton. This provisional unit has been adopted with a view to ensuring that all anti-pneumococcus serum (Type I.) that is available in Great Britain and Northern Ireland shall be titrated on a common basis. Details of the preparation of the standard serum and of comparative tests that have been carried out with it will be found in an article by Hartley, Parish, Petrie and Wilson Smith (19383). These authors point out that the use of a common standard for the titration of anti-pneumococcus serum (Type I.) would render the results obtained in laboratories and hospitals throughout the world directly comparable. The writer is able to testify from personal experience, in collaboration with Dr. W. T. J. Morgan, to the valuable aid afforded by the standard in routine and experimental work (Morgan and Petrie, 1933). The standard preparation is available to all who desire to make use of it, by application to the Department of Biological Standards, The National Institute for Medical Research, London, N.W.3. Attempts have been made from time to time to titrate the specific antibody by means of a precipitation test. Wilson Smith (1932) has shown that the method is applicable to natural anti- pneumococcus serum Type I. ; it has not, however, been found to give regular results when concentrated sera are used. 128 SERUM TREATMENT OF LOBAR PNEUMONIA REFERENCES ARMSTRONG, R. R. 1931. Brit. Med. J., 1, 214. 3 1932. Brit. Med. J., 1, 187. ARMSTRONG, R. R., and Jounson, R. S. 1931. Brit. Med. J.,1, 931. » 9” 1932. Brit. Med. J., 2, 662. Barpwin, H. S., and Ceci, R. L. 1926. J. Amer. Med. Ass., 87,1709. Barpwin, H. S., and Ruoapes, D. R. 1927. Amer. J. Med. Sci., 174, 191. Brake, F. G. 1932. Ann. Int. Med., 5, 673. Britisu MebpIicAL Assoc. MEETING. 1932. Discussion on Serum Treatment of Pneumonia, Lancet, 2, 519. BuLrowa, J. G. M., RosenBrLUTH, M. B., Park, W. H., and COOPER, G. 1928. J. Amer. Med. Ass., 90, 1349. CeciL, R. L., BaLpwiN, H. S., and LARSEN, N. P. 1927. Arch. Int. Med., 40, 253. Ceci, R. L., and Brake, F. G. 1920. J. Exp. Med., 32, 1. CeciL, R. L., and PLummMER, N. 1930. J. Amer. Med. Ass., 95, 1547. - 5 1932. J. Amer. Med. Ass., 98, 779. CeciL, RusserL LL. 1932. Brit. Med. J., 2, 657. Core, R. 1929. J. Amer. Med. Ass., 93, 741. CowaN, J., et al. 1930. Lancet, 2, 1387. ’s ” 1932. Lancet, 2, 8. EpiNnsurcH. Physicians to the Royal Infirmary. 1930. Lancet, 2, 1390. Ferron, L. D. 1928. J. Infect. Dis., 43, 531. - 1930. J. Amer. Med. Ass., 94, 1893. Francis, T., Jr. 1933. J. Exp. Med., 57, 617. GOODNER, K. 1929. J. Immunol., 17, 279. GrrrriTH, F. 1929. * A System of Bacteriology,” 2, 201. HarTrEY, P., Paris, H. J., PETrIE, G. F., and WiLsoN SMITH. 1933. Lancet, 2, 91. HEIDELBERGER, M., and Avery, O.T. 1923. J. Exp. Med., 38, 73; 1924, ibid., 40, 301. KesseL, L., and Hyman, H. T. 1927. J. Amer. Med. Ass., 88, 1703. Krumwiepe, C., and VavLentiNg, KE. 1916-1919. Coll. Studies Bureau Labs.,N.Y., 9, 307. Locan, W. R., and SmeaLy, J. T. 1932. Brit. Med. J., 1, 188. Morcan, W. T.J.,and Petrie, G. ¥. 1933. Brit. J. Exp. Med., 14, 323. NevureLp, F., and ScanNitzer, R. 1928. ‘ Handb. der path. Mikroorg.,” 4, 966. Parisu, H.J. 1930. J. Path. Bact., 33, 729. Park, W. H., and Cooper, G. 1928. J. Amer. Med. Ass., 90, 1349. Park, W. H., Burrowa, J. G. M., and RosensLUTH, M. B. 1928. J. Amer. Med. Ass., 91, 1503. PETRIE, G. F., and MorcanN, W. T. J., 1931. Brit. J. Exp. Path., 12, 447, " ”" 1932. Brit. J. Exp. Path., 18, 96, REFERENCES 129 Raia, A., PLumMver, N., and Suvrrz, S. 1931. Amer. J. Dis. Child ., 42, 57. RosenBriTa, M. B. 1928. J. Amer. Med. Ass., 90, 1349. SaBIN, A. B. 1930. J. Inf. Dis., 46, 469. SasiN, A. B. 1933. J. Amer. Med. Ass., 100, 1584. Smita WinsonN. 1932. J. Path. Bacl., 35, 509. Surrirr, W. D., and IF'inranp, M. 1931. J. Amer. Med. Ass., 96, 1465. TreEvVAN, J. W. 1930. J. Path. Bact., 33, 739. VaLenTINE, F. C. O. 1933. Lancet, 1, 22. R.A. VACCINES. CHAPTER XI THE SERUM TREATMENT OF CEREBROSPINAL FEVER INTRODUCTORY AN ANALYSIS OF THE REASONS GIVEN FOR THE OCCASIONAL INEFFICACY OF THE SERUM . . The ideal scheme of clinical investigation Flexner’s account of the results obtained during the early y years of serum treatment. . . . . . . The variable results of serum treatment . Certain technical details concerning the preparation of the serum that may influence its efficacy . . : . The importance of employing appropriate strains The agglutinin content of commercial sera as an index of their therapeutic potency. . . The preparation of het sera in inter- epidemic periods. % The alleged importance of an antitoxic component in the serum ; The choice of a multivalent or a monotypical serum. The technique of serum production and its relation to the therapeutic results . . 3 . The concentration of anti- -meningococcus serum The reinforcement of the therapeutic action of the serum by the addition of fresh human serum : . The problem of the fulminating case . . ‘ v : The mode of action of the serum . d . : DosSAGE AND MODES OF ADMINISTRATION OF THE SERUM ’ Intrathecal route ” . Combined intrathecal and intravenous or intramuscular routes Intracisternal route . ’ a ’ . Intraventricular route . THE SERUM TREATMENT OF CEREBROSPINAL FEVER IN INFANTS AND YOUNG CHILDREN . 2 ’ . ‘ i THE SERUM TREATMENT OF CHRONIC MENINGOCOCCAL BACTERIZEMIA ’ ’ ’ ’ . : : : Introductory PAGE 130 131 133 133 134 135 135 136 137 138 139 139 140 ANTI-MENINGOCOCCUS serum has been used in the treatment of cerebrospinal fever during the past quarter of a century. A 130 VARIABLE RESULTS OF SERUM TREATMENT 131 perusal of the original sources of information on the subject is likely to leave the reader with the conviction that the serum constitutes a specific and efficacious, if somewhat variable, remedy. The early reports of Flexner, Dunn, Claude Ker and Gardner Robb are worth consulting, because these workers were familiar with the too often tragic picture of the disease in the period before,1906 when the serum began to be employed, and were thus able to appreciate the mitigation of the symptoms and the reduction in the mortality from 70 or 80 per cent. to about 30 per cent. which followed its use. The general opinion to-day is that the serum, when it is given at an early stage of the illness, controls the infective process and helps to bring it to an end. The illness is shortened and terminates not infrequently by crisis; the occurrence of complications and sequele is less common than when no serum has been given ; relapses, too, are not so often seen ; and it is rare for the infection to smoulder for months as formerly happened in the absence of specific treatment. In spite of this conclusion, it must be admitted that especially of late years serum therapy has appa- rently at times been ineffective. Thus it is stated that in some European countries treatment by serum seems to have become almost completely inefficacious, as in Denmark and to a less extent in Sweden. In Germany a cautious and even pessimistic view is taken of its value ; Bieling and Meyer, for example, do not regard it as the principal therapeutic agent, but merely as an auxiliary to be classed with lumbar puncture, optochin and other remedies for the relief of the symptoms. Again, Prausnitz lays stress upon the fact that meningococcal strains that have been obtained from various countries reveal intricate and ill-defined antigenic relation- ships, and argues that for the present ‘“ the prospects of producing anti-meningococcus sera of practical value are rendered highly doubtful by the existence of this antigenic multiplicity.” An Analysis of the Reasons given for the Occasional Inefficacy of the Serum The epidemic prevalence of cerebrospinal fever in the last decade in the United States has furnished opportunities for acquiring o—2 132 SERUM TREATMENT OF CEREBROSPINAL FEVER experience of the treatment of the disease by means of a specific serum, and of reviewing afresh the numerous problems that are bound up with the methods of preparation and administration of the serum. The American records show an increasing incidence of the disease from the year 1925, when 1,859 cases were reported, to the year 1929, when the number rose to 9,660 cases. During 1928 and 1929 there were considerable outbreaks in some of the larger cities. In Detroit, for example, an outbreak began in the spring of 1928 and reached its peak in May, 1929 ; in this period there were 867 cases and 430 deaths—a fatality-rate of 50 per cent. (Norton and Gordon, 1930). These authors comment on the high death-rate, and conclude that either the local strain of meningo- coccus was of exceptional virulence or that the therapeutic sera employed were lacking in the appropriate specific antibody ; yet it is stated that 62 epidemic strains and 33 carrier strains were all agglutinated by a 1 in 400 dilution of a multivalent anti-meningo- coceus serum obtained from the horse (Norton and Broom 1930). The high death-rate in the Detroit outbreak seems to show that for some unknown reason the serum treatment of cerebrospinal fever occasionally fails under present conditions; and a study of the literature brings to light similar experiences elsewhere in the United States and also in other parts of the world. McCoy (1929) refers to the assertion that in recent years commercial sera have proved at times to be ineffective, and points out that success- ful results following treatment by the serum are not always published. With regard to the opinion which is held by some authorities that the use of multivalent sera has reduced the mor- tality from 75 per cent. to about 25 per cent., McCoy states that he is not aware of any series of cases that has established the value of specific serum treatment from the point of view of a rigid statistical analysis. The correctness of this statement can be verified by a survey of the published records. Nevertheless, the statistical data that are available are sufficiently impressive in spite of defects in their collection. A useful table is that given by Wadsworth (1931), which assembles from a variety of sources the data concerning groups of serum-treated cases and of cases not so treated. This table shows that 36-8 per cent. of 9,760 ANALYSIS OF THE EARLY RECORDS 133 serum-treated patients died and 74-3 per cent. of 1,313 control patients ; a ratio of 1 : 2 in favour of the serum-treated cases. The Ideal Scheme of Clinical Investigation. We may suppose that the ideal scheme of clinical investigation would comprise four series of patients who would be comparable with each other as far as possible, and that the method of treatment to which each group was subjected would be as follows : the first to receive general treatment only ; the second to be treated by lumbar puncture and spinal drainage ; the third to receive normal horse serum intra- thecally ; and the fourth to receive specific serum treatment intrathecally. In such a scheme the serological type of the infecting coccus would be determined in each case, and the type distribution would be taken into account in the final analysis. There is, however, little likelihood, in practice, of the fulfilment of these conditions. Here we may remark that spinal drainage by means of repeated lumbar punctures, apart from serum treatment, is not generally thought to exercise a beneficial influence upon the disease, although Foster and Gaskell (1916) considered it to be of some value. From his experience in Edinburgh, Ker concluded that temporary relief was occasionally obtained by repeated lumbar punctures and the withdrawal of varying amounts of spinal fluid, but that this method of treatment did not appear to modify the subsequent course of the illness. Dunn (1908), too, employed lumbar puncture alone during two years without being able to lower the fatality- rate, and Flexner (1917) referred to the observations with a similar significance which were made during the period 1904-1910 by Netter, Goeppert, and others. Flexner’s Account of the Results obtained during the Early Years of Serum Treatment. Flexner’s account of the results of serum treatment in 1,300 cases of cerebrospinal fever, which was published in 1913, remains an outstanding contribution to the subject. The serum was prepared at the Rockefeller Institute and was distributed in the United States and Canada, and in several European and Asiatic countries. Of 1,294 patients who received the serum, 400 died (30-9 per cent.). A table is given which shows the increase in mortality in proportion to the delay 184 SERUM TREATMENT OF CEREBROSPINAL FEVER in administering the serum, and other tables present an analysis of the serum-treated cases from other points of view. The data as a whole certainly seem to justify Flexner’s conclusion that the serum was an effective curative agent, although their evidential value would have been considerably strengthened if a comparable analysis of the control cases had been given. The statement is made that the mortality among the controls did not differ essentially from that which prevailed in various countries at the same time, namely, about 70 per cent., but no evidence is forth- coming that observations on the control cases were recorded in a comparable manner. with those in the serum-treated group. Details of dosage and of the methods of administering the serum are not given. In a later article (1928), Flexner reiterates his belief that the serum has been of great service in the treatment of the disease, and he estimates that the death-rate has been reduced from 65 or even 90 per cent. to 25 per cent. or less by its use. The Variable Results of Serum Treatment. Tables have been drawn up by several authors which bring together the results of serum therapy obtained by different workers who used sera from different sources (Blackfan ; Worster-Drought and Kennedy ; and Friedemann). Friedemann’s table is instructive, because the data are arranged according to the therapeutic results as judged by the percentage mortality in the groups ; the mortality-rates vary from the exceptionally low figure of about 15 per cent. (Dopter’s cases) to 75 per cent., a figure which may be regarded as equal to the death-rate of a control group. The most striking data that have been published within recent years are those of Wadsworth (1932), who reports a mortality-rate of 18-4 per cent. among 742 patients of all ages. These figures do not include patients who lived for less than 24 hours after the first dose of serum was given to them, nor patients with secondary and mixed infections of the meninges or intercurrent infections which were the immediate cause of death, nor instances of the chronic form of the disease in children, who died after an illness lasting for two or three months ; the exclusions were those adopted by Flexner in his early work. The serum that was used in this IMPORTANCE OF APPROPRIATE STRAINS 185 group was prepared under Wadsworth’s direction in the Labora- tories of the New York State Department of Health. Results indicating a similarly favourable experience have been reported from Roumania (Cantacuzéne, 1931). Certain Technical Details concerning the Preparation of the Serum that may influence its Efficacy. The Importance of employing appropriate strains. Friedemann has discussed the cause of the variable results which have been obtained in different countries with anti-meningococcus serum, and concludes that it is to be found in the presence or absence in the sera of antibodies which are effective against the local epidemic strains. Most bacteriologists are agreed that the four type-specific races of Gordon and Murray are not sharply defined, but that, on the contrary, they show a considerable amount of antigenic over- lapping. Types I. and III.—sometimes referred to as Group I.— are closely related, and Types II. and IV. (Group II.) likewise form an inter-related group. The boundaries between the main groups cannot be regarded as mutually exclusive (W. M. Scott, 1917 ; Kirkbride and Cohen, 1932 ; Branham, 1932). During epidemic times it is important to arrange for the determination of the prevalent types, and to take steps for the inclusion of fresh repre- sentative types in the immunizing doses that are to be used in the preparation of the serum. It is not difficult to find evidence that points to the necessity for this procedure. Thus an American stock of serum with an excellent reputation failed in the treat- ment of the disease among soldiers in Great Britain during the war, whereas good results were obtained by the substitution of sera prepared in England from the epidemic strains. A similar experience has been recorded from South Africa ; and Felix and Yunowich noted that in Palestine the efficacy of batches of sera derived from different countries seemed to be related to their agglutinating properties towards the local strains of meningo- coccus. In the Belgian Congo Brutsaert (1931) found that the clinical results showed considerable improvement when a serum was prepared on the spot from local strains which were isolated during the outbreak. Lack of specificity, as judged by agglutina- tion tests, is not, however, a complete explanation, because certain 186 SERUM TREATMENT OF CEREBROSPINAL FEVER commercial sera that were used in some of the recent American outbreaks were unsatisfactory, although they possessed agglutinins for the local strains (Norton and Gordon ; and Smithburn et al.). An important factor in the production of an effective therapeutic serum is probably the antigenic quality of the immunizing strains, as indicated by the ‘roughness’ or smoothness” of the colonies. Appearances of this kind have not hitherto been easy to demonstrate, but recent work tends to show that the meningo- coccus possesses rough and smooth variants (Rake, 1931 ; Enders, 1932 ; Petrie, 1932; Maegraith, 1933). Rough cultures are likely to be less potent antigens than smooth freshly isolated cultures. It is, however, of interest to note that the serum prepared by Wadsworth, which seems to have given good clinical results, is made by the use of six representative strains, two each of Types I., II. and III., which have been cultivated on laboratory media for more than 13 years. The Agglutinin Content of Commercial Sera as an Index of Therapeutic Potency. Branham, Taft and Carlin (1930) examined 155 strains of the meningococcus that were isolated from patients during the recent epidemics in the United States; 140 of them were isolated from the cerebrospinal fluid : the method of classifi- cation adopted was that of Gordon and Murray. They found that 71 per cent. of the strains belonged to Types I. and III. ; the serological resemblance between these types is so close that they may be regarded as practically identical. All the 155 strains were tested for agglutination with multivalent therapeutic sera that had been prepared by eight manufacturers; it was found that 91 per cent. were agglutinated by these sera and that they con- formed to one or other of Gordon’s four types; the remaining strains (9 per cent.) were not agglutinable by the commercial sera and did not fall within Gordon’s classification. These results signify that failures in the use of therapeutic sera in the United States cannot be accounted for by defective representation of the type-specific antigens among those that were used in the production of the sera. The question may be asked whether the agglutination test is really a measure of the therapeutic value of a serum, and the PREPARATION IN INTER-EPIDEMIC PERIODS 137 answer is that the test cannot be accepted as a guide to its potency, since poorly agglutinating sera may be of value in treatment, and, conversely, sera with a high agglutinating titre may be inefficacious. For example, Neal and her co-workers (1928) note that in their experience the agglutination titre of different batches did not run parallel with their therapeutic activity. Thus two batches of serum with an almost identical titre were tested clinically, and the results showed that one was much more potent than the other. Again, an old serum preparation with a very poor agglutinin titre gave good results in practice. A laboratory test whereby the titre of the curative antibodies can be measured is not yet available. The Preparation of Therapeutic Sera in Inter-epidemic Periods. During epidemics of the disease it is not difficult for those who are responsible for the production of the serum to obtain fresh strains of the meningococcus for immunizing purposes; on the other hand, in inter-epidemic periods, when only sporadic cases occur, freshly isolated strains are not always easily obtainable, and thus doubt must arise whether the serum is sufficiently inclusive. Branham, Taft and Carlin (1930) in their paper on the distribution of the types of meningococcus in the United States, give an interesting table which compares the frequency of the types in the epidemic years 1918-1919; the epidemic years 1928-1929 ; and the non-epidemic years 1921-1922. The figures show : (1) that a redistribution of the types had taken place among the epidemic strains ; and (2) that the sporadic strains include a high proportion which do not conform to the well-recognized types. The experience of these workers agrees with that of Murray (1929), who mentions that during the past few years he has examined 15 strains from sporadic cases in England, and in this number found only one that was * typical.” The significance of these observations in relation to the preparation of anti-meningococcus serum during non-epidemic periods is obvious. Sera that have been prepared during inter-epidemic periods may possess little therapeutic value when a fresh outbreak occurs. This was the experience in the early months of the recrudescence of the disease in England, which has led to the inquiry that is being con- 188 SERUM TREATMENT OF CEREBROSPINAL FEVER ducted by the Ministry of Health into the results of serum treat- ment (1931). The question has been raised whether a contingency of this kind may be met by collecting a surplus stock of serum during an epidemic period and preserving it under proper conditions of storage in a dry stable form and in sufficient amount to allow time for the production of a fresh stock. The present author and his colleagues have tested the feasibility of carrying out such a plan and have reason to know that a satisfactory product can be obtained. An alternative method is to prepare a sufficient stock of antigens in a dry stable form ; but this plan is not suitable for coping with a sudden emergency, because it entails a delay of several months while the horses are being immunized. The Alleged Importance of an Antitoxic Component in the Serum. The meningococcus possesses an endotoxin, and Gordon (1920) believes that the ability of a sample of serum to neutralize the endotoxin is the true measure of its potency. Unfortunately, the results of later researches on the preparation and the characters of the endotoxin have been disappointing, and thus attempts to titrate the antitoxin content of the serum are not likely to be successful until further information is forthcoming. There is some evidence that a limited degree of neutralization can be obtained in tests with susceptible animals, such as mice and young guinea-pigs, but in the view of Murray (1932) the data do not carry conviction. Work by Ferry, Norton and Steele (1931) has brought them to the conclusion that each of the various types of the meningococcus forms a toxin in young broth cultures which may cause a local congested area when it is introduced into the human dermis ; they think, too, that a toxin exists which is common to all the types. An antitoxic serum can be produced in the horse, which is capable of neutralizing the erythrogenic action of the toxin in the skin. The specific toxins are considered to have no relation to the endotoxin. Experiments in monkeys showed that the intrathecal injection of living meningococci caused a fatal form of cerebrospinal meningitis, and that the intraperitoneal injection, at a critical stage of the illness, of the antitoxin derived from the horse was followed by recovery in many of the animals. These experiments are of great MULTIVALENT AND MONOTYPICAL SERA 189 interest, but they await confirmation. The value of the thera- peutic results in the monkey experiments would have been increased if the ordinary antibacterial serum had been given to a control group. Flexner (1907) carried out experiments on monkeys, and admitted that they were inconclusive owing to the small number of animals that he used. The specific serum appeared to have a protective action, but so also did normal serum, The Choice of a Multivalent or a Monotypical Serum. Gordon (1920), as the result of his researches during the war on the menin- gococcal endotoxin, strongly advocated the employment of sera which are prepared by immunizing horses with single types, and not, as is the usual method, with a variety of strains. He believed that each type of meningococcus possesses an endotoxin which is peculiar to itself, and that strains within the boundary of a single type may yield different endotoxins. As has already been stated, the clinical value of a particular serum is correlated, in his view, with its anti-endotoxin content, and he described a method of determining the presence or absence of this property by the injection of serum-toxin mixtures into mice. Monotypical sera were distributed during the year 1918 to Army medical officers for clinical use, and the results of treating 276 patients were analyzed by Hine (1920). Actually there were 141 patients who received adequate treatment, and of these 27 died (19-1 per cent.). In a discussion on the subject, both Arkwright and Ledingham spoke in favour of the use of multivalent sera. At the present time monotypical sera are seldom used, and even if they were available, a multivalent serum should be given at once, without waiting for a bacteriological report on the infecting type, if a turbid fluid in which meningococei are present is obtained by lumbar puncture. In the United States the Control Authority renders it obligatory upon serum manufacturers to issue only multivalent sera. It may be recalled that the good results in Wadsworth’s series of cases followed the use of a multivalent serum ; the serum which has given equally satisfactory results in Roumania was prepared against many races of meningococcus. The Technique of Serum Production and its relation to the Thera- 140 SERUM TREATMENT OF CEREBROSPINAL FEVER peutic Results. The serum that was employed in the treatment of the 1,300 cases analyzed by Flexner was prepared by immunizing horses with subcutaneous injections of fresh cultures. Intravenous injections were tried, but they ten led to give alarming reactions, and were accordingly discontinued (Flexner and Jobling, 1908). Nevertheless, the usual practice at the present time is to immunize horses by means of intravenous doses of killed or living cocci. Since there is no way of estimating the potency of the serum, the scheme of dosage and the choice of appropriate intervals for withdrawing the blood are empirical and vary in different labora- tories. The work of Stanley Griffith during the war on the preparation of anti-meningococcus serum indicated that intra- muscular injections were inferior to the intravenous method in respect of the production of agglutinins and opsonins; and, moreover, they had the disadvantage of causing large painful swellings which sometimes broke down. But during the war MacConkey prepared at the Lister Institute a particular batch of serum which gave valuable results in treatment, by inoculating horses intramuscularly with the immunizing material. The Concentration of Anti-meningococcus Serum. Neal, Jackson, Appelbaum and Banzhaf (1928) obtained encouraging results with a globulin solution prepared by Banzhaf. From the description of the process of purification and concentration appended to their account, it seems probable that the portion of the globulin fraction which was isolated corresponded to the so-called ‘¢ Felton ” fraction in anti-pneumococcus serum. The mortality in the first 24 patients who received the concentrated preparation was only 12-5 per cent. ; the authors note that most of them responded quickly to the treatment. It is of interest, too, that although the agglutination titre of these preparations was much higher than that of the serum from which they were made, the agglutinin content did not correspond with their therapeutic activity. They state that some lots of the antibody solution were unsatisfactory, probably because of the poor quality of the original serum. Smithburn and his colleagues express a preference for a concentrated prepara- tion—in consideration of its smaller volume—when it is to be given intrathecally to children. CONCENTRATION OF THE SERUM 141 During the past few years a concentrated globulin solution has been prepared at the Serum Department of the Lister Institute, and has been employed in the treatment of epidemic cases of the disease. The method consists in discarding the albumin fraction, which is unlikely to contain appreciable amounts of antibody ; the “Felton” and the pseudoglobulin fractions are retained. There has not as yet been enough experience of its use to pronounce definitely upon its therapeutic value, but the clinical results suggest that the mode of its preparation has not brought about any loss of potency. The lack of an experimental method of titrating the essential antibodies in the serum makes it impossible to carry out comparative tests with different preparations, except on the basis of carefully recorded clinical observations. A brief reference may be made to an attempt to titrate anti- meningococcus serum by a method which appears to make use of a novel immunity reaction (Shwartzman, 1929). This author found that when a dose of meningococcal extract was given intra- cutancously to a rabbit, a subsequent intravenous inoculation of the extract gave rise to a local reaction in the skin at the site of the first injection ; this, however, could be prevented by the addition of a specific serum to the intravenous dose of the extract. The work is still in the experimental stage, and the exact signifi- cance of the reaction is not yet fully understood. Murdick and Cohen (1933) have investigated the distribution of the antibody in the serum that neutralizes the substance which is responsible for the local skin reaction of Shwartzman, and have found that, together with the agglutinins and precipitins, it is associated ‘with the water-insoluble globulin. The Reinforcement of the Therapeutic Action of the Serum by the addition of Fresh Human Serum. The experiments of McKenzie and Martin (1908) showed that substances which are bactericidal to the meningococcus are present in increasing amounts in normal human serum, the serum of acute and chronic cases of cerebrospinal fever, and the serum of patients who have recently recovered from the disease ; in their opinion the lethal effect upon the cocci is brought about by a combination of a thermostable immune-body with a labile complement. Acting on this knowledge, they pro- 142 SERUM TREATMENT OF CEREBROSPINAL FEVER ceeded to treat 14 acute cases of the disease with serum obtained from convalescent patients, and 8 of them recovered; 2 patients were treated with their own serum, and both recovered. In 1916 Fairley and Stewart tried the effect of adding fresh human serum obtained from convalescent patients to anti-meningococcus serum before giving it intrathecally ; 7 out of 10 patients recovered. On the basis of experimental work which showed that the addition of fresh normal human serum to anti-meningococcus serum increases its opsonic and bactericidal properties, Kolmer, Toyama and Matsunami (1918) formed the opinion that in those cases which are resistant to the action of the serum from 2 to 5 c.cm. of fresh sterile human serum should be added to the dose of the specific serum before it is injected. The treatment of a patient in this way has been reported by Bunim and Wies (19383). The infection (Type IL.) persisted in spite of the ordinary serum treatment ; in view of the favourable condition of the patient this was discontinued, with the result that a relapse set in. The symptoms quickly abated when an intrathecal dose of 15 c.cm. of fresh normal human serum, followed next day by a dose of the ordinary serum, was given to the patient. It is probable that the technical difficulties which are inherent in the method have hindered its trial on a large scale. The Problem of the Fulminating Case. The fulminating type of case, in which a fatal issue takes place within 24 hours of the onset of the illness, is not likely to be influenced favourably by even a highly potent serum. The epidemic in Indianapolis (Smithburn et al, 1930) was attributable to Type III. meningo- coccus, and was characterized by a large proportion of such cases (57 per cent. of 144 patients) ; cutaneous haemorrhages were noted in 69 per cent; of 119 patients 64 per cent. yielded a positive blood culture ; and the fatality-rate was 64 per cent. These per- centage figures are consistent with each other, and indicate that a bacterizzmia was the dominant factor and that in face of it the serum was powerless as a means of treatment. The Mode of Action of the Serum. The reader will have already surmised that one reason for the unsatisfactory position of anti- meningococcus serum as a specific remedy is the want of precise DOSAGE AND MODES OF ADMINISTRATION 143 knowledge of its mode of action in restraining the infection. A probable conjecture is that an antibacterial component plays an essential part in the treatment, and it may also be that the serum is able by some means, whether specific or not, to neutralize the endotoxin. The qualities of the serum which, in the opinion of Murray, it is desirable to estimate are in the order of their im- portance : (1) its power of neutralizing endotoxin ; (2) its power of promoting the phagocytosis of virulent cocci in the presence of complement ; and (8) its bacteriolytic power. Dosage and Modes of Administration of the Serum Intrathecal Route. Daily injections of from 30 to 60 c.cm. for adults and from 10 to 20 c.cm. for infants are recommended, and at least four doses in all should be given ; the amount of the dose should be less than that of the cerebrospinal fluid which is with- drawn ; and the serum should be warmed to body temperature and introduced into the spinal canal by the gravity method at the usual site for lumbar puncture. In order to lessen the risk of a relapse, two or three additional doses of serum should be given after the meningococei have disappeared from the cerebrospinal fluid ; and it is advisable to perform several lumbar punctures with the object of promoting drainage of the fluid after the serum treatment has been stopped. According to Neal, the average sporadic case requires 6-10 doses of serum ; these cases are considered by her to be less severe than those which occur in times of epidemic prevalence. Early administration of the serum is of great importance, since there is abundant evidence that the fatality-rate is least among patients whose treatment by serum is begun within the first three days of the illness. Full details of the technique of administration of the serum are given in the Memorandum on cerebrospinal fever which has been issued by the Ministry of Health (1931). Combined Intrathecal and Intravenous or Intramuscular Routes. Herrick (1918) maintained that a bacteriemia is often present and that the meningitis is a secondary complication of the disease ; he therefore recommended that the intrathecal injections should be supplemented by large doses of serum given intravenously. 144 SERUM TREATMENT OF CEREBROSPINAL FEVER The general opinion, however, is not favourable to this practice, except in cases that are predominantly bacterizmic. Neal (1916 1919) is critical of the data on which Herrick based his belief that intravenous treatment improved the clinical results; other workers have seen dangerous reactions follow the method in some patients in spite of negative cutaneous and conjunctival tests for hypersensitiveness (Smithburn et al) ; and Blackfan thought that in two patients the intravenous injections predisposed to a fatal issue. Banks (1931) has used the method in a small number of cases, and has been favourably impressed by the results; in his experience immediate serum reactions do not occur and even large doses are well tolerated, in this respect contrasting with the reactions that may follow the intravenous injection of diphtheria antitoxin. Nevertheless, the relatively slow course of the disease with the gradual production of antibodies in the blood may increase the tendency to severe allergic reactions, when the serum is given intravenously at the end of the first week or later in the illness. If the physician should wish to supplement the intrathecal doses, intramuscular injections possess the advantage of seldom causing reactions of this kind, and are doubtless almost as effective as intravenous doses. Intracisternal Route. Of late years, and particularly in the United States, the method of injecting the serum into the cisterna magna—the subarachnoid space bounded by the inferior surface of the cerebellum, the posterior surface of the medulla, and the occipito-atlantal ligament—has been employed to a considerable extent. It appears to be free from danger, but should not be undertaken without previous practice on the cadaver. A descrip- tion of the technique is given by several authors (Purves-Stewart, 1925 ; Fantus, 1926 ; Brain and Strauss, 1930). The introduction of serum by this route is believed by some physicians to be neces- sary when the condition of spinal subarachnoid block exists. A description of the mechanism of spinal block in cerebrospinal fever will be found in a paper by Stookey, Elliott and Teachenor (1930). The condition is most often due to the presence of a thick exudate in the spinal canal, or to subarachnoid adhesions. It may be diagnosed when only a few cubic centimetres of an extremely INTRACISTERNAL ADMINISTRATION 145 purulent fluid which has a low pressure are obtained by lumbar puncture, and when at the same time a copious flow of fluid under tension is obtained by cisternal puncture. Ebaugh (1925) prefers alternate injections of the serum by the cisternal route and by lumbar puncture ; eight patients who were in an extremely critical condition on admission to hospital were thus treated, and five of them recovered. A still more thorough tech- nique is described by Bennett (1929): if the cerebrospinal fluid is fibrinous, he irrigates the subarachnoid space with Ringer’s solution by flushing the space through the needle in the cisterna, and also by allowing the solution to flow between the needle in the lumbar theca and the one in the cisterna. He regards cisternal puncture as an easier method in competent hands than lumbar puncture, and thinks it is better tolerated by the patient. To a patient with fulminating meningitis he gave fifteen injections, the total dosage amounting to 219 c.cm. ; of these injections eleven were cisternal, three spinal, and one intravenous; complete recovery followed. A series of 69 cases, with a fatality-rate of 17-4 per cent., has been reported by Fox (1929) ; patients with the fulminating form of the disease who died within a few hours of admission to hospital are not included in the series. The average amount of serum given to each patient was 190 c.cm.; 19 per cent. of the total amount of serum administered was given intravenously, 24 per cent. intra- muscularly, 24 per cent. intrathecally, and 33 per cent. intra- cisternally. There was an average of five lumbar and five cisternal injections for each patient. A comparison between the results of serum treatment by the cisternal and the usual lumbar route has been made by Goldman and Bower (1931). They treated 48 patients by the lumbar route with a fatality-rate of 52 per cent.; and 43 patients by the cisternal route with one of 25-5 per cent.; patients who died within 24 hours after their admission to hospital are not included in these figures. Some workers believe that thorough irrigation of the spinal canal with saline is a valuable adjunct to serum treatment, and that the additional trouble is justified by the improved results which follow. Thus Whittingham and his colleagues (1931) 146 SERUM TREATMENT OF CEREBROSPINAL FEVER preferred this plan to the method of producing an increase in the intrathecal pressure by giving a general anwsthetic, as recom- mended by Flack, and were impressed by its beneficial effects in toxic and comatose patients. Intraventricular Route. This method has not been much used, and it is doubtful whether the results are encouraging. Neal prefers the ventricular to the cisternal route when there is obstruc- tion to the flow of the cerebrospinal fluid ; she hopes by this means to avoid the risk of pressure upon the medulla. A large experience of cerebrospinal fever in New York has led her to advise the very cautious introduction of the serum by lumbar puncture when, owing to a spinal block, there is no flow of fluid ; if this plan does not succeed, resort may be had to ventricular puncture. She favours conservative rather than such intensive methods of serum treatment as intrathecal injections given every 8 or 12 hours, or injections by other routes. In young infants ventricular puncture is best performed through the anterior fontanelle. Lyon (1932) thinks he has obtained good results by introducing serum into the ventricles and at the same time withdrawing fluid from the spinal subarachnoid space ; the method is particularly useful in infants with a patent anterior fontanelle. For details of the technique the reader should consult the articles by Purves-Stewart (1925), Mackay (1925), and Fantus (1926). The Serum Treatment of Cerebrospinal Fever in Infants and Young Children Opinion is unanimous that the disease is almost always fatal in infants less than one year old, when serum is not given to them. Among the large number of cases of meningococcal meningitis observed by Neal and Jackson in New York there were twenty-five among infants within the first three months of life. The treatment included daily injections of serum by the lumbar route, and, when there were definite signs of block, ventricular or cisternal injections were also given ; of 23 of these patients, 11 died (47-8 per cent.). McLean and Caffey (1926) treated 11 children, who were suffering from the sporadic type of the disease and who had SERUM TREATMENT OF INFANTS 147 symptoms of obstruction of the subarachnoid space, by intro- ducing serum into the ventricles and the cisterna ; eight com- pletely recovered. On the other hand, McKhann (19380) found that, although the symptoms often diminished in severity when serum was given alternately into the ventricles and into the lumbar subarachnoid space, death ultimately followed from chronic hydrocephalus. Benjamin (1929) recommends early intensive treatment by the intravertebral routes combined with intravenous and intramuscular injections. The tendency at the present time is to pursue energetic treat- ment in critical cases. For example, Peet (1926) gives details of the treatment of a girl, aged eight years, to whom 790 c.cm., of serum were administered : 560 c.cm. by eighteen lumbar punctures ; 155 c.em. by six cisternal punctures; and 75 c.cm. by four ventricular punctures ; the child made a perfect recovery. In the opinion of this author cisternal and ventricular punctures and injections will do much to prevent hydrocephalus. The benefit derived from injections by these routes may conceivably be due in part to the drainage of the cerebrospinal fluid, and this makes it difficult to apportion the share of each factor in the final result. That drainage of the fluid may do good apart from serum treat- ment is shown by the case of a child of five months who was under the care of Mackay ; she performed twenty-one ventricular punctures, from 50 to 100 c.cm. of fluid being withdrawn at each puncture, the last on the fifty-third day of the illness. A consider- able leak took place on one occasion through the puncture wound, and from that time the condition of the child began to improve and the circumference of the head to diminish. A method has been reported from Germany which gives further evidence of the tendency to institute drastic treatment for the disease in children. In brief, it consists in removing as much as possible of the cerebro- spinal fluid by lumbar puncture and replacing it by air. Warmed serum is then injected until air can no longer be aspirated. The advantages of the method are said to be the removal of the infecting organisms with their toxins and the circumstance that the serum escapes dilution by the cerebrospinal fluid, and is thus enabled to come into better contact with the affected portions of the brain 148 SERUM TREATMENT OF CEREBROSPINAL FEVER and the spinal cord. The statement is made that the technique is simple, and the treatment well borne by the children (Siegl and Sollgruber, 1926 ; and Holtz, 1928). The Serum Treatment of Chronic Meningococcal Bacterizemia In the year 1926 Friedemann and Deicher described a chronic form of meningococcal infection which begins with the signs of rhinitis or bronchitis, and is accompanied by an intermittent fever, arthritis, and an eruption which resembles erythema nodosum ; meningitis is generally absent, but may occur late in the illness. The condition of the patient is good even when the cocci can be cultivated from the blood; the prognosis, too, is favourable, notwithstanding that the illness may last for some months. An organism which was isolated from the blood of a patient was agglutinated by a Type II. serum (Graves, Dulaney and Michelson, 1929) ; and a strain from the blood of a patient who was under the care of Marlow (1929) was agglutinated by a multivalent serum, but here the test was inconclusive owing to the tendency to spontaneous agglutination of the cocci in saline. The disease is not a common one, and it is probably overlooked from time to time. Vesell and Barsky (1930), who have reported a case which did not recover, state that the treatment should consist in the intravenous injection of anti-meningococcus serum, namely, 20-40 c.cm. doses, every 12 hours, the condition of the patient being taken into account in estimating the dosage. The first- mentioned patient (Graves et al.) received no benefit from the administration of the serum, although large amounts of it were employed. A fatal case of chronic meningococcal bacterizemia associated with endocarditis has been reported by Stevenson (1931); treatment by intramuscular doses of serum, of which a total volume of 275 c.em. was used, was ineffectual. On the other hand, Master (1931) has had three cases of this condition, com- plicated probably with endocarditis, in which the serum seemed to have done good. In a case described by Riven and Appelbaum (1931) intravenous serum treatment caused the blood cultures to become sterile and the symptoms to disappear completely. A CHRONIC MENINGOCOCCAL BACTERIEMIA 149 similar case with intermittent fever lasting for several months and characterized by a recurring rash and arthritis was permanently cured by a single intramuscular injection of serum (Takerka and Leitner, 1931). The intravenous injection of trypaflavine (0-60-75 gm. for an adult) is held by some to be a more effective agent for the purpose of sterilizing the blood than the serum (Bétzei, 1930 ; Priet, 1930). A careful study of strains that have been isolated from the blood of patients suffering from chronic meningococcal bacteri- emia seems to be worth making. Special attention should be paid to their group and type relationships, to their antigenic nature in respect of smoothness or roughness, and to their power of producing a toxemia in susceptible animals. REFERENCES ARKWRIGHT, J. A. 1920. Brit. Med. J., Sept. 18th. Banks, H. S. 1931. Lancet, 1, 747. BensaMmiN, BE. L. 1929. Arch. of Ped., April, p. 252. Ref. in Brit. Med. J., Epit., Sept. 7th, 1929. Bex~eTT, A. E. 1929. J. Amer. Med. Ass., 93, 1060. BieLING, R., and Meyer, F. 1932. * Heilsera und Impfstoffe in der Praxis.” Leipzig. Brackran, K. D. 1921. J. Amer. Med. Ass., 76, 36. ” 1922. Medicine, 1, 139. Borzel, A. 1930. Med. Klin., Jan. 24th, p. 127. Ref. in Brit. Med. J., Epit., April 12th. Brain, W. R., and Strauss, E. B. 1930. * Recent Advances in Neurology,” London. Branuam, S. E. 1932. J. Immunology, 23, 49. Brana, S. E., Tarr, C. E., and CArLIN, S. A. 1930. Pub. Health Rep., 45, 1131. BruTsAERT, P. 1931. Ann. de la Soc. Belge de Méd. Trop., Mar. 31st, p. 22. Bun~im, J. J., and Wis, F. A. 1933. J. Amer. Med. Ass., 100, 178. CANTACUZENE, J. 1931. Bull. de U Off. Internat. d’ Hyg. Publ., 23, 658. Dunn, C. H. 1908. J. Amer. Med. Ass., 51, 15. Epavch, F. G. 1925. J. Amer. Med. Ass., 85, 184. E~xpers, J. F. 1932. J. Bact., 23, 93. FamrrLey, N. H., and Stewart, C. A. 1916. Quarant. Service Pub- lication No. 9, Commonwealth of Australia. Fantus, B. 1926. J. Amer. Med. Ass., 87, 755. FeLix, A., and YuNowicH, R. 1924. J. Immunol., 9, 193. Ferry, N. S. 1932. J. Immunol., 23, 315, 325. 150 SERUM TREATMENT OF CEREBROSPINAL FEVER Ferry, N. S., NorTON, J. F., and Steele, A. H. 1931. J. Immunol., 21, 293. FLexNER, S. 1907. J. Exp. Med., 9, 142, 169. IW 1913. J. Exp. Med., 17, 553. 5 1917. ‘Mode of Infection, Means of Prevention and Specific Treatment of Epidemic Meningitis.” Rockefeller Institute Medical Research. a 1928. J. Amer. Med. Ass., 91, 21. FLEXNER, S., and JoBLiNG, J. W. 1908. J. Exp. Med., 10, 141. Foster, M., and GaskerL, J. E. 1916. Cerebrospinal Fever.” Cambridge. Fox, M. J. 1929. Wisconsin Med. .J., 28, 13. Ref. in J. Amer. Med. Ass., 92, 685. FrIEDEMANN, U. 1926. Handb. der exp. Therapie Serum- und Chemotherapie. Edit. Wolff-Eisner, Munich. FriEDEMANN, U., and DEIcHER, H. 1926. Deut. med. Wschr., 52, 733. GorpMmAN, T., and Bower, A. G. 1931. Amer. J. Med. Sciences, 181, 414. Gorpon, J. E., and Norton, J. F. 1930. J. Prev. Med., 4, 339. GorpoN, M. H. 1920. Med. Res. Counc. Spec. Rep. Ser., No. 50. Graves, W. R., DuraNey, A. D., and MicHeELsoN, I. D. 1929. J. Amer. Med. Ass., 92, 1923. GRIFFITH, A. STANLEY. 1920. J. Hyg., 19, 33. Herrick, W. W. 1918. J. Amer. Med. Ass., 71, 612. Hing, T. G. M. 1920. Med. Res. Counc. Spec. Rep. Ser., No. 50. Hovrz, K. 1928. Arch. f. Kinderheilk, Nov. 27th, p. 293. Ref. in Brit. Med. J., Epit., Mar. 16th, 1929. Ker, C. B. 1908. Edin. Med. J., 1, 306. KIrkBRIDE, M. B., and CongN, S. M. 1932. Amer. J. Hyg., 15, 444. KoLMER, J. A., Toyama, T., and MaTsunami, T. 1918. J. Immunol., 3, 157. LepiNneuam, J. C. G. 1920. Brit. Med. J., Sept. 18th. Lyon, G.M. 1932. Amer. J. Dis. Children, 43, 572. Mackay, H. M. M. 1925. Med. J. and Record, Apr. 15th. MaeGrAITH, B. G. 1933. Brit. J. Exp. Path., 14, 227. Marrow, F. W. 1929. J. Amer. Med. Ass., 92, 619. MASTER, A. M. 1931. J. Amer. Med. Ass., 96, 164. McCov, G. W. 1929. Pub. Health Reports, 44, 1595. McKenzig, I., and MarTIN, W. B.M. 1908. Brit. Med. .J., 2, 1342. 5 : » 1908. J. Path. & Bact.,12, 539. McKnann, C.F. 1930. New Eng. J. Med., Mar. 13th, p- 520. Ref. in Brit. Med. J., Aug. 9th, 1930. McLEAN, S., and Carrey, J. P. 1926. J. Amer. Med. Ass., 87, 91. Murpick, P. P., and CoHEn, S. M. 1933. J. Immunol., 24, 531. Murray, E. G. D. 1929. Med. Res. Counc. Spec. Rep. Ser., No. 124. 3 1932. J. Bact., 28, 90. Near, J. B. 1916-1919. Coll. Studies Bureau Labs., N.Y., 9, 13. Neaw, J. B,, and Jackson, H. W. 1927. J. Amer. Med. Ass., 88, 1299. REFERENCES 151 NEAL, J. B., Jackson, H. W., and ArpELBAUM, E. 1926. J. Amer. Med. Ass., 87, 1992. NEAL, J. B., Jackson, H. W., APPELBAUM, E., and Banzuar, E. J. 1928. J. Amer. Med. Ass., 91, 1427. Norton, J. F., and Broom, N. H. 1930. J. Prev. Med., 4, 355. Norton, J. F., and Gorpon, J. E. 1930. J. Prev. Med., 4, 207. Peer, M. M. 1926. J. Amer. Med. Ass., 86, 1818. PrrriE, G. F. 1932. Brit. J. Exp. Path., 13, 380. PrauvsniTz, C. 1929. * Mem. on Internat. Stand. of Therap. Sera and Bact. Products,” p. 41. Prier, J. 1930. Thése de Paris, No. 476. Ref. in Brit. Med. J., Epit., June 21st. PURVES-STEWART, J. 1925. Lancet, 2, 1159. Rake, G. 1931. Proc. Soc. Exp. Biol. and Med., 29, 287. “Review of Certain Aspects of the Control of Cerebrospinal Fever.” Ministry of Health, London, 1931. RIVEN, S. S., and AppELBAUM, A. A. 1931. Ann. Int. Med., 4, 1387. RoBB, A. GARDNER. 1908. Brit. Med. J., 1, 382. Scorr, W. M. 1917. “Rep. Local Gov. Board on Cerebrospinal Fever.” London. SHWARTZMAN, G. 1929. J. Amer. Med. Ass., 93, 1965. SIEGL, J., and SOLLGRUBER, K. 1926. Arch. f. Kinderheilk, Aug. 7th. Ref. in Brit. Med. J., Epit., Nov. 20th, 1926. SviTHBURN, K. C., et al. 1930. J. Amer. Med. Ass., 95, 776. Stevenson, W.D. H. 1931. Brit. Med. J., 2, 1173. Stookey, P. F., ELuiorr, B. L., and TEACHENOR, F. R. 1930. J. Amer. Med. Ass., 95, 106. TakerkA, H., and LEITNER, N. 1931. Deutsche Med. Woch., 57, 717. VeseLn, H., and Barsky, J. 1930. Amer. J. Med. Sci., 589. WapsworTH, A. 1931. Amer. J. Hyg., 14, 630. 55 1932. Trans. Ass. Amer. Physicians, 47, 161. WADSWORTH, A., and KIRKBRIDE, M. B. 1926. Amer. J. Hyg., 6, 507. Warrrincaam, H. E., KivpaTrICK, J. M., and GrrrriTHS, E. W. B. 1931. Brit. Med. J.,1,1101. Worsrer-DroucHT, C., and KENNEDY, A. M. 1919. * Cerebrospinal Fever.” London. WricHT, I. S., DE SancTis, A. G., and SHEPLAR, A. 1929. Amer. J. Dis. Children, 38, 730. CHAPTER XII ANTI-ANTHRAX SERUM THE treatment of anthrax in man by means of a specific serum has been tried in many countries during the past thirty-five years, but in spite of the experience thus gained there still remain gaps in our knowledge of the subject. Excellent recent summaries of the biology of B. anthracis and its immunological relations will be found in the articles by Eurich and Hewlett (1930) and by Sobernheim ; the writer is indebted to their studies of the disease for some of the details mentioned below. Anthrax exists primarily as a disease of animals, and man is attacked while engaged in occupations that bring him into contact with infected animals or their products. Human anthrax is not a common disease in Great Britain ; in 1927 there were 35 cases with three deaths. From 1919 to 1926, 632 cases were reported in the United States, and among them there were 177 deaths (28 per cent.) ; during the years 1923-1925 there were 406 cases in Germany. In man the disease may assume three clinical forms ; these are determined by the site of entry of the bacillus. A malignant pustule may appear either in the skin or in the alimentary tract, or the bacilli may be inhaled and infect the lungs (wool-sorters’ disease). Recovery from intestinal and from pulmonary anthrax is rare, and thus the treatment of these varieties by serum is not likely to lead to a favourable issue ; it is chiefly in the treatment of cutaneous anthrax that the administration of the serum is beneficial. . Experimental animals can be protected against a virulent infection by immunizing them with attenuated cultures, but it is not easy to demonstrate specific antibodies, such as bacteriolysins, agglutinins, tropins or complement-binding substances in their 152 FACTORS INFLUENCING SERUM TREATMENT 153 blood. During immunization precipitins appear in the serum of the animal but, according to Sobernheim, they provide no index of its protective or curative value. There is no definite proof of the existence of a specific toxin and, indeed, we have not as yet a clear understanding of the pathogenic action of the anthrax bacillus. An immune-serum may be presumed to exert an antibacterial or, in more general terms, an anti-infectious action, and thus it is important to know whether B. anthracis comprises a number of specific types ; up to the present serological races have not been identified (Eurich and Hewlett). Gratia (1924) noted two types of anthrax colony, which may correspond to the “rough” and “ smooth ’ variants of Arkwright ; of the two, the rough variety was the more virulent. The exact significance of his observations has still to be worked out, but it would seem that account should be taken of them when horses are being immunized for the produc- tion of a therapeutic serum ; the immunization should be com- pleted with cultures of the bacillus that possess full virulence. The serum that was prepared in Siena by Sclavo from asses has been in use for the treatment of human anthrax since the year 1895, and it is perhaps the best known of all. On the whole, clinical reports speak in its favour (Sabolotnyi, 1926; Hodgson, 1928, 1929). Eurich employed it in 300 cases ; the mortality was 5 per cent. In the opinion of Eurich and Hewlett three points should be considered in comparing the statistics of serum treatment : (1) the site of the pustule ; (2) the stage of the disease when treat- ment was begun; and (3) the source of the infection. A bacterizemia is especially liable to appear when the pustule is situated in an area such as the neck. The authors just cited state that anthrax in man, when contracted from East Indian wool, appears to be less severe than when it is contracted from other sources, such as Persian wool, probably owing to the slighter degree of contamination of the wool from the East Indies. They suggest that the exceptionally good results obtained by Sclavo with his serum may be due to the circumstance that many of the cases arose from contact with pigs’ bristles ; these are not heavily contaminated with the bacillus. There is reason to believe that the results are better when operative treatment is not undertaken. 154 ANTI-ANTHRAX SERUM The analysis in the accompanying table indicates that serum treatment alone is followed by fewer deaths than when the combined treatment is given. TasLe II Analysis of 1,105 Cases of Cutaneous Anthrax treated by various methods. The data represent the official figures up to the year 1929, as supplied by the Factory Department, Home Office, London. Method of Treatment. Cases. Deaths. Mortality-rate (Percentage.) Serum only : : 378 25 66 Excision only . : 418 48 115 Excision and serum 272 32 11'8 No special treatment 37 21 568 Kraus and his colleagues have denied a specific réle to anti- anthrax serum, and have affirmed that equally good results follow from the employment of normal ox serum, but other workers, including Sobernheim, do not share their views. Sordelli, Harispe and Beltrami (1928) prepare the serum from horses or mules by subjecting them to a prolonged intravenous immunization ; they use capsulated strains of B. anthracis which have been cultivated on serum agar. The serum obtained in this way possesses a high precipitin content. It is able to protect rabbits if it is given to the animals before the test-dose of culture, but it fails to protect when given at the same time as the culture and naturally also when given after the test-dose. These authors assert that the antibody is associated with the euglobulin fraction : 6 mgm. of this protein, when given intraperitoneally to guinea-pigs, protected them against a test-dose of 20 minimal lethal doses of anthrax culture; on the other hand, the American workers, Eichhorn, Berg and Kelser (1917) have stated that the pseudo- globulin fraction of the serum contains the protective antibody. Here the observations of Rosenhol (1929) may be cited; he PREPARATION OF THE SERUM 155 obtained preparations of the portion of the serum globulins that is thrown out by the addition of distilled water to the serum ; this fraction of the serum proteins—the euglobulin—contained the whole of the precipitins which were present in the original serum. Brocq-Rousseu, Staub and Urbain (1926) immunized horses intravenously with asporogenous bacilli which had been killed by treatment with alcohol and ether. They alternated the doses with injections of virulent bacilli and obtained a highly protective serum. Besredka has furnished experimental evidence which, he thinks, supports the hypothesis that the cutaneous tissues alone are susceptible to the infection, and that immunization by the intra- cutaneous route is particularly effective. Brocq-Rousseu and Urbain tested this method in their immunization of horses and found that immunity was produced, although no demonstrable immune-bodies appeared in the serum. The method has also been tried by Kudrjawzew and Romanow (1929), who regard it as a valuable adjunct to the subcutaneous and intravenous routes, for by its means they obtained highly active sera within a short time. Sobernheim begins the immunization with attenuated cultures, by employing them in the manner of the Pasteurian vaccines, or by using a simultaneous serum-vaccine technique. When some immunity has been established the cautious employment of virulent cultures is begun and is continued until 24 or 36 agar slopes are tolerated. In Sobernheim’s experience the subcutaneous inoculation of the cultures yields results that are quite as good as those obtained by the intravenous route. A new technique for the preparation of a highly protective serum has been described by Urbain (1931). When the horse has reached the final stage of immunization, a series of subcutaneous injections of killed B. pyocyaneus (Pseudomonas pyocyanea), killed anthrax bacilli, and living anthrax bacilli are given. By this means the serum attains a high titre of agglutinins and protective antibodies. Lusztig (1926), Sobernheim, and other workers have described methods of titrating the protective antibody in susceptible animals, such as the guinea-pig or the rabbit, but it is improbable that an exact estimation of the protective value of the sera can be obtained by any of these methods. A consequence of this uncertainty is 156 ANTI-ANTHRAX SERUM that it is not possible to attach much importance to statements of dosage. The instructions given for the use of Sclavo’s serum recommend that 30 or 40 c.cm. should be injected subcutaneously, and that, after 24 hours have elapsed, a similar dose should be repeated if the local lesion or the general state of the patient has not improved. In severe cases 10 c.em. should be given intra- venously and repeated after two or three hours; in addition, subcutaneous doses should be given. Two important principles that are applicable to all forms of specific serum treatment should be kept in mind : (1) the adminis- tration of the serum at the earliest possible moment ; and (2) the choice of the intravenous route in grave cases which are first seen by the practitioner at a late stage of the disease. REFERENCES Brocq-Rousseu, Staus, and UrBaiN, A. 1926. Ann. Inst. Past., 40, 595. Ei1cnnorN, BERG, and KELser. 1917. J. Agric. Research, Vol. 8, cited by Sobernheim (1926). Eurich, F. W., and HewLeTT, R. T. 1930. “A System of Bacterio- logy.” London, H.M. Stationery Office, 5, 439. GraTIA, A. 1924. C. R. Soc. Biol., Paris, 90, 369. Hobeson, A. E. 1928. Lancet, 2, 594. Hooason, A. E. 1929. Brit. Med. J., 2, 667. Kraus, R., and BELTrAMI, P. 1921. Z. Jf. Immunitdtsforsch., 31, 93. Kupriawzew, G., and Romanow, D. 1929. Centralbl. Jf. Bakt., Abt. I. Orig., 110, 164. Luszric, A. 1926. Centralbl. f. Bakt., Abt. I. Orig., 98, 492. Rosennor, H. P. 1929. Centralbl. f. Bakt., Abt. I. Orig., 111, 92. SABOLOTNYI, S. S. 1926. Centralbl. J. Bakt., Abt. 1. Orig., 99, 53. SoBERNHEIM, G. 1926. * Handb. der exp. Ther. Serum-u. Chemother.,” Miinchen, p. 358. SoBERNHEIM, G. 1929. ¢ Handb. der path. Mikroorg.” (Kolle, Kraus, Uhlenhuth), 3, 1146. SorpEeLLI, A., HarisPe, C., and BeLTrami, P. 1928. C. R. Soc. Biol., November 9th, p. 1423. Ursain, A. 1931. C. R. Soc. Biol., 107, 477. CHAPTER XIII ANTI-TYPHOID SERUM Tue treatment of typhoid fever by the administration of a specific serum is on a far less satisfactory basis than the prophy- lactic use of the corresponding vaccine. And yet researches on the preparation of an anti-typhoid serum for therapeutic purposes have been carried out during the past thirty-five years. In the closing years of the last century Bokenham in London and Tavel and Krumbein in Berne immunized horses with typhoid cultures. But there has never been complete agreement upon the value of anti-typhoid sera in clinical practice. The essential difficulty appears to be the want of precise knowledge of the specific bacterial elements which take part in the attack of the typhoid bacillus upon its host, and which may or may not act as antigens in the processes that are concerned with the production of a state of partial or solid immunity. Controversy, in particular, has gathered round the question whether the typhoid bacillus contains a specific toxin which is capable of producing an antitoxin, such as, for example, the dysentery (Shiga) toxin and antitoxin. Some workers believe in the need for an antitoxic serum, whereas others think that the serum acts chiefly as an antibacterial agent. The sera prepared by Macfadyen (1906) and Besredka (1906) were probably not given an adequate clinical trial. Chantemesse (1907) published the results he obtained from the use of a serum derived from horses which had received prolonged immunization with heat-killed broth cultures of virulent strains of the bacillus. During a period of six years (1901-1907) the mortality from typhoid fever in the Paris hospitals was 17 per cent. in 5,621 cases, but at his own hospital it was only 4-3 per cent. in 1,000 cases. These results were encouraging, and Chantemesse attributed the benefit conferred by his serum to an opsonizing and antitoxic 157 158 ANTI- TYPHOID SERUM action. Nevertheless, this explanation has not been accepted as a wholly convincing one owing to the minute doses—* quelques gouttes ”—of the serum which were employed in treatment ; a second dose was rarely given. A. E. Wright (1906) suggested that Chantemesse’s serum contained a residuum of the typhoid antigen which was used for immunizing the horses, and that in this way it functioned as a bacterial vaccine and not in virtue of any specific protective antibodies. The chief work on the preparation of an anti-typhoid serum during the past ten years has been done by French serologists, and particularly by Rodet (1929, 1931); he immunizes horses by means of intravenous injections of young broth cultures. The serum he obtains is said to lessen the fever and the other toxemic symptoms, to ward off the complications of the disease, and te reduce the mortality. A later report gives information on the results of the serum treatment of about 300 patients. Early administration, that is, within the first five days, had the effect of shortening the pyrexial period, and yet for some unexplained reason relapses were frequent. The serum caused a disappearance of the toxic symptoms, and brought about a feeling of well-being. The death-rate in a group of 169 bacteriologically confirmed cases was 11-2 per cent. Grasset and Gory assert that they have had good clinical results with a serum which is obtained by hyperimmunizing horses with formolized autolysates of typhoid bacilli. Grasset continued his researches in Johannesburg and found that some patients failed to benefit by the use of a serum which had been prepared from European strains. He believes that the South African strains are not serologically identical with these, although the dissimilarity is less than is implied in differentiation into types. The serum is both antitoxic and anti- bacterial, and its therapeutic activity is stated to reside in the pseudoglobulin fraction. Treatment during the early stage of the illness, namely, the first twelve days, affords the maximum thera- peutic results ; for example, 32 (91 per cent.) out of 85 cases showed definite benefit from the serum. A serum has been prepared which is effective against the typhoid bacillus and also against B. paratyphosus A and B. Grasset hopes to give detailed statistics THERAPEUTIC EFFECTS 159 later, but makes the statement that early serum treatment diminishes the duration of the illness to less than half the average period and considerably reduces the number of deaths. Reinthaler (1928) has given a trial to an anti-endotoxic serum prepared by Kraus, and believes that it modifies the severity of the symptoms and shortens the illness. A recent contribution by Gross (1930) from the Behring Institute in Marburg describes results that he has obtained in his experimental work on the specific toxin of the typhoid bacillus. By selecting appropriate strains he prepares a necrotizing toxin which is lethal to mice and which, when it is used to immunize horses, gives rise to a specific antitoxin. This serum is said to exert not only a prophylactic, but a curative action in mice and to contain agglutinins, complement-binding and bactericidal bodies. Reports on the clinical use of this serum will be awaited with interest. The serum from convalescent patients has occasionally been used in practice, but there is at present no certain knowledge of its effects; an obstacle in the way of its employment is that patients who are recovering from typhoid fever cannot well spare the blood required for this purpose. REFERENCES BESREDKA, A. 1905. Ann. de U'Inst. Past., July 25th. " 1906. Ann. de I'Inst. Past., February. BokenuHAM. Cited in WaLker, E. W. A. 1901. J. Path. and Bact., ». 250. CHANTEMESSE, A. 1907. * Sérothérapie de la Ficvre Typhoide,” Paris. GRrASSET, E. 1930. J. Med. Ass. S. Africa, 4, 380. ” 1931. C. R. Soc. Biol., 106, 810. "» 1931. Proceedings 18th Congress of Hygiene, Paris. Grasset, E., and Gory, M. 1927. C. R. Soc. Biol., 97, 1211. Gross, Hans. 1930. Centralbl. f. Bakt., I. Abt. Orig., 117, 187. MACFADYEN, A. 1906. Proc. Roy. Soc. B., 77, 548. REINTHALER, K. 1928. Wien. klin. Wochenschr., 41, 947. Roper, A. 1929. Paris Méd., July 27th, p. 81. % 1931. Lyon Méd., August 23rd, p. 201 ; August 30th, p. 225. Tavern and KrumBeIN. Cited in WaLker, E. W. A. 1901, J, Path. and Bact., 7, 250. WricHT, A. E. 1906. Clin. Journ., May 16th, CHAPTER XIV THE SERUM TREATMENT OF PLAGUE THERE are three clinical types of plague in which serum treat- ment has been employed, namely, bubonic plague, with the bacilli localized in the bubo; a bacterizemic form in which the bacilli have passed through the lymphatic barrier into the blood- stream ; and a pneumonic form in which the primary site of the invasion is in the lungs. It will be recalled that the plague bacillus is characterized by a capacity for rapid and widespread multiplication in the tissues of its host, and that the bacilli, when they die and become autolysed, liberate an endotoxin within the tissues which, if it accumulates in sufficient amount, causes the death of the patient from toxemia. These considerations naturally have a bearing on the methods of production and titration of anti-plague sera. G. Dean, Rowland, and MacConkey, who worked many years ago in the serum laboratories of the Lister Institute, prepared an antitoxic serum from horses and carried out experiments with it. MacConkey found that the antitoxin was associated with the pseudoglobulin fraction of the serum proteins, and that it could be concentrated by the usual methods. The serum that was prepared by Rowland and MacConkey was tested in Bombay by the Plague Research Commission (1912). There was a difference in the fatality-rate of about 10 per cent. in favour of the serum- treated cases, when compared with the control group, a result which was regarded as of doubtful significance. Later experience, however, appears to be favourable to this form of treatment, when the serum is given early in large doses intravenously. Some of the accounts speak of an amelioration of the symptoms due to an antitoxic action of the serum (Barrett Heggs, 1923, and others). The results that have been reported recently by Naidu and 160 THERAPEUTIC EFFECTS 161 Mackie (1931) encourage the hope that it may be possible to prepare a serum which will bring about a cure even in patients in whom a considerable degree of bacterizemia is present. The authors admit that the number (43) of cases they have treated is too small to justify a definite conclusion, but they cite supporting experimental evidence which indicates that their serum has a potent action in protecting laboratory animals. They obtained the serum by immunizing cattle in preference to horses, and the immunization was effected by giving to them serial intravenous doses of a highly virulent strain in the living state. The invariably fatal issue of pneumonic plague explains the view which is commonly held that serum treatment is of no avail in this form of plague. Nevertheless, there are indications that life is prolonged by its use, and that some of the symptoms are mitigated within a few hours of its administration, a result which suggests that the serum has an antitoxic action. The early administration of large doses of the serum by the intravenous route is essential if good effects are to be looked for. As the present writer (1929) has already pointed out, further research on the preparation of anti-plague serum is necessary before its clinical use can be put on a sound basis. In particular, it is desirable to agree upon a test for the measurement of antitoxin content and of protective antibodies in terms of standard units. REFERENCES These will be found in the following general article on Plague. Perrie, G. F. 1929. “A System of Bacteriology,” London, H.M. Stationery Office, Vol. 3, p. 137. Naipu, B. P. B., and Mackie, F. P. 1931, Lancet, 2, 893, R.A. VACCINES, 6 CHAPTER XV ANTI-CHOLERA SERUM THE treatment of cholera by means of a specific serum has not so far yielded results which can be regarded as conclusive. For this reason it is unnecessary to refer in detail to the clinical observations which have been made in various countries on the effects of serum prepared in different laboratories. Information of this kind will be found in several recent summaries (Hahn, 1926 ; Hetsch, 1927 ; Kolle and Prigge, 1927 ; Kraus, 1928 ; and Harvey, 1929). Here we may confine the discussion to a con- sideration of some of the difficulties that stand in the way of an effective serum therapy of the disease. The first question is whether the cholera vibrio is a well-defined specific organism which can be regarded as the cause of all choleraic infections. The typical V. choleree, which is obtained from patients during an epidemic of cholera, is everywhere regarded as its cause. But the matter is not as simple as it appears, for there is a group of cholera-like vibrios which are associated with choleraic infections and which closely resemble the true cholera vibrio, except that they are serologically distinct from it and, moreover, show a marked diversity of antigenic behaviour among themselves. Mackie (1929) has given the name V. paracholerce to this group, although Kraus had previously applied the term to a group of vibrios which were discovered by Gotschlich. The latter worker, in the year 1905 at El Tor in Sinai, isolated from the bodies of six pilgrims, who had died of a dysenteric infection which had not produced any of the symptoms of cholera, the strains that were named para- cholera vibrios in 1909 by Kraus. Recent views on the kinship of these strains are summarized in the following statement. They are considered by some workers (Abdoush, 1982; van Loghem, 1932) to be serologically identical with the typical V. cholere ; on the other hand, Gohar (1932) has shown that the “ O antigens 162 GENERAL CONSIDERATIONS 163 of the two races are dissimilar ; Doorenbos (1932) believes that the El Tor vibrio is merely V. cholere which has become contaminated with bacteriophage ; and Mackie regards the El Tor vibrio as a variant of V. cholerce which is characterized by reduced virulence, since it has not been found during times of epidemic prevalence in India. Next, it is important to know whether the true V. cholere constitutes a homogeneous group, or whether a number of sero- logical races exist, which are strictly type-specific. Douglas (1921) takes the view that all the true cholera strains belong to a single serological type, and Topley and Wilson think it improbable that races exist whose specific antigen, as determined by the polysac- charide of Landsteiner and Levine (1927), will be found to differ from each other. Abdoush (1982) and Gohar (1932) were unable to find any serological difference among strains that had been isolated in various parts of the world. In opposition to this experience, workers in Japan recognize three serological types among the indigenous strains , of which one is the typical strain of Koch. A good deal of controversy has centred upon the nature of the toxin of V. choleree, and agreement has not yet been reached. The general belief at the present time is that the toxin is a labile endotoxin which is not easily obtained free from the bacterial bodies, and which is not lethal in the small amounts that are characteristic of the so-called ¢ exotoxins,” such as those of the bacillus of diphtheria and of tetanus ; the corresponding antitoxin is regarded as being also relatively feeble. Kraus, on the other hand, argues that the cholera vibrio secretes a true exotoxin in broth cultures, and that an antitoxic serum can be prepared from it. This author believes, further, that an antitoxic serum which neutralizes the exotoxin of the El Tor vibrio is likewise capable of neutralizing the toxic action of the typical cholera vibrio. The question of the need for an antibacterial component remains an open one. It is not difficult to prepare a serum of this kind which is effective in experimental infections, but it has been suggested that a similar serum would be ineffective in man owing to lack of the requisite complement action. 6—2 164 ANTI-CHOLERA SERUM The cholera toxin appears to cause desquamation of the intestinal epithelium, with the result that the vibrios are able to penetrate into the submucosa ; again, the specific toxin which is liberated from the culture-mass in the lumen of the gut is readily absorbed through its damaged mucous membrane. If we accept this view of the pathology of cholera, it seems rational to aim at the production of a therapeutic serum which will contain both an antibacterial and an antitoxic component. REFERENCES AspousH, Y. B. 1932. Brit. J. Exp. Path., 13, 42. Doucras, S. R. 1921. Brit. J. Exp. Path., 2, 49. DoorexBos, W. 1932. Ann. Inst. Past., 48, 457. Gonar, M. A. 1932. Brit. J. Exp. Path., 13, 371. Haun, M. 1926. * Handb. der exp. Ther. Serum- u. Chemotherapie,” Miinchen, p. 341. Harvey, W. F. 1929. ‘ A System of Bacteriology,” London, H.M. Stationery Office, 4, 421. Herscn, H. 1927. ¢ Handb. der path. Mikroorg.,” Jena, 4, 125. KorLe, W., and Pricer, R. 1927. * Handb. der path. Mikroorg.,” Jena, 4, 1. Kraus, R. 1928. * Handb. der path. Mikroorg.,” Jena, 2, 623. LANDSTEINER, K., and LEVINE, P. 1927. J. Exp. Med., 46, 213. Locum, J. J. van. 1932. Zeitschr. f. Hyg., 114, 20. Mackie, T. J. 1929. “A System of Bacteriology,” London, H.M. Stationery Office, 4, 424. Torrey, W. W. C., and WiLsoN, G. S. 1929. ‘ The Principles of Bacteriology and Immunity,” London, 1, 329. CHAPTER XVI THE PROPHYLAXIS OF MEASLES BY MEANS OF SERUM PAGE INTRODUCTORY 5 > . : 2 : » . 165 Tue PropPuYLACTIC VALUE OF SERUM OBTAINED FROM (CONVA- LESCENT PATIENTS . . . . 166 The conferment of full protection by the serum. ‘ . 166 Mitigation of the attack by means of the serum . ‘ . 167 The duration of the immunity conferred by the serum . . 168 The use of the serum as a diagnostic agent. ‘ ; . 168 The preparation and storage of ** convalescent > serum . 168 The organization of supplies of the serum . 169 Tue Propuyracric VALUE OF NORMAL ADULT HuMAN SERUM . 170 A SUMMARY OF EVIDENCE RELATING TO THE VALUE OF HUMAN SERUM IN THE PROPHYLAXIS OF MEASLES . 172 Tur CONTROL OF EPIDEMICS OF MEASLES BY MEANS OF “ Con- VALESCENT’ AND NORMAL ADULT SERUM ’ . 173 Tue Curative VALUe or HuMaAN IMMUNE-SERUM 174 THE USE OF SERA OBTAINED BY THE IMMUNIZATION OF ANIMALS | 174 Introductory Measvres is a highly contagious disease, especially of early child- hood, the cause of which remains as yet undiscovered. In the acute stage of the illness the virus is present in the blood-stream and in the naso-pharyngeal secretions. The disease is extremely infective in the catarrhal stage, and the causal agent is carried from one person to another in droplets of secretion from the upper respiratory passages. Measles is not found spontaneously in the lower animals, and the infection is apparently transmissible only to monkeys. The incubation period in man lasts usually for 10 or 11 days. Bronchopneumonia is the most important and fatal complication, particularly in children under three years of age, and it accounts for the high death-rate (70 per cent.) in this age- group. Recovery from the disease is associated with a solid immunity, for second attacks are very rare. This brief description 165 166 THE PROPHYLAXIS OF MEASLES provides the clue to the successes and the limitations of the em- ployment of immune-serum in the prophylaxis and treatment of measles. The ground may be cleared at once by defining the limitations : (1) an effective serum for the prophylaxis or treat- ment of measles has not, as yet, been prepared from animals ; and (2) although sera of human origin have a definite value as prophylactic agents, they have little or no curative effect. The Prophylactic Value of Serum obtained from Convalescent Patients The Conferment of full Protection by the Serum. The first successful attempts to protect against the infection of measles by means of human convalescent serum were made in the year 1916 at Tunis by Nicolle and Conseil (1918). Park and Zingher obtained similar results about the same time, and many later workers have confirmed the value of the method. When a child has been exposed to the infection, and it is sought to protect him against the risks to which he is liable if he should be attacked by the disease, there is a choice of methods : either a full but tem- porary protection against the impending illness may be aimed at, or it may be preferable to arrange for incomplete protection, so that the illness will be of so mild a character as to remove any risk to life and yet a lasting immunity is conferred. We may first consider the circumstances that make it advisable to protect fully children who are exposed to the risk of infection. When the child is in a poor state of health and cannot receive proper nursing at home, and particularly when the age is below three years, a dose of serum should be given which will confer a complete—but transient—immunity. The postponement of an attack of measles to a later age by this means minimizes the risk of a severe illness during early childhood which might prove to be fatal. Children who are suffering from tuberculosis, rickets, diphtheria, whooping cough or scarlet fever should be given a full dose of serum soon after exposure to the infection. When measles is epidemic, the infection may break out in the wards of a hospital or an institution where children are being treated. Such an USE OF CONVALESCENT SERUM 167 occurrence constitutes a difficult administrative problem, for immediate isolation of the contacts, a measure which succeeds in checking other types of outbreak, does not aid in its solution. Measles is said to be responsible for more cross-infection in hospitals than any other infectious disease (Gunn, 1928), and, moreover, the illness resulting from it in children who are congre- gated in schools and hospitals is likely to be a severe one. In circumstances of this kind it is best to arrange for the immediate administration of a dose of serum which will give complete pro- tection, and thus obviate the necessity for quarantining the ward. The effective dose for this purpose depends upon : (1) the age of the child ; (2) the state of its health; and (3) the interval between the time of exposure to the infection and the time when it is proposed to give the serum : this should not be more than five days. The amounts of serum that are recommended by various authors differ to some extent, but an analysis of 39 numerical statements gives, as an average, 5 c.cm. as the minimum dose and 12 c.cm. as the maximum ; these amounts are probably generous estimates and agree with those given recently by Gunn (19321), This worker has found a minimum dose of 5 c.cm. of serum to be necessary, and he determines the dose for children above three years of age by multiplying the age of the child by two and administering the resulting number of cubic centimetres. The serum, unlike an alien serum, seldom produces febrile or other reactions. Mitigation of the Attack by means of the Serum. Where healthy children over five years of age are concerned, the temporary nature - of the immunity conferred by convalescent serum is a disadvantage. In 1923 Debré and Ravina published a modification of the method which produces an attenuated infection and, in conse- quence, a permanent immunity. This result is obtained by deliberately delaying the administration of the serum till a later stage of the incubation period, that is, until the sixth or seventh day after exposure to the infection. The subsequent illness is so mild that it may be recognizable with difficulty as an attack of measles. The incubation period may be prolonged; the temperature is slight and lasts for a day or two only ; the catarrhal symptoms are 168 THE PROPHYLAXIS OF MEASLES mild or absent; and Koplik’s spots are few or they may be absent. The rash, if detectable, is less confluent, and the macules are smaller than usual. Complications are rarely seen in the modified form of the disease. A suitable dose is from two and a half to six cubic centimeters, given on the sixth or seventh day after exposure to the infection. The Duration of the Immunity Conferred by the Serum. The limits, as estimated by various authors, are two weeks and ten weeks. If the serum is given before exposure to the infection the immunity lasts from two to four weeks ; if it is given early in the incubation period with the intention of conferring complete pro- tection, some degree of active immunity may be superimposed upon the passive immunity, and, if this happens, the patient will be protected perhaps for a few months; the modified attack confers a lasting immunity (Silverman, 1928). The use of the Serum as a Diagnostic Agent. When a dose of serum is given near the end of the incubation period, the course of the disease is not influenced, but the Debré phenomenon will be seen ; this consists of a blanched area at the site of injection, which contrasts with the surrounding rash. A small intracutaneous dose of the serum produces the same effect. The blanching does not occur if the test is carried out after the rash has appeared (Debré and Ravina, 1923). The Preparation and Storage of ‘‘ Convalescent’ Serum. Blood is withdrawn from the patient from 7 to 10 days after the temperature has become normal; the limits mentioned by various authors are the 4th and the 21st day. Park and Freeman (1926) and Debré and Joannon (1926) have given details of the technique and of the precautions that must be observed. It is not usually advisable to collect blood from children under 10 years of age, and adult convalescent patients are not often available. Gunn states that in the London County Council (lately Metro- politan Asylums Board) hospitals in London, patients above the age of seven years with measles numbered less than 2 per cent. The selected donors should have had a typical attack of un- complicated measles, and should be free from syphilis, malaria or tuberculosis. They should be kept under observation for several PREPARATION AND STORAGE OF SERUM 169 days to make certain that they are not incubating any other infectious disease. From 500 c.cm. to 600 c.cm. of blood may be taken from an adult. The blood can be prevented from clotting by the addition of potassium oxalate solution and the red cells allowed to deposit by gravity or spun down in a centrifuge, or the serum may be separated after clotting. The pooled serum from at least three donors should be used in order to ensure an average potency. The ordinary amount of antiseptic is added, the serum is filtered, and sterility tests are carried out before it is issued. Sutherland and Anderson (1928), in their work at Manchester, obtained blood from nurses in other institutions in the city who had had the misfortune to contract measles. Gunn (1928) used the serum from 21 convalescent donors, all of them under the age of 10 years, and most of them less than six years old ; from 20 to 60 c.cm. of blood were withdrawn from each patient, and three or more samples were pooled. The serum prophylaxis of measles has been successfully applied in the Federated Malay States by Kingsbury (1927); he obtained blood from convalescent adults 4 to 10 days after defervescence; in his opinion, the addition of 0-5 per cent. phenol and double filtration of the serum remove the risk of the transmission of malaria. If the serum is stored in an ice-chest, it retains its potency for at least twelve months. The serum, whether it is given for the purpose of securing complete or partial protection, should be injected intramuscularly. The Organization of Supplies of the Serum. The difficulty of obtaining serum when measles becomes epidemic makes it neces- sary to arrange for its systematic collection, storage and distribu- tion. W. S. C. Copeman (1927) has discussed the possibility of dealing with epidemics of measles in this way; he cites the experience in Paris where in 1925, at the instance of the Academy of Medicine, two centres for the collection and storage of serum from cases of measles were established. At one of these centres— the Children’s Hospital —10 litres of serum, representing 2,000 protective doses, were collected in the first five months, a supply which proved to be adequate. During an epidemic at Syracuse, New York State, which was 170 THE PROPHYLAXIS OF MEASLES investigated by Silverman (1928), a quantity of nearly 7 litres of blood was collected from twenty-one convalescent donors. Nabarro and Signy (1929) acknowledge the collaboration of Dr. R. Massingham of the London Fever Hospital, who collected a large amount of blood from adult measles patients ; many volunteers gave 200-300 c.cm. of their blood and felt no ill effects. In Leningrad, Mendelewa (1929) collected in three months from 292 adult donors 53 litres of blood ; this quantity yielded 30 litres of serum. It appears, however, that it is not always easy to persuade patients who are convalescent from measles to contribute their blood for the benefit of others. Thus Park has stated that in New York adult convalescents generally refuse to be bled, and McNalty, in the discussion on Copeman’s paper, affirmed his belief that donors would be difficult to find, and mentioned that in his experience none of the parents of patients who had recovered from polio- myelitis sanctioned the withdrawal of blood for the treatment of others. Buchanan (1927) doubts whether many parents will agree to the inoculation of their children with convalescent serum. The scheme outlined by Copeman on the basis of experience of the method in other countries includes the enlightenment of parents on the danger of measles in children under three years of age, a trained staff to carry out the practical details of the scheme, and a campaign through the medium of the Press for furthering the spread of useful information on the subject. Silverman, in the outbreak already mentioned, followed a plan of this kind, and found that the scheme worked well, except for an occasional shortage in the personnel. There was not a single death in 898 persons who reccived the serum, and in the 297 patients for whom records were available not one suffered from bronchopneumonia, and only two had ear complications ; these were perhaps explic- able by the circumstance that the serum had been given to them near the onset of the attack. The Prophylactic Value of Normal Adult Human Serum The difficulty of obtaining blood from convalescent patients may be overcome to some extent by the use of blood from healthy USE OF NORMAL ADULT HUMAN SERUM 171 adults with a clear history of measles. The immunity conferred by an attack of measles is of such an enduring character that the serum of persons who have had measles 20 or 30 years before still contains protective substances. Degkwitz (1920) was apparently the first to test this protective action in the prophylaxis of measles and to obtain evidence of its efficacy ; others have confirmed his observations. Some prefer to use the blood of parents or near relatives, and this procedure possesses obvious advantages : parents are likely to be willing to provide blood for their own children and a Wassermann test should not be necessary. The method is simplified by using the whole blood ; 1 c.cm. of a 10 per cent. solution of sodium citrate in a 10-c.cm. syringe prevents the clotting of this volume of blood. Barenberg, Lewis and Messer (1930) compared the results of using adult whole blood, which was usually taken from the parents, with those following the injection of convalescent serum. They consider that adult blood is less effective than blood which is taken from patients; on the other hand, it is easier to obtain. Harries (1930) obtained a supply of pooled serum from 11 or 12 probationer nurses who had measles in childhood. He gave a dose of 10 c.cm. to 54 susceptible children who had been exposed to measles, with the result that only two showed an attenuated form of measles after an unusually long incubation period ; the other children escaped the infection. Adult serum is less effective per unit volume than convalescent serum—roughly in the proportion of one to two. The available data indicate that an adequate dose of convalescent serum is from 5 to 12 c.cm., and that from 12 to 25 c.cm. of adult serum and from 25 to 50 c.cm. of whole adult blood are needed to confer full pro- tection ; to produce an attenuated attack the dose may be halved and given later in the incubation period. In the experience of Gunn (1932 2) the results have not been quite as good as those from convalescent serum. The attack was prevented in 99 per cent. of the cases if adult serum was given before the third day; if given after the third day an attenuated attack followed, but after the eighth day no effect was obtained. He thinks there is reason to hope that adult serum may yet be concentrated, so that smaller doses will suffice, and that the loss of protective antibody which 172 THE PROPHYLAXIS OF MEASLES occurs during storage may be obviated by arranging for the serum to be desiccated and kept as a dry powder in cold storage until required. Burn (1931) found that the results of giving serum from adults who had measles in infancy to some 70 children for the pur- pose of mitigating the attack werc comparable to those obtained by the use of convalescent serum. THe gave 8 c.cm. as an average dose, and the day of administration of the serum was, on the average, 5-5 days from the appearanc: of the rash of the patient who was the source of the infection. A Summary of Evidence relating to the Value of Human Serum in the Prophylaxis of Measles A survey of the numerous publications on the subject reveals a striking unanimity of opinion upon the efficacy of human antiviral serum in preventing or mitigating an attack in susceptible persons who have been exposed to the infection. Gunn (1928) states that of a total of 8,500 published cases more than 97 per cent. were success- fully protected; his own results confirm this figure. Only a few references need be given here in illustration of the general experi- ence. Park and Freeman (1926) gave 4-10 c.cm. of plasma taken from convalescents to 433 children; 91 per cent. were fully protected, 7 per cent. had modified measles, and 2 per cent. an unmodified attack. During an outbreak in an institution, Haas and Blum (1926) treated 174 children with plasma from con- valescent patients that had been collected from one to four months after defervescence and stored from two weeks to four and a half months before injection ; 51 per cent. of the patients were protected for three months or more, and even when the plasma failed to protect, the illness was modified in every case. The inocu- lated group showed an incidence of 53 per cent. as compared with 84 per cent. in the uninoculated group; there were decidedly fewer cases of bronchopneumonia in the serum-treated group. The use of the method in the tropics has been reported on by Kingsbury (1927), who succeeded in controlling a number of outbreaks of measles on rubber plantations in the Federated Malay States; he concludes that the injection of convalescent CONTROL OF EPIDEMICS 173 serum is a prophylactic measure of real value. Sutherland and Anderson (1928) believe that convalescent serum is a safe and trustworthy prophylactic remedy if an adequate dose is given within the first three days of the incubation period. Gunn (1928) obtained a protection rate of 95-7 per cent. in 69 susceptible children. Mendelewa (1929) has given an account of work on the specific prophylaxis of measles which was carried out during three years at the Institution for Mother and Infant Welfare in Leningrad. When convalescent serum, adult serum, or citrated -whole blood was given to 689 susceptible children, 578 remained well ; 42 of the cases that occurred in spite of the protective dose were abortive, 57 mild, and 7 of ordinary severity ; there were 5 deaths (0-7 per cent.) as compared with 17 per cent. deaths in 188 non-immunized children. The Control of Epidemics of Measles by means of ‘ Convalescent *’ and Normal Adult Serum The benefits conferred upon the individual child by giving an antiviral serum in the circumstances we have described above can scarcely be doubted, and yet the question whether an epidemic of measles can be limited or suppressed by the widespread use of the serum is not easily answered. The interesting article by Stocks (1981) on the control of measles epidemics should be read by everyone who is concerned with the problem. He points out that the temporary nature of the immunity which is conferred by the early administration of an immune-serum and the protracted course of the epidemic render it improbable that schools can be kept free from invasion for more than a brief period by means of serum prophylaxis. An analysis of outbreaks of measles which he has undertaken shows that the susceptible home contacts and the five-year-old children are the groups to which attention should be paid in attempting to modify the character of an epidemic. He regards the immunization of all home contacts with convalescent or parents’ serum as the most practical procedure, but thinks that the total volume of an epidemic cannot be reduced by more than about 13 per cent. by this means. Finally, he discusses the 174 THE PROPHYLAXIS OF MEASLES propriety of encouraging the spread of the infection at family gatherings, care being taken to follow the exposure by adequate protection by means of serum. Reference to the practical considerations and difficulties of controlling outbreaks has also been made by Newman, Okell, and others in a discussion on the prophylaxis of measles (Newman et al., 1932 ; Picken, 1931). The Curative Value of Human Immune-Serum Most physicians agree that, if the administration of the serum: is delayed until the onset of symptoms, the course of the disease and the severity of the attack are not modified. The use of Sera obtained by the Immunization of Animals Although complete proof is wanting, knowledge that has been gained from clinical and experimental observations is consistent with the belief that the cause of measles is a filter-passing virus ; a chapter is devoted to measles in the volume on virus diseases in the recently published * System of Bacteriology ” (McCartney, 1930). Within the past ten years a number of coccal organisms have been put forward as the causal agent of the disease, and a ‘“ specific ” serum has been prepared from some of them. A full description of these bacteria and of clinical experience with the sera produced from them need not be given here, because their etiological significance has not been confirmed. Tunnicliff (1917), Ferry and Fisher (1926), and other workers have cultivated from the throat secretions and from the blood of patients a diplococcus which produces small green colonies on blood agar. The characteristic features of the strains differ to some extent, and particularly in regard to their oxygen require- ments, but there is reason to think that they represent essentially the same micro-organism. The cultures are said to produce a toxin which gives a characteristic reaction in the skin of susceptible children and so distinguishes them from immune children ; Park, Williams and Wilson (1927) were, however, unable to confirm this statement. Smith (1929) examined cultures belonging to SERA OBTAINED FROM ANIMALS 175 the group, and could not find that they possessed differential characters which separated them from similar cocci obtainable from persons who were not suffering from measles ; he thinks, in short, that they form part of the normal bacterial flora of the respiratory passages. Sera have been prepared from animals by immunizing them with Tunnicliff’s coccus and also with the coccus of Ferry and Fisher, but the clinical results are generally regarded as being inconclusive. Degkwitz, working in Munich, immunized sheep by inoculating them with filtrates of naso-pharyngeal secretions from patients who were suffering from measles and with tissue cultures that had been implanted with the ‘virus’ of the disease. This serum has been extensively tried, but the clinical reports of its use are almost without exception unfavourable. Readers who desire further information on the employment of animal sera in measles should consult the articles by McCartney (1927 and 1930). REFERENCES BARENBERG, L. H., LEwis, J. M., and Messer, W. H., 1930. J. Amer. Med. Ass., 95, 4. BucHaNAN, G. See Copeman, W. S. C. 1927. Proc. Roy. Soc. Med., 20, 1609. Burn, M. 1931. Lancet, 1, 1135. CoreMAN, W. S. C. 1927. Proc. Roy. Soc. Med., 20, 1609. DeBRE, R., and Joannon, P. 1926. ‘La Rougeole ; épidémiologie, immunologie, prophylaxie.” Paris (Masson et Cie). DEBRE, R., and Ravina, J. 1923. Bull. Soc. Méd. Hop., 47, 226. Deckwitz, R. 1920. Z. Kinderheilk., 25, 134 ; 1928, ibid., 45, 365. Ferry, N. S., and Fisugr, L. W. See under McCARTNEY, J. E., 1930, and Gunn, W., 1928. GunN, W. 1928. Lancet, London, 2, 690. a 1932.1 Brit. Med. J.,1,183. 45 1932.2 Lancet, 1, 675. Haas, S. V., and Brom, J. 1926. J. Amer. Med. Ass., 87, 558. Harries, E. H.R. 1930. Med. Officer, Feb. 15th, p. 77. KiNGsBURY, A. N. 1927. J. Hyg. 27,1. LetaeMm, W. A. 1930. Med. Officer, Feb. 8th, p. 61. McCArRTNEY, J. E. 1927. Lancet, 1, 93. 4 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 276. MENDELEWA. J. A. 1929. Arch. f. Kinderheilk., 89, 119. 176 THE PROPHYLAXIS OF MEASLES NaBarro, D. N,, and SieNy, A. 1929. Lancet, 1, 1106. 5 “ 1931. Brit. Med. J., 1, 12. NEwMAN, G., etal. 1932. Brit. Med. J., 2, 260. NicoLLE, C., and ConstIL, E. 1918. Bull. Soc. Méd. Hép., 42, 336. OkEeLL, C. C. 1932. Brit. Med. J., 2, 397. Park, W. H. 1928. ‘Newer Knowledge of Bacteriology and Immunology,” Chicago, p. 938. Park, W. H., and FrReemAN, R. G., Jr. 1926. J. Amer. Med. Ass., 87, 556. Park, W. H., WiLLiams, A. W., and WiLson, M. 1927. Amer. J. Pub. Hith., 17, 460. Park, W. H., and ZINGHER, A. See ZINGHER, A. 1924. J. Amer. Med. Ass., 82, 1180. Picken, R. M. F. 1931. Lancet, 2, 1380. SILVERMAN, A. C. 1928. J. Amer. Med. Ass., 91, 1786. Smith, J. 1929. J. Hyg., 28, 363. Stocks, P. 1931. Brit. Med. J., 2, 977. SuTHERLAND, D. S., and ANDERSON, J. S. 1928. Lancet, 2, 169. TuNNICLIFF, R. See under McCarrNEY, J. E., 1930, and Gunn, W., 1928. CHAPTER XVII THE SERUM TREATMENT OF ACUTE ANTERIOR POLIOMYELITIS A GENERAL ACCOUNT OF THE DISEASE ; v . 3 Tue IMPORTANCE oF EARLY DiacNosis IN THE SERUM TREAT- MENT OF POLIOMYELITIS . v . , " v . TE EMPLOYMENT OF HUMAN SERUM IN THE TREATMENT OF POLIOMYELITIS ’ ; # . . : " . The experimental foundations for the use of human serum Evidence that supports the therapeutic value of * convalescent” and human immune-serum . 3 . The therapeutic value of normal human serum . ' . The curative dose and the route of administration of human immune-serum . 3 ‘ ’ v 3 3 ' A summary of criticisms directed against the use of human antiviral serum . g . . ’ > ; ‘ General conclusion upon the use of human antiviral serum THE PREPARATION, STORAGE AND ORGANIZATION OF SUPPLIES OF HUMAN ANTIVIRAL SERUM ‘ ’ ’ y . 3 PROPHYLAXIS BY MEANS OF AN ANTIVIRAL SERUM FROM MAN OR rrOM THE HORSE ’ - » ’ ’ : 3 ANTIVIRAL SERA OBTAINED BY IMMUNIZATION OF THE HORSE AND OTHER ANIMALS . ’ . ’ The production of an antiviral serum in the horse. Clinical experience with antiviral serum from the horse TaE TITRATION OF ANTI-POLIOMYELITIS SERUM ’ Tae MODE OF ACTION OF ANTI-POLIOMYELITIS SERUM . . Trae USE oF ROSENOW’S ANTI-STREPTOCOCCUS SERUM IN THE TREATMENT OF POLIOMYELITIS . 7 . . A General Account of the Disease PAGE 177 179 181 181 182 184 185 186 188 188 190 191 191 194 195 196 196 POLIOMYELITIS is a communicable disease of the central nervous system, which is caused by a filterable virus, whose chief attack is upon the ganglion cells of the grey matter of the spinal cord and the medulla ; cellular degeneration and small hemorrhages are the characteristic lesions. A permanent paralysis or paresis of one or more limbs is an occasional sequel, but the disease is not often 177 178 THE SERUM TREATMENT OF POLIOMYELITIS fatal ; paralysis of the respiratory muscles is the usual cause of death. Most of the cases are among children under five years of age, and the highest frequency is found in the second year of life. In the Northern Hemisphere the incidence of the disease is mainly in the months of July, August and September. The disease is usually spread by droplets of naso-pharyngeal secretion from abortive cases or carriers. The patient in the stage of pyrexia or paralysis is apparently incapable of transmitting the disease. When the virus is deposited on the nasal mucous mem- brane of a susceptible individual it ascends along the branches of the olfactory nerve to the olfactory lobes, and thence to the grey matter in the brain stem and cord ; the general view has been that the virus passes along the lymphatics of the nerves, but Fairbrother and Hurst (1930) contend that it travels by way of the axis cylinders and later work supports their opinion. In Sweden many outbreaks have occurred during the past fifty years. An interesting account has been given by Davide (1928) of an epidemic of poliomyelitis which he observed in a sparsely populated region in Northern Sweden. The disease began in the spring of 1925, and for some time the clinical features resembled those of influenza and included vomiting and catarrhal symptoms. In nearly all the patients stiffness of the nape of the neck was present as well as pains in the buttocks and the calves of the legs ; a high proportion of the cases belonged to the benign or abortive type. Towards the end of the summer the disease assumed a more serious form with the occurrence of paralysis ; it sometimes ended fatally. During the autumn the epidemic again changed its character, and intestinal symptoms with violent diarrhcea were common ; stiffness of the neck was still a persistent feature. At this stage of the epidemic the illness usually lasted for only two or three days, and the symptoms were mild; in a few cases, however, severe paralysis occurred. The people of the district called the mild form, which was not complicated by paralysis, the “ stiffneck disease ”’ (“ maladie de la nuque ”). The experience of Davide did not confirm the statement, which is sometimes made, that in epidemics of poliomyelitis it is rare to see more than one case in a family ; he found that, as a rule, all the children in a IMPORTANCE OF EARLY DIAGNOSIS 179 family were attacked ; most of them, however, showed the symptoms of the abortive form. Similar observations have been made by Paul and Trask (1932). The Importance of Early Diagnosis in the Serum Treatment of Poliomyelitis The incubation period lasts, on the average, for about a week (from three to 14 days), and the preparalytic stage for 48 to 72 hours. Since it is believed that serum treatment is likely to be effective only when it is given before the symptoms of paralysis have appeared, the importance of losing no time in reaching a diagnosis in a suspected case is manifest. The following account of the early symptoms and signs is derived from articles by Collier (1927), Aycock and Luther (1928) and Batten and Wyllie (1929). First, we shall mention the general symptoms that are asso- ciated with the infective process; next, the symptoms that are referable to irritation of the meninges; lastly, and of great importance, the characters of the cerebrospinal fluid that are diagnostic of the disease. The onset is usually sudden with fever, headache, and signs of gastro-intestinal disturbance, such as vomiting, constipation or diarrhea. Drowsiness and a desire to be left alone are often observed. The face is flushed, the expression is an anxious one, and there is often pallor about the nose and mouth ; the mucous membrane of the throat may be slightly injected. The temperature is generally under 102° F., and the fever lasts for two to four days. In 100 consecutive cases observed by Allen Starr, 69 began with fever and 381 had no fever. The prostration is greater than the accompanying pyrexia would indicate, and the pulse is more rapid than might be expected from the temperature. A rash is not uncommonly observed and may simulate scarlet fever ; in some patients there is profuse sweating. Symptoms of meningeal irritation are present. Thus muscular twitchings are common, and there is frequently a somewhat coarse tremor when the child moves. Attempts at voluntary movements are characterized by a general unsteadiness. There may be 180 THE SERUM TREATMENT OF POLIOMYELITIS retention or incontinence of urine lasting for about a day. The rigidity of the neck is quite noticeable, but is usually not as marked as in patients with meningitis. The patient can flex the head on the neck, but cannot easily bend the neck on the shoulders, so that when the head is brought half-way forward, resistance is encountered and he complains of pain. A sign which is even more constant and characteristic than stiffness of the neck is stiffness of the spine, which prevents the head from being bent down towards the knees—the negative knee-kissing sign—(Aycock and Luther, 1928). When placed in the sitting position, the child is unable to fold the arms, and prefers to distribute the body-weight through the arms, which, together with the spine, form a tripod (Amoss’s sign). Kernig’s sign is not as a rule present at this stage, but the deep reflexes are often increased ; at a later stage of the illness they are diminished. A cerebral tache can almost always be elicited. Diplopia may be complained of and nystagmus may be present. The examination of the cerebrospinal fluid obtained by lumbar puncture is the final step in the diagnosis. The fluid is usually under moderately increased pressure and its withdrawal relieves the headache and the muscular and neuritic pains ; it is clear to the naked eye, but shows a faint haziness by transmitted light— the ground-glass appearance referred to by Zingher. There is an increase in the cell count, the numbers varying from 20 to 800 per cubic millimetre. At an early stage the blood shows an increase in the polymorphonuclear leucocytes, and these cells may pre- dominate in the cerebrospinal fluid, but later they are replaced by lymphocytes. There is a normal percentage of chlorides and glucose, but the globulin content is increased. Bacteria are not found. According to Collier, similar findings are met with in the cerebrospinal fluid only in encephalitis lethargica, but in this disease polymorphonuclear cells are never seen. It has been recom- mended that the examination of the fluid should be made at the bedside while the puncture needle is left in situ, and that, if the findings are consistent with a diagnosis of poliomyelitis, a thera- peutic dose of serum should be administered through the same needle. The intrathecal injection of any kind of serum, for USE OF HUMAN IMMUNE-SERUM 181 example, normal horse serum, normal human serum or convales- cent serum from poliomyelitis patients, gives rise to a cellular reaction which results in an increase of polymorphonuclear leucocytes in the cerebrospinal fluid. The Employment of Human Serum in the Treatment of Poliomyelitis In order to make the subsequent discussion clear we may state that human serum from three sources has been used for this purpose : (1) the serum from patients who are in the stage of convalescence from an acute attack; (2) that from persons who have passed through an attack some time previously—even up to 10 or 20 years ; and (3) the serum of normal adults with no history of having suffered from the disease. The serum from the first group may be referred to as *“ convalescent serum * and that from the second group as * human immune-serum ** or human antiviral serum.” The loose application of the term ‘ convalescent serum * to the second group gives rise to confusion and should be avoided, for writers on the subject do not always make clear the precise nature of the serum they have employed. The Experimental Foundations for the use of Human Serum. Landsteiner and Popper in 1909 found that monkeys are suscep- tible to the virus of poliomyelitis, and soon afterwards Flexner and Lewis and other workers showed that the disease can be transmitted through a series of monkeys, apparently without end. The symptoms and the course of the disease in the monkey closely resemble those of poliomyelitis in children, and the histo- logical changes in both are exactly the same (E. W. Hurst, 1929). The virus, when mixed in vitro with convalescent serum from man or monkey and injected intracerebrally, fails to produce the experimental disease in the monkey. When monkeys are inocu- lated intracerebrally or intranasally with the virus of poliomyelitis, the intrathecal injection of convalescent serum obtained from human patients or monkeys will protect them from the paralysis that would otherwise follow ; the nasal route of infection is more easily blocked by the action of the serum than the cerebral route. Again, an intrathecal injection of 2 c.cm. of a convalescent serum 182 THE SERUM TREATMENT OF POLIOMYELITIS into a monkey will protect the animal from the effects of an intracerebral injection of virus made four days later. Lastly, if 15 c.cm. of convalescent serum are administered intravenously to monkeys 24 hours before a suitable dose of virus is injected intracerebrally the animals are fully protected. This result may seem surprising in view of the fact that in normal circumstances, when immune-bodies are inoculated into the blood-stream, they do not reach the cerebrospinal fluid, since they are unable to pass the barrier of the choroid plexus. The explanation is that the injury caused by the inoculation of virus material into the brain of the monkey breaks down the barrier set up by the meninges and the choroid plexus. We owe these experimental results to Flexner and his colleagues ; an account of them will be found in papers by Flexner (1928 and 1929) and by Flexner and Stewart (1928). Evidence that Supports the Therapeutic Value of * Convalescent ’’ and Human Immune-serum. Zingher (1917), in New York, used the serum from convalescent patients and from persons who had passed through an attack of the disease from one to several years before, and even up to thirty years or more. He noted a temporary exacerbation of the meningeal symptoms after the injection. Of 54 patients who were treated with immune-serum in the preparalytic stage, 44 remained free from paralysis and 5 of the 10 who showed paralysis completely recovered. Aycock and Luther (1928) treated 106 patients in the preparalytic stage with convalescent serum. There was only one death (0-9 per cent.), as compared with a mortality-rate of 14 per cent. in a series of cases in the year 1927 in Massachusetts which were untreated with serum. Careful measurements of the amount of paralysis were made by the orthopedic staff of the Harvard Infantile Paralysis Commission ; and according to the numerical system of rating adopted, the average degree of paralysis in the treated series gave a value of 19 as compared with 63:6 in 482 untreated cases. Among the treated patients, 5-7 per cent. were attacked by paralysis of a severity which corresponded to the two most severe grades in the classification adopted by the Commission, whereas 46 per cent. of the untreated group had paralysis of a USE OF HUMAN IMMUNE-SERUM 183 like severity. The extent and the severity of the paralysis varied with the interval that elapsed between the onset of the disease and the time of treatment ; patients who received serum on the first day showed much less paralysis than those who were treated on later days. A series of 126 cases has been reported on by Ayer (1929), who treated most of them intrathecally with con- valescent serum or serum from immune donors; the treatment was attended by most gratifying results. The considerable epidemics that have occurred within late years in Canada have given opportunities for the trial of immune- serum. McEachern and his colleagues used it extensively in 1928 in the Manitoba epidemic. They employed the pooled serum derived from six or eight donors who had suffered from the disease from a few months to as long as 33 years before. The intramuscular route was used almost exclusively, and they regard this method of administration as sufficiently efficacious to justify its choice when an epidemic is in progress. The dose was 25 c.cm. when given in the preparalytic stage, and it was repeated if it was thought to be necessary. Of 74 patients who received one or more doses of serum in the preparalytic stage, more than 90 per cent. made a complete recovery, 7 per cent. showed residual paralysis, and there were no deaths. A group of 54 patients received no serum, and of these 26 per cent. recovered, 63 per cent. showed paralysis, and 11 per cent. died. The medical men who had experience of the Manitoba epidemic were convinced of the value of human immune-serum in the treatment of the disease. Cushing, in Montreal, and Hector, from his experience of the disease in New Zealand, are also strong advocates of the use of human immune-serum in the treatment of poliomyelitis. A contribution by Netter (1930), which was prompted by the recent epidemic in Alsace, reaffirms the therapeutic value of serum from persons who have already had the disease ; Netter’s experi- ence dates from the year 1915, when he treated several patients with ¢ convalescent ”’ serum. He injects either the serum or twice the volume of citrated whole blood intramuscularly, and gives a total dose of from 60 to 90 c.cm. of the serum. The disease has been prevalent in Victoria, Australia, since the 184 THE SERUM TREATMENT OF POLIOMYELITIS year 1925. An account of its treatment in the preparalytic stage by means of human immune-serum has been given by Macnamara and Morgan (1932); they give details of 133 cases which were thus treated. They conclude that the results of the treatment were excellent, as judged by the low mortality-rate, the low average degree of paralysis, and the slight incidence of the severe forms of paralysis. They recommend that it should be admini- stered as soon as the examination of the cerebrospinal fluid has confirmed the clinical diagnosis, and they think that it may be given to patients within 24 hours after the onset of paralysis if the fever continues. But, in agreement with the general opinion, they believe that the treatment is of no avail when the paralysis has become stationary. The Therapeutic Value of Normal Human Serum. When serum from convalescent patients or from immune donors is difficult or impossible to obtain, normal human serum may be used. This is a rational procedure, for all the evidence tends to show that persons who have never suffered from an attack have yet acquired an immunity in childhood, either by the reception of sub-infective doses of the virus or as the result of unrecognized abortive attacks. The immunity is of long duration, for second attacks of polio- myelitis are almost unknown. The extent of a naturally acquired immunity in a family group or a community can be determined by protective tests in monkeys, but the expense of this procedure is the reason for the somewhat scanty information on the subject. Shaughnessy, Harmon and Gordon (1930) carried out a number of tests of this kind and came to the conclusion that the serum of family contacts and of normal children and adults possesses an equal and even a greater capacity to neutralize the virus than the serum of persons who have recovered from an attack ; but that the serum of infants seems to be almost lacking in this property. The figures they give are small ; 86 sera were tested and these were divided into seven groups. Since only five children under two years of age were tested, and two of these showed some neutralizing power, the conclusion they draw from this group can only be a tentative one, although, doubtless, it harmonizes with clinical experience. Aycock and Kramer DOSAGE AND MODES OF ADMINISTRATION 185 (1930) found a high immunity-rate (83 per cent.) among urban mothers and their new-born infants, with a correspondence between mother and child. They think that their results indicate a passive transmission of immunity from mother to child. They found evidence also for the existence of a widespread immunity among persons with no history of having had the disease; the serum of 18 out of a total of 21 such persons neutralized the virus. Similar conclusions have been reached by Brodie (1932), who found that the serum of 17 (59 per cent.) of 29 normal adult city residents possessed an antibody content which he estimated to be about 90 per cent. of that of human immune donors. Moreover, early cases of the disease appeared to respond as favourably to the normal serum as to that of the immune donors. Some additional evidence that the serum of healthy persons who have been in contact with cases of poliomyelitis contains antiviral bodies has been furnished by Fairbrother and Brown (1930). The Curative Dose and the Route of Administration of Human Immune-serum. Although it is not difficult to demonstrate the presence of a specific antiviral substance in a sample of human serum, whether from a convalescent patient or from a normal person, there is no exact method of titrating the potency of such sera; the circumstance that the monkey is the only test animal available for the purpose imposes obvious limits in work of this kind. The dosage recommended by various authors is merely a volumetric one, and may be taken as an indication of the amount which is considered to be adequate. The workers in the United States prefer to give the serum both intrathecally and intra- venously. From 10 to 20 c.cm. should be given at the earliest possible moment intrathecally, and this should be followed immediately afterwards by a dose of from 40 to 200 c.cm. intra- venously ; the intravenous dose need not be repeated, but, if it is thought desirable, the intrathecal dose may be repeated on two or three successive days. There need be no fear of giving the serum intravenously, since, in contrast with horse serum, the introduction of human serum directly into the circulation is rarely followed by unpleasant sequela ; it should be warmed to body temperature and injected slowly. The dose depends to some 186 THE SERUM TREATMENT OF POLIOMYELITIS extent upon the age of the patient ; children up to two years of age may be given the smaller amounts mentioned above. The table which summarizes the observations of Macnamara and Morgan shows that for the last 47 cases mentioned in it the average first dose—by the combined intrathecal and intravenous routes— was about 60 c.cm., and that a second dose of about 40 c.cm. was given in six of these cases. The serum may conveniently be filled into 25 c.em. ampoules, so that, when the appropriate amount has been given intrathecally, the remainder of a total volume of 50 c.cm. can be administered at once by the intravenous route. If the symptoms do not subside within 18 hours an addi- tional dose of 50 c.cm. may be given intravenously. As Flexner and Stewart (1928) have pointed out, there is as yet no certain knowledge of the relative advantage of the intrathecal and the intravenous routes, nor do we know whether either alone may not give as good results as those obtained by the combined method. Moreover, as already stated, Netter and the Canadian workers prefer the intramuscular route for its administration. Most clinicians, however, agree that serum treatment is of no avail when the signs of paralysis have appeared. A Summary of Criticisms directed against the use of Human Antiviral Serum. In the epidemic of the year 1916 in New York, the efficacy of intrathecal doses of serum from persons who had recovered from the disease was tested by Neal, Du Bois and Abramson ; they obtained such discouraging results that they did not feel justified in continuing the method. A severe reaction often followed the introduction of the serum, and they thought that the acute aseptic meningitis caused by the irritant action of the serum upon the slightly inflamed meninges might be harmful, although they admitted that other workers believed that benefit may result from the phagocytic activity associated with the cellular reaction. Kellogg (1929), in a critical article on the present status of convalescent serum therapy, justly argues that the case for treatment of this kind is less secure in poliomyelitis than it is for the prophylaxis of measles by ¢ convalescent * serum. He thinks that the evidence is not convincing, and, although he appreciates CRITICAL CONSIDERATIONS 187 the difficulty of arranging for a satisfactory series of control cases, he enters a plea for controls which are as far as possible comparable with the serum-treated group of cases. A similar attitude is adopted by Morse (1929), who nevertheless believes that immune-serum should be given, but that an open mind, tinged with a healthy scepticism, should be kept when estimating the results. Brain and Strauss (1930) suggest that the good effect of the serum treatment is partly due to the spinal drainage which precedes it, and that the use of any kind of serum intrathecally is likely to alter the permeability of the meninges, and so influence the course of the infection. There is experimental evidence in favour of this view, for in the year 1918 Flexner and Amoss showed that antiviral substances pass from the blood of immune monkeys into the cerebrospinal fluid if the permeability of the ‘ meningeal-choroidal complex ’ has been increased by an aseptic inflammation, such as that which is produced by an intrathecal injection of normal horse serum. These authors state that in man immune-bodies may be detected in the circulation as early as the third day of an attack of poliomyelitis, and they suggest that the diversion of immune-bodies brought about in this manner may explain why some patients seem to react favourably to intrathecal injections of normal horse serum. A critical note has recently been sounded by Kramer and his associates, and by Park. These workers have come to the con- clusion that, when a control group of patients is reserved for comparison with the serum-treated patients, a significant difference is not apparent in the clinical results. Park makes the comment that this is not surprising in view of the circumstance that, when the nerve cells are invaded by the virus of poliomyelitis, they are beyond the range of action of the serum, and that specific immune- sera have not been shown to possess much value in other diseases that are caused by a filterable virus. While admitting the general validity of these statements, the reader may consider whether an immune-serum may not be capable of exerting a preventive action by protecting the contiguous healthy cells from attack. He should consult the articles by Kramer and by Park, and also weigh the 188 THE SERUM TREATMENT OF POLIOMYELITIS arguments that have been advanced in opposition by Macnamara (1932). General Conclusion upon the use of Human Antiviral Serum. The survey of the clinical experience which has been sketched above leads to the conclusion that the serum should be given without delay in those cases in which the diagnosis is confirmed by an examination of the cerebrospinal fluid before the signs of paralysis have appeared. The method is based on sound experimental evidence, although perhaps the clinical evidence is not as well supported on the statistical side as could be wished } it is naturally difficult for those who have become convinced of the value of the method to withhold the serum from a control group of patients. Sporadic cases are likely to be overlooked until the diagnosis is made certain by the appearance of the characteristic paralysis. Early diagnosis is of the greatest importance, and this depends largely upon the intelligent observation of the carly symptoms by the parents and the readiness with which they seek medical advice ; a judicious publicity campaign does good during an outbreak by helping to maintain the requisite alertness of mind. Even during epidemic prevalence the signs and symptoms of the disease are so variable in their character and severity, and the mild forms are so difficult to diagnose with certainty, that a correct judgement upon the effect of any kind of treatment is difficult. And even when the diagnosis in the preparalytic stage can be regarded as certain the physician has no means of predicting whether paralysis will follow or, if it appears, whether it will take a mild or a severe course. In the opinion of Flexner (1928), the variable and indeterminaté factors that influence the course of the disease make it difficult to estimate the effect upon it of therapeutic agents. The Preparation, Storage, and Organization of Supplies of Human Antiviral Serum Flexner (1928) has advised that in choosing * convalescent > donors, their age, body-weight, and physical condition should be noted. In the selection of donors of serum in V ictoria, Macnamara and Morgan adopted the criterion of demonstrable PREPARATION AND STORAGE OF SERUM 189 residual paralysis. From 300 to 500 c.cm. of blood may be taken from persons between 13 and 16 years of age, and from 500 to 1,000 c.cm. from adults. This amount may be removed from professional donors once every three weeks if their condition remains good. The blood is allowed to clot in the slanting position, is kept at room temperature for half an hour, at 37° C. for one hour, and in the cold room for 24 hours. The serum is removed, and if necessary it is spun to free it from red blood corpuscles. A Wassermann test and a test for sterility are carried out, and 0-3 per cent. tricresol is added. Another plan is to receive the blood into the requisite amount of potassium oxalate solution and to deal with the plasma in the usual way. This method is suitable only when the blood can be manipulated immediately and the plasma clotted without delay, since otherwise trouble may be experienced owing to secondary clotting. Moreover, the shaking of the blood in transport appears to favour hemolysis in a proportion of the samples. There is some evidence that the serum retains its antiviral property for a considerable time when it is stored in the ice-chest. Burnet and Macnamara (1929) found no proof of diminished activity after three years, and Netter (1930) makes a similar statement. The organization of supplies of human antiviral serum should not offer much difficulty, and steps should be taken to procure the serum and to maintain the supply during the non-epidemic periods. The New York Academy of Medicine arranged for adult victims of infantile paralysis to contribute serum in readiness for a possible outbreak in children in the summer of 1929. Twenty-five adults, 17 of whom were women, most of them crippled by the disease, responded, and almost all refused the compensation which was offered to them. A quantity of serum was obtained which was sufficient to treat a large number of cases. Cushing (1930) states that at Montreal during a recent epidemic cripples in distant towns and villages wrote to the Health Department offering to come at their own expense to supply serum. More than 8 litres of immune-serum were used during the Manitoba epidemic. Leake (1929) has urged that the supply and distribution of immune-serum should be organized on a permanent basis, so that 190 THE SERUM TREATMENT OF POLIOMYELITIS the scheme may be ready to operate at once when an outbreak is detected. : Prophylaxis by means of an Antiviral Serum from Man or from the Horse An immune-serum of human origin, in addition to its use as a curative agent, may be given for the purpose of protecting those who have been exposed to the risk of infection when an epidemic is in progress. Flexner and Stewart (1928) have suggested a sub- cutaneous dose of 10 c.cm. for children and one of 20 c.cm. for adults, which may be repeated after four or six weeks if the danger is still present. In their opinion the incidence of the disease is so haphazard that the efficacy of prophylactic injections is not likely to be easily determined, but they point out that the practice will help to allay the anxiety of parents and other relatives. In the course of his epidemiological observations on an outbreak in Northern Sweden, Davide (1928) attempted to estimate the pro- phylactic value of convalescent serum. He obtained blood from patients on the tenth day, at the earliest, after the total disappear- ance of the fever, and used the separated serum, to which 0-5 per cent. phenol had been added ; he made certain that the Wassermann reaction was negative. For his test he selected persons who were not older than 25 years of age. Seventy-three persons were inocu- lated, of whom one was subsequently attacked, whereas, of the control group, fourteen were attacked by the abortive or the typical form of the disease. Davide regards these results as merely suggestive of protection conferred by the serum, and states that he finds it by no means easy to form a definite judgement upon the value of the method. In view of the difficulty of obtaining convalescent serum at the beginning of an epidemic, Moro (1930) advises that normal adult blood or serum should be given prophy- lactically, and that the injections should be confined to children between six months and five years old—the most susceptible age group. In urban districts, where most adults are immune, at least 20 c.cm. of the parents’ blood should be injected into a muscle ; in rural districts, where only about half of the adults are immune, the blood of adult town-dwellers should be given. PROPHYLAXIS BY MEANS OF SERUM 191 The extensive epidemic of 1931 in New York City gave an opportunity for testing the prophylactic value of human anti- viral serum (Flexner, 1933). Several thousand children received a prophylactic injection of 30 c.cm. of parental whole blood, with the result that the incidence of the disease was believed to be lower among them than among those who had not been inoculated. An addendum to the monograph on poliomyelitis (1932) furnishes suggestive information regarding the prophylactic value of normal adult blood. An outbreak of the disease occurred in the autumn of 1932 at Bradford, Pa., in the United States. Brebner, who visited the town when eight cases had already been notified, urged the necessity of inoculating every child under 15 years of age with 10-20 c.cm. of parental whole blood. His advice was adopted wholeheartedly, and 1,300 children were so treated. During the inoculation period of 25 days there were 32 additional cases among children who had not received the blood, but none among the inoculated children. An antiviral serum which is obtained by immunizing horses with the virus, as described below, may be used instead of human immune-serum for the prophylaxis of the disease. The advan- tages which it has over a human immune-serum are that an adequate supply of it is obtainable with much less difficulty, and that it is possible to ensure a higher content of antiviral substance, as estimated in monkeys, than is present in the average pooled human serum. A supply of antiviral serum from the horse has recently been produced at the Lister Institute, Elstree, and is available for prophylactic use. Moreover, a considerable quantity of this serum has been desiccated and is kept in cold storage, so that it can be converted into the liquid form and prepared for issue within a short time, should an outbreak occur. Antiviral Sera obtained by the Immunization of the Horse and other Animals The Production of an Antiviral Serum in the Horse. Flexner (1910) was apparently the first worker to immunize a horse with the virus. The result was disappointing, for he found that after 192 THE SERUM TREATMENT OF POLIOMYELITIS many months of treatment with large doses of filtrates that con- tained the active virus the serum of the horse had acquired no antiviral property. It is evident that this particular animal chanced to be refractory to immunization with the virus, an experience which has been noted by other workers in recent years (Fairbrother and Morgan, 1930, 1931). In the Huxley Lecture of the year 1912, Flexner stated that the horse and the sheep could be made to yield antiviral sera which, like human and monkey convalescent sera, were capable of conferring passive immunity. In a later publication (1928), Flexner suggested that sera prepared from animals such as the horse and the sheep, which are not them- selves subject to experimental poliomyelitis, are therapeutically inefficacious. Neustaedter and Banzhaf (1917) immunized a horse subcutane- ously with a trypsinized filtrate of a human brain and cord, and afterwards with a filtrate of the brain and cord of monkeys. The treatment seems to have been an intensive one, and it caused a febrile reaction after each dose. Two days after a dose of 300 c.cm. of a filtered 5 per cent. suspension of monkey cord and brain the horse was unable to stand up; its temperature became sub- normal and it died the next day. Experiments showed that the serum of this horse possessed protective properties for monkeys, when these were inoculated intracerebrally with serum-virus mixtures. This serum was used with doubtful success in a number of human cases by Weyer, Park and Banzhaf (1929). These workers immunized a horse by means of intravenous injections of virulent material, and obtained a serum which was somewhat more potent than the average human convalescent serum. Banzhaf prepared a refined product from it, and found that the antiviral substance was associated with the pseudo- globulin fraction of the serum, and that the globulin solution, when injected intrathecally into monkeys, conferred an immunity, which lasted a considerable time, against the intranasal instillation of the virus (Rhoads, 1931). Rhoads (1931) tested a globulin solution supplied to him by Park and was unable to confirm wholly the results of Weyer, Park and Banzhaf ; he suggested that the discrepancies in the experimental results were due to a differ- ANTIVIRAL SERUM FROM THE HORSE 193 ence in potency of the strains of virus that were employed in the tests ; the virus used by Rhoads seemed to be more certainly lethal than that used by Park and his co-workers. In 1918 Pettit in the Pasteur Institute continued the work of Neustaedter and Banzhaf, at first with a sheep and later with a horse. He found that his serum possessed protective properties against the virus. Stewart and Haselbauer (1928) tested a sample of Pettit’s anti-poliomyelitis horse serum and concluded that it neutralized the virus only occasionally. Fairbrother (1930) immunized a horse by repeated intramuscular inoculations of increasing doses of a potent virus and obtained an antiviral serum. Later, in co-operation with Morgan (1930, 1931), he inoculated three horses with intravenous and intramuscular doses of filtrates that contained the virus, with the result that the serum of one horse acquired definite antiviral properties, whereas that of the others yielded no appreciable evidence of antiviral activity. The immune-serum was fractionated, and the distribution of the protective substance among the different fractions was ascertained by the injection of protein-virus mixtures into the brain of monkeys. The results indicated that the protein which was precipitated at a relatively low concentration of ammonium sulphate—the so-called euglobulin fraction—was much more potent in antiviral action per unit weight of protein than that which was precipitated at higher concentrations of this salt. The evidence suggested that in the serum of this horse there was a general distribution of the protective substance throughout the serum proteins, since carefully fractionated preparations of the pseudoglobulin and albumin possessed definite, although slight, virus-neutralizing properties (Morgan and Fairbrother, 1930). This serum has been used in the treatment of a few cases in England, but it has not been possible, so far, to draw any conclusion as to its efficacy. Schultz and Gebhardt (1931), in California, have also reported on the production of an antiviral serum in the horse, but up to the present we have no information regarding its clinical value. Howitt and her associates (1931) immunized two goats and a sheep with spun unfiltered 10 per cent, suspensions of virus R.A, VACCINES: 7 194 THE SERUM TREATMENT OF POLIOMYELITIS material, and have treated 12 patients in the preparalytic stage with serum from this source ; a dose of 50-100 c.cm. was given to them intramuscularly. They gained the impression that good results were obtained. Clinical Experience with Antiviral Serum from the Horse. The serum obtained from horses by Pettit is the only one that has been tried on a considerable scale. A survey of the reports on its use in the preparalytic stage indicates that the evidence in its favour is inconclusive. Stewart and Haselbauer (1928) could not satisfy themselves that the beneficial results cited were really due to the serum, and they criticize the inclusion of types of cases which were, in their opinion, little likely to be benefited by a specific anti-poliomyelitis serum, namely, paralytic cases of some standing and cases of myelitis which, when judged by clinical criteria, were certainly not poliomyelitis. Neustaedter (1981) takes the view that the diagnosis of poliomyelitis is prac- tically impossible in the preparalytic stage, and that therefore an estimate of the therapeutic value of a specific serum is also impossible in this stage. In his opinion the efficacy of the serum can be tested only in frankly paralytic cases, and he believes that the French clinicians have amply demonstrated its value in such cases. He thinks that intrathecal injections should not be given, since they invariably produce violent reactions accompanied by a temperature of 104°-106° F., and even convulsions and coma ; Pettit and also Park have likewise reported reactions of this kind. Neustaedter recommends intravenous and intramuscular injec- tions, with a dose for adults of 20-30 c.cm. of unconcentrated serum ; the dose should be repeated daily or every two days until the temperature is normal or until ten injections have been given ; the corresponding dose of a concentrated serum is 5 c.cm. He con- cludes that the efficacy of Pettit’s serum has been shown in more than 60 cases of poliomyelitis with definite paralysis. There is no indication that any sort of control series was instituted, and further it is obvious that in a disease like poliomyelitis the prognosis in individual cases of paralysis is very difficult to estimate. More- over, the opinion of Neustaedter and of the French observers is not supported by the general experience that serum treatment in TITRATION OF ANTI-POLIOMYELITIS SERUM 195 established cases of paralysis is of no avail. The efficacy of a specific serum produced in the horse will remain, like that of human immune-serum, a matter for argument, until evidence on a larger scale has accumulated. The Titration of Anti-poliomyelitis Serum There is no agreed method of carrying out the titration of the protective substance in an immune-serum, from whatever source it may be obtained. The subject is, indeed, in a very unsatis- factory state, for it is scarcely possible to compare the results obtained by different workers. Some of the difficulties that surround the subject may be indicated here. First, there is the necessity for using an expensive test animal—the monkey. Again, although the monkey-passaged virus retains its virulence for a long time, it has been stated that occasional fluctuations occur which are not predictable (Flexner and Amoss, 1918); the impor- tance of controls is thus clearly evident. Further, in the recorded work the quantitative expression of the lethal power of the test dose of virus often lacks precision ; for example, the approximate number of minimal lethal doses it contains is not always men- tioned. Loose statements are occasionally met with, such as that a certain dilution of serum protected against an unspecified or vaguely defined test-dose; or that so many parts of serum neutralized one part of virus; or that a concentrated serum. neutralized more than 100 parts of the poliomyelitis virus in vitro. Until agreement is reached upon methods of titration that will give comparable and approximately accurate results, research on specific sera, both in the laboratory and in hospitals, will undoubtedly be hampered. Some workers use the intracerebral method of injection for titrating serum-virus mixtures, but this produces varying degrees of trauma of the brain substance. To obviate trauma, Flexner and Rhoads (1929) advised injecting the virus into the cisterna magna of the test animal, a route which has the advantage of bringing about an experimental infection with constancy without causing injury to the brain and cord. Rhoads (1931) states that —2 196 THE SERUM TREATMENT OF POLIOMYELITIS the intracisternal method has now been superseded by intranasal instillation of the virus; this procedure causes no trauma, and is the usual portal of entry of the virus in man. The primary and indeed the essential requisite for the titration of anti-poliomyelitis sera is the provision of a standard serum, and the adoption by its means of a standard unit of protective sub- stance against the virus. This unit, whether it applies to the standard serum or to any sample of specific serum it is desired to titrate, will be determined in a suitable test animal according to a recognized technique. A widespread and, if possible, universal acceptance of such a unit will assist in deciding upon the proper dosage and mode of administration of antiviral sera. The Mode of Action of Anti-poliomyelitis Serum Little information is available regarding the mode of action of specific antiviral sera. When the virus is exposed to the action of the serum it is not killed, but is only ““ held in some sort of an ineffective combination,” as was shown by the cataphoresis experiments of Olitsky, Rhoads and Long (1929). There is no proof that, as Neustaedter has suggested, the serum owes its protective property to an antitoxin. The use of Rosenow’s Antistreptococcus Serum in the Treatment of Poliomyelitis Rosenow (1930) has advocated the use of an antistreptococcus serum which is prepared by immunizing horses with a strain of streptococcus which he has cultivated from the throat, the brain, the spinal cord and the cerebrospinal fluid of typical cases. He asserts that the clinical results are good; even progressive paralysis may be arrested in many cases, and the death-rate and the incidence of residual paralysis are definitely lowered. A globulin preparation is available, and the intravenous and intra- muscular routes are recommended. The serum is said to have a toxic effect on the meninges, and it is advised, therefore, that it ROSENOW’S ANTISTREPTOCOCCUS SERUM 197 should not be given by the intrathecal route. The views advanced by Rosenow have received some support from clinicians, but, on the whole, it must be considered that there is no solid ground for regarding this serum as a specific therapeutic remedy which is able to influence the pathogenic action of the causal agent of polio- myelitis, that is, the specific filterable virus. Flexner and his colleagues, and more recently Fairbrother, have given reasons for doubting the importance of the streptococcus in the etiology of the disease, and the same authorities have shown that the serum, when it is tested in monkeys, has no antiviral properties. REFERENCES The following monograph on poliomyelitis gives a good account of the disease from every point of view and contains a full bibliography. “ PorLiomvyeritis ~ (International Committee for the Study of Infantile Paralysis, organized by Jeremiah Milbank), Baltimore, The Williams & Wilkins Co., 1932. A useful summary of modern practice in the diagnosis and treatment of the disease is given in : * Memorandum on Acute Poliomyelitis.” Ministry of Health, London. H.M. Stationery Office. 1932. Amoss, H. L. 1921. J. Amer. Med. Ass., 76, 110. Avcock, W. L., and Kramer, S. D. 1930. J. Exp. Med., 52, 457. 1930. J. Prev. Med., 4, 189. i " 1930. Ibid., 4, 201. Avcock, W. L.,and Luruer, E. H. 1928. J. Amer. Med. Ass.,91, 387. Avcock, W. L., Luruer, E. H., and Kramer, S. D. 1929. Ibid., 92, 385. AYER, W. D. 1929. Amer. J. Med. Sci., 177, 540. Barren, F. E., and WyLLig, W. G. 1929. * Diseases of Children,” ed. by Thursfield, H., and Paterson, D., London, p. 684. Brain, W. R., and Strauss, E. B. 1930. °° Recent Advances in Neurology,” London, p. 370. Bropie, M. 1932. J. Exp. Med., 56, 507. Burner, F. M., and MACNAMARA, J. 1929. Med. J. Australia, Dec. 14th, p. 851. COLLIER, J. 1927. Brit. Med. J.,1, 751. CusHING, H. B. 1930. Brit. Med. J., 2, 524. Davipe, H. 1928. Bull. de UOff. Internat. Hyg. Publique, 20, 74. FaBer, H. K. 1931. J. Amer. Med. Ass., 96, 935. FAIRBROTHER, R. W. 1930. Brit. J. Exp. Path., 11, 43. FAIRBROTHER, R. W., and Brown, W. G. S. 1930. Lancet, 2, 895. FAIRBROTHER, R. W., and Hurst, E. W. 1980. J. Path. & Bact., 33, 17. ss 3 198 THE SERUM TREATMENT OF POLIOMYELITIS FAIRBROTHER, R. W., and Morcan, W. T. J. 1930. Brit. J. Exp. Path., 11, 298. 1931. Lancet, 2, 584. FLEXNER, S. 1910. J. Amer. "Med. Ass., 55, 1105. ’ 1912. Brit. Med. J., 2, 1261. ” 1928. J. Amer. Med. Ass., 91, 21. ” 1928. Ibid. 91, 2014. * 1929. Bull. N.Y. Acad. Med., Second Series, §, 252. 1933. Brit. Med. J.,1, 132. FLEXNER, S., and Amoss, H. L. 1918. J. Exp. Med., 28, 11. FLEXNER, S., and Ruoaps, C. P. 1929. Proc. Nat. Acad. Sci., 15, 609. FLEXNER, S. and STEWART, F.W. 1928. New Eng. J. Med., 199, 213. vi 1928. J. Amer. Med. Ass., 91, 383. Hector, C. M. 1927. Brit. Med. J., 1, 624. Howirr, B. F., Suaw, KE. B., THELANDER, H., and Limper, M. 1931. J. Amer. Med. Ass., 96, 1280. Hurst, E. W. 1929. J. Path. and Bact., 32, 457. Kerroce, W. H. 1929. J. Amer. Med. Ass., 93, 1927. KrAMER, S. D., Avcock, W. L., Soromon, C. I., and THeENEBE, C. L. 1932. New Eng. J. Med., 206, 432. LANDSTEINER, K., and Popper, E. 1909. Z. fiir Immuniltdtsf., Teil I., Orig., 2, 377. LEAKE, J. P. 1929. Public Health Reports, 44, 1819. McEAcHERN, J. M. 1930. Brit. Med. J., 2, 524. McEAcHERN, J. M., Cuown, B., BELL, L.. G., and McKeNzie, M. 1929. Canad. Med. Ass. J., 20, 369. McEAcHERN, J. M., CaHowN, B., BELL, L. G., and McKenzie, M. 1929. Canad. Pub. Hlth. J., 20, 235. MACNAMARA, J. 1932. Lancet, 2, 1353. MACNAMARA, J., and MorGaN, F. G. 1932. Lancet, 1, 469, 527. Morcan, W. T. J., and FairBroTHER, R. W. 1930. Brit. J. Exp. Path., 2, 512. Moro, E. 1930. Klin. Woch., 9, 2383. MoRrsE, J. L. 1929. New Eng. J. Med., 200, 1236. Near, J. B.,,Du Bois, P. L., and ABramson, H. L.. 1916-1919. Coll. Stud. Bureau Labs., Dept. Health, N. York, 9, 77. NETTER, A. 1930. La Presse Méd., 38, 1169. NEUSTAEDTER, M. 1931. J. Amer. Med. Ass., 96, 933. NEUSTAEDTER, M., and Banzuar, KE. J. 1917. Ibid., 68, 1531. Onitsky, P. K., Ruoabs, C. P., and Lo~G, P. H. 1929. J. Exp. Med., 50, 273. Park, W. H. 1928. ‘ Newer Knowledge of Bacteriology,” Chicago p. 941. ’s 1932. J. Amer. Med. Ass., 99, 1050. Pauw, J. R., and Trask, J. D. 1932. J. Exp. Med., 56, 319. PeTrTIT, A. 1930. Monde Médical, No. 781. Ruoaps, C. P. 1931. J. Exp. Med., 53, 123. RosenNow, E. C. 1930. J. Amer. Med. Ass., 94, 777. REFERENCES 199 Scauvrrz, E. W., and GEBuarDT, L. P. 1931. Proc. Soc. Exp. Biol. Med., 28, 412. SuauvcHNEssY, H. J., Harmon, P. H., and Gorpbon, F. B. 1930. Proc. Soc. Exp. Biol. and Med., 27, 742. Stewart, F. W., and HASELBAUER, P. 1928. J. Exp. Med., 48, 449. WEYER, E. R., Park, W. H., and Banzuar, E. J. 1929. Amer. J. Path., 5, 517. ZINGHER, A. 1916-1919. Coll. Stud. Bureau Labs., Dept. Hlth., New York, 9, 158 5; 1917, J. Amer. Med. Ass., 68, 817. CHAPTER XVIII THE VACCINE AND SERUM TREATMENT OF CERTAIN DISEASES IN MAN CAUSED BY FILTER-PASSING VIRUSES PAGE RECENT WORK ON ACTIVE IMMUNIZATION AGAINST VIRUS DISEASES 200 Variola . : , : : ' ’ . ‘ . 201 Rabies . . : 3 ; 3 : . 201 Rocky Mountain Fever ¥ ’ : : 2 : . 202 Typhus Fever . . : . . . . . . 202 Yellow Fever . 2 : ‘ 4 L204 THE SERUM TREATMENT OF VIRUS DISEASES # ¥ : . 206 Chicken-pox . : ' . v : . 206 Post-vaccinal Encephalitis. ? 3 § : hy . 207 Mumps . : . ’ ’ . . > ’ . 208 Psittacosis : y : 3 . : . : . 209 Typhus Fever : . : . : A . 210 Yellow Fever . ¥ 2 : ’ ’ ’ ; . 210 Dengue Fever . . . . > . . . . 211 Ix this chapter are grouped together a number of infectious diseases, the causal agent of which is believed to be a filter-passing micro-organism. The fact that our knowledge of the nature of virus infections is of recent date furnishes the explanation why no considerable progress has as yet been made in this part of the field of vaccine and serum therapy. RECENT WORK ON ACTIVE IMMUNIZATION AGAINST VIRUS DISEASES The chief difficulty that confronts the experimental worker who is studying the conditions for the production of an active immunity to virus diseases is the obvious one that, unlike the bacteria, it is impossible at present to prepare and utilize pure cultures of members of the group of viruses. Two crucial questions in the immunology of virus diseases call for a definite answer: (1) Are the processes of immunity that are evoked by natural or experimental virus infections strictly comparable with those which are engen- dered by bacteria and their antigens ?; and (2) can a specific 200 VARIOLA : RABIES 201 immunity be established by the use of killed preparations of the virus ? The answers appear to be that there is no essential difference between the immunity mechanism which is set in motion by viruses and by bacteria when they are introduced into the body of an animal ; and that, in conformity with experience in bacterial immunity, the most efficient vaccine against a virus disease is the living attenuated organism, although the killed virus may in some instances have a prophylactic value. In the account that follows, mention will be made of the few virus diseases that attack man against which it has been possible to confer an active immunity. Variola Vaccination against small-pox is the best instance of the employment of a virus which has become attenuated by passage through another species of animal, and which is utilized as an agent for the production of an active immunity against the infection in man. The survey of smallpox and vaccinia by the late Dr. I. R. Blaxall (1930) renders it unnecessary to enter here into details of vaccination procedures. Rabies Therapeutic immunization of a person who has been bitten by a rabid animal is of the active type, although it is begun after the infection has taken place, that is, in the incubation period ; this lasts from one to two months, the length of time depending upon the position of the bite through which the virus has entered. There are various forms of immunizing preparations, which contain the virus in the living or dead state, but the basis of all of them is the fixed virus derived by passage through the brain and the spinal cord of the rabbit ; the term * fixed virus ” was given by Pasteur to the rabbit-passaged strain with an incubation period of six or seven days. According to Harvey and McKendrick (1930), the success which follows the use of a dead virus preparation in the prophylaxis of rabies, for example, Semple’s carbolized vaccine, disproves the statement that a living virus is necessary for the production of an active immunity. Research is proceeding at the Pasteur Institute at Kasauli in India and elsewhere with 202 VACCINES AND SERA IN VIRUS DISEASES the object of ascertaining the best technical methods for preparing an antigen which will act both rapidly and efficiently (Cunningham, Malone and Craighead, 1933). Rocky Mountain Fever Rocky Mountain spotted fever is an acute infectious disease whose symptoms and course strongly resemble typhus fever; in the former, however, there is a greater tendency to haemorrhages and gangrene. The causal agent is Dermacentrovenus rickettsii (Wolbach) ; and its transmission to man is by means of the wood- tick, Dermacentor venustus (vel D. andersoni). The guinea-pig is highly susceptible, and when it is inoculated with the contents of an infected tick its fate depends upon whether the insect was engorged or empty. In the first event the guinea-pig contracts the disease, but if the tick was empty an immunity without illness is conferred. A feed of blood will cause the mitigated strain in the tick to become virulent, and the contents of *‘ reactivated ” ticks when treated with phenol in order to kill the virus, are used as a prophylactic agent. It is stated that up to September, 1928, more than 4,000 persons had received the vaccine and that the results were encouraging ; the protection, however, does not last long and the vaccine deteriorates rather rapidly (Spencer, 1929). A valuable summary of current knowledge of the disease and its prevention has been written by Parker (1933). He advises that persons who are exposed to the infection to any considerable degree should be vaccinated. The result of seven years’ experience has been that a vaccine prepared by the United States Public Health Service from infected ticks affords complete protection to the average person against the milder strains, and that it affords a sufficient degree of protection against the lethal strains to mitigate to a great extent the severe symptoms and to ensure recovery. The protection lasts for one tick season only, and therefore revaccination at yearly intervals is necessary. Typhus Fever The widespread prevalence of typhus fever in several European countries during the war gave an impetus to researches into its TYPHUS FEVER 203 etiology and immunology. Arkwright (1930) has pointed out that as the result of this work three important sets of observations have been firmly established : (1) the presence of the virus in the blood and organs of man and experimental animals during the acute stage of the illness; (2) the presence of the virus in the gut of pediculi which have imbibed the blood of typhus patients : the gut of infective lice contains large numbers of a micro-organism, Rickettsia prowazeki, which is doubtless the form assumed by the virus in the louse ; and (3) the discovery by Weil and Felix that the blood serum of typhus patients agglutinates certain strains of B. proteus, one of which is the well-known ““ X.19.” The relation- ship of the blood virus, Rickettsia prowazeki, and the X strains of B. proteus has not yet been elucidated ; there are investigators who are inclined to believe that all three forms represent different phases of the same micro-organism (Felix, 1930, 1933). Epidemic typhus is a louse-borne disease, although in some endemic centres other insects, such as ticks and fleas, are suspected of being the insect carriers. Weigl (1930) prepared a vaccine for human use from the intes- tines of lice that were inoculated by his method of introducing the infective material per anum by means of a fine capillary pipette ; 0-5 per cent. phenol is added to the material. His stock suspen- sions consist of 100 louse intestines per c.cm., and this amount contains 5,000 million Rickettsize. By the use of this vaccine Weigl was able to protect his laboratory assistants, and also men in the Public Health Service who were frequently exposed to the infection. The preparation of the vaccine demands a highly skilled technique, and it is costly to produce. During the past few years Zinsser and Castaneda (1931, 1932, 1933) have carried out researches on the virus of the Mexican variety of typhus fever. They have utilized a method devised by Mooser of bringing about Rickettsia lesions in guinea-pigs by giving to them an intraperitoneal dose of the virus, and have found that the morbid changes can be enhanced by employing scorbutic animals. Later, they made use of a subcutaneous injection of benzol in olive oil with the object of depressing the activity of the leucocytes and thus further increasing the virulence of the 204 VACCINES AND SERA IN VIRUS DISEASES organisms. Still later, they exposed male white rats to the action of X-rays and immediately afterwards gave to them an intra- peritoneal dose of a suspension of a virus-infected tunica vaginalis —a tissue which is particularly prone to a concentrated invasion by the virus. The rats were killed on the fourth or fifth day and the peritoneal exudate, the peritoneal washings, and the tunica were treated with a 0-2 per cent. solution of formalin in saline in order to kill the virus. Each rat yielded a suspension of about 15 c.cm. of washed Rickettsize, the density of which was almost equal to typhoid vaccine in respect of the number of organisms it contained. A vaccine prepared in this way produces complete immunity in guinea-pigs to a subsequent infection with the Mexican virus when the test-dose of virus is a moderate one (100-250 infective doses). They noted that a vaccine prepared from the Mexican organisms confers only a partial protection against the European (Breinl) strain. Quite recently these authors immunized a horse with the rat vaccine and found that its serum agglutinated the louse vaccine of Weigl, which is made from a European strain, at an end-point which was similar to its agglutination titre for B. proteus, X.19 ; as a result of the immunization, the Weil-Felix reaction increased from a titre of 1/40 to one of 1/160. The serum did not, however, protect against the European virus, and the authors are unable to explain why the protection test should have failed when the agglutination reaction was positive. Dyer, Workman, Rumreich and Badger (1932) have prepared a vaccine from infected fleas (Xenopsylla cheopis), and have obtained some evidence of the production of immunity in guinea- pigs by its use. They hope to secure improved results by employing a more potent virus. Yellow Fever The experiments of Hindle (1930) in 1928-1929 showed that the inoculation of a dead virus preparation into a susceptible animal was followed by a high degree of immunity. He used the liver and spleen of a monkey which was killed when moribund from the YELLOW FEVER 205 disease ; these were ground and extracted with distilled water, the suspension filtered through fine muslin, and formalin, phenol or glycerine added. Aragio employed this type of vaccine in the epidemics of 1928-1929 in Rio de Janeiro. Findlay and Hindle (1931) note that the passive immunity which is produced by the injection of an immune-serum disappears in one or two weeks. When the serum is given promptly, for example, within 24 hours of an accidental infection in man, it may avert an illness, but it has little effect after the symptoms have appeared. These workers found that a subcutaneous or intraperitoneal inoculation of a mixture of the virus and a specific serum produces a more durable immunity than that which is conferred by the serum alone. They think it probable that a slow liberation of the virus takes place ; although the amount set free is insufficient to cause the disease, it is able to stimulate the production of the specific antibody. Theiler (1930) showed that yellow fever virus lost its virulence for monkeys after it had been passaged through mice. According to Sawyer, Kitchen and Lloyd, the virus, after 100 or more passages in mice, is probably of low virulence for man, but it is still capable of causing mild attacks of yellow fever, so that it seems advisable to attenuate it still further or to give it together with a potent immune-serum. They arranged for the immunization of 15 persons by means of a single injection of a dried mixture of the living fixed mouse virus and human immune-serum, with additional injections of sufficient immune-serum to ensure protection. They ascertained that the immunity in these persons rose within a few weeks to a degree corresponding to that which is reached after a natural attack, and concluded that the immunizing reaction was caused by an actual, although modified, infective process. Findlay (1933) gives an interesting description of the results of immunizing 23 men and 2 women in London by the method of Sawyer, Kitchen and Lloyd (1932). He regards the simultaneous injection of an immune-serum as a necessity, in view of the fact that the vaccination of monkeys by the mouse-fixed virus alone may be followed by encephalitis ; on the other hand, Sellards and Laigret (1932) advocate the use of the mouse-fixed virus alone for 206 VACCINES AND SERA IN VIRUS DISEASES vaccinating man. With one exception, the local and general reactions in the group of persons immunized by Findlay were comparatively benign ; details are given of the symptoms in the subject who reacted badly, and the view is taken that it is difficult to find a satisfactory explanation of his illness. The virus-serum method seems to be of value, because in Findlay’s experience an accidental infection has not occurred among the staff who are working with the virus in his laboratory, although all of them were non-immune prior to vaccination : a result which contrasts with the morbidity among a group of laboratory workers before the method was introduced. There are two desiderata in its prepara- tion : (1) a reduction in the amount of the immune-serum that is necessary for each person inoculated; and (2) the elimination of the foreign mouse-brain protein. Recent experiments that have been carried out in monkeys by Findlay show that the injection of 1 c.cm. of immune-serum into their skin, when followed one hour later by virus material into the same area, leads to a high degree of immunity in the animals ; the procedure does not cause a rise of temperature. THE SERUM TREATMENT OF VIRUS DISEASES Chicken-pox After an attack the patient acquires a solid and lasting immunity. The serum collected from convalescent patients has been used as a prophylactic agent by physicians in the United States. In 1923 Blackfan, Peterson and Conroy found that a transient immunity to varicella was conferred upon susceptible children by the intramuscular injection of 5 c.cm. of convalescent serum. Weech (1924) and Mitchell and Ravenel (1925) formed a good opinion of the value of this method of prophylaxis. Gordon and Meader (1929) state that in their experience a high proportion of exposed persons are protected if they are treated with 10 c.cm. of a pooled adult serum which has been obtained within one month of the appearance of the rash. Serum, when taken from persons after five months have elapsed since their illness, protects only one-third of the children to whom it is given. Although chicken- POST-VACCINAL ENCEPHALITIS 207 pox is a comparatively harmless disease, attempts to provide protection are justified, according to Gunn (1982), in view of its long incubation period, which necessitates a prolonged quarantine of infected hospital wards, and also of the extreme contagiousness and high resistance of the virus, which frustrate measures that may be taken for isolation of the contacts. This author gave pooled convalescent serum to 48 presumably susceptible children at intervals varying from the first to the sixth day after exposure to the infection, and had a protection-rate of about 27 per cent. He considers this serum to be far inferior to convalescent measles serum as a prophylactic agent, and advises that, in order to compensate for the deficiency, liberal doses of it should be given. Post-vaccinal Encephalitis In some European countries since the year 1924 a nervous disease of the nature of acute disseminated encephalomyelitis has been observed in rare instances to follow vaccination. The symptoms and the pathological changes in the brain and cord are indistinguishable from those which are found in the similar disease that occasionally complicates smallpox, measles and, perhaps, other virus infections. Detailed information on the clinical, pathological and epidemiological features of the post- vaccinal form will be found in an article by Greenfield (1930) and in the Reports of the Vaccination Committee (1928, 1930). Here it may be noted that the nervous symptoms follow the operation of vaccination after an interval of 10 to 14 days, that most of the patients are of school age, and that there is a tendency to familial incidence. The Committee take the view that the epidemiological features point strongly to the existence of some individual and local predisposition, and that the cause of the disease is still obscure. In 1929 a child who was apparently suffering from this condition was treated, on the suggestion of Dr. M. H. Gordon, by an intra- thecal injection of the serum of the mother, who had been vaccinated along with her child ; the effect of the serum seemed to be good. Hekman, in Holland, about the same time, tried 208 VACCINES AND SERA IN VIRUS DISEASES independently a similar method of treatment, with results that constitute, in the opinion of the Vaccination Committee, a powerful argument for the use of human anti-vaceinial serum at the earliest possible moment after the onset of the illness. Ledingham, Morgan and Petrie (1931) have reported on the preparation, concentration and content in antiviral body of a specific serum prepared from the horse. A concentrated preparation of this serum is available for the treatment of cases of post-vaccinal encephalitis, but so far it has not been employed in a sufficient number of cases to justify a definite conclusion upon its efficacy. Mumps In 1908 Granata showed that the intravenous injection of the “sterile ” filtered saliva of patients in the acute stage of mumps produces a three days’ fever in rabbits. In 1913 Nicolle and Conseil transmitted the virus to monkeys. M. H. Gordon, in 1914, inoculated monkeys intracerebrally with the filtered buccal secretion of patients in the acute stage of the illness ; a meningitis followed which killed some of the animals. In 1916 Wollstein showed that the infection was transmissible to cats when they were inoculated with similar material. The immunity in man is usually life-long. More than fifteen years ago Hess used the blood from con- valescent patients as a prophylactic remedy in a Home for Infants in New York during a severe epidemic ; the whole blood was given intramuscularly to 17 contacts, and all escaped the infection. Regan (1925) gave to 81 children an average amount of 8 c.cm. of serum within a period varying from the first to the sixth day after exposure to the infection, with the result that only one child became ill; 11 were discharged before the incubation period ended, and 69 showed no signs of the disease. The blood was taken from adult donors from the 10th to the 20th day of the illness. He suggests that the best method may be to give the serum late in the incubation period, evidently with the object of allowing a modified attack to take place. During an epidemic of mumps in 1927 in Odessa, Skrotskiy treated 179 children prophylactically MUMPS : PSITTACOSIS 209 with 5-15 c.cm. of convalescent serum administered subcuta- neously ; all but two of the children, who had a mild attack, escaped. An account of observations on the effect of con- valescent serum in checking an institutional outbreak is given by Barenberg and Ostroff (1931). In two unprotected wards with 41 susceptible children half of them caught the infection, whereas in seven wards in which there were 189 susceptible children, half of whom were given a prophylactic dose of serum, the incidence was 26-6 per cent. An alleviation of the symptoms was noted as a result of the dose of serum, and the authors estimated that the duration of the epidemic was shortened from a period of five or six months to two months. The results obtained by Gunn (1932) in a group of 21 serum cases and 22 controls in hospital wards were unsatisfactory, but in his opinion justified a further trial with larger doses of the serum. Attempts have been made by the use of convalescent serum to lessen the incidence of orchitis as a complication in mumps, especially in outbreaks among young adults, as, for example, in military barracks; evidence that indicates a reduction in the number of cases with orchitis is given by de Lavergne and Florentin (1925), Teissier (1925), and Iversen (1930). Psittacosis Psittacosis is a disease of parrots which is communicable to man and which is caused by a filterable virus. The prevalence of the disease in the year 1930 in Great Britain and other countries has given opportunities for experimental investigations, and these have thrown considerable light upon its clinical features and the nature of the causative agent (Bedson, Western and Simpson, 1930 ; M. H. Gordon, 1930; Krumwiede, McGrath and Oldenbusch, 1930 ; Rivers and Berry, 1931). In man some of the symptoms resemble those of influenza, with pulmonary signs at an early stage of the illness, whereas others, such as epistaxis, vomiting, consti- pation or diarrhcea with abdominal distension, are suggestive of typhoid fever (Fisher and Helsby, 1981). The infection is transmissible experimentally to mice, guinea-pigs, rabbits, and monkeys. Immune-sera can be prepared which neutralize the 210 VACCINES AND SERA IN VIRUS DISEASES virus when they are injected into the skin of a guinea-pig (Bedson), but convalescent serum from patients appears to have little or no protective value. Rivers and Berry (1931) were unable to find protective antibodies in convalescent human sera when these were tested in mice and rabbits. Typhus Fever The immunity after an attack in man is of a fairly durable character. Felix has stated that protective properties appear in the serum of man, the monkey, and the guinea-pig, namely, those species which give a febrile reaction to the virus, but that they are absent in the rat and the rabbit ; in them the reaction takes the form of a cryptic infection. Accordingly, he thinks it probable that attempts to produce a protective serum in horses and other large animals will be useless with the exception, perhaps, of the donkey, for in this animal a febrile reaction to the virus sometimes occurs (Nicolle and Conseil). An immune-serum would probably be used only as a prophylactic agent ; Arkwright states that con- valescent sera and immune horse sera have not been successful as a means of cure, and Decourt and Sallard (1930) have not been able to find that convalescent serum possesses undoubted prophy- lactic and curative properties. Research has not yet made clear the relationship of the typhus virus to B. proteus X.19, and to Rickettsia prowazeki—the infective form of the virus in the louse, but it is probable that the virus has a complex antigenic structure. Difficulties of this kind stand in the way of the preparation of a curative serum. Yellow Fever An attack of yellow fever leaves the patient with a solid immunity, and in harmony with this observation there is the fact that his serum can be shown experimentally to possess protective bodies up to at least seven years after recovery from the illness. Marchoux and Simond (1906) gave convalescent serum to patients and thought that it lessened the severity of the symptoms. Pettit, Stefanopoulo and Frasey (1928) prepared a serum from horses YELLOW FEVER : DENGUE FEVER 211 and baboons by inoculating them with infective material from monkeys dead of yellow fever; they believed that it protected monkeys and that it exerted a curative effect if given early. Frasey and his co-workers state that in the horse the serum acquires protective properties at the end of a three-months’ period of weekly inoculations. In a review of the subject, Hindle (1930) expresses the opinion that, having regard to the rapidity of the course of the disease and to the extent of tissue destruction which has already taken place before the diagnosis has been made, it is doubtful whether serum will prove to be beneficial in the treatment of yellow fever in man. Dengue Fever Dengue fever is a mosquito-borne disease which is caused by a filterable virus. The disease is rarely fatal, but frequently produces much discomfort. The virus has a possible relationship to yellow fever and probably affects man only. An attack is followed by an immunity of variable duration. Convalescent serum has no protective action when given prophylactically or simulta- neously with the virus, and thus it appears that specific serum therapy is not likely to be efficacious in the treatment of the disease (Burton Cleland, 1930). This conclusion is supported by the work of Simmons, St. John and Reynolds in the Philippine Islands, for specific antibodies—with the possible exception of a complement-fixing antibody—were not demonstrable in serum that was obtained during the febrile and post-febrile stages of dengue fever. REFERENCES Active Immunization against Virus Diseases ARKWRIGHT, J. A. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 393. 3 Braxarn, F. R. 1930. ‘ A System of Bacteriology,” London, H.M. Stationery Office, 7, 84. CunNINGHAM, J., Marone, R. H., and CRAIGHEAD, A. C. 1933. Indian Med. Research Mem., No. 26. Dyer, R. E., Workman, W. G., RumreicH, A., and BADGER, L. F. 1932. Publ. Hlth. Reps., 47, 1329. 212 VACCINES AND SERA IN VIRUS DISEASES FeLx, A. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 412. Fevix, A. 1933. Trans. Roy. Soc. Trop. Med. & Hyg., 27, 147. Finbray, G. M. 1933. Bull. de UOffice Internat. Hyg. Publique, 25, 1009. Finpray, G. M., and HixpLE, E. 1931. Brit. Med. J., 1, 740. Gye, W. E., and LepiNngHAM, J. C. G. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 23. Harvey and McKENDRICK. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 198. i HinpLe, E. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 449. PARKER, R. R. 1933. Archives of Path., 15, 398. SAwYER, W. A., KITCHEN, S. F.,and LLoyp, W. 1932. J. Exp. Med., 55, 945. SELLARDS, A. W., and LalGrer, J. 1932. C. R. Acad. Sei., 194, 1609 and 2175. THEILER, M. 1930. Ann. Trop. Med. and Parasitol., 24, 249. Weer, R. 1930. “ Die Methoden der Aktiven Fleck-Fieber Immu- nisierung,” Cracow. ZINSSER, H., and CastaNnepa, M. Ruiz, 1931. J. Exp. Med., 53, 493. ss ” 1931. J. Immunol., 21, 403. » » 1932. Proc. Soc. Exp. Biol. and Med., 29, 840. 5 : 1933. J. Exp. Med., 57, 381. » 5 1933. J. Exp. Med., 57, 391. Chicken-pox Brpson, S. P. 1930. “A System of Bacteriology,” 7, 165. London. Brackran, K. D., Pererson, M. F., and Conroy, F. C. 1923. Ohio State Med. J., 19, 97. GorbON, J. E., and MeapER, F. M. 1929. J. Amer. Med. Ass., 93, 2013. MircueLL, A. G., and RAVENEL, S. F. 1925. Arch. Pediatr., 42, 709. WeEcH, A. A. 1924. J. Amer. Med. Ass., 82, 1245. GuNN, W. 1932. Brit. Med. J., 1, 183. Post-vaccinal Encephalitis GREENFIELD, J. G. 1930. “A System of Bacteriology,” 7, 133. ’ London. LepiNenam, J. C. G., Morcan, W. T. J., and PETRIE, G. F. 1931. Brit. J. Exp. Path., 12, 357. Report of the Committee on Vaccination. 1928. Ministry of Health, H.M. Stationery Office. Report of the Committee on Vaccination. 1930. Ministry of Health, H.M. Stationery Office. (This Report contains a full bibliography.) REFERENCES 213 Mumps BARENBERG, L. H., and OsTrOFF, J. 1931. Amer. J. Dis. Child., 42, 1109. Gorpon, M. H. 1914. Rep. Loc. Govt. Board Publ. Hilth., No. 96. 4 1927. Lancet, 1, 652. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 273. GRANATA, S. 1908. Bull. Inst. Past., 6, 1093. Gunn, W. 1932. Brit. Med. J., 1, 183. Hess, A. F. 1915. Amer. J. Dis. Child., 10, 98. Iversen, P. 1930. Ugeskrift for Laeger, 92, 167. pE LAVERGNE, V., and FLoreNTIN, P. 1925. Bull. de I' Acad. de Méd., Paris, 93, 362. NicorLLe, CH., and ConseiL, E. 1913. C. R. Soc. Biol., Paris, 75, 217. REGAN, J. C. 1925. J. Amer. Med. Ass., 84, 279. SKROTSKIY. 1929. Odessa Med. J., 4,8. Ref.in J. Amer. Med. Ass., 93, 345, 1929. TEISSIER. 1925. Bull. de I Acad. de Méd., Paris, 93, 369. WorrLsTEIN, M. 1921. J. Exp. Med., 34, 537. on 1916. J. Exp. Med., 23, 353. Psittacosis BeDSON, S. P., WESTERN, G. T., and Simpson, S.L. 1930. Lancet, 1, 235, 245. Fisuer, H. R., and HeLsBY, R. J. 1931. Brit. Med. J., 1, 887. Gorpon, M. H. 1930. Lancet, 1, 1174. KrumwieDpe, C., McGrath, M., and OLpENBUSCH, C. 1930. Science, v1, 262. Rivers, T. M., and Berry, G. P. 1931. J. Exp. Med., 54, 105. » 32 1931. J. Exp. Med., 54, 129. i931. J. Exp. Med., 54, 119. Rivers, T. M., BErrY, G. P., and SprUNT, D. H. 1931. J. Exp. Med., 54, 91. Typhus Fever ARKWRIGHT, J. A. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 393. Drcourt, P., and SALLARD, J. 1930. Rev. Méd. et Hyg. Trop., 22, 200. Felix, A. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 393. N1coLLE, C., and Constr, E. 1925. Arch. Inst. Past., Tunis, 14, 355. Yellow Fever Frasey, V., PeErriT, A., and STEFANOPOULO, G. 1930. Rev. méd. [frangaise, 11, 361. Hixpre, E. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 449. 214 VACCINES AND SERA IN VIRUS DISEASES Marcnoux, E., and Smvonp, P. L. 1906. Ann. Inst. Past., 20, 16, 104, 161. PerTIiT, A., STEFANOPOULO, G., and Frasey, V. 1928. C. R. Soc. Biol., Paris, 99, 1114. Dengue Fever CLELAND, J.B. 1930. “ A System of Bacteriology,” 7, 376. London. Simmons, J. S. S., St. Joun, J. H., and REvyNorps, F. H. K. 1931. Philipp. J. Sci., 44, 1. CHAPTER XIX THE EMPLOYMENT OF VACCINES AND SERA IN VETERINARY PRACTICE INTRODUCTORY SwiNE FEVER SWINE ERYSIPELAS CATTLE PLAGUE v . . HEMORRHAGIC SEPTICEMIA OF CATTLE . FooT-AND-MOUTH DISEASE ANTHRAX BLACKLEG ’ ’ ; BovINE PLEURO-PNEUMONIA . TETANUS ’ " . AFRICAN HORSE SICKNESS Doc DISTEMPER CANINE JAUNDICE. LamB DYSENTERY : . : = Braxy Sueep Pox . ' 2 . HEMORRHAGIC SEPTICEMIA OF SHEEP Fowr CHOLERA FowL Pox . ’ . THE STANDARDIZATION OF VETERINARY BIOLOGICAL Probucrs . Introductory PAGE 215 217 220 222 22¢ 224 226 228 228 229 230 232 234 235 236 236 236 237 237 238 WHEN the material for this book was being prepared for publication the suggestion was made to one of the authors (G. F. P.) that an account of the use of vaccines and sera in veterinary work might be added with advantage. A review of the subject has convinced him that a good deal may be learned from the study of the modes of production and employment of agents that serve for the prophylaxis of animal diseases, although 215 216 VACCINES AND SERA IN VETERINARY PRACTICE veterinary practice obviously differs in several respects from medical practice. Thus, in dealing with animals, methods of immunization are permissible which entail the risk of sometimes causing death in members of the species that happen to possess exceptional susceptibility. A good instance is the widely practised, and, in general, highly effective method of simultaneous inocula- tion, whereby an active immunity is conferred by the living infective agent under the protection given by a simultaneous injection of a specific serum. But in certain diseases, as for example African horse sickness, the balance between protection and infection is occasionally a delicate one owing to individual variations in susceptibility, and thus death may ensue in some of the vaccinated animals. Moreover, in this and other diseases that are prevalent in tropical and semi-tropical countries dangerous parasites such as piroplasms and trypanosomes may be unwittingly introduced along with the virus material, or the immune-serum may be toxic to some of the vaccinated animals owing to the production of isolysins during the immunizing process, or again a dormant infection in the recipient may be reawakened by the febrile reaction which follows the injections. Risks of this kind are held to be justifiable in veterinary practice by the economic gain which accrues in the aggregate; they are naturally never incurred where human lives are concerned. It is proposed to discuss here, with respect to each disease, the steps that may be taken to combat it with the aid of a vaccine, or a specific serum, or the method of simultaneous inoculation with the living virus or bacterium and the specific serum. The reader who is unfamiliar with the subject should note that in veterinary practice the use of specific sera is confined largely to their prophylactic effect; a curative action is seldom in question and is thus of secondary importance at the present stage of veterinary science. Another point of difference is that the veterinarian has the advantage over his medical colleagues in being able to employ the animal species which is subject to a particular infection as the producer of the serum, and also as the test animal for the purpose of estimating its potency. The numerical records of veterinary experience with vaccines SWINE FEVER 217 and sera are less suited to the application of statistical methods than the corresponding medical data, and, moreover, a good deal of the published information is widely scattered, and must be searched for in Governmental and Colonial Reports that are not readily accessible. The aim of the writer has not been deflected by these considerations, for he merely desires to give to the medical reader who is interested in the vaccine and serum treat- ment of disease a simple outline which will enable him to draw a comparison between medical and veterinary practice. Swine Fever (Peste porcine : Virusschweinepest) Swine fever is an infectious disease of pigs which is caused by a filterable virus. In the acute form of the disease the symptoms and lesions are those of a haemorrhagic septicemia, and in the chronic form a secondary necrotic process gives rise to the elevated ulcerated patches—the characteristic * buttons ”—in the large intestine. These are probably caused by a secondary infection with Salmonella suipestifer ; in a similar way, Pasteurella suiseptica is often present in the pneumonic lesions of swine fever. In the year 1903 de Schweinitz and Dorset proved that the disease could be transmitted by means of the filtered bacteria-free serum of a diseased pig. The virus is not transmissible to any other species of animal. As early as the year 1898 Preisz showed that a considerable proportion of pigs that had been exposed to the natural infection could be protected if they were inoculated with serum obtained from animals which had recently recovered. The protective methods that are now employed include the use of a hyper- immune serum ; this is obtained by giving serial doses of virulent material to animals that are already immune, and by bleeding them at appropriate intervals. There are three methods of employment of the serum : (1) it may be given alone to produce a temporary passive immunity ; (2) it may be administered together with a dose of the virus—the method of simultaneous inoculation ; and (3) it may be given as a curative agent, 218 VACCINES AND SERA IN VETERINARY PRACTICE The serum alone may be given to healthy contact > animals on infected premises, but the resulting immunity lasts, on the average, for only 14 days. In the opinion of Gerlach (1931) a spontaneously acquired active immunity from a mild or unrecog- nized infection contracted under the guard of the serum undoubtedly occurs, but he regards such an occurrence as infrequent. Uhlenhuth and his collaborators (1928) share this view. The method of simultaneous inoculation is the one that is the most extensively practised at the present day, as in the United States and in Germany. The discussion on the subject at the International Veterinary Congress in London in 1930 made it clear that, in the opinion of competent authorities, the value of the simultaneous method is indisputable. The immunity it produces is a lasting one. Dorset referred to the experience in the United States and mentioned that the method has been used exclusively for more than 15 years; that one million litres of the serum are now produced annually ; that 20 million pigs are protected in this way every year; and that the losses had declined in 1929 to 3 per cent., in spite of less stringent traffic regulations. In his view a full dose of active virus and a liberal dose of serum should be given on opposite sides of the body ; the excess of serum does not tend to shorten the immunity. Nusshag believes that the method yields less satisfactory results when applied to sucking pigs, but Gerlach recommends the inoculation of all healthy contacts, including the sucklings and the pregnant sows. There is general agreement concerning the advisability of choosing for the immunizing process a standard virus, which is maintained at a constant level of virulence under expert super- vision, rather than the strain of virus which is responsible for the localized outbreak that happens to be under observation. This raises the question whether local strains may not fall into different specific groups. Fortunately, from the practical point of view, there is little evidence for a plurality of viruses in swine fever, although the East African and Argentine strains apparently resist the action of alien hyperimmune sera. The explanation which is preferred at the moment favours the theory of differences in SWINE FEVER 219 virulence and not that of specific variations ; further research is needed in order to elucidate the interrelationship of the various strains. The serum has a definitely curative action when it is adminis- tered to animals in the early febrile stage of the illness. Hutyra and Koves state that it will protect up to six days after an artificial infection. When large doses are given intravenously, 50-75 per cent. of the animals are saved and acquire an active immunity (Uhlenhuth, Miessner, and Geiger, 1928). The diagnosis of swine fever, and especially of its chronic and latent forms, has an obvious bearing on the successful employment of serum therapy in the disease, and it is significant that at the recent Congress in London the discussion centred round the co-related topics of diagnosis and inoculation. In the United States and Germany the preparation of the virus and of the hyperimmune serum is under governmental control. A highly protective serum is obtainable in no other animal than the pig; the risk that is associated with the introduction of foreign protein into the horse, for example, is one of the causes why a potent serum cannot be obtained from this animal. Dorset uses pigs three months after they have undergone simultaneous inoculation, and Uhlenhuth employs either naturally or artificially immunized pigs. Virulent blood or filtered urine or serum or organ extracts may be used, and given either subcutaneously or intravenously in the immunizing process. A good account of the technical details will be found in the article by Uhlenhuth, Miessner, and Geiger (1928). There is no standard method of titration of the serum. According to Eberson the antiviral body is present in the globulin and not in the albumin fraction, so that the serum can be concentrated if desired. In the immune-sera investigated by him the pseudoglobulin formed only a very small part of the total globulins; most of the protective antibody is, therefore, present in the euglobulin fraction. In conclusion it should be noted that S. suipestifer and P. suiseptica are unable to initiate and maintain the epizootic spread of swine fever, and that accordingly immune-sera prepared by the use of cultures of these bacteria or their products cannot control 220 VACCINES AND SERA IN VETERINARY PRACTICE the epizootic, however beneficial they may be as therapeutic agents. The belief is gaining ground that S. suipestifer, when it is present as the sole infective agent, may cause considerable morbidity in large piggeries. There does not, however, seem to be any easily available evidence that will permit an estimate to be formed regarding the efficacy of a specific serum against S. suipestifer. Swine Erysipelas (Rouget : Rotlauf) Swine erysipelas is an infectious disease which is prevalent in Great Britain and on the Continent of Europe, but which is less familiar to veterinarians in the United States of America. The clinical forms include: (1) an acute bacterieemia with a high fatality-rate ; (2) a subacute, urticarial form (‘diamonds ”); and (8) chronic forms which may show a characteristic vegetative endocarditis affecting chiefly the mitral valve of the heart. The causal agent is Bacillus erysipelatis suis (Erysipelothriz rhusio- pathie), a Gram-positive, non-motile, non-spore-bearing organism with notable powers of survival in saprophytic conditions. A specific serum can be prepared in horses or cattle or buffaloes by the intravenous injection of living cultures of graduated virulence ; these animals possess a natural resistance to the infection, but they are nevertheless subject to considerable reactions in the course of the immunization. In Germany the titration of the serum is carried out in mice and a simultaneous test is made with a standard serum issued by the State Control Institute ; in France the pigeon is used as the test animal. The mode of action of the serum as a protective agent is not fully understood. In the natural infection the bacilli are often found to be ingested by the leucocytes, and yet proof is wanting that opsonins play a part in the mechanism of immunity. The potency of the serum is well retained if it is kept in suitable conditions of storage at a low temperature. According to Ruppel and Ornstein the protective antibody is associated with the pseudoglobulin fraction of the serum proteins. SWINE ERYSIPELAS 221 The serum may be used : (1) to confer a temporary immunity when circumstances render this desirable ; (2) as a curative agent ; and (3) together with the culture in order to produce an active immunity—the so-called sero-vaccination. In accordance with the general experience a passively conferred immunity lasts for only a short time—from 10 to 15 days. This method has a limited application and is seldom employed on the Continent, but it is used at the present time in England (Edwards, 1931). The serum gives excellent results when it is administered to diseased pigs, and, according to experience in Germany, is able to save 80 or 90 per cent. of the animals (cited by Preisz). The method of sero-vaccination has proved to be a very satisfactory one. A freshly prepared mixture of the serum and culture is administered, and this is followed a fortnight later by a dose of culture alone. The resulting immunity lasts from four to five months. The reader who desires more detailed information is advised to consult the article by Edwards (1931) and by Preisz (1928). The bacillus of swine erysipelas occasionally gives rise to infections in species other than the pig. Thus it has caused extensive epizootics in field-mice in California; outbreaks of polyarthritis in lambs in England and in Europe ; and outbreaks among various species of birds, for example, fowls, ducks, and pigeons. In a bacterieemic fowl disease of this nature which was reported from Prague, an anti-swine-erysipelas serum, when used for prophylaxis and treatment, was decidedly effective (cited by Edwards). Man also may become infected when brought into contact with the disease in swine. The recorded cases are mostly from Germany, and the patients were butchers, veterinary surgeons, and laboratory attendants. The disease in man is known as * erysipeloid,” and it may run an acute or chronic course. The entry of the infective agent is usually by skin wounds on the hands, but may be by way of the digestive tract from the ingestion of diseased meat. There is good evidence that the specific serum against swine erysipelas exerts a strikingly favourable action in these cases, and that it prevents generalization of the infection. 222 VACCINES AND SERA IN VETERINARY PRACTICE Cattle Plague (Peste bovine : Rinderpest) Cattle plague is an acute, contagious, and often fatal disease which is chiefly incident upon cattle, although in some parts of the world buffaloes and wild ruminants are liable to the infection. The smaller experimental animals and man are immune to the disease. The symptoms are those of an acute septicemia, and the infective agent is considered to belong to the group of filter- passing viruses. There is reason to believe that the virus is closely associated with the leucocytes of the blood and that it may indeed reside within them. The protective action of the serum of cattle that have recovered from the disease is an old observation which has been amply verified. The potency of convalescent serum is not high, but it is easy to prepare a hyperimmune serum. The early investigators "gave considerable and repeated doses of virulent blood to immune animals ; but it was found later that a single massive dose was sufficient to produce a strongly protective serum. When working at Muktesar in India, Edwards showed that a potent serum was procurable as the result of a single injection, without reinforcing doses, on condition that the animal manifested a mild but undoubted reaction. The modes of employment of the hyperimmune serum are precisely similar to those which are used for controlling swine fever and foot-and-mouth disease. The simultaneous method, whereby a durable immunity is produced by the injection at the same time of virulent material and a sufficient dose of serum to modify the attack, is the one that is most often used. In Nigeria, Egypt, and India the results have been satisfactory, and according to Kearney it is the best method at the present time for suppressing cattle plague in countries in which the disease is indigenous. Certain disad- vantages attach to it, but they can be avoided. Thus there is a risk of conveying protozoal infections in the virulent blood. The transmission of pathogenic trypanosomes is prevented by adding a small quantity of tartar emetic to the citrate solution into CATTLE PLAGUE 223 which the virus-containing blood is drawn (Kearney). Where tick-borne diseases are prevalent, the virus-producers must be kept free from ticks from the time of birth ; Walker’s experience in Kenya has shown that there is no difficulty in rearing virus- makers that are “ clean ” in this respect. Further, sucking calves are not easily immunized, although, according to the experiments of Edwards, the immunity, after a symptomless initial reaction, lasts to a useful degree for two years. For the control of the disease in India this author recommends the widespread employ- ment of the simultaneous method. He suggests the use of a virus which has been adapted to goats after passage through the rabbit, and which has been prepared at the site of the outbreak ; by this means protozoal contaminations are avoided. It is possible that in the near future serum prophylaxis will be superseded by the employment of suitable vaccines prepared from virulent material, but the evidence for their value, although promising, is not yet conclusive. The curative action of the serum is slight, so that beneficial results are not likely to be obtained unless very large doses are given at the earliest possible moment after the appearance of the symptoms. Hartley (1914) investigated the distribution of the antiviral body in the serum, and found that it was associated chiefly with the euglobulin fraction of the serum proteins and that it could be separated by dialysis. The active fraction of the serum does not seem to possess any marked viricidal action when tested in vitro. For further details concerning the use of cattle plague serum the reader should consult the articles by C. Todd (1930), Kearney (1931), and Curasson (1931), together with the discussion on the subject that is reported in the Proceedings of the International Veterinary Congress (1931). Hamorrhagic Septiceemia of Cattle (Pasteurellose bovine : Hdamorrhagische Septikimie der Rinder) The Pasteurelloses are met with as epizootic diseases, and they affect diverse species of animals, among which, from the point of 224 VACCINES AND SERA IN VETERINARY PRACTICE view of the veterinary practitioner, cattle, fowls, and swine are the most important representatives. A good account of the bacteriology of the Pasteurella group and of the interrelation of the strains that are peculiar to each animal species is given by Schiitze (1929). This author concludes from his survey of the subject that the Pasteurellas should be regarded as a collection of essentially identical strains, although each has become adapted to its particular host. This group of diseases is sometimes spoken of as that of the haemorrhagic septiceemias, a term which is expres- sive of two of its chief characteristics. Animals are not easily vaccinated against any member of the Pasteurella group, nor are the results of passive immunization always satisfactory. P. boviseptica, the causal agent of hemorrhagic septicemia of cattle, is probably the same bacillus as that which is found in « harbone,’’ a similar disease of the buffalo. Information on the serum treatment of this disease of cattle has come mostly from India, where the losses from the natural infection may be severe. Holmes obtained a specific serum by immunizing cattle and buffaloes by the simultaneous method. The serum confers a passive immunity which protects at least 90 per cent. of the animals for a period of about four weeks. Foot-and-mouth Disease (Fiévre aphtheuse : Maul- und Klauenseuche) Foot-and-mouth disease is a highly contagious febrile disease of cattle, pigs, sheep, goats, and other ungulates, for example, reindeer and camels, which is characterized by the formation of vesicles in the mouth and on the feet, and is caused by a filterable virus; this discovery was made in the year 1897 by Loeffler and Frosch. A great deal of work on the virus has been done of late years in England and on the Continent; and the investigations have been signally furthered by the use of the guinea-pig as the experimental animal. The susceptibility of the guinea-pig to the virus and its value as a test animal were demonstrated by Waldmann and Pape (1920, 1921). In Great Britain and the United States outbreaks of foot-and- FOOT-AND-MOUTH DISEASE 225 mouth disease are kept under control by strict measures for slaughtering affected animals and their immediate contacts ; but in France, Germany and Italy there are no such measures in force, and consequently opportunities have been afforded in these countries for testing the value of specific prophylactic and curative agents. An active immunity against the virus can be produced by the inoculation of a formolized vaccine prepared from the tissues or serum of infected animals, but this method has not passed beyond the experimental stage. The serum obtained from convalescent cattle or from those which have recovered and have had their immunity increased by the injection of virulent material will passively protect susceptible animals for a period of about a fortnight. Some years ago in Germany convalescent serum from cattle was given in amounts of 1 c.cm. per kgm. of body-weight to animals which were ill and to those which had a temperature, although they showed no other signs of the disease. The serum was given to animals in the epizootic area which were healthy and without fever, and at the same time they were vaccinated by rubbing infective saliva into the mucous membrane of the mouth. A decided disadvantage attaching to the use of convalescent serum is the circumstance that samples from different animals show wide variations in their protective value. Thus, in 1926, Waldmann and Trautwein (1928), on account of a scarcity of hyperimmune serum, prepared and tested 6,000 litres of convalescent serum, and found that some of the individual samples were so weakly active as to be valueless ; convalescent sera possess as a rule a titre which is six to twenty times weaker than hyperimmune sera. Another difficulty that is encountered in the use of convalescent sera is the fact that at least three types of virus are associated with foot-and-mouth disease, and that, as shown by cross- immunity tests in guinea-pigs, swine and cattle, these types arc immunologically distinct. The method of hyperimmunization which is carried out at the present day in Germany by Waldmann is based on that which was originally devised by Loeffler; it takes the form of the administration to cattle, possessing a basal immunity, of serial R.A. VACCINES, 8 226 VACCINES AND SERA IN VETERINARY PRACTICE subcutaneous doses of centrifuged and unfiltered lymph derived from pigs that are infected for the purpose. The serum is a trivalent one, and is tested in guinea-pigs for antibody content in the following way. Vesicles are produced on the sole of the foot by means of intracutancous inoculation with the virus ; these are normally followed by generalized lesions. The titration of the serum is effected by ascertaining the dose of it which, when given subcutaneously, will prevent generalization of the infection. The hyperimmune serum has a threefold application for the limitation of outbreaks of the disease. It may be given as a prophylactic to healthy animals in a dose of 20 c.cm. per 112 pounds of body-weight, or as a curative agent to affected animals, or it may be employed simultancously with the living virus as a means of producing a sustained immunity. The protective action of the serum, when it is given alone, is of brief duration—about a fortnight—and it is asserted, especially in Germany, that, as a rapidly acting and convenient prophylactic, the serum has proved its usefulness in circumstances where the degree of concentration of the infection is not very great; its effects are, however, not easily disentangled from those which are attributable to the energetic preventive measures, such as segregation, disinfection, and passive protection of the less immediate contacts, that always accompany serum prophylaxis. The serum modifies the symptoms when it is given in large doses as soon as possible after the signs of infection become manifest. At the recent Congress in London Cominotti reported in very favourable terms upon his experience in Italy of the use of a hyperimmune serum. He mentioned that the intravenous method of giving it was particularly effective in animals which were in the septiceemic stage of the disease—that is, before the appearance of the local lesions. Anthrax (Charbon : Milzbrand) An active immunity can be produced in susceptible animals by the use of vaccines prepared according to the Pasteurian methods, ANTHRAX 227 A vaccine, which consists solely of spores, has been employed since the year 1920, especially in Russia ; it has also been given an extensive trial in Australia, South Africa, North America, and Japan. Excellent results with this vaccine have been reported from South Africa by Viljoen, Curson, and Fourie (1928), who give a detailed account of the method of preparing it and of using it under field conditions. Great care must be taken in choosing strains that have been properly attenuated and that possess a high immunizing value. The authors experienced no difficulty in producing a reliable spore vaccine for cattle and sheep. A suitable vaccine for equines is made from specially selected strains ; these yield a vaccine which is a good deal milder than the vaccine for cattle and sheep; goats require a still weaker vaccine. The advantages of the spore vaccine are that the content of living organisms remains fairly uniform for many months, and that deterioration on keeping affects the resistant spore form of the anthrax bacillus to a less degree than the vegetative form. The immunity conferred by the spore vaccine lasts for about 12 months. The simultaneous injection of vaccine and specific serum— sero-vaccination—has the advantage over the purely passive immunity conferred by anti-anthrax serum of inducing a solid and lasting immunity. The injections are made subcutaneously at sites that are as distant as possible from each other. This method has been applied to a small extent for the protection of cattle, sheep, horses, and swine with good results. If the dose should be repeated the homologous serum, that is, a serum which has been prepared in the same species, ought to be used, in order to avoid the anaphylactic effects that may otherwise follow. When immediate protection is required for animals that are exposed to the risk of contracting the disease, injections of serum, when given alone, have proved their value. The immunity appears to last longer than might be expected—sometimes for as long as two or three months (Sobernheim). The serum is also capable of acting as a curative agent; in severe cases large doses should be given intravenously. There is evidence that the animals can be saved even when a bacterieemia 8—2 228 VACCINES AND SERA IN VETERINARY PRACTICE is present. It may be stated, however, that the serum is not much used in practice, because the affected animals succumb so rapidly that the diagnosis is generally made post mortem. The article by Sobernheim (1929) should be consulted for further details. Blackleg (Charbon symptomatique : Rauschbrand) The serum treatment of this disease of young cattle occupies a secondary place as compared with the prophylactic methods that have been employed to limit its ravages. Recent work by Leclainche and Vallée has provided a safe and éfficient vaccine by making use of formolized cultures of the causal bacterium, B. chauwvei (Bosworth, 1931). There appears to be no special difficulty in preparing either an antitoxic serum or an antibacterial serum for prophylactic or curative use, but in practice such sera are not often employed. Experimental work has shown that cattle with well-marked local lesions resulting from the injection of virulent cultures of B. chauvei quickly recover when they are treated with a large dose of the specific serum, thus averting a certainly fatal termination. Bovine Pleuro-pneumonia (Peripneumonie contagieuse : Lungenseuche der Rinder) Pleuro-pneumonia is a contagious disease which is met with only in bovines, and is caused by a highly pleomorphic micro-organism, of which the smallest forms are capable of passing through candle filters ; the systematic position of the causal agent is still obscure. The lesions that result from the infection are sufficiently indicated in the name that is given to the disease. As a rule, only one lung is affected, and in acute cases the inflamed portion, which is round and firm, is either of small size or invades almost the whole of a lobe. According to Walker (1930), the Arabs and Moors of North Africa have for centuries practised a method of preventive inocula- TETANUS 229 tion which consists in taking pleural lymph from an infected animal and rubbing it into the scarified skin of the forehead of the animals that are to be protected. Even before the Pasteurian epoch a method of vaccinating cattle by the insertion at the root of the tail of virulent lymph was extensively used with good results (Willems, 1852). The specific organism can be readily cultivated in serum broth, and continued subcultivations have the effect of attenuating its virulence. Attenuated cultures are now being utilized for the purpose of conferring an active immunity upon susceptible herds. The vaccine is injected at the usual site, namely, at the root of the tail ; it may cause a local lesion, but there is no risk to life. This method of prophylaxis is employed in East Africa, South Africa, and in Australia. Tetanus (Tétanos : Tetanus (der Starrkrampf’) ) The horse is the species that is most susceptible to tetanus ; cattle, sheep, and goats are less often attacked. The disease may follow such operations as castration, spaying, and docking, and it is known that the infection is more common in some districts than in others, and even in certain farms in these districts. The prophylactic value of tetanus antitoxin in veterinary practice is undoubted. It should be given before surgical operations are undertaken, and when wounds of any kind have become contaminated with earth or dung-soiled bedding. The dose should not be less than 3,000 International units (1,500 U.S.A. units), and it may be increased or repeated according to the severity and degree of infection of the wound. The effect of the serum is short-lived, and it is worth noting that a more durable immunity can be produced by immunizing the animal by means of a formolized toxin (*‘ anatoxine ”). The present writer is able from personal experience to attest the value of active immunization in protecting horses that are under treatment for the production of therapeutic sera. When admitted to the stables each horse receives an intramuscular dose of 50 c.cm. of tetanus toxoid to which 2 per cent. of alum has been added ; two or three months later the dose is repeated, but with 230 VACCINES AND SERA IN VETERINARY PRACTICE 0-1 per cent. of alum added ; thereafter a dose is given every six months. The Therapeutic Substances Regulations (1931) enjoin on manufacturers of sera that they shall arrange for the horses to be actively or passively immunized in order that enough tetanus antitoxin may be present in the blood to protect them during the whole period of their use as a source of serum. When the symptoms of tetanus have appeared the likelihood of benefit ensuing from the administration of the antitoxin is small, especially in the horse, which is the most susceptible of all animals to the effects of the toxin, as is apparent when the lethal dose of toxin is calculated per gramme of body-weight. Notwith- standing, a large dose of antitoxin should be given intravenously with the least possible delay : not less than 100,000 International units. Jensen analysed the information relating to 541 horses, and found that there was a reduction in the mortality when large doses of antitoxin had been given intravenously. Magnesium sulphate has been recommended as a method of treatment or as an adjuvant to the action of the antitoxin, but although there is no doubt regarding its antispasmodic action in tetanus, it probably does no more than mask the symptoms without influencing the course of the disease, African Horse Sickness (La peste du cheval : Pferdesterbe) African horse sickness is a sporadic or enzootic or, on occasion, epizootic disease with a high death-rate, which is caused by a filterable virus. The mule is less susceptible to the infection than the horse, and the ass is highly resistant. The rainfall is the dominant factor in the spread of the disease, but the exact mode of its transmission in nature is still unknown. There are four clinical forms : (1) the horse sickness fever ; (2) the pulmonary form (known in South Africa as Dunkop) ; (3) the anasarcous or cardiac form (Dikkop); and (4) the mixed form. Fdematous effusions into the pleural and pericardial cavities are the chief signs of the disease post mortem. Animals that have recovered from the natural disease possess AFRICAN HORSE SICKNESS 231 considerable immunity, and are spoken of as * salted.” Never- theless, a breakdown in the immunity may follow a subsequent natural or experimental infection. The work that has been done on the subject by Theiler and his co-workers shows that the degree of immunity is a variable one, and that it is relative to the virulence of the test strain of virus and the dose of it that has been administered. The outcome of an experimental test is not therefore easily predictable ; details will be found in the article by Theiler (1930). The serum of horses, mules, and asses that have recovered has no protective action, but when these animals are hyperimmunized their serum acquires antiviral properties. The immunization of horses, according to Theiler’s method, consists in the intravenous injection of the virus together with the requisite amount of a hyperimmune serum-—not less than 0-5 c.cm. per kgm. of body- weight—and of another intravenous injection of virus and serum-— not less than 0-75 c.em. of serum per kgm. of body-weight after an interval of a few days; two strains of virus are used. Theiler states that the deaths from the inoculations amount to 3 per cent. ; this is considered to be a satisfactory result. The steps that are taken to produce a hyperimmune serum are as follows. Horses are first rendered immune, and then they are hyper immunized by the transfusion of about 10 litres of virulent blood from a sick animal: an amount that may represent 10,000,000 minimal lethal doses. The serum from 4 or 5 per cent. of the horses becomes haemolytic from the production of isolysins. However, the danger of toxic symptoms from the inoculation of an isolytic serum can be averted by testing in vitro the serum of each horse for the presence of a hemolysin. Attempts have recently been made at the Onderste Poort Laboratory near Pretoria by Du Toit and Alexander (1930) to immunize horses by means of a formolized virus ; the spleen pulp of an infected animal is the basis of the immunizing preparations. They conclude that it is possible to produce a solid immunity by the subcutaneous injection of preparations of virulent spleen which have been submitted to progressively lower concentrations of formalin. 232 VACCINES AND SERA IN VETERINARY PRACTICE The precise conditions for the inactivation of the virus and for the retention of the immunizing properties of the product have not yet been determined. Dog Distemper (Maladie du jeune dge des chiens : Hundestaupe) The investigations that have been carried out during the past few years, and, in particular, the experimental work of Laidlaw and Dunkin, have brought convincing proof that the essential causal agent of canine distemper is a filter-passing virus. In spite of the early work of Carré (1905), which furnished evidence for this view, the associated bacterial invader, B. bronchisepticus, was for long looked upon as the primary cause of the infection. Other bacteria, for example, streptococci, may also play a secondary part, but, in the opinion of Laidlaw, dogs that have been artificially immunized against the virus are resistant to bacterial infections of this kind, and will remain healthy, even when they are kept in contact with naturally infected dogs. The problem of the prophylaxis and treatment of distemper by means of a specific vaccine or serum is simplified in practice by the circumstance that, so far as is known, only one strain of virus exists. ’ The high degree of natural immunity which follows an attack of the disease suggests that a vaccine, when suitably prepared from experimental material and with an adequate virus content, can be successfully utilized for the conferment of an active immunity ; the evidence in favour of the value of such a vaccine is conclusive. Laidlaw and Dunkin have prepared a vaccine by making a 20 per cent. suspension in saline of the organs of experi- mentally infected dogs and by adding to this 0-1 per cent. of formaldehyde. A dose of this vaccine is followed a week later by a small dose of living virus in the form of a suspension of the spleen of an infected ferret. Attempts had been made to produce a specific serum, but it is doubtful whether a highly potent product was obtainable until the careful work of Laidlaw and Dunkin revealed not only the DOG DISTEMPER 233 best immunizing procedure but also a method for concentrating the antiviral body and methods for testing the potency of the serum. The following account briefly summarizes their work. Dogs that have been artificially immunized are kept for at least a month after the subsidence of the symptoms. Twenty c.cm. of a 20 per cent. suspension of a potent dog virus are then injected subcutaneously or intramuscularly, and the same dose is injected next day into the opposite side of the body. The maximal antibody response to this treatment is on or about the seventh day, and the animal is then bled out under full anwxsthesia. The method of concentrating the antibody follows the procedure that has been described by Felton for the separation of the protective fraction in anti-pneumococcus serum. This portion of the serum proteins is a globulin which is insoluble in water, has an isoelectric point of about pH 5-8, and is soluble in weak acid or alkali and in water which has been saturated with carbon dioxide. The bulk of the distemper antibody is associated with this fraction. The complement-fixing property of the serum appears to reside in the fraction which contains the protective antibody ; it is probable that the technique of complement fixation affords a measure of potency. The serum may be titrated on compound- bred puppies of the same litter from a stock which has been free from distemper for five or six generations; it is given simultaneously with the virus or as late even as three days after the infecting dose. The results of attempts to titrate the serum by using ferrets have been disappointing, but at least the experi- ments have shown that 0-1 c.em. of a good serum is able to neutralize 10,000 infective doses of the virus. The hyperimmune serum or the purified antibody may be used, as has already been described for similar sera, in three ways : (1) to confer temporary protection; (2) to confer a lasting pro- tection by utilizing the simultaneous method ; and (3) to treat sick animals. The first method may be of value in certain circum- stances, when only a brief protection is needed; the passive immunity begins to disappear after nine days. The second method 234 VACCINES AND SERA IN VETERINARY PRACTICE confers a solid and durable immunity, and a point of interest in the technique is that, as with swine fever, the administration of a considerable excess of the specific serum does not interfere with the process of immunity. The curative effect of the serum is manifest when it is given at any stage in the incubation period and at the time of the primary rise of temperature. Laidlaw and Dunkin adduce instances which suggest that potent sera will prove to be efficacious even in the later stages of the disease. When a rapid effect is desired, the serum should be injected intravenously. Canine Jaundice (La jaunisse des chiens : die Gelbsucht des Hundes) Canine jaundice (yellows) is prevalent in all parts of Great Britain, and is especially incident upon young sporting dogs in foxhound and breeding kennels. Apart from the mild cases the mortality is a high one; in the experience of Okell, Dalling, and Pugh (1925), who were the first to demonstrate the leptospiral nature of the infection, at least 95 per cent. of the affected animals die. The following notes are abstracted from the published work of these authors. The canine strain of leptospira agrees in all respects with the rat strain of Leptospira icterohemorrhagice, and it is almost certain that dogs are infected from contamination of their food and bedding by rats; virulent leptospiras are eliminated in the urine of a considerable proportion of healthy rats. A preventive and curative serum can be prepared by immunizing the horse with rat strains of the leptospira ; this is the method which is used for obtaining the specific serum for the treatment of spirochatal jaundice or Weil's disease, the analogous infection in man. There is evidence that the serum is effective as a prophylactic when it is given to dogs that are exposed to the natural infection, but the protection does not last for more than a, few weeks. Laboratory tests have proved that dogs, when they are infected with a dose which represents many minimal lethal doses, can be saved by administering the serum intraperitoneally as late as the fourth day after infection. Field trials show that LAMB DYSENTERY 235 the serum, when given early in the disease, has a curative action ; this may take place, even when jaundice has appeared. Lamb Dysentery Lamb dysentery is a serious intestinal disease which attacks lambs within a few days of their birth. In Great Britain it is endemic in Northern England and Southern Scotland. The etiology of the disease is still obscure. The work of Gaiger and Dalling (1923) led them to believe that the symptoms could be ascribed to the joint pathogenic action of an anaerobe of the B. welchii group and of a coliform bacillus. Dalling and his colleagues showed later that a prophylactic dose of B. welchii antitoxin, when administered to young lambs, diminishes the fatality from about 20 per cent. to about 1 per cent. A similar disease affecting sheep on the Romney Marsh in Kent has been investigated by McEwen, who has isolated an anaerobe, B. paludis, which has cultural and serological affinities with B. welchii and the anaerobe of lamb dysentery. Passive immunization of the lambs by means of serum is not always feasible in the conditions of practice in the field, and the active immunization of the mothers has been shown to be a simpler and an equally effective measure. The reader will find details of the method and an account of the results in Chapter XXV. (page 442); it may suffice to mention that the work has been carried out chiefly by Dalling and his collaborators, who now use a formol-culture as the immunizing agent. A dose of the vaccine is given to the ewes in the autumn and a second dose in the spring a week or two before parturition. The immunization of the lambs takes place through the medium of the colostrum and the immunity, although a transient one, is sufficient to prevent them acquiring the disease. The lack of agreement concerning its causation has stood in the way of preparing an effective specific serum. Thus Bosworth (1928) doubts whether the action of any one of the bacteria hitherto described or any combination of them affords a complete explanation of the etiology of the disease. 236 VACCINES AND SERA IN VETERINARY PRACTICE Braxy (Bradsot : Bradsot) A brief account of this disease of young sheep and its preven- tion is given in Chapter XXV. (page 445), so that we may merely note here that the infective agent is believed to be Vibrion septique, a well-known member of the group of anwmrobes, and that good results seem to have been obtained in prophylaxis by the use of formolized cultures. Sheep Pox (Clavelée : Schafpocken) Sheep pox is an infectious epizootic disease characterized by a papulo-vesicular eruption not only of the skin but of the mucous membrane of the respiratory tract and genitalia. It has some resemblance to human smallpox and may occasion a considerable mortality—from 10 to 50 per cent. As with all the animal pock diseases the causal agent is a filterable virus. A general account of it has been given recently by Blaxall (1930) and by Bridré (1931). The immunity conferred by a natural attack is a persistent one, but it may disappear after some years. It has long been known that the serum of sheep that have recovered from an attack has a preventive and curative action. Immune sheep can be hyper- immunized with the virus, and the serum thus obtained exerts a decided prophylactic effect on animals that are exposed to the natural infection ; the immunity lasts from two to three weeks. The serum has a curative action when it is administered soon after the symptoms make their appearance. Haemorrhagic Septiceemia of Sheep (Pasteurellose ovine : Himorrhagische Septikimie der Schafe) This disease is caused by a micro-organism of the Pasteurella group (P. oviseptica) and it takes the form of a pneumo-enteritis. The experimental evidence shows that rabbits can be protected by the use of a specific serum, and experience under field conditions FOWL CHOLERA: FOWL POX 237 appears to be in favour of serum treatment or a simultaneous method which combines both active and passive immunity. Fowl Cholera (Choléra des poules : Gefliigelcholera) The causal agent (Pasteurella aviseptica) has been studied for many years ; Pasteur isolated it in the year 1880 for the first time, and attempted to control the disease by using as a vaccine cultures that had been attenuated by passage through rabbits. A full account of the characters of this bacterium will be found in the monograph by Manninger (1928). Fowl cholera serum is prepared from horses or cattle, which are immunized either subcutaneously or intravenously with increasing doses of broth cultures or agar suspensions, after a temporary immunity has been conferred on them by means of the specific serum. In Germany the serum is subject to State control, and the white mouse is used as the test animal. The mode of action of the serum is not quite clear, but it probably includes both extracellular lysis and phagocytosis. The serum, when used for the control of an outbreak, has a curative and protective action, although the passive immunity lasts only from one to four weeks. A simultaneous method which combines both active and passive immunity has been tried, but, in Manninger’s opinion, very irregular results have been obtained. The reason for this is to be found in the difficulty of estimating the relative dosage of vaccine and serum ; an excess of antibody interferes with the production of an active immunity. Fowl Pox (Variole aviare : Gefliigelpocken) Fowl pox is a contagious disease caused by a filterable virus, and is characterized by ‘* eruptive lesions on the unfeathered parts of the head, by yellow cheesy membranes in the mouth, and by an oculo-nasal discharge (Doyle, 1931). It causes serious losses in the poultry industry everywhere. Recent articles by Doyle 238 VACCINES AND SERA IN VETERINARY PRACTICE (1931) and by Findlay (19380) should be consulted for further details. Birds that have recovered from an attack acquire a considerable immunity, although the serum has but little antiviral action. Dalling, Allen and Mason (1925), and Findlay (1928) have succeeded in hyperimmunizing fowls and thus obtaining an antiviral serum, which has been used prophylactically and curatively by the former authors with good results. Others have been less fortunate, and Doyle, for example, states that he has made many attempts to produce a protective serum by hyperimmunizing fowls, but that in no instance did the serum confer any appreciable protection against either a natural or an artificial infection. Recent work indicates that the key to the successful prophylaxis of fowl pox is to be found in the relationship between this disease and pigeon pox. Pigeons possess a high degree of resistance to fowl-pox virus, whereas fowls are susceptible to pigeon-pox virus, but the resulting infection produces relatively mild lesions and is rarely fatal. Doyle and Minett (1927) investigated the property of pigeon-pox virus in protecting fowls against fowl-pox virus. An extension of this work by Doyle (1930) led him to conclude that pigeon-pox virus confers upon fowls a solid immunity to the natural infection with fowl pox, and that when this method is employed in conjunction with hygienic measures it is likely to bring about a rapid and substantial reduction in the incidence of the disease. The Standardization of Veterinary Biological Products (Sera, Vaccines, and Diagnostic Agents) This important subject occupied the attention of the Inter- national Veterinary Congress which was held in 1930 in London, and the existing circumstances as regards standardization were reviewed in reports by Eichhorn of New York, Mohler of Washington, and Flueckiger of Berne. All of them urged the need for creating, if possible, an International Body for the purpose of extending the control of prophylactic and therapeutic products STANDARDIZATION OF VETERINARY PRODUCTS 239 for veterinary use, and for pursuing research into the methods of standardizing and titrating these products. Opinion in the Con- gress was unanimous that an organization of this kind should be planned, and at the final session the following resolution was passed :— “That the attention of the :Office International des Epizooties de Paris’ be drawn to the urgent necessity of a prompt consideration of the question of the standardization of biological products.” There is no room for doubting that the control of veterinary pro- ducts according to standards that possess national or, preferably, international authority would further the interests of the stock- owner, the veterinary practitioner, and the community as a whole. The present writer can testify to the great advantages that have accrued from the work of the League of Nations Standardization Commission, so far as the production of serum from the medical aspect is concerned. The information that is given in the articles by Eichhorn and by Mohler conclusively proves the necessity for legislative control of the preparation of veterinary sera, vaccines, and diagnostic agents. REFERENCES Nore. No attempt has been made to furnish a complete list of references under the various subjects because the general accounts that are given in ** A System of Bacteriology,” London ; * Handbuch der pathogenen Mikroorganismen.” (Kolle, Kraus, and Uhlenhuth) ; and Proceedings of the 11th International Veterinary Congress, London, 1931, provide detailed references to recent publications. Swine Fever Dorset, M. 1931. Proc. Internat. Veter. Congress, London, 3, 76. Dorset, M., Geicer, W., et al. 1931. Ibid. 1, 322. Dorset, M., and Houck, U. G. 1931. Farmers’ Bulletin, No. 834, U.S. Dept. Agric. EBerson, F. 1915. J. Inf. Dis., 17, 339. Garroway, I. A. 1930. ** A System of Bacteriology,” London, H.M. Stationery Office, 7, 302. Gricer, W. 1931. Proc. Internat. Veter. Congress, London, 3, 76. Nussmac, W. 1931. Ibid., 3, 95. Skipymore, D. I. 1927. * Comparative Values of Types of Anti-Hog- Cholera Serum,” Circular No. 11, U.S. Dept. Agric. 240 VACCINES AND SERA IN VETERINARY PRACTICE UnreNnurh, P., MiessNEer, H., and GeiGer, W. 1928. < Handb. d. path. Mikroorg.,” 9, 281. Swine Erysipelas Epwarps, J. T. 1931. “A System of Bacteriology,” London, H.M. Stationery Office, 8, 379. Prersz, H. von. 1928. < Handb. d. path. Mikroorg.,”” 6, 449. Cattle Plague ArsrECHT, B. 1927. << Handb. d. path. Mikroorg.,” 9, 29. CurassoN, G., Kearney, W., et al. 1931. Proc. Internat. Veter. Con- gress, London, 1, 413. HarTLEY, P. 1914. Mem. Ind. Civ. Vet. Dept., No. 3. KearNEY, W. 1931. Proc. Internat. Veter. Congress, London, 8, 637. Toop, C. 1930. ‘A System of Bacteriology,” London, H.M. Stationery Office, 7, 284. Pasteurelloses Grisser, K. 1929. < Handb. d. path. Mikroorg.,” 6, 835. Huryra, F. vox. 1928. [Ibid., 6, 483. MANNINGER, R. 1928. [Ibid., 6, 529. Scutrze, H. 1929. “A System of Bacteriology,” London, H.M. Stationery Office, 4, 446. Wasusurn, H. J. 1930. Farmers’ Bulletin, No. 1018, U.S. Dept. Agric. Foot-and-mouth Disease A~NprEWS, W. H., and LieNIi:RES, J., ef al. 1931. Proc. Internat. Veter. Congress, London, 1930, 1, 194. MarrLanp, H. B. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 60. WarbpmanN, O., and TravrweiN, K. 1928. < Handb. d. path. Mikroorg.,” 9, 189. Anthrax Eurich, F. W., and Hewrerr, R. T. 1930. “A System of Bacterio- logy,” London, H.M. Stationery Office, 5, 439. SOBERNHEIM, G. 1929. < Handb. d. path. Mikroorg.,” 8, 1041. Wasnsur~, H. J. 1931. Farmers’ Bulletin, No. 784, U.S. Dept. Agric. ViLJoEN, P. R., CursonN, H. H., and Fourie, P.J. J. 1928. 13th and 14th Reports of the Director Vet. Educ. and Res. Dept. Agric., Union of S. Africa, p. 431. Blackleg Bosworru, T. J. 1931. Proc. Internat. Veter. Congress, London, 3, 199. Monvrer, J. R. 1930. Farmers’ Bulletin, No. 1355, U.S. Dept. Agric. Varig, H., et al. 1931. Proc. Internat. Veter. Congress, 1, 348. REFERENCES 241 Bovine Pleuro-pneumonia WaLker, J. 1930. A System of Bacteriology,” London, H.M. Stationery Office, 7, 322. WiLLeMms, L. 1852. Rec. Méd. vet., 3 S., 9, 401. Tetanus KisLEr, M. 1928. < Handb. d. path. Mikroorg.,” 4, 1027. Fiioes, P. 1929. “A System of Bacteriology,” London, H.M. Stationery Office, 3, 298. African Horse Sickness Du Torr, P. J., and ALEXANDER, R. A. 1930. ‘16th Rep. Director Vet. Serv. and Anim. Industry, Union of S. Africa,” p. 85. Du Torr, P.J., and Nerrz, W. O. 1932. 18th Rep. Director Vet. Serv. and Anim. Industry, Union of S. Africa,” p. 35. TueiLer, A. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 362. Dog Distemper Carri:, H. 1931. Proc. Internat. Veter. Congress, London, 3, 176. Dunkin, G. W. 1931. Ibid., 3, 184. Dunkin, G. W., et al. 1931. Ibid., 1, 335. Eicunor~, A. 1930. J. Amer. Vet. Med. Assoc., 76, 146. Hinz, W. 1931. Proc. Internat. Veter. Congress, London, 3, 157. Lamraw, P.P. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 232. Lambraw, P. P., and Dunkin, G. W. 1931. J. Comp. Path. and Therap., 44, 1. ScuroepER, H. 1927. « Handb. d. path. Mikroorg.,” 9, 145. Canine Jaundice Lawrence, C. J. M., and Okerr, C. C. 1929. Lancet, 2, 327. OkELL, C. C., Daruing, T., and Puen, L. P. 1924. Veter. Record, November 29th. oi " 1925. Veter. Journ., 81, 3. OxkeLL, C. C., Puch, L P., and Darrin, T. 1925. Proc. Roy. Soc. Med., 18, 17. Lamb Dysentery DaLLiNG, T., Bosworti, T. J., et al. 1928. Veter. Record., October 6th. Danning, T., Mason, J. H., and Gorpon, W. S. 1927. J. Comp. Path. and Therapeutics, 40, 217. Hare, T., and GLynN, BE. 1927. J. Path. and Bact., 30, 473. 242 VACCINES AND SERA IN VETERINARY PRACTICE Braxy and Certain Allied Diseases BenneTTs, H. W. 1932. Council for Sci. and Indust. Res., Australia, Bull. No. 57. Duncan, N. 1932. Ann. Inst. Past., 48, 604. McEweN, A. D. 1932. Veter. Journ. 88, 180. McEweN, A. D., and RoBerts, R. 5. 1931. J. Comp. Path. and Therap., 44, Part 1. »» 5 1932. 1Ibid., 45, Part 3, p- 212. Roserts, R. S., and McEwen, A. D. 1931. 1bid., 44, Part 3. Turner, A. W. 1930. Council for Sci. and Indust. Res., Australia, Bull. No. 46. Sheep Pox Braxarr, F. R. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 141. Briori:, J. 1931. Proc. Internat. Veter. Congress, London, 3, 12. Fowl Cholera ManNINGER, R. 1928. ¢ Handb. d. path. Mikroorg.,” 6, 529. Fowl Pox DarriNg, T., Mason, J. H., and GorponN, W. S. 1929. Brit. J. Exp. Path., 10, 16. Dovre, T. M., 1930. 1bid., 43, 40. o 1931. Proc. Internat. Veter. Congress, London, 3, 675. 1930. J. Comp. Path. and Therap., 43, 40. DovLe, JT. M., and MiNETT, F. C. 1927. J. Comp. Path. and Therap., 40, 247. FinprLay, G. M. 1930. “A System of Bacteriology,” London, H.M. Stationery Office, 7, 259. ZURrRUKzOGLU, ST. 1928. < Handb. d. path. Mikroorg.,” 9, 379. Standardization of Veterinary Products Eicnnorn, A. 1931. Proc. Internat. Veler. Congress, London, 3, 236. FLUECKIGER, G. 1931. Ibid., 3, 249. GraUB, E., et al. 1931. Ibid. i 352 and 452. Monvrer, J. R. 1931. Ibid., 3, 259. PART II VACCINE THERAPY CHAPTER 1 THE PREPARATION OF A VACCINE ACTIVE immunization against infective disease has been practised for many years and since its inception the nature of the material used for immunization has been an outstanding problem. In the Middle Ages pus from a smallpox lesion was used to inoculate healthy individuals, but this method had obvious drawbacks. Then Jenner introduced a new principle, namely, the use of an attenuated virus. From the time of Jenner until the advent of Pasteur no advance was made. Pasteur used pure cultures of bacteria which by certain procedures he had attenu- ated. so that when injected into susceptible animals they did not kill, but, on the other hand, rendered the animals immune to subsequent infections with virulent cultures. This ushered in the modern era of prophylactic vaccine therapy, and after the striking success of Pasteur’s famous demonstration at Pouilly le Fort there was no question as to the efficacy of vaccine therapy. Authorities might quarrel about the details of the process, but the principle was established for all time. The whole of Pasteur’s work was done with living attenuated vaccines, and as there were certain risks attached to the inoculation the method was applied to human disease only in a few cases, notably rabies. Salmon and Theobald Smith (1884-1886), working with the hog cholera bacillus, were the first to show that by the use of dead 243 244 THE PREPARATION OF 4 VACCINE cultures some immunity could be established in animals. It was, however, due to the work of Haffkine, Pfeiffer, Kolle and especially Wright that the modern method of prophylactic inoculation of man with dead cultures was developed, and this was followed by Wright's introduction of killed cultures for the treatment of established infections. Much controversy has taken place in the past as to whether dead vaccines are as effective as living attenuated vaccines, and this question has in recent years been revived in connection with the BCG vaccine for tuberculosis. With the attenuated anthrax vaccine there have been many instances in which animals contracted the disease as the direct result of the inoculation, and while this did not prevent the use of such a vaccine for the immunization of animals it made the system impracticable for man. Kven apart from the possibility of the attenuated virus regaining some of its virulence, there are other dangers inherent in the use of living vaccines, as has been exemplified in the recent disaster in Lubeck in connection with the immunization of children against tubercle with BCG. In this instance an official inquiry showed that the deaths were not due to the vaccines having regained virulence, but that in the process of manufacture of the vaccine in Lubeck a virulent culture was accidentally substituted for the avirulent one, with the result that the children were infected instead of being protected. Human nature being what it is, there is always the remote possibility of some such error when dealing with living vaccines, and, so long as these vaccines are used, disasters will occur. The element of safety in the use of killed vaccines, even when made under * rush * conditions, is well brought out in the experience with typhoid vaccine in the British Army in the war. Harvey in the Medical Research Council’s “ System of Bacteriology ” states :— “ Fifty million c.em. of vaccine were issued from the Royal Army Medical College, Millbank, and inoculated into several millions of men, and not a single case was reported of any accident attributable to want of care in the preparation of the vaccine.” The simple vaccine consists of a standardized suspension of bacteria which has been sterilized by heat at a temperature just SENSITIZED VACCINES 245 sufficient to kill the microbes, diluted to a convenient strength for use, and to which has been added some antiseptic to preserve its sterility. This is the type of vaccine which is most extensively used at the present day. Various attempts have been made to modify the vaccine in certain respects. The vaccine, consisting as it does of a suspension of bacterial bodies, contains certain toxic substances, and when large doses ¢ reaction,” which may be anything from a slight malaise to high fever with the usual accompaniments. It has been shown that synchronous with this ¢ 2 are injected there follows what is known as a reaction there is in some cases at least a diminution in the protec- tive power of the body—the negative phase. Modifications of the vaccine have been made with the object of reducing the toxicity and so allowing larger doses to be administered without discomfort. ‘« Detoxication ’’ by Alkalies and Acids. Lustig and Galeotti (1897), for the production of anti-plague serum, used a plague bacillus vaccine which had been dissolved in caustic soda and precipitated by hydrochloric acid. By this procedure they were enabled to inoculate large amounts of bacterial protein into the animals used for the production of the serum. The first Plague Commission, however, found that the serum produced in this way was inferior to that produced by Yersin by the inoculation of untreated bacilli. This method has been revived by Thomson (1919) in connection with gonococcus, and has been extended by him to a large number of vaccines for therapeutic purposes. While it is true that the protein of a very large number of bacteria treated in this way can be injected without discomfort to the patient, it is not known how far the treatment has destroyed the antigenic value of the vaccine. Sensitized Vaccines. These were introduced by Besredka. The living bacteria were mixed with an excess of immune serum, allowed to stand for a time and were then washed free from the serum and injected into the patient. In this way the toxic properties of the vaccine were supposed to be neutralized while the antigenic properties remained. This was further modified by the use of dead bacteria treated with immune serum. and in this 246 THE PREPARATION OF 4 VACCINE form the sensitized vaccine has been used on a considerable scale. The argument in its favour was that larger quantities could be injected without the production of a negative phase, but, as with the detoxicated vaccine, it is impossible to say how much of the original vaccine remains active after treatment with the serum. Many workers have shown that the injection of such sensitized vaccines into animals results in a very poor yield of antibodies. Armstrong (1925) showed that when sensitized pneumococcus vaccine was administered to animals there was a small but very rapid rise in the protective power of the serum and this he explained as being due to the dissociation of the immune serum from the bacterial bodies. The later increase in antibodies in the serum of the vaccinated animals due to active immunization was slower and less in the case of the sensitized than in that of the ordinary simple pneumococcal vaccine. Formolized Vaccines. Loewenstein, Glenny, Ramon and others have shown that when a toxin is treated with formalin it loses its toxic properties while retaining its antigenic power; in other words, it ceases to be a toxin, but when injected into an animal it stimulates the production of antitoxin. Such toxin, which has been detoxicated by formalin, is generally referred to in English literature as toxoid, but in France, following Ramon’s nomen- clature, the name ‘“ anatoxine ” is used. This will be referred to later in connection with immunization against diphtheria. The same principle has been applied to whole cultures. The broth culture or the saline suspension of microbes from an agar culture is incubated with formalin (0-2 to 0-5 per cent.). This procedure kills vegetative bacteria, and, where an exotoxin exists, converts this into a toxoid which is not poisonous, but retains its antigenic properties. Such a vaccine has been called an ana- culture, or formol-culture, and has been used with success. Dalling has published the results of inoculating sheep with a formol-culture of a special strain of B. welchii, which prove that lamb dysentery was almost eradicated in the inoculated flocks (see Chapter XIX., Part 1). This method has also been applied to Shiga’s dysentery bacillus which produces in culture a powerful toxin, so that vaccines made in the ordinary way are so toxic that it has proved to be ’ DEFATTED VACCINES 247 impracticable to administer them in doses sufficient to immunize man against this type of dysenteric infection. Ramon and Dumas (1925) treated the toxin with formalin and found that its toxic properties were much reduced, but its immunizing powers remained. Durand (1925) obtained similar results with *“ ana- cultures.” He was able to inject a dose of 1,500 million formolized bacilli in man without reaction, whereas a dose of 500 million heat-killed bacilli gave a violent reaction. These results have been confirmed by Wherry and Bowen (1925). Wherry and his colleagues in a later paper (1928) describe the favourable results which they have obtained with anacultures of typhoid bacilli in the treatment of typhoid fever (see p. 278). Diaplytes (Defatted Vaccines). Douglas and Fleming (1921) showed that when bacteria were extracted with acetone their antigenic capacity was not diminished and some of them by this procedure became easily dissolved by trypsin. Douglas, in a later paper (1921) demonstrated that a tryptic digest of acetone extracted bacteria furnished a good antigen. He argued that when a vaccine is administered the antigenic substances only become active when they have been liberated by some process of digestion or dissolution of the microbes, and that by predigestion a vaccine could be obtained in which the antigen was already in solution. Dreyer (1924) * defatted ” bacteria by first boiling them in formalin and then extracting them with acetone. This resulted in the tubercle” bacillus losing its acid fastness and staphylococci and streptococci becoming gram negative. At the same time, the bacteria became easily soluble in trypsin. The defatted bacteria were suspended in salt solution, and this suspension constituted the vaccine. It was claimed by Dreyer that tubercle vaccine made in this way could cure tubercu- losis in guinea pigs, and at first the clinical trials in man appeared to be favourable. More extended observations, how- ever, seemed to show that these defatted vaccines (diaplytes) had no advantage over the ordinary vaccines. The antigens appear to be extraordinarily stable, as even boiling with strong formalin does not destroy them. Tubercle diaplyte vaccine proved to be as least as toxic to a tuberculous patient as was a 248 THE PREPARATION OF A VACCINE bacillary emulsion. Experimentally, it was found that a tubercle diaplyte vaccine produced in animals an anti-tuberculous serum superior in its content of antibodies to that produced by means of bacillary emulsions. Lipo-vaccines. In these the bacteria were suspended in an oily medium. This was done so that the vaccine substance would be slowly absorbed and the stimulus to the body to produce antibodies would be prolonged, thus approaching more nearly the condition in an actual infection and at the same time avoiding the necessity for repeated injections of prophylactic vaccines. Le Moignic and Pinoy (1916) and Achard and Foix (1916) reported favourably on the use of vegetable oils as a vehicle for suspending bacterial vaccines. Whitmore and his co-workers (1918) found that typhoid vaccines suspended in oil gave rise to an even better production of agglutinins than those suspended in saline. At the same time, the reaction was very mild, so that the three original doses of the saline vaccine could be combined in one dose without producing any unpleasant results. This type of vaccine was largely used in the United States Army during the war. Mennonna (1927) reported unfavourably on typhoid lipo- vaccines. Up till 1920 the Italian Army was inoculated with ordinary vaccines, but since that date lipo-vaccine has been used. There has been a marked rise in the incidence of typhoid fever since the introduction of the lipo-vaccine. Pneumococcal lipo-vaccines were used by Cecil and Vaughan (1919) in their work on the prophylaxis of pneumonia ; this is discussed on p. 303. There can be no doubt that vaccines suspended in an oily medium do confer immunity, but the available evidence goes to show that they are less effective than the ordinary vaccine suspended in saline solution. Vaccines Killed by Chemical Substances. The French who, before the war, did not favour vaccines, have since used them extensively. Following the lead of Vincent, who used enteric vaccines killed with ether, they have used vaccines killed with various chemicals, notably ether, iodine, and sodium fluoride. It BACTERIAL EXTRACTS 249 is supposed that some of these chemicals, in addition to killing the bacteria, detoxicate them to some extent. Although very popular in France, they donot seem to have been extensively used elsewhere. Bacterial Extracts and Filtrates and Digests. A large variety of extracts and filtrates of bacteria have been used as vaccines. The general idea underlying these preparations is that when the antigen is in solution it can react immediately with the body cells, so that the immunizing process commences without the delay which is entailed when the antigen has to be dissolved out of the intact bacteria. There is no doubt that a large variety of bacterial extracts are antigenic and can function as vaccines, but there is as yet no definite evidence that they are superior, or even equal, to the simple bacterial vaccine. Gratia (1930) has used bacteria dissolved by means of a ferment secreted by certain streptothrices or moulds. These organisms are commonly found in the air, and some of them produce ferments which have a remarkable lytic effect on dead bacteria. Gratia states that such mycolysates have lost much of their toxic qualities while their antigenic characters are preserved. The author has confirmed Gratia’s observations. In animals it was found that a vaccine of Shiga’s dysentery bacillus dissolved in this way was still toxic, but considerably less so than was the original vaccine. A staphylococcus mycolysate had apparently an immunizing power equal to that of a staphylococcus vaccine of the same strength. The same was true of a typhoid mycolysate, except that in this case the whole of the flagellar antigen was destroyed in the lysis of the typhoid bacilli. Thus, following the injection of a typhoid mycolysate (and the same applies to typhoid bacilli dissolved with trypsin), the agglutinins which appear are of the granular or somatic type, and there is no production of the flocculent or flagellar agglutinins. Ferry and Fisher (1924) have claimed that salt solution washings of 24-hour growths of B. typhosus are higher in antigenic properties than the corresponding bacterial suspension or the washed organisms. Their conclusions rested mainly on the results of agglutination and complement fixation tests made on the serum of rabbits inoculated with these vaccines. They showed definitely 250 THE PREPARATION OF A VACCINE that the supernatant fluids, when injected into rabbits, induced the formation of a considerably larger yield of agglutinins than did the bacterial deposit. These results, were, however, obtained before the relative importance of the flagellar and somatic antigens were properly appreciated. It will be pointed out in the next chapter of this volume that in the case of a flagellated organism like B. typhosus the antigen contained in the flagella has little or no importance in connection with immunity to infection. It might be expected that if a saline suspension of a flagellated organism is shaken violently for a few minutes and then centrifuged the supernatant fluid would contain considerable amounts of flagellar substance so that it is not surprising that Ferry and Fisher found that the supernatant fluid was antigenically very potent, but as they were concerned with flagellar agglutinins their results have little practical value as an indication of the immunizing power of a vaccine. The figures which Ferry and Fisher have published as regards the complement fixation test show that the supernatant fluids were inferior in antigenic content to the bacterial bodies. Vaccines made from the supernatant fluid of centrifuged broth cultures or saline suspensions of bacteria have been used for some years, but no clinical results are forthcoming. Horder and Ferry (1926) claim that they are free from toxic substances but this conflicts with the findings of Bigger (1927) and others who have found that the supernatant fluid from a saline suspension of a toxigenic staphylococcus contains large amounts of toxin. There is no doubt that these vaccines do contain some antigen but on the available evidence the useful antigenic content is less than that of the bacterial bodies. Autolysates. These have been recommended as vaccines, and with some bacteria, such as staphylococcus, they are probably effective, but sometimes the breaking-down process proceeds too far and the immunizing power of the vaccine is lost. This has been shown to be the case with the pneumococcus, an organism which undergoes autolysis very readily. A completely autolysed pneumococcus culture has been found to make a very poor protective vaccine. Much more work requires to be done before the process of ANTIVIRUSES 251 autolysis can be recommended in the preparation of vaccines except in special instances. Solution of Bacteria by means of a Bacteriophage. It has been shown by d’Herelle and others that bacteria dissolved by bacterio- phage preserve their antigenic power and are efficient as vaccines. Bacteriophage therapy has been extensively used with the idea of propagating the bacteriophage and giving it the opportunity to act on the infecting bacteria and cause their dissolution. In practice, however, the bacteriophage cannot be separated from the products of the bacteria on which it has propagated itself in culture, so that any administration of bacterio- phage is combined with that of a highly antigenic bacterial solution. Thus all treatment with bacteriophage is a combination of bacteriophage therapy proper and vaccine therapy. Flu (1929), by the use of plague vaccines dissolved by means of bacteriophage, has obtained very striking results in the prevention of experimentz]l plague in rats (p. 293). Antiviruses. These were introduced by Besredka (1919-1924). They are made by growing organisms for some weeks in broth until the medium will no longer permit the growth of the particular organism. Besredka concluded that filtrates of such cultures contained a specific inhibitory substance to which he gave the name “ Antivirus.” He has stated that animals inoculated subcutaneously or intradermally or poulticed on their shaved skin with staphylococcal or streptococcal antiviruses arc resistant to inoculations of virulent cultures introduced 24 to 48 hours later. No antibodies were formed, but the immunity was general and not confined to the treated area. The questions raised by Besredka are admirably discussed by Ledingham (1931) in the Medical Research Council’s * System of Bacteriology ” (Vol. 6, p. 60), and it is unnecessary to go into the question fully here. Many workers have come to conclusions that differ from those of Besredka. It has been shown that by the use of broth or other substances results can be obtained similar to those following the use of antivirus. The question of specificity is also disputed. There are, however, many favourable clinical 252 THE PREPARATION OF A VACCINE reports on the use of antivirus, and Schweinburg’s observation (1928) is of some importance. He was preparing antivirus for use by certain physicians and he states that when on one occasion he substituted nutrient broth for antivirus without the physicians’ knowledge they complained that the «“ vaccine 7 did not act. All these different methods of preparing vaccines have their enthusiastic advocates. Certain methods seem to be definitely inferior to the simple vaccine for general purposes, but with others it is difficult or impossible to say that any one is better than the others. In most cases the simple vaccine (a killed standardized suspension of bacteria in normal saline) appears to be efficient, and it must be remembered that any complication of the method of preparation increases the possibility of contamination. It would appear, however, on a priori grounds, that if it were possible to obtain all the useful antigenic components of the bacteria in solution and at the same time accurately standardize the antigenic content, this would form the best type of vaccine. It would allow the actual effective dose of the vaccine to be more accurately measured, in that it would do away with any uncer- tainty as to the time taken in different individuals to dissolve the antigens out of the bacterial bodies injected. Such vaccines, as we have seen, exist, but it is not yet certain that they contain all the antigenic components or that they can be accurately standardized. It seems likely, also, that the addition of chemicals, and especially formalin, for the purpose of reducing the toxicity of certain vaccines will be more general than it has been in the past. Autogenous and Stock Vaccines An autogenous vaccine is one prepared from cultures of the particular microbe which is infecting an individual patient and which is used for the treatment of that individual. A stock vaccine is intended for use in patients other than those who were the source of the cultures. When the infection is a definite one, and when it is certain that the microbe recovered from the patient is really the infecting AUTOGENOUS AND STOCK VACCINES 253 agent, there is little doubt that in most cases an autogenous vaccine has theoretically an advantage over a stock vaccine. The freshly isolated culture is almost certain to be in the “S ” phase (see Chapter IL), and is antigenically active. Then the antigen will be identical with that acting in the body from the natural infection ; there will be no antigenic differences such as might exist if the vaccine were made from another strain of the same microbe. No steps need be taken to increase or diminish the specificity of the vaccine; it matters not if the vaccine has extreme specificity, as it is only prepared for the purpose of acting on the one infection by the particular strain of bacterium from which the vaccine is made. A much less satisfactory reason why autogenous vaccines are frequently used is that many patients have a strong objection to having injected into them microbes which are derived from someone else. The difficulty with autogenous vaccines arises especially when the nature of the infection is more or less obscure. In the case of infections of the alimentary or respiratory tracts, sometimes the nature of the infecting organism is clear, but this is by no means always the case, and the methods of determining the nature of the infecting organism are far from perfect. In these regions of the body there are normally many bacteria, and cases occur where there is obviously some infection but in which no bacteria can be found other than the types normally present. The difficulty arises mainly in the case of the streptococci and the organisms of the B. coli group. These we all harbour normally, but it is certain that streptococci of the non-haemolytic types and members of the B. coli group are responsible for many of the infections of the alimentary and respiratory tracts. There is no certain means of knowing which are the pathogenic and which are the saprophytic forms of these bacteria ; indeed, it is probable that there are no absolutely saprophytic forms, and that all of these microbes are, in certain circumstances, capable of invading the body. In such circumstances an autogenous vaccine may casily be made from the wrong microbe, when it would be useless except for its non-specific effect. It is in such cases that the skill and experience of the bacteriologist come into play. There is little 254 THE PREPARATION OF 4 VACCINE difference between a skilled and an unskilled bacteriologist in the actual preparation of a simple vaccine, but there is a vast difference when it comes to selecting the infecting bacterium from the usual mixture which is obtained from an intestinal culture. Methods of Choosing the Infecting Organism. By Cultural Methods. Frequently this is done merely by choosing the microbe which is most numerous in the culture. This is the simplest but the least effective method. An examination of a stained film of the infected material should never be omitted, as by such an examina- tion the right culture medium can be chosen, and some idea of the proportion of the different bacteria actually present can be obtained. If this is omitted, then an unsuitable medium may be used and the organism really responsible for the infection may be completely missed. Wright (1915) introduced the method of pyo-sero-culture. In this a pilot volume of pus was drawn up a capillary tube and was followed by a series of volumes of the patient’s serum. The serum was therefore infected with whatever pus adhered to the wall of the tube and the implants became less and less with each succeed- ing volume of serum. The organism selected was the one which grew out in the greatest number of volumes of serum. This method was extended by Solis Cohen (1927), who substi- tuted the patient’s blood for serum as the culture medium. He employed two methods; in the first, dilutions of the infective material were drawn into a capillary tube and then expelled, leaving a ““ wash ” on the sides of the tube. The tubes were then filled with blood from the patient’s finger and incubated. In the second method the infected material was planted on a suitable culture medium and also on the bottom of a sterile tube into which was placed 2 or 3 c.c. of patient’s blood. The implanted plate and blood were incubated for 24 hours, when the former was placed in an ice chest to restrain further growth, and the latter was planted on another plate of the same culture medium. After another 24 hours the two growths were compared, and in choosing the strains from which to make the vaccine particular attention was paid to those which grew out in the patient’s blood. By this method the organisms chosen would belong to the class STOCK VACCINES 255 which have been termed by Wright * serophytes,” that is, those which will grow out freely in unaltered human serum. There is not sufficient evidence to warrant the assumption that the use of blood from the patient enables a better selection to be made than normal human blood. The use of human blood as a culture medium certainly prevents the appearance of large numbers of saprophytes, but it is not justifiable to assert that the particular strain or strains of bacteria which can flourish in the patient’s blood are the actual infecting microbes. By Serological Methods. In the case of members of the B. coli group the agglutination reactions of the patient’s serum may be tested with the different types of B. coli isolated, and if a particu- larly high titre of agglutination is discovered this type is selected as the infecting organism. This method has drawbacks, in that many individuals presumably uninfected agglutinate one or more strains of B. coli, or it may be that the individual has recovered from a B. coli infection and is now infected with some other microbe. The agglutination reaction with B. coli, like that of the typhoid bacillus, probably persists for a considerable time after recovery from the infection. The opsonic index has frequently been used in the past to distinguish infections, especially with B. coli. This gives consider- able information, but is very laborious. Stock Vaccines. Vaccines designed strictly for prophylactic use must of necessity be stock vaccines. In the treatment of infections stock vaccines must be used (a) in cases in which it is impracticable to isolate the infecting organism although the nature of the infection may be known, e.g., gonorrheeal arthritis or surgical tuberculosis, and (b) in cases in which it is desired to commence vaccine treatment at once without waiting for the preparation of an autogenous vaccine. In practice they are very frequently used for economic reasons, and in large clinics it is common to use stock vaccines for the treatment of a great many infections and only to use autogenous vaccines for such patients as fail to improve satisfactorily. The nature of a stock vaccine will depend on the homogeneity or otherwise of the antigenic character of the particular microbe 256 THE PREPARATION OF 4 VACCINE from which it is made. In the case of the typhoid bacillus the vaccine may be made from a single strain—and, indeed, in the past almost all anti-typhoid vaccines have been so made—as the different strains of this bacillus are antigenically identical, except, as we shall see (Chapter I1.), some of them are non-motile and do not possess the flagellar antigen. With pneumococcus and other organisms the case is quite different in that there are certain different types which are antigenically distinct, and, although it has been shown that the type specificity of pneumococcus vaccines is not absolute, it is desirable that in the preparation of a stock vaccine of such an organism the vaccine should contain representa- tives of many different types. Apart from convenience, the chief point in favour of stock vaccines for the treatment of infections is that they contain a large number of strains of organisms which have been selected from cases in which the infection was obvious. They are therefore made from pathogenic organisms of the type which it is desired to use, whereas in an obscure or difficult case an autogenous vaccine may sometimes be made of a harmless weed. REFERENCES AcHARD, CH., and Foix, Cu. 1916. C. R. Soc. Biol., 19, 209. ARMSTRONG, R. R. 1928. Proc. Roy. Soc., Series B, 98, 533 BrsreDpkA, A. 1919. Ann. Inst. Pasteur., 33, 301, 557, 882. 5 1921. Ann. Inst. Pasteur., 35, 421. * 1924. Ann. Inst. Pasteur., 38, 565. BiGGER, J. W., Boranp, C. R., and O'Meara, R. A. 1927. J. Path. Bact., 40, 261, 271. Crcin, R. L., and Vavenan, H. F. J. Exp. Med., 29, 457. DoucLas, S. R. 1921. Brit. J. Exp. Path., 2, 175. Darring, T. 1928. Vet. Jour., 84, 640. DrevEer, G. 1923. Brit. J. Exper. Path., 4, 146. Duranp. 1925. C. R. Soc. Biol., 92, 159. FENNEL, E. A. 1918. Jour. Am. Med. Assn., 70, 427. Ferry, N. S., and Fisner, L. W. 1924. Brit. J. Exp. Path., 5, 185. Fru, P. C. 1929. Zbl. f. Bakt. Abt. 1 Orig., 118, 473. GrLENNY, A. T., and SUpmerseN, H. J. 1921. J. Hyg., 30, 176. GRATIA, A. 1930. C. R. Soc. Biol., 104, 1058. Harvey, D. 1929. “A System of Bacteriology,” 4, 15, London, H.M. Stationery Office. HorpEer, T., and FErry, N.S." 1926. Brit, Med. J., 2, 177. REFERENCES 257 Lepincuam, J. C. G. 1931. “A System of Bacteriology,” London, H.M. Stationery Office, 6, 60. Lx MoigNac and PiNxoy. 1916. CC. R. Soc. Biol., 79, 201, 352. Lustic and Gavrorri. 1897. Deutsche Med. Woch., 23, 227. MENNONNA, G. 1927. Ann. d’Ingiene, 37, 373. Prrerson, W. F. 1918. J. Am. Med. Assn., 70, 427. Ramon and Dumas. 1924. Paris Med., 53, 480. Ramon, G. 1923. C. R. Acad. Sec., 177, 1338. SCHWEINBURG, F. 1928. Z. ImmunForsch., 58, 53. Sovris-Conen, M. 1927. Brit. Jour. Exp. Path., 8, 149. TunomsonN, D. 1919. Lancet, 2, 1102. Wierry, W. B., and Bowen, J. A. 1925. J. Inf. Dis., 37, 125. Wuerry, W. B., LE Branc, T. J., Fosiay, L., and Taomas, R. 1928. J. Inf. Dis., 43, 189. Wricnr, A. E. 1915. Brit. Med. Jour., 2, 629. R.A. VACCINES. 9 CHAPTER II BACTERIAL VARIATION IN REGARD TO IMMUNITY BacTeERrIAL variation has played a part in immunology since the time of Pasteur, who made his first anthrax vaccines by growing his cultures in such a way that he produced a variant which had lost much of its virulence and in which other changes had occurred. Its significance as regards active immunity has, however, only been properly appreciated during the last few years, and it is largely owing to the work of Arkwright that certain fundamental facts have come to light. Until these facts were known, that is, till a few years ago, stock vaccines were made in many laboratories from old cultures, the antigenic characters of which were not investigated. (Probably this happened more frequently in those laboratories which were not attached to hospitals, as in such laboratories freshly isolated cultures were not so easy to obtain.) From the knowledge which we now possess it is certain that some of these vaccines must have been very poor immunizing agents, and it is probable that some of the disappointing results which were obtained with vaccines have been due to the cultures from which the vaccines were made having undergone variation so that they had lost the antigens which were necessary to produce active immunity. Bacteria when freshly isolated from infected tissues are generally in a state in which they contain their full complement of ¢ protective ”’ antigens, and for this reason vaccines made from such freshly isolated cultures are efficient, whereas vaccines made from the same strains of bacteria after months or years of sub- culture outside the body may be useless. R and S Variation The most important bacterial variation from the point of view of vaccine therapy is that described by Arkwright (1921) as 258 R. & S. VARIATION 259 “rough ” and ‘ smooth.” Arkwright noticed that in cultures of various organisms there appeared colonies which differed from the normal in that they had a rough appearance, whereas the normal colony had a smooth, shiny surface—hence the names “ rough ” and “smooth * applied to these types. In later work other changes were found in these variants; the rough type clumped when suspended in normal saline and when grown in broth it formed a sediment. At first the extreme importance of this variation was not appreciated, but further work by Arkwright and others revealed that the change in the appearance of the colony was correlated with an alteration in the constitution of the bacterial cell, which resulted in a modification of its antigenic properties and a loss of virulence. The “rough > and ‘ smooth » forms were originally described by Arkwright in regard to B. dysenterice, B. paratyphosus B, B. typhosus, and B. pseudotuberculosis rodentium. Griffith (1923) showed that a similar variation occurred in pneumococci, and it has now been demonstrated in such a variety of bacteria that it may be regarded as a general phenomenon. It has been found that the visible rough ” change in the colony is not an invariable accompaniment of the change in the constitution of the microbe, and the terms * rough ” and * smooth ” have been superseded by the letters “R” and “8.” The “8S” type is the normal pathogenic form, and the “R” type is the degenerate avirulent variant. In regard to pneumococci, it was shown by Griffith and others that the “ R > forms were without capsules, that they had lost their soluble specific substance, and that all type-specific characters had disappeared, so that all “ R * pneumococci were identical no matter from what type they had originally been derived. The freshly isolated organism is almost invariably of the *“S” type, but in subculture it tends gradually to degenerate into the “RR” type. This degradation occurs rapidly with some bacteria and much more slowly with others. The culture medium used has sometimes a marked effect on the rapidity of the change (see whooping cough, p. 334). 260 BACTERIAL VARIATION IN IMMUNITY It has been found that very rapid degencration to the “ R ” form can be produced in virulent bacteria by growing them in a homologous immune serum, and this method is now frequently used to produce such variants. The transformation of *“ R *’ into “87 forms does not occur so readily, but in many cases it can be accomplished. The observation that virulent *“S* bacteria grown in an immune serum rapidly degenerate to the “ R *> form may have a very important significance in recovery from an acute infection. After a time the infected individual develops a con- siderable degree of general immunity, and the bacteria have perforce to grow in the body fluids, which contain immune bodies. In many infections these immune bodies have no direct lethal action on the bacteria, but it is not unlikely that by virtue of the growth of the bacteria in the presence of these immune bodies such bacteria undergo to a greater or less extent an “ R ” variation, when they lose their invasive properties and are casily destroyed by the phagocytes. So far as vaccine therapy is concerned, the most important point is that the “ R ” variant is useless as a protective vaccine. This was shown by Arkwright (1927) in regard to B. typhosus, and it has been confirmed in the case of other microbes by Ibrahim and Schiitze (1928), Topley (1929) and others. Topley utilized the method of experimental epidemiology. Mice were inoculated with various types of B. aertrycke vaccine and were put into infected cages along with control uninoculated mice. Animals inoculated with vaccines containing the *S ** antigen showed a considerable amount of immunity, but the animals which were inoculated with vaccines containing “R” (but no “S”) antigen were not protected to a greater extent than mice inoculated with an indifferent vaccine (staphylococcus). In view of these results, it is essential that in the preparation of vaccines ““ R” variants should be rigidly excluded. Grinnell’s (1932) work on typhoid vaccine is of great interest in this connec- tion. He has pointed out that nearly all the anti-typhoid vaccine prepared in the United States is made from one strain of typhoid bacillus (Rawlings), which was originally isolated by Sir Almroth Wright in 1900. This bacillus had therefore been grown on H. & 0. VARIATION 261 artificial medium for thirty-three years. Grinnell obtained 12 cultures of this strain from different laboratories which produced anti-typhoid vaccine and compared these with a recently isolated virulent culture. As regards their cultural characters, one had undergone complete * R > variation, and the others were inter- mediate between “ R” and “S.” It was found that all these « Rawlings strains had lost their virulence for mice—only two mice dying out of 60 injected intraperitoneally with 0-1 c.c. of an 18-hour broth culture, as against 38 out of 40 injected with the same quantity of the recently isolated strain. Vaccines were made from all the Rawlings ” strains and from the recently isolated strain. Mice were immunized with these vaccines and then tested by injecting them with the living virulent strain. The mice which had received the * Rawlings ”* vaccines showed no protection—the death rate was the same (87 per cent.) as in the non-immunized control animals—but of those which had received the vaccine made from the recently isolated culture only 3 per cent. died. This work makes it clear that these old ‘ Rawlings’ strains of B. typhosus are unsuitable for the preparation of vaccines and the inference is that most of the anti-typhoid vaccine used in the United States of recent years has been deficient in protective power. 3 ‘“H’ and ‘‘ O ”’ Variation Theobald Smith and Reagh (1903) showed that there were differences in the agglutination reactions between motile and non- motile forms of the same organism. They obtained a non-motile strain of B. suipestifer, and found that when they immunized animals against it the serum of these animals agglutinated it in a fine granular form in contrast to the loose woolly floccules which formed when an animal was immunized with and its serum tested against the motile form. They propounded the theory that the motile form contained two antigens, one in the flagella and the other in the body of the microbe, while the non-motile form contained only the somatic antigen. Beyer and Reagh (1904) investigated the properties of these antigens and found that the 262 BACTERIAL VARIATION IN IMMUNITY flagellar antigen was destroyed by heat very much more easily than was the somatic antigen. The practical significance of the flagellar and somatic antigens and antibodies was not realized by Theobald Smith and his co-workers, and for about twenty years their work remained practically unknown and without any influence on subsequent research. Our present knowledge of this type of variation is the outcome of the work of Weil and Felix (1916) on the agglutination of B. proteus X19 by the serum of typhus fever patients. They differentiated two types of colony in this proteus bacillus culture— one the usual spreading type and the other discrete. These types were labelled ““ H*' (Hauch) and ““ O ” (ohne Hauch) respectively. Animals immunized with the “ 0” type agglutinated both types in the fine granular form, but when the animals were immunized with the “ H ™ type their serum agglutinated the “ O ** type bacillus in the same manner, but the “ H” type was agglutinated in loose floccules. At first it was not recognized that the *“ H > type was motile and flagellated and that the “ O ” type had no flagella and was non-motile, but when this was discovered (Foetten, 1919 ; Braunt Schiffer, 1919) Smith and Reagh’s results fell into line with those of Weil and Felix. It is now well established that the flagella of a motile organism contain an antigen which is separate and distinct from the antigens contained in the body of the microbe. The letters *“ H > and “0,” although originally used in regard to the appearance of the colony, have now been transferred to the antigens. The flagellar antigen is now referred to as ““ H >” and the somatic as *“ 0.” The “07 (somatic) antigen is remarkably stable, and is hardly at all affected by boiling for two hours. The “ H* (flagellar) antigen is destroyed by boiling, by weak acids and alkalies, by alcohol, or by digestion with ferments (trypsin, etc.). In cultures grown on carbolic acid agar the *“ H* antigen disappears and only the “0” antigen is developed. Importance of *“ H™ and * 0" Variation in Vaccine Therapy. The ordinary virulent motile organism, say, of the Salmonella group, contains both “H™ and “0” antigens; the non-motile variant contains only the *“ O ” antigen. The important practical H. & 0. VARIATION 263 question is what part these antigens play in the immunizing process against infection. Research in this direction at first concerned “Hand * O antigens only (Felix. 1924, Felix and Olitzki, 1926), but has subsequently been combined with investigations into the nature of the response to antigens contained in the “R™ and “S§ variants. These “ RR” and “S 7 variants are independent of the motility of the microbe, and it has been shown that the “H ” (flagellar) antigen is, in motile organisms, common to both “R” and “S 7 types, and that it is in their somatic antigens that these differ. When “ R ” variation takes place, the somatic “0” antigen is lost and its place is taken by a different antigen, which is called the somatic *“ R * antigen. An animal immunized with the “ R ” form of a motile bacillus produces antibodies to the “ H ** antigen which affect “ R ** and “S ” forms alike. Thus an animal inoculated with a motile “ R ”’ form of B. typhosus will give the ordinary floccular agglutination with both “R” and “S” types. The somatic agglutination reactions of these types would, however, differ widely. It has already been noted that the *“ R” variant is incapable, when used as a vaccine, of immunizing animals against infection. What, however, is the relation of the *“ H ”” and * O ** antigens to immunity ? The experiments which were quoted in relation to the value of the “R > variants have here to be reconsidered. Arkwright (1927) showed that he obtained no immunity to B. paratyphosus A by inoculating with a vaccine made from a flagellated * R > culture of this organism, although good immunity was obtained with a vaccine made from an *“S” culture, whether flagellated or not. The flagellated ““ R™ culture contained the “H” antigen of B. paratyphosus A (together with the “RR” somatic antigen), and as this gave no immunity, and as with the “§ * vaccine the same degree of immunity was obtained, whether the “ H flagellar antigen was present or not, it was argued that the “ H >’ antigen had little or nothing to do with the immunizing process against infection. This was supported by Topley’s experiments, in which mice immunized with different types of vaccine were placed in infected cages. He found that no immunity to B. aertrycke was conferred by any vaccine which did not contain 264 BACTERIAL VARIATION IN IMMUNITY the “O°” somatic antigen of this organism. Vaccines which con- tained the *“ H ” antigen of B. aertrycke but which lacked the “0” antigen conferred no protection against the natural infection. The work of Springutt (1927) and of Ibrahim and Schiitze (1928) on the immunization of mice with B. aertrycke vaccines indicated that although the ““ H ” antigen combined with the “ R > somatic antigen produced no active immunity, a combination of the *“ H antigen with the “0” somatic antigen gave better immunity than the “ O * antigen by itself. Schiitze (1930) has since made the definite statement that the presence or absence of the “ H > or flagellar antigen does not influence the prophylactic value of such vaccines. He attributes the diminution in the value of the vaccine after prolonged heating to 100° C. to a partial destruction of the “0” antigen. This work, and further researches by Felix (1924, 1926) and others, makes it clear that it is the antigen contained in the bodies of the smooth type of organism which is mainly concerned in the production of active immunity against infection. The importance of the *“ O ” antigen in this respect has been brought out in another way. It has been shown that B. typhosus and B. enteritidis (Gaertner) have a common *“ O ” antigen (Weil and Felix, 1920). Herzog and Schiff (1922) have recorded that in a food-poisoning outbreak in a hospital which contained a certain number of typhoid convalescents these all escaped infection, and they were the only patients who had eaten the infected food who did so. These patients, being immune to the typhoid bacillus by reason of their recent attack, were apparently immune to infection with an organism different to the typhoid bacillus but containing the same somatic antigen. Direct experimental methods of immunization of animals have confirmed Herzog and Schiff’s observation. Topley (1929) and Schiitze (1930) immunized mice with B. paratyphosus B and then exposed them to infection or actually infected them with B. aertrycke (which has the same “O ”” antigen). They obtained just as good protection with the vaccine of B. paratyphosus B as they did with the homologous B. aertrycke vaccine. It is clear from this work that the antigen contained in the IH. & 0. VARIATION 265 bodies of the smooth type of organism is the one which is most important in the production of immunity. If the © O 7 antigen alone were concerned in immunity certain practical considerations would follow. The ¢“ O ” antigen is very stable ; it is not destroyed by boiling, by weak acids or alkalies, by alcohol or by digestion, all of which destroy the flagellar antigen. Efficient vaccines of microbes like the typhoid bacillus could then be made in a great variety of ways, and especially they could be sterilized by boiling. The whole of the conditions necessary for immunity are, however, not yet quite clear, and, as has been insisted on by Arkwright, it would be unwise to ignore the fact that substances which are destroyed by boiling may play some rdéle in immunity. Further support of the plea that vaccines should not be over-heated is given by Iyengar (1926). He tested the immunizing power of vaccines of P. avisepticus, which had been heated to different temperatures, and found that vaccines heated to 80°C. had considerably less immunizing power than those heated to 60° C. or less. In the case of anti-plague vaccine, also, the immunogenic antigen is heat-labile (Schutze, 1932). Bacterial variation has also to be considered in regard to the tests which are applied to ascertain whether immunity has developed as a result of the administration of vaccines. Many decisions have been taken on the results of serological tests of the inoculated animal. An animal inoculated with an antigen produces antibodies to that antigen, whether it be the “HH” flagellar antigen, the “ R ”” somatic antigen, or the *“ O ”” somatic antigen. In the past, attention has been largely centred on the floccular agglutination reaction. The ordinary Widal test, per- formed with formolized or carbolized suspensions, discloses almost solely “ H » agglutinins (Felix and Olitzki, 1928), and the *“ H * antigen has been shown to have little bearing on the immunity produced. It was largely as a result of such observations that it was laid down that the anti-typhoid vaccine in the British Army should not be heated to a higher temperature than 53° C. and, on similar evidence, it was decided that after a period of three months the immunizing power ol anti-typhoid vaccine diminished. As these 266 BACTERIAL VARIATION IN IMMUNITY conclusions took no account of the © O°" antigen, which has been shown to be the important factor in protective inoculation, they are quite unwarranted. In the regulations governing the commerical production of vaccines in the United States of America, an official test is laid down for the potency of anti-typhoid vaccine. To pass this test the vaccine must, when injected into rabbits, provoke the pro- duction of agglutinins to a degree not materially less than a stan- dard typhoid vaccine. This test was introduced before the importance of the “0” agglutinin was appreciated and any vaccine can pass the test which contains a sufficiency of the “H,” or flagellar, antigen. Thus a vaccine of a perfectly rough flagellated typhoid bacillus would successfully pass the test, although, according to ali the experimental evidence, such a vaccine would be quite devoid of protective power (cf. Pope, 1711). The observations of Grinnell (see p. 260) on the ** Rawlings ” strain of B. typhosus might be recalled in this connection. The work which has been discussed in this chapter clearly shows that bacterial variation must be considered in connection with vaccine therapy, and in particular in the preparation of vaccines. Old stock cultures must be looked upon with suspicion, as there is always the possibility that they have undergone a “RR” variation with loss of immunizing power. Autogenous vaccines are invariably made from freshly isolated cultures, and it is possible that some of the superiority which is ascribed to such autogenous vaccines depends on the fact that the bacteria have not been subcultured sufficiently long for them to have undergone variation. Stock vaccines should, where possible, be made of recently isolated cultures, but where this is impracticable the cultures should be maintained in such a manner that they retain their virulence and full antigenic capacity. REFERENCES ARKWRIGHT, J. A. 1921. Jour. Path. and Bact., 24, 36. ol 1926. Jour. Path. and Bact., 29, 318. 1927. Jour. Path. and Bact., 30, 345, 566. 1924. Brit. Jour. Exp. Path.. 5, 104. REFERENCES 267 ARKWRIGHT, J. A. 1929. Lancet, 2, 963. ARKWRIGHT, J. A., and GoyLE. 1924. Brit. Jour. Exp. Path., 5, 104. BevER and Reacn. 1904. Jour. Med. Res., 10, 89. FeLix, A. 1924. J. Immunol., 9, 115. Fenix, A., and Ovrrrzki, L. 1926, ibid., 11, 31 ; 1928, J. Hyg., 28, 55. GrirriTH, F. * Ministry of Health Rep.,” 1923, No. 18. Herzog, F., and Scurrr, F. 1922. Z. ges. exp. Med., 29, 412. Irani, H. M., and Scutrze, H. Brit. Jour. Exp. Path.. 9, 353 IvENGar, K. R. K. 1926. Ind. Jour. Med. Res., 13, 547. Lepinenam, J. C. G., and ScutTze, H. 1931. “A System of Bacteriology,” London, H.M. Stationery Office, 6, 93. Orcutt, M. L. 1924. Jour. Exp. Med., 40, 43, 627. Pore, ALEXANDER. 1711. *° Essay on Criticism,” line 115. Scutrze, H. 1930. Brit. Jour. Exp. Path., 11, 34. ” 1932. Brit. Jour. Exp. Path., 13. 284. Smith, T., and Reach, A. L. 1903. Jour. Med. Res , 10, 89. Serincur, E. 1927. Z. ImmunForsch, 52, 25. Torrey, W. W. C. 1929. Lancet, 1, 1337. Wein, E., and Feuix, A. 1917. Wien. klin. Wschr., 30, 1509. CHAPTER III LOCAL IMMUNIZATION MaixLy by reason of the prominence given to the theories of Besredka, much work has been done recently on this subject. Besredka has put forward the hypothesis that different microbes have specific affinities for different tissues, and that to prevent infections by such microbes the most important aim is to immunize the sensitive tissue. He regards infection with certain microbes, such as B. anthracis, the staphylococci and the streptococci, as especially cutaneous infections, and with others such as the typhoid and dysentery and cholera bacilli as essentially intestinal infections. The specific affinities of bacteria are well known, but much controversy has taken place in regard to Besredka’s contention that an immunity to the cutaneous microbes can only be satis- factorily attained by application of bacterial products to the dermis, and that immunity to intestinal infections is due to a local immunity of the intestine to the infecting bacterium. His theories are largely based on his work on anthrax infections. He has maintained that infection with anthrax is very much easier vid the skin than by any other route. This contention has stimu- lated a considerable amount of research. Some workers have corroborated Besredka’s findings, whereas others have not been able to support his claims. A carefully carried out, series of obser- rations by Sobernheim and Murata (1924) showed that the dose of anthrax bacilli necessary to infect was least by the intramuscular route and highest by the intravenous or intraperitoneal routes ; between these came the intracutaneous and subcutaneous doses. It would appear that the most favourable route for infection by different bacteria is not the same in all animals, so that the method which is most effective in one animal may be unfavourable in another species. 268 LOCAL IMMUNIZATION 269 Following on his finding that animals are more susceptible to anthrax infection by the skin than by any other route (which, as can be seen from the evidence given above, is disputed), Besredka experimented on the immunization of animals against anthrax. and he states that a much more solid immunity is obtained, and with smaller doses, if the bacilli are applied to or injected into the skin than if they are given by any other route. The available evidence shows that cutaneous inoculation against anthrax confers immunity, and in veterinary practice in France Besredka’s methods have had considerable vogue. Nicolas (1925) has reported that the results of cutaneous vaccination of the horses in the French army in Africa have been excellent, and that it is safer than the older method, in that the fatalities due to inoculation did not occur. (It must be remem- bered that these anthrax vaccines are living attenuated cultures, not dead cultures, such as are used in human immunization.) Velu (1927), in Morocco, as a result of an extensive trial in sheep, states that the cutaneous method of immunization has many advantages. Other observers have confirmed these results. Ledingham (1931) suggests that the immunity conferred is a general immunity due to the persistent application of the antigen to a relatively tolerant tissue rather than a local immunity of the “ one and only vulnerable site.” As regards intestinal organisms, Besredka maintains that the best method of immunization is to give the vaccine by the mouth so that the bacterial substances come in contact with the intestinal mucosa and set up a local immunity which prevents subsequent invasion of the body by these organisms. Besredka does not aim at bringing about the appearance of the usual antibodies in the blood, nor does he consider them of any significance in the local immunity of the intestine to diseases such as typhoid fever or dysentery. It is an old belief that vaccines given by the mouth give rise to a certain amount of immunity. Loeffler, as far back as 1906, obtained immunity to B. typhi murium (B. aertrycke) by feeding mice on dead cultures of the microbe. This observation has been repeatedly confirmed. Besredka has combined his enteric vaceines with bile in order 270 LOCAL IMMUNIZATION to modify the permeability of the intestinal mucosa. Whether bile is essential to the immunization seems doubtful. By the use of a dead vaccine combined with bile he has shown that he can obtain immunity to paratyphoid infections in animals. This immunity develops rapidly, and is said to be independent of the production of the usual antibodies. The result of such experiments led him to conclude that the immunity is a local one of the intes- tinal mucosa, and is not dependent on the presence of antibodies in the circulating blood. The existence of local immunity in the intestinal cells in such cases cannot be proved, but much work has been done on the question of whether or not antibodies appear in the blood. A large number of reports have been published showing that antibodies do appear in the blocd after the oral administration of enteric organisms (Shiga, 1908 ; Courmont and Rochaix, 1911 ; Webster, 1922 ; Hoffstadt and Thompson, 1929, ete.). Webster, working with mouse typhoid, found that immunity could be established by oral administration of vaccines, but that there was no evidence that it was due to a local immunity of the intestinal mucosa. Pijper and Daw (1930) have shown that the ingestion of a vaccine prepared from a flagellated typhoid bacillus results in the appearance in the blood of somatic (“ O ’) agglutinins only. The flagellar antigen is apparently destroyed in the alimentary canal. The author has found that a typhoid vaccine digested with trypsin and other ferments likewise produces, when injected into man and other animals, only “0” (somatic) agglutinins. It is possible, therefore, that the absence of any response to the flagellar antigen when the vaccine is administered by the mouth is due to the destruction of this antigen by the digestive ferments in the alimentary tract. Practical Administration to Man of Vaccines by the Mouth. Many people have a strong dislike of being pricked with a needle, and if as good immunity could be obtained by swallowing a vaccine it would be easier to organize immunization on a large scale. Typhoid vaccine per os has been extensively used. and there have been favourable reports. LOCAL IMMUNIZATION 271 Oral administration of T.A.B. (typhoid-paratyphoid) vaccine has been used extensively in South Africa, especially among native workers in the mines, who, according to Cluver (1929), objected to subcutaneous injection. Sometimes the vaccine was used as a suspension which was taken in teaspoonful doses, some- times it was made into pills. Three doses, each of 45,000 million bacilli, were given combined with ox bile in keratin-coated pills. Cluver reports that following the administration of this vaccine the incidence of typhoid fever has been very greatly reduced. No comparative observations were made with the vaccines admini- stered subcutaneously. : In the prophylaxis of cholera extensive observations were made in India by Russell (1927). The figures obtained are here summarized. \ . : ase Nature of Vaccine. | No. Oassnpt Deaths. ene Moi per cent. Three doses bile vac- | cine per os. " 4,932 18 4 0-36 22.2 Cholera vaccine, * sub- cutaneous . . | 25,645 90 27 0-35 30-0 Controls untreated . | 36,649 711 217 1-94 39 * 17,160 received one inoculation, 8,485 received two inoculations. In this comparative trial of cholera bili-vaccine (oral) and the ordinary cholera vaccine (subcutaneous) the figures obtained show that both conferred a high degree of immunity. Russell expressed the opinion that the subcutaneous method was superior, and he was able to show, for example, that five days after a single subcutaneous dose of cholera vaccine the immunity was about as high as that present three days after a full course of the oral vaccine. Vaccination by the mouth against dysentery was begun by Shiga, who obtained good results in certain asylums where the disease was endemic. Nicolle and Conseil (1922) used a limited number of volunteers, who were immunized ” by the ingestion 272 LOCAL IMMUNIZATION of four doses of 100,000 millions of dead Shiga bacilli on an empty stomach. Fifteen and 18 days afterwards they were given 10,000 and 20,000 millions of live Shiga bacilli. The two treated men remained well and the two controls contracted dysentery. Certain field experiments have been carried out. In a barrack epidemic at Versailles out of 1,070 unvaccinated men 297 contracted dysentery (27-7 per cent.), while of 546 soldiers who had been vaccinated orally only 42 contracted the disease (7:6 per cent.). Data from other sources have been recorded giving essentially the same results. From the recorded results there is no doubt that immunity can be produced by the administration of vaccine by the mouth. The dose required is much larger than is necessary by the subcutaneous route, and it is unlikely that the results will be so uniform. At present there seems to be no reason to give up the subcutaneous method except, as has been said before, in those cases where the individual's temperament is such that it is impossible to administer vaccines by injection. REFERENCES BESREDKA, A. 1927. * Local Immunization,” London. 5 1930. Bull. Inst. Past., 28, 19. Review with Biblio- graphy. BuSrREDKA, A., and Bassecnrs., 1918. Ann. Inst. Past., 32, 193. CLuver, E. 1929. Lancet, 1, 1302. CourmonT and Rocuarx. 1911. C. R. Acad. Sciences, 152, 797, 1027. Horrsrapr, R. E., and Tuomrson, R. L. 1929. Amer. Jour. Hyg., 9, 1. E Lepineuam, J. C. G. 1931. « Pneumococcus, Type I. . 1,000 3 » si Thy 17, . 1,000 * I. . 500 » The second and third doses were twice as strong. von Sholly and Park (1919-1920), who also used large doses, obtained entirely negative results following a large series of inoculations of mixed vaccine. On the other hand, a favourable series of observations is recorded by Eyre and Lowe (1918). They inoculated 1,000 men of the New Zealand Expeditionary Force in England with two doses of a mixed vaccine at the end of March, 1918, and observation was kept on these until August 18th of the same year. The vaccine was made from freshly isolated cultures obtained locally. The criterion they used was admission to hospital with respiratory disease, and comparison was made with the rest of the New Zealand Forces in England who were uninoculated. The figures obtained were these :— Admission to Hospital April 5th to August 18th. 1,000 inoculated men : . ” : 12 Average per 1,000 of 19,000 uninoculated men of the New Zealand Forces . : 73-1 MIXED VACCINES IN COLDS 319 Their serological investigations seemed to show that the average New Zealander was normally less immune to the organisms associated with respiratory catarrh than was the average British soldier. There are also several small series where the vaccine was apparently successful. Two of these may be referred to :— Simey (1921) quotes the case of a public school for girls where inoculation had been carried on for four years. In this case all the inoculated girls apparently became immune, for they did not miss one day, but among the uninoculated a considerable number con- tracted colds and influenza and two had pneumonia. Lempriere (1929) records his experiences at Haileybury School. He states that his results, although far from perfect, are more than good enough to justify continued effort, particularly in the case of known susceptibles to catarrhal infection. All the serious complications of *“ colds ”” occurred in the uninoculated. The number of cases, however, in each series is small, and the figures are not such as would satisfy statistical tests. Most of the published figures, therefore, seem to indicate that the results obtained by indiscriminate inoculation of the popula- tion against colds are indefinite. On the other hand, a large number of practitioners, and many of these are skilled observers, are convinced that it is possible to prevent “ colds ” by means of vaccines. Of course, the opinions of such practitioners are not capable of exact analysis, like the adverse figures quoted above, but they are not without evidential value. The population dealt with in the three negative series mentioned above and that on which the practitioners form their opinion are widely different. The first represents the whole populace, but the second only that section who suffer so frequently or severely from ‘colds ” that they seek inoculation as a preventive. It would appear that the general public, so far as colds are concerned, can be divided into three classes. The first and most numerous class is that in which the individuals only get occasional colds—perhaps one each winter—and who rarely worry about it or seek medical advice. Their immunity is apparently at a high level, as shown by the fact that they are only rarely infected, 320 PREVENTION OF THE COMMON COLD although very frequently exposed to infection—indeed, this class is the one most frequently exposed, as they usually take no precautions and do not mind coming into close contact with persons suffering from colds. It might be possible to increase their immunity somewhat, so that even if they contracted a virus infection there would be greater resistance to the associated bacteria, but this benefit would be offset by a certain number of cases in which there is exposure to a virulent infection in the few days after inoculation when the bacterial immunity may be temporarily reduced. This class is not likely to benefit by prophylactic vaccination. The second class consists of a not inconsiderable body of individuals who have a chronic infection of some portion of the respiratory tract. During the © quiescent ” periods the symptoms may be so slight that they are accepted as part of the normal course of events. This chronic infection has more or less frequent exacerbations which the sufferer labels as * colds.” The third class consists of those who, while no chronic infection can be demonstrated, yet suffer frequently from colds, and here it can only be assumed that they are naturally less immune to the primary infection. It may well be that many of these are actually suffering from a chronic infection. The diagnosis of such a condition is extremely difficult. It has been shown that a large proportion of the population normally harbour bacteria of pathogenic types, and it is very probable that a definitely patho- logical condition exists much more frequently than is generally recognized. In this connection Shope’s work on swine influenza (1932) is of great interest. He has shown that in this disease the primary infection is a virus, and that when animals are infected with this virus alone the result is merely a transient fever. When, however, the virus is combined with hamophilic bacteria, the true disease is reproduced in its entirety. He has shown also—and this is of the utmost importance— that inoculation with virus alone of pigs which were carriers of this heemophilic bacillus reproduced, not the simple virus disease, but the complete syndrome of swine influenza. This observation that a virus infection of normal animals (who MIXED VACCINES IN COLDS 321 do not carry the catarrhal bacteria) results merely in a transient illness, while in the animal with a bacterial infection a much more serious illness results, might be applied to the problem of the common cold. If, as seems probable, the same conditions apply, then any procedure which tends to rid a bacterial carrier of his infection should be of material assistance, especially in diminishing the severity of the cold, and theoretically it affords justification for the use of bacterial vaccines, even if the common cold is primarily due to a virus. As mixed vaccines have been almost universally used for the prevention of * colds,” there is lacking, in the case of most of the organisms concerned, definite proof of the power of vaccines of the individual microbes to protect man against infections of such microbes. The pneumococcus, however, which is probably the most common bacterium found in so-called colds is an exception, for there is ample evidence that a pneumococcus vaccine can protect laboratory animals and man against pneumococcal infec- tion. This is discussed in the chapter on lobar pneumonia (p. 298). With most vaccines the immunity conferred has been purely type- specific, but there are observations which showed apparently some wider specificity whereby men were protected against the hetero- geneous Group IV. pneumococci by vaccines of Types 1, 2, and 3. Now the pneumococci found in colds almost all belong to Group IV., which differ from the definite types and from each other in their serological relationships. In the case of * colds,” therefore, a vaccine which is species-specific would be ideal. Day (1933) has described such a vaccine. This is made from a partially autolysed culture, and when injected into mice it protects them against all types. It is with the second and third classes mentioned above that we are mainly concerned in a consideration of the question of pro- tection against colds by means of vaccines. Assuming that the primary infecting agent of the infectious “ common cold *’ is a virus, and that a vaccine of that virus does not exist, the third group is, so far as vaccine prophylaxis is concerned, in almost the same position as the first (immune) group. The only difference is that, as colds are more frequent, it R.A, VACCINES. al 322 PREVENTION OF THE COMMON COLD is more important for these individuals to be immunized against the respiratory bacteria so that if possible the severity of the attack may be lessened. In such cases, as there is no chronic infection and probably no increased sensitiveness to the bacteria concerned, there is no reason why the dose of the vaccine should be modified. It is of considerable importance, however, to recognize the second class, namely, those suffering from chronic infections. These persons doubtless contract virus colds in the same way as other people, but it is likely that most of the illnesses which they call *“ colds ” are merely exacerbations of the chronic infection. The best results of vaccine therapy are obtained in this class, but many of these sufferers have acquired a hypersensitiveness to one or more constituents of the mixed vaccine so that the injection of the large doses commonly recommended for prophylaxis may induce a focal reaction accompanied by the symptoms of one of the patient’s usual colds. If a chronic infection is recognized it is expedient to commence the inoculations with a dose of one- quarter to one-half the usual prophylactic dose. The season of the year at which the inoculations are carried out is of some importance. It has been found that at midsummer the incidence of respiratory catarrhal bacteria is distinctly less than it is in winter. Individuals inoculated in, say, September, before the ““ cold ” season has commenced, are less likely to be carrying these catarrhal bacteria than they are later in the year when they have doubtless been in contact with individuals suffering from acute catarrhs. There is no information as to how many carriers of the cold virus exist, nor is there any information as to whether this virus in a carrier can be awakened to activity by the appear- ance of a bacterial infection. Following the ordinary large doses which are used for prophylaxis, there is likely to be a temporary depression of bacterial immunity, and if such vaccines are given in the winter months to individuals who are carrying the virus, then during this temporary depression a bacterial infection may supervene, which, in its turn, may rouse the virus to activity, so that an ordinary cold results. As a result of experience, it has been found wise, when inocula- RESULTS OBTAINED IN HOSPITAL PRACTICE 323 < tions are commenced during the ‘ cold season, to commence with a small dose of vaccine and then proceed with the usual prophylactic doses. Composition of Prophylactic Vaccines against Respiratory Catarrhs. In almost all cases the stock vaccine used is a mixed one. Some vaccines contain representatives of all the types of organism associated with catarrhal conditions; others contain only the principal offenders, namely, pneumococcus, influenza bacillus, and streptococcus. A mixed vaccine of the latter type was largely used during the influenza epidemic of 1918-1919, and it has continued to be used since, both for the prevention of minor respiratory catarrhs and the more serious complications of influenza. In cases with chronic infections autogenous vaccines are frequently used. Here, however, they are used for treatment of the existing infection and for the prevention of exacerbations of such infections, which are usually called colds. Results obtained with Prophylactic Vaccines in Hospital Practice. It must be remembered that the patients who present themselves for inoculation are those who are more than usually susceptible to ““ colds 7” and ** catarrhs.”” Many of them have chronic bacterial infections. It is impossible to obtain comparable controls, and thus the results have to be assessed according to the opinion of the physician. In a large proportion of such cases there is a reduction in the number of *“ colds ”” and likewise in their severity. A certain number of patients report that after two or three inoculations they have been completely free from colds for one or more years, but it seems impossible to claim that such results are other than chance ones. In some cases, on the other hand, no benefit results from the vaccine. It has been found not infrequently that when a stock vaccine has failed and there is a chronic infection an autogenous vaccine is successful. In all such cases a more extended series of inocula- tions is necessary. In all the extensive series of prophylactic inoculations of random samples of the population which have been cited, three doses only of vaccine were given, but in those cases with chronic infections the question of prophylaxis is not so important as treatment of an existing infection, and if a longer 11-2 324 PREVENTION OF THE COMMON COLD course of vaccine treatment is given to such patients many successes can be recorded. So far as is known, the duration of immunity is short (not more than three months) and, therefore, if the vaccine is admini- stered in the autumn it should be repeated in the following January. Probably a better method is to administer a dose each month during the winter. Treatment of ‘‘ Colds '’ by Means of Vaccines The author has for many years been working in a laboratory where mixed stock anticatarrhal vaccines are constantly available. ‘roughness 7 of the throat was noticed a ¢ Whenever the first small dose of vaccine was administered. A second dose was often given after a few hours, and another next morning. In most cases this was successful in aborting the cold, although not in- frequently the herpetic attack which so often accompanies a cold appeared. This method, although useful to laboratory workers and others in an institution where vaccines are easy of access and where there is no financial consideration, is hardly suitable in practice where the patient does not seek advice till the infection is well established. The dose used is 0-1 e.c. of the mixed cold vaccine made at St. Mary’s Hospital. This may not be the best combination, but it appears to be effective. After the *“ cold ” has become established, in most cases vaccines have little effect except when the acute coryza passes on to a purulent rhinitis or a bronchitis lasting for several weeks. In such cases the duration of the infection can be materially reduced by vaccine therapy. Of course, this conclusion can only be a matter of opinion, as individual cases vary so much that no reliable statistical evidence is likely to be obtainable. The usual mixed stock ‘“ anti-cold ”’ vaccines may be used in minute doses, or the sputum or nasal mucus may be examined and an autogenous vaccine made. Small doses (say 10 million pneumococci, influenza bacilli or M. catarrhalis) may be given every day for three or four CHRONIC BRONCHITIS 325 days, and if that does not suffice then double the dose or more may be given on alternate days. Chronic Bronchitis The bacteriology of chronic bronchitis is essentially the same as that of the acute catarrhal infections, except that there is as yet no indication of a virus infection. The principal infecting microbes are B. influenzw, pneumococcus and streptococcus, with occasional infections of Gram-negative cocci, Friedlander’s bacillus and staphylococci, but in cases complicated with severe bronchiectasis there may be in addition a large and varied saprophytic flora. Stock or autogenous vaccines may be used, and it is probable that there is no condition in which vaccine therapy is more satis- factory to the patient. Autogenous vaccines are the most satisfactory. The nature of the infection can best be ascertained by the examination of a stained film of the sputum. If this examination is omitted and the sputum is simply placed on a medium like blood agar, then in a considerable proportion of the cases the influenza bacillus, which is probably the most common infection, will be completely missed. The stock vaccines employed are usually mixed vaccines similar to those used for the prevention of respiratory catarrhal infections, but preferably weaker. One that has been used successfully since 1910 has the following formula :— B. influenze . ‘ . 50 millions per c.c. Pneumococei : » . 50 \ ’s Streptococci : . . 10 5 Staphylococci. . . 200 " 5 M. catarrhalis : . 25 \ a B. septus ‘ ‘ . 25 ” " B. friedlanderi : . 25 2 This is given in doses commencing with 0-1 c.c. and increasing by 0-1 or 0-2 c.c. at intervals of from three to seven days up to 1 e.e. Some prefer a vaccine of the more common microbes only, and 326 PREVENTION OF THE COMMON COLD a suitable stock vaccine of this nature is the one which was recom- mended by the Army authorities for the prevention of influenza. Its composition is as follows :— B. influenze : ‘ . 60 millions per c.c. Pneumococci . : . 200 25 ys Streptococci : : . 80 ” vs This is used in doses similar to the more comprehensive vaccine described above. Some prefer a stronger vaccine, containing up to 1,000 million pneumococci and half that number of influenza bacilli, but it is probably wiser that the treatment should be commenced, at any rate, with the weaker vaccine. One advantage of these less comprehensive vaccines is that they give much less local reaction. A disadvantage is that they have no specific action on an infection other than one caused by the three organisms included in the vaccine. REFERENCES Day, H. B. 1933. Jour. Path. and Bact., 36, 77. Evre, J. W. H., and Lowe, E. C. 1918. Lancet, 2, 484. FERGUSON, F. R., DAVEY, A. F. C,, and TorLey, W. W. C. 1927. J. Hyg., 26, 98. FLEMING, A., and MacLEAN, I. H. 1930. Brit. Jour. Exp. Path., 11, 127. Foster, G. B. 1917. J. Inf. Dis., 21, 451. Hovre, J. 1932. J. Path. and Bact., 35, 817. JorpaN, E. O., and Suare, W. B. 1921. J. Inf. Dis., 28, 357. Kruse, W. 1914. Miinch med. Woch., 81, 1547. Lemprikreg, L. R. 1929. Brit. Med. J., 1, 973. MiLLs, K. C., SHIBLEY, G. S., and Docugez, A. R. 1930. J. Exp. Med., 52, 701. OLiTsky, P. K., and McCarTNEY, J. BE. 1923. J. Exp. Med., 28, 427. RoBERTSON, R. C., and Groves, R. L. 1924. J. Inf. Dis., 34, 400. Scumipr, P. 1920. Deutsch. med. Woch., 46, 1181. Snore, R. 1931. J. Exper. Med., 54, 373. SiMEY, A. J. 1921. Lancet, 2, 1051. TuaomsoN, D., and Twuomson, R. 1932. ‘The Common Cold,” London. Torrey, W. W. C., and others. 1930. °° Ministry of Health Reports on Public Health,” No. 58. voN Suorry, A. I., and Park, W. H. 1919-1920. J. Immunology, 6, 103. WEBSTER, L. T., and Crow, A. D. 1932. J. Exp. Med., 55, 445. CHAPTER XI INFLUENZA Many of the questions which have been discussed in connection with the common cold apply also to the problem of epidemic influenza. Bacteriology This has been admirably dealt with by Scott (1981) in the * Medical Research Council System of Bacteriology,” and by Dible in ‘““ Recent Advances in Bacteriology ” (1932), and it is unnecessary here to give more than a brief summary. Two views as to the primary infecting agent are held. One is that Pfeiffer’s influenza bacillus is responsible, and the other is that it is due to a filter-passing organism. B. influenze was accepted as the primary infection from 1892, when it was first described by Pfeiffer, until the pandemic of 1918-1919, when many workers became sceptical and search was made for another cause. Whether or not B. influence is the primary infection, it is agreed that in influenza as it appeared in the 1918-1919 epidemic this organism was constantly present in large numbers, and that it was an important pathogenic agent, especially in the pulmonary complications. That it played a part in the infection was shown by the fact that agglutinins and complement-fixing substances to it were found in the serum of patients suffering from influenza, but un- fortunately these tests are of no value in determining whether it was the primary infection, as they would equally appear if it was secondary to some other invader. The pathogenicity of this bacillus to monkeys was shown by Blake and Cecil (1920). Cultures of a virulent strain were instilled into the mouth and throat of twelve monkeys. All of these after a few hours became ill and showed coryza, fever, cough, etc., 327 328 INFLUENZA somewhat similar to true influenza. In two of the monkeys a clinically recognizable pneumonia developed. Cecil and Steffen (1921) made further studies on human volunteers. They used a culture derived from the peritoneum of a monkey, and this was instilled into the nose or throat. Most of the volunteers after a few hours contracted an illness which lasted two to ten days, and which resembled a mild attack of influenza. The influenza bacillus is, however, commonly present in all types of catarrhal infection of the respiratory tract, and is fre- quently the only or the predominant microbe in very chronic infections. Furthermore, bacilli which cannot by our present methods be distinguished from B. influenze are to be found in the mouths and throats of all healthy individuals (Fleming and Maclean, 1930). The alternative to Pfeiffer’s bacillus as the primary infective agent in epidemic influenza is a filter-passing virus. The observa- tions on this point during the great epidemic of 1918-1919 were inconclusive, and since then the indefiniteness of the disease “influenza ” has militated against any clear results being obtained. Olitsky and Gates (1921) isolated from the throats of influenza patients a minute filter-passing bacillus (B. prewmosintes), which they were able to cultivate and with which they were able to infect rabbits. They believed it to be the primary infective agent, which damaged the lungs and permitted infection with B. influenza and the other microbes responsible for the severe complications. Later work has shown that similar bacilli are present normally in the throat. The relation of these to B. pnewmosintes and the relation of B. pnewmosintes to influenza have yet to be fully worked out. Long, Bliss and Carpenter (1931) have recently used the same methods which were successfully employed by Dochez in connec- tion with the common cold. Three chimpanzees which had been kept isolated from possible infection were inoculated with bacteria- free filtrates of washings of the nose and pharynx of persons in the early stage of influenza. The inoculations were followed, after a short incubation period, by an influenza-like disease characterized especially by fever, prostration, and leucopenia. The symptoms produced in these animals were very different from those occurring VACCINES FOR PREVENTION OF INFLUENZA 329 in Dochez’s chimpanzees inoculated with filtrates from individuals suffering from the common cold. Shope’s work on swine influenza, cited above (p. 320), is of great interest in relation to the human disease. He showed that the primary infection is a virus, but a simple infection with the virus results in a transient fever, whereas a combined infection of the virus and influenza bacillus produces the true syndrome of swine influenza. It is tempting to compare these results with human influenza in 1918, when the summer wave was a two-day fever without complications, and the autumn wave was much more serious, with a high incidence of pulmonary complication and a relatively high mortality.! Use of Vaccines for the Prevention of Influenza and its Complications Whatever may be the nature of the primary infection in in- fluenza, all are agreed that the serious complications, especially bronchopneumonia, are wholly or in large part due to bacterial infections. The bacteria involved are chiefly B. influence, pneumococci, and streptococei, and vaccine prophylaxis has in the main been directed against these three organisms. The results obtained with vaccines have not been uniform. 1 Since this was written a very important communication has been made by Wilson Smith, Andrewes, and Laidlaw, in which they have shown that filtered bacteria-free washings from the throats of patients suffering from influenza (the 1933 epidemic) give rise to a febrile influenza-like disease in ferrets. They found that the disease passed from ferret to ferret by contact and they were able to transmit the disease through a long series of animals by means of suspensions of the nasal mucosa. Animals which had recovered from the disease were absolutely immune against fresh infection ; their serum, and also the serum from human convalescents, was capable of neutralizing the virus. An interesting point which these workers have brought out is that this influenza virus is identical with, or closely allied to, Shope’s virus of swine influenza. The latter gives rise to the same disease in ferrets and after recovery from an attack of Shope’s virus disease the animals are absolutely immune to the human virus. Animals which have recovered from the human virus disease are not, however, completely immune against Shope’s virus. This work provides strong evidence that the primary infection in influenza is a filter- passing virus and at the same time opens up a wide field for investigation by showing that a small and inexpensive animal. the ferret, is susceptible to the disease and can be used in the study of the problems of influenza. 330 INFLUENZA Two important negative reports have been made by von Sholly and Park (1921) and Jordan and Sharp (1921). Both of these relate to periods after the great influenza pandemic of 1918-1919. In both, large groups of individuals belonging to institutions were inoculated and adequate controls were available in people living under the same conditions. A mixed vaccine was given in three doses and the results were observed during an epidemic of influenza occurring in 1920. In both of these investigations it was found that no immunity had been conferred by the vaccine. In von Sholly and Park’s series the vaccine was given about three months before the epidemic, and in the other series the interval was about one month less. Three months is about the limit of time within which one might expect any immunity from an influenza vaccine, as it is well known that the immunity conferred by an attack of the disease is short lived. There is no record of the nature of the cultures used in the preparation of the vaccine—whether they were freshly isolated or old laboratory cultures. In the latter half of 1918 the British Army Authorities recom- mended the use of a mixed vaccine of the following composition : B. influenze . 60 millions. Pneumococcus . 200 os Streptococcus . 80 5 This was used on a large scale among the troops in England and the returns obtained were collected by Leishman (1920), who summarized in the table given below such returns as were con- sidered trustworthy. Ratios per 1,000 Incidence of Strength. Incidence of | pulmonary attack. complica- Deaths, tions. Inoculated : 15,624 14-1 1-6 0-12 Uninoculated 43,520 47-8 13-3 2.25 VACCINES FOR PREVENTION OF INFLUENZA 331 The observations were made between November, 1918, and April, 1919. Of the inoculated about one-half received only a single dose of 0-5 c.cm., while the other half received an additional dose of 1 c.cm. of the mixed vaccine. Eyre and Lowe (1919) have reported results obtained among men of the New Zealand Expeditionary Force in England. Their figures are : Inoculated. Uninoculated. Average total strength . 16,104 5,700 Incidence of influenza ‘ 1:3 9%, 419, Mortality of all cases . ; 0-26 9%, 2.2 9% These figures indicate that the vaccine conferred some pro- tection. The reduction of the mortality among the inoculated is striking. A large number of other reports favourable to vaccine prophy- laxis have appeared, for example, by Minaker and Irvine (1919), and Wynn (1920). Many of these reports, however, are seriously defective from a statistical point of view. The results published by Duval and Harris (1919) may be given in more detail. They used a freshly prepared vaccine of B. influenzee only, and gave doses containing 1,000, 500, and 1,000 millions respectively at intervals of three days. The figures which they obtained are given in the following table :— Percentage No. No. attacked. attacked. Inoculated . : 3,072 102 3-3 Not inoculated . 866 375 43-8 The inoculations were carried out at the height of an epidemic. It is stated that the controls consisted of such people as refused 332 INFLUENZA inoculation, and as all the inoculations were carried out within six days it would appear that the infection risk was about equal in the two groups. The results show a remarkably high incidence among the controls and apparently a very marked degree of protection among the inoculated group. Since the great epidemic of 1918-1919 there has been no opportunity of testing a vaccine on undoubted epidemic influenza. It is true that epidemics labelled influenza occur, but there is no certainty that they are the same disease as existed during the pandemic which ended in 1919. Since the use of the vaccines described is aimed chiefly at the prevention of the serious complications of influenza, and especially bronchopneumonia, the recent results from the gold mines of South Africa (p. 305) may again be referred to. During the past few years the bacteriology of pneumonia, as found in these mines, has been changing from an almost pure pneumococcal flora to a mixed flora of pneumococcus, influenza bacillus, and strepto- coccus, somewhat similar to that found in influenzal broncho- pneumonia. From the preliminary results now published it appears that considerable protection against these mixed infections can be obtained by the use of a mixed vaccine. The position remains that two well-controlled series of observa- tions made after the great epidemic of 1918-1919 gave absolutely negative results, whereas many observations made during the epidemic indicated that some protection resulted from the adminis- tration of vaccines. The incidence of the disease was less in the inoculated groups, but the chief result was a great diminution in the mortality. Many practitioners with extensive experience were very enthusiastic about the value of vaccines during the great epidemic, and, while their evidence has not the value of properly controlled experiments, it cannot be lightly thrown aside. Preparation of Anti-influenza Vaccine. As B. influenza, pneumococci and streptococci are the common bacteria asso- ciated with severe influenza, it is reasonable to incorporate these in a mixed anti-influenza vaccine. B. influenzw can be administered in doses of 500 or 1,000 millions without inducing any reaction. Wright, Lister and others have shown that man REFERENCES 333 tolerates well doses of 1,000 millions or more of pneumococcal vaccine. A suitable anti-influenza vaccine therefore may contain 500 to 1,000 millions of B. influenza, 1,000 millions pneumococci and 100 millions streptococci. There is no knowledge as to the degree of species-immunity which is conferred by a vaccine of a single strain of B. influenze, but as these bacilli differ widely antigenically it is safer to incor- porate in the vaccine a large number of freshly isolated strains. Such a vaccine might be administered in doses of 0-25, 0-5 and 1 c.cm. at intervals of four to seven days. The resulting immunity cannot be expected to last more than three months. REFERENCES Brake, F. G., and Ceci, R. L. 1920. J. Exp. Med., 32, 691. CeciL, R. L., and STEFFEN, G. J. 1921. J. Inf. Dis., 28, 201. Dire, J. H. 1932. ‘ Recent Advances in Bacteriology,” London, Pp. 256. Duvar, C. W., and Harris, W. H. 1919. J. Immunol. 4, 317. Evre, J. W. H., and Lowe, E. C. 1919. Lancet, 1, 553. FLEMING, A., and MacrLeaN, I. H. 1930. Brit. J. Exp. Path., 11, 127. JorpaN, E. O. and Snare, W. B. 1921. Jour. Inf. Dis., 28, 357. Leisnvan, W. B. 1920. Lancet, 1, 366. Long, P. H., Briss, E. A., and CARPENTER, H.M. 1931. J. Am. Med. Ass., 97, 1122, MINAKAR, A. J., and Irvine, R. S. 1919. Jour. Am. Med. Ass. 72,847. Ouirsky, P. K., and Gates, F. L. 1921. J. Exper. Med., 33, 125, 261, 373, 713 ; 34, 1. Suore, R. 1931. J. Exper. Med., 54, 373. Scorr, W. M. A System of Bacteriology,” London, H.M. Stationery Office, 2. 352. SyrrH WILSON, ANDREWES, C. H., LaiprLaw, P. P. 1933. Lancet 2, 66. von SnorLy, A. I, and Park, W. H. 1921. J. Immunol., 6, 103. WynN, W. H. 1920. Lancet, 2, 313. CHAPTER XII WHOOPING COUGH Tuis is a disease in which prophylactic inoculation might be expected to give excellent results. One attack of the disease confers a life-long immunity and, if Bordet’s bacillus is the primary infecting agent. it is reasonable to assume that prophylactic inoculation with a vaccine of this bacillus will confer a consider- able degree of immunity. However, the results obtained have been very contradictory. Reasons for these contradictory results will be discussed in connection with the antigenic structure of the bacillus. Quite recently some evidence has been brought forward to show that whooping cough may be a virus disease, but there is as yet no definite proof of the existence of such a virus, while the evidence for the etiological importance of Bordet’s bacillus is very strong. Antigenic Structure of B. pertussis As with other bacteria, it is of considerable importance to the vaccine therapist to ascertain whether the different strains of B. pertussis are antigenically identical. As far back as 1910 Bordet and Sleeswyck showed that inoculation of animals with old stock strains of this bacillus, grown on agar, did not result in the production of good agglutinating sera for freshly isolated strains. These workers considered the nature of the culture medium to be of great importance in regard to antigenic capacity. Bacilli grown on rich blood medium differed antigenically from those grown without blood, but even when grown on blood agar the bacilli were shown by Bordet (1913) gradually to degenerate in toxicity and immunizing power. The importance of this observa- tion apparently escaped general notice for many years. Krum- wiede, Mishulow and Oldenbusch (1923) differentiated two types 334 ANTIGENIC STRUCTURE OF B. PERTUSSIS 335 by agglutination tests, and Hayano (1923) found four serological types. Similar results have been reported elsewhere. Among the Danish workers, to whom we are indebted for much of our knowledge regarding whooping cough, Kristensen (1922 and 1927) found that all the strains he isolated belonged to a single type. A very important communication on this subject is that of Leslie and Gardner (1931), which serves to throw light on these different findings and probably also on the variable results which have been obtained in practice with vaccines of B. pertussis. They analysed the antigenic properties of 32 strains of this bacillus obtained from different sources and found that all fell into four well-marked groups, which they termed Phases I.. IL, III, and IV.” All of the freshly isolated strains, whether isolated in England or Den- mark. were in Phase I. and were toxic for guinea-pigs. The stock laboratory strains were almost without exception in Phases III. and 1V.. and were non-toxic. They further showed that the two American serological types were identical with Phases IIL. and IV. (type A = Phase IV. and type B = Phase II1.). Leslie and Gardner were able by various cultural methods to show that a culture from a single colony can produce in turn all the antigens characteristic of the four phases, thus showing that the different “types,” which had been described, are merely variants of the same bacillus. They believe that Phases I. and IL correspond to the * smooth” pathogenic form of other bacteria and Phases IIL and IV. to the * rough’ saprophytic form. In culture there is a tendency for the bacilli to pass into Phases III. and IV., but if sufficient fresh blood is incorporated in the medium the original phase persists for long periods in culture. It is sometimes impos- sible to bring about a reversion from Phase IV. into the pathogenic forms. The antigenic relationships of “R™ and “S77 variants are described elsewhere (p. 258), and it has been shown that vaccines of the “$7 form have immunizing powers, whereas vaccines of the *“ R ” form are deficient in this respect. Leslie and Gardner have obtained results indicating that an efficient prophylactic vaccine can only be made from bacilli in the patho- genic Phase (I.). This is of the utmost practical importance in the preparation of a vaccine. There is no doubt that many of the 336 WHOOPING COUGH vaccines in the past were made from old stock cultures. (An exception is the Danish vaccine, as in that country whooping cough was diagnosed by culture and a plentiful supply of freshly isolated strains was at hand.) This in all probability accounts for the very variable results which have been obtained with the vaccine here and in America, and for the good results which the Danish workers have got, especially in the Faroe Islands. Preparation of the Vaccine. In view of the older work of Bordet and the recent observations of Leslie and Gardner, the vaccine should be made as soon as possible after isolation of the cultures, and the cultures should have been continuously grown on fresh blood agar. Old stock cultures which have been kept for a long time on serum agar or some other bloodless medium are unsuitable owing to the probability that they have changed antigenically into Phases III. or IV. This recommen- dation really applies in greater or less degree to all vaccines, but it is desirable to mention it specially in connection with B. pertussis, as cultures of this organism have not in the past been as easy to obtain as cultures of many of the other organisms from which vaccines are prepared, and in many laboratories the asy path has been taken and stock vaccines have been made from old cultures. Apart from this, there are no special recommendations in the preparation of simple B. pertussis vaccines. Mixed Vaccines. For the prevention of whooping cough a simple B. pertussis vaccine may be used or this microbe may be combined with the usual secondary invaders to form a mixed vaccine in the same way that mixed vaccines are prepared {rom the various bacteria associated with other respiratory catarrhal conditions. It was originally shown by Bordet and it has been amply confirmed that in the early stages of whooping cough B. pertussis may be present in large numbers and in almost pure culture, but that very soon secondary invaders appear. These secondary invaders are in almost all cases B. influenze and pneumococci. There is no published evidence to show that mixed vaccines are superior for prophylactic purposes to the simple vaccine, but for RESULTS OF PROPHYLACTIC INOCULATION 337 treatment it is reasonable to assume that it would be wise to attempt to immunize against the complete infection rather than against merely a portion of it. Results of Prophylactic Inoculation The reported results are conflicting, but in most cases some immunity has been observed. It will serve no good purpose to go through in detail the individual results, as there is no record of the nature of the cultures from which the vaccines were prepared, and, as has been shown in connection with the antigenic structure of B. pertussis, this is all-important. As an instance, the stock vaccine which has been made for the last 20 or more years at St. Mary’s Hospital consisted, until recently, of old stock cultures with a variable quantity of more recently isolated strains. In view of Leslie and Gardner’s work, the vaccine must have varied in its immunizing properties largely according to the content of recently isolated strains, and this would account for the varying results obtained by individual users of the vaccine. Needless to say, this vaccine and in all prob- ability most stock vaccines now are made from B. pertussis in Phase I. In large cities it has been found difficult to obtain reliable figures relating to protection against whooping cough. The Danish workers, however, have been favoured in having in the Faroe Islands an isolated community on whom valuable observations could be made. Every few years epidemics of whooping cough occur, and in these epidemics practically every unprotected person contracts the disease. Madsen has reported on an epidemic occurring in 1923. Over 2,000 individuals were inoculated with vaccines of B. pertussis. There was one district in which only 2 out of 108 non-vaccinated persons escaped infection, as against 20 per cent. of those vac- cinated, who remained uninfected. Of the whole 2,094 vaccinated individuals only 5 died, while there were 18 deaths among 627 persons who were not vaccinated. The incidence and the mor- tality, therefore, were much reduced, and the opinion is expressed 338 WHOOPING COUGH that the disease was much less severe among the vaccinated. Kristensen and Larsen (1926) have shown that immune bodies (complement-fixing substances) appear in the serum of inoculated individuals, just as they do after an attack of the disease. The results obtained in 1923 have been confirmed in a more recent epidemic in the Faroe Islands (Madsen, 1932). The figures reported by Madsen are given in the following table : Vaccinated. Non-vaccinated. 1832. 446. Number. Per cent. Number. | Per cent. Not attacked . v 458 25 8 2 Mild cases . : 4 1,236 3 225 50 Moderate cases J 29 1:6 170 38 Severe cases . 3 8 0-4 35 8 Fatal cases . . . 1 — 8 2 From these figures it seems quite clear that the vaccine had a definite influence in protecting against the attack, and in modifying the disease so that it was much milder in the vaccinated indi- viduals. This is brought out even more clearly if the * moderate cases are neglected, as in the following table : Vaccinated. Non-vaccinated. Number. Per cent. Number. Per cent. Not attacked or mild cases . % * 1,694 98 233 52 Severe or fatal cases 9 0-5 43 10 These latest figures show clearly that inoculation with a vaccine of B. pertussis does in susceptible individuals confer some immunity against an attack of whooping cough and that if an inoculated person should contract the disease the severity of the attack is markedly diminished. Pierret (1932) states that 90 per cent. of success is obtained by giving three injections of 250 millions B. pertussis on alternate days for prophylaxis. In cases where there was the slightest TREATMENT BY VACCINES 339 fear of the existence of whooping cough, doses were given every day to abort the disease. He attaches some importance to the method of killing the bacteria constituting the vaccine, pre- ferring a fluorized vaccine, and states that much larger doses must be given (up to 4,000 millions) if the vaccine is killed by heat. He sums up : “It can be said that anti-whooping cough vaccine, if it could be administered to all contacts, would stifle epidemics in hospitals and villages, and would render whooping cough one of the least important epidemic diseases, instead of being, as at present, one of the most contagious and serious of all.” Pierret is more optimistic than the more northern observers, perhaps because there is some difference in the whooping cough seen in France and that prevalent in the northern climate of the Faroe Isles. Vaccines in the Treatment of Whooping Cough Vaccine therapy in whooping cough was first carried out by Freeman in 1909, and he reported that the vaccine-treated cases recovered more quickly. Various unfavourable reports have appeared (Hess (1914), von Sholly, Blum and Smith (1917), Paterson and Smellie (1922), (Howell, 1930), (Thomson, 1930) ), but on the other hand there are a very large number of reports showing good results following the use of vaccines. Only some of the more important or recent can be referred to. An interesting one is that of Nicolle and Connor (1913), who used a living vaccine. They injected this subcutaneously in doses of 400 to 2,000 millions every two or three days, and they claimed that 78 per cent. of the cases were cured in from three to twelve days. Herrman and Bell (1924), as a result of a study of 300 cases, have expressed views very favourable to vaccine treatment. They state that in some 25 per cent. of the cases the improvement was so marked ¢ that there was no doubt as to the specific effect.” Bloom (1925) has recorded the results of thirteen years’ experience. He uses a mixed vaccine of B. pertussis and B. influenze in doses of 5,000 millions of the former, and 3,500 millions of the latter on alternate days at first, and later on every 340 WHOOPING COUGH third or fourth day until the prominent symptoms abate. He has found the average duration of the disease to be 24 days and states that vaccine therapy is the most efficient form of treatment. Leitner (1930) considers that vaccine therapy represents a great advance in the crusade against whooping cough. He gives six to eight large doses at intervals of 24 or 48 hours. He, like most other workers, emphasises the importance of commencing the treatment as early as possible. Kaupe (1930) used doses of 2,000, 4,000 and 6,000 millions in children and nurslings in the very early stages of whooping cough, and reports very favourable results, the children being practically cured 10 to 12 days after the first injection. Pierret (1932) is enthusiastic as regards vaccine therapy up to the third week of the disease, ¢ that is, during the catarrhal stage, and before the whoop becomes established, or when the Bordet- Gengou organism is still easily demonstrated.” . . . “In the least promising cases the nature and duration of the coughing attacks are modified and the disease develops more mildly and is free from complications. Vomiting usually ceases after the third or fourth day of the treatment and sometimes even after the evening of the day on which the first injection is given. In favour- able cases one sometimes witnesses a real suppression of the disease, which ends suddenly in from two to four days.” He states that he has “never seen the usual complications of whooping cough in patients treated early.” As in the case of prophylactic inoculation, Pierret goes further than our English workers, but it is to be hoped that our results in the future will equal those he has obtained in France. It is very difficult from a study of the reports to assess the true value of vaccine therapy. Some competent observers have obtained absolutely negative results, whereas others are enthu- siastic. As many stock vaccines have in the past been made from old and probably degenerate cultures, it seems that the nature of the vaccine is tne most likely explanation of the very different results which have been obtained. Now that the importance of using freshly isolated cultures is more fully appreciated by the manufacturers of stock vaccines, it may safely be assumed that DOSAGE OF VACCINE 341 the results in the future will in general be better than those which have been obtained in the past. It would appear that it is especially in the carly stages of the infection that the best results are to be obtained with vaccines, and the evidence supports the conclusion that vaccine therapy commenced early does cut short the attack. Dosage of the Vaccine. With different observers this has been very variable. However, B. pertussis vaccine can be injected in large doses into patients without reaction. Among those who have reported favourable results, Luttinger (1915-1917) commenced with 250 millions, and the dose was doubled on alternate days, four doses in all being given. Bloom (1925) gave 5,000 millions B. pertussis and 3,500 millions B. influenze on alternate days till the most prominent symptoms abated, and then lengthened the interval to three or four days. Pierret (1932) gives 250 millions of fluorized bacilli every day for three days, then the same dose on alternate days, till six in- jections have been made, and finally one to three additional injections of the same dose. Of these, only Bloom used a mixed vaccine, but as there is almost constantly in the later stages a secondary infection of B. influenza and pneumococci, there seems to be a case for the combination of these bacteria with B. pertussis in a vaccine for therapeutic use. B. pertussis vaccine gives very little reaction, and doses up to 4,000 or 5,000 millions can be given to children without difficulty. For prophylaxis, a suitable scale of dosage for children of different ages is shown in the following table : 3rd Dose in 1st Dose. 2nd Dose. millions of B. pertussis. Under 1 year . . 400 800 1,600 1 to 2 years : : 800 1,600 3,200 3 to 5 years 3 * 1,200 2,400 4,000 5 to 10 years . 1,600 3,200 4,000 Over 10 years . : 2,000 4,000 4,000 4 342 WHOOPING COUGH For the treatment of whooping cough the dose of vaccine cannot be regarded as by any means settled. Luttinger used 250 millions, Kaupe 2,000 millions, and Bloom 5,000 millions, so that it can be seen that within a large range of dosage favourable results have been obtained. It remains for future workers to determine accurately the optimum dose. It may be that for the treatment of whooping cough it is better to combine vaccines of the secondary invaders with B. pertussis vaccine. An appropriate mixed vaccine of this type might contain B. pertussis 500 millions, B. influenzee 250 millions, and pneumococcus 20 to 100 millions. Such a vaccine could be given every day or every two days to a child of five or six years in doses of from 0-2 c.c. to 1 c.c.! REFERENCES Broom, C.J. 1925. Arch. Pediat., 41, 13. Borber, J. 1913. Zentralb. Bakt. Paras. Abt. 1, Orig., 66, 276. BorbeT, J., and SLEEswyck. 1910. Ann. Inst. Pasteur., 24, 476. FREEMAN, J. 1909. Brit. Med. Jour., 2, 1064. Havano, M. 1923. Japan Med. Wld., 3, 104. Herrman, C., and Bern, T. 1924. Arch. Pediat., 41, 13. Hess, A. F. 1914. J. Am. Med. Ass., 63, 1007. Kavrr, W. 1930. Med. Klinik., 26, 1735. Kristensen, M. 1922. Comm. Inst. Sérothérap. de I' Etat Danois., 13, No. 2. 2 1927. C. R. Soc. Biol., 96, 355. KRISTENSEN, M., and LARSEN, S. A. 1926. C. R. Soc. Biol., 95, 1110. KrumwieDpe, C., ef al. 1923. J. Inf. Dis., 32, 22. LerrNer, P. 1930. Jahrbuch f. Kinderh., 129, 343. Leste, P. H., and GarpNER, A. D. 1031. J. Hyg., 81, 423. LurTiNGER, P. 1917. J. Am. Med. Ass., 68, 1461. » 1915. New York Med. Jour., 101, 1043. MADSEN, TH. 1932. Centenary Meeting, Brit. Med. Ass. "5 1925. Boston Med. Surg. J., 192, 50. Nicorrr, C., and Connor, A. 1913. C. R. Acad. Se., 156, 1849. Paterson, D., and SMELLIE, J. M. 1922. Brit. Med. Jour., 1, 713. Prerrer, R. 1932. Proc. Roy. Soc. Med., 1329. Trissier, P., Renny, J., Rivanier, E., and CamBAsseEDES, H. 1929. J. Physiol. et Path. Gen., 27, 549. . VON SHoOLLY, A. I., BLuMm, J., and Smith, LL. 1917. J. Am. Med. Ass., 68, 1451. ! Cockshut (Brit. Med. J., 1933, 2, 819) considers these doses too small and states that he has obtained extremely good results in the treatment of whooping cough by the use of doses 4 to 10 times those recommended. S CHAPTER XIII ACUTE RHEUMATIC FEVER Much attention has recently been given to the bacteriology of this disease, and certain important results have been obtained. Since Poynton and Paine originally isolated streptococci from certain rheumatic lesions, this group of organisms have been especially associated with acute rheumatism. Different observers, however, have incriminated different types of streptococci, and this has caused considerable confusion. Poynton and Paine isolated non-h@molytic streptococci; Streptococcus viridans has been isolated (Cecil, Nicholls and Stainsby, 1929, etc.), while Small (1927) and Birkhaug (1927) have described streptococci of the indifferent type. There is also accumulating a considerable body of evidence in favour of haemolytic streptococci being associated with acute rheumatism. There have been different conceptions of the nature of the infection, e.g. :— 1. That it is a generalized infection. 2. That there is a streptococcal focus and that the rheumatic symptoms are due to toxins absorbed from that focus. 3. That by absorption of some streptococcal product from an infected focus the tissues have acquired a peculiar hypersensitive- ness to streptococci or their products, and the rheumatic symptoms are allergic in character. The variety of streptococci which have been associated with rheumatic fever makes it difficult to believe that this disease is a generalized infection. It is against all bacteriological teaching that a definite clinical entity should have a varied microbic etiology. The theory that it is a toxic manifestation hardly helps in this respect unless we assume that very varied streptococci can produce the same toxin—a hypothesis for which there is no 343 344 ACUTE RHEUMATIC FEVER experimental foundation. The allergic theory is more helpful as it seems possible that the sensitizing protein may be common to a variety of streptococci so that patients infected with one strepto- coccus may become sensitized, not only to that one, but to all others which possessed the same sensitizing protein. The allergic theory was propounded by Swift and his co-workers (1928-1929), who showed that repeated intradermal, subcutaneous or intra- muscular injections of streptococci give rise in animals to great hypersensitiveness to streptococci and their products. They showed also that intravenous injections desensitize the animals. It has been noticed by various observers that some 10 to 20 days after an acute streptococcal sore throat it is not uncommon for acute rheumatic symptoms to develop, and attention has been drawn to the similarity in the time relation between this and serum sickness. Non-haemolytic Streptococci in Acute Rheumatic Fever. Strepto- cocci of the viridans type, similar to those associated with chronic rheumatic conditions, have repeatedly been isolated from lesions in rheumatic fever. An important communication in this con- nection has appeared by Cecil, Nicholls and Stainsby (1929). By a special technique they isolated streptococci from blood cultures in 35 of 60 patients with rheumatic fever. Of these streptococci, 33 were of the viridans type, while one was haemolytic and one was indifferent. From 66 control bloods, streptococci were isolated in only a single instance. With regard to the joints, they obtained streptococci from the synovial fluid in five out of seven acute rheumatic joints examined, while 13 controls all gave negative results. Birkhaug (1927) isolated an indifferent streptococcus from various lesions in rheumatic fever patients. From most of his cultures he obtained a toxic filtrate. He gave himself 1 c.c. intramuscularly in the forearm and into the wrist joint, and this was followed by an attack of acute polyarticular rheumatism. When tested by intradermal injection of dilutions of the filtrate, the majority of patients with a definite history of rheumatic fever showed a distinct hypersensitiveness. In a later communication, Birkhaug (1929) has stated that rheumatic fever patients exhibit a NON-HAEMOLYTIC STREPTOCOCCI 345 hypersensitiveness, not only to his indifferent streptococcus, but also to streptococci of the viridans type. Small (1927) isolated a similar indifferent streptococcus which he has called streptococcus cardio-arthritidis. Small carried out treatment of 282 patients with a serum and vaccine made from this streptococcus. Acute cases treated with serum sometimes rapidly improved. Joint pains and choreic movements ceased within a week. Frequently, however, relapses occurred after a few weeks, when a vaccine of the streptococcus was used to produce a more lasting active immunity. It was found that patients with deep-seated rheumatic infections had a low opsonic index to the streptococcus and the opsonic content of the serum was found to run more or less parallel with the clinical condition, being low in the acute stage and then rising and remain- ing high during convalescence. The patients with a low opsonic index were found to be very sensitive to the vaccine. The initial dose of the vaccine was 1,000,000 cocci, and the doses were repeated every five to seven days. To avoid what he terms the * Secondary Reaction,” which, when it occurs, is at its height 10 to 14 days after the injection, he prefers a soluble antigen consisting of a bacteria-free filtrate of a saline suspension of the streptococci which had been allowed to stand for seven days in a refrigerator to extract. Small’s results with a combination of serum in the acute stage followed by vaccine to prevent relapses are favourable, but further results must be obtained before the treatment can be held to be generally effective. Hemolytic Streptococci in Rheumatic Fever. Mention has already been made that it has frequently been noted that it is not uncommon for rheumatic fever to follow an acute hzmolytic streptococcal sore throat. Parish and Okell (1927) showed that if rabbits which were passively immunized with hemolytic strepto- coccus antitoxin were infected with virulent haemolytic strepto- cocci they developed subacute joint lesions. Todd (1932) has found that patients suffering from rheumatic fever have in their serum a greatly enhanced content of streptococcal anti-hzemolysin. This was found also in patients recovering from definite haemolytic 346 ACUTE RHEUMATIC FEVER streptococcal infections (puerperal fever, etc.). Todd’s series of rheumatic cases is not a very large one, but if by more extended observations it is confirmed that all patients with rheumatic fever have a high titre of anti-heemolysin in their blood, this should go a long way towards establishing the association of haemolytic streptococci with the disease. Collis, Sheldon and Gray have investigated the sensitivity of the skin to extracts of haemolytic streptococci, and they have found that patients with acute rheumatism are definitely hypersensitive. It must be remembered that Birkhaug has found that these patients are hypersensitive to various non-haemolytic streptococci. Recently (1932) Coburn and Pauli have published several papers on the relation of haemolytic streptococci to acute rheumatism. They support the work of Glover and Griffith, and Collis in England, and bring forward much new evidence that rheumatic fever is a sequel to a hwxmolytic streptococcal infection of the throat or elsewhere. They have shown that the haemolytic streptococci associated with rheumatic fever are, like the scarlatinal strepto- cocci, of different serological types. Their observations have led them to the conception * that the specificity of the rheumatic response, like the reaction in certain other streptococcal diseases, depends not entirely on the character of the parasite, but is related to some individual mechanism of the rheumatic subject.” They support Todds work on the presence of streptococcal anti-heemolysin in the serum of rheumatic fever patients, and extend it by showing the presence of other antibodies (agglutinins, compiement-fixing bodies and precipitins) to haemolytic strepto- cocci. Coburn and Pauli have established a strong case for rheumatic fever being a manifestation of a haemolytic streptococcal infection. Vaccine Treatment with Hemolytic Streptococci Wilson and Swift (1931) used intravenous injections of strepto- cocci with the object of densensitizing the patients. Their preliminary report covers a four-year period of observation from 1927 to 1930—on 172 children, the majority of whom were under TREATMENT WITH HAEMOLYTIC STREPTOCOCCI 347 ten years of age. Approximately one half of the children were treated by intravenous inoculation, while the other half, which formed a comparable group, served as controls. The initial dose was 250,000 streptococci, and at weekly intervals the amount was doubled until a maximum dose of 10 million was reached, after which this dose was repeated several times. In 50 cases a second course of treatment was administered. Focal reactions were not observed and general reactions were slight and infrequent. In the inoculated group 45 per cent. were free from recurrence for periods of from sixteen months to two years after treatment as against only 18 per cent. in the control group. Wilson and Swift consider that it is very probable that this diminished incidence of recurrence has a definite relation to the intravenous vaccination. Collis and Sheldon (1932) have treated a number of children with intravenous injections of stock haemolytic streptococcal vaccines. Most of these were treated as out-patients. Although there were among these out-patients a few mild reactions in the early part of the treatment, they agree with Wilson and Swift that such intravenous therapy can be used without undue risk in an out-patient clinic. Weekly doses were given commencing with 200,000 cocci followed by 500,000, and then continued as follows : 1, 21, 5, 10, 15, 20 millions, and then by 10 million increases up to 100 millions. With cases of rheumatic carditis no difficulty was experienced, but six cases of convalescent chorea were found to be liable to unpleasant reactions. These chorea cases were very sensitive to streptococcal extracts. The treatment had no effect on the vague pains of ¢ subacute rheumatism,” but there is little evidence that this condition is associated with hemolytic streptococci. Seven acute cases of active rheumatic heart disease were treated with intravenous injections of streptococci, and of these the treat- ment had to be discontinued in three owing to the occurrence of unpleasant reactions with the early doses. The remaining four received up to 70 millions. They all improved and had no relapses within one year. Collis and Sheldon note that in those that improved the hamolytic streptococcal infection of the throat rapidly disappeared, while in those in whom treatment had to be 348 ACUTE RHEUMATIC FEVER discontinued the tonsils were persistently infected with haemolytic streptococci. The skin reaction to extracts of haemolytic streptococci was followed in 25 children. These were originally all strongly positive, but after treatment 16 of the 25 were completely negative. Cases are cited to show that there is a parallelism between clinical improvement, desensitization and the presence of immune bodies in the serum. These preliminary results are very encouraging, and this method of intravenous inoculation may prove extremely useful in the treatment of acute rheumatism. Intravenous Inoculation of Vaccines. The fact that intravenous inoculations of streptococci can be carried out safely in an out- patient clinic is of considerable practical importance. This has been stated by Swift and his colleagues and confirmed by Collis and Sheldon in connection with haemolytic streptococci in acute rheumatism, and Wetherby and Clausen have found the same to apply to a non-hxmolytic streptococcus in chronic rheumatism. It is well known that there is a vast difference in the reaction following the intravenous injection into man of streptococci and of bacilli, such as B. typhosus or B. coli. Injection of the latter, even in comparatively small doses (say 25 or 50 millions), are almost always followed by typical protein shock, indeed, such inoculations form one of the most popular and effective means of inducing protein shock. Similar doses of streptococci, however, inoculated intravenously are usually followed by no reaction whatever, although, as stated by Collis and Sheldon, if the patient is hyper- sensitive to streptococci, reactions may follow. With some other bacteria the reaction following intravenous injection of killed vaccines is inconsiderable, and in such cases vaccine therapy by the intravenous route may be more effective than by the usual subcutaneous injection. The author has used this method with vaccines of the acne bacillus in the out-patient department. This vaccine gives hardly any reaction, and the results appeared to be distinctly better than when the vaccine was given by the subcutaneous route. Much work remains to be done on this subject. REFERENCES 349 The practical drawback to intravenous inoculation in the out- patient department is the occurrence of occasional reactions consisting of fever with sometimes a rigor and nausea, or even vomiting. Although these reactions may not often occur, they may in certain circumstances be very distressing to patients treated in an out-patient department. REFERENCES Birknavc, K. E. 1927. J. Inf. Dis., 40, 549. ” 1929. J. Inf. Dis., 44, 363. Braprey, W. H. 1931. Quart. Jour. Med., 1, 79. Ceci, R. L., Nicoorts, E. E., and Stainssy, W. J. 1929. J. Exp. Med., 50, 617. . CoBURN, A. F., and Paurr, R. H. 1932. J. Exp. Med., 56, 609, 633, 651. Corrs, W. R. F., and SurLpbon, W. 1932. Lancet, 2, 1261. Corrs, W. R. F. 1931. Lancet, 1, 1341. GLOVER, J. A., and Grirrrr, F. 1931. Brit. Med. Jour., 2, 315. Paris, H. J., and Oxerr, C. C. 1927. J. Path. and Bact., 30, 521. SCHLESINGER, B. 1930. Arch. Dis. Children, 5, 411. SmarLy, J. C. 1927. Am. J. Med. Sc., 173, 101. os 1928. Am. J. Med. Sc., 175, 638. SueLpoN, W. 1931. Lancet, 1, 1337. Swrrr, H. F., DERICK, L., and Hircucock, C. H. 1928. Trans. Assoc. Amer. Physicians, 43, 192. Swirr, H. F., and Derick, L. 1929. J. Exp. Med., 49, 615. ” # 1929. J. Exp. Med., 49, 883. Swirr, H. F., Hrrcucock, C. H.,, McEwEN, C., and DERICK, L. 1931. Amer. Jour. Med. Sc., 181, 1. Toop, E. W. 1932. Brit. Jour. Exper. Path., 13, 248. WitsoN, M. G., and Swirr, H. F. 1931. Amer. Jour. Dis. Children, 42, 42. CHAPTER XIV CHRONIC RHEUMATIC CONDITIONS Tue term “ Chronic Rheumatism » has been used popularly to include very many painful conditions, some of which are totally different pathologically from those to be discussed, but the phrase * Chronic Rheumatic Conditions » is convenient in the discussion of the various forms of non-specific infective arthritis, together with fibrositis and perineuritis, which, so far as is known, are etiologically similar. *“ Chronic Rheumatic Conditions * is there- fore used to include fibrositis, rheumatoid arthritis, osteo-arthritis, arthritis deformans, and villous arthritis. * Typical cases of the above types of arthritis differ in their morbid appearances, but great similarities are present in all, and a case belonging in its early stages to one type may in its later stages develop into another ”* (Willcox, 1926). As the etiology and the methods of vaccine treatment of these different clinical conditions are the same, it is convenient to deal with them together. We are not here concerned with chronic infective arthritis due to a specific organism such as the tubercle bacillus or the gono- coccus. Such cases are treated with vaccines of the specific organism, but the general lines of treatment remain the same. Sometimes, however, cases are labelled gonorrheeal arthritis merely because they follow soon after a gonorrhceal infection ; when they are treated with a gonococecal vaccine they do not improve although they may subsequently derive much benefit from a streptococcal vaccine. In such cases it may be assumed that the relation of the arthritis to the gonorrhceal infection is merely one of time, and that the condition is really a rheumatic one. The treatment of these common and often crippling chronic rheumatic conditions by means of vaccines has engaged the atten- 350 BACTERIOLOGY 351 tion of many workers—indeed, no one practising vaccine therapy can avoid the problem—but unfortunately there is yet no abso- lutely clean-cut method which can be applied successfully to all cases. Good results have been obtained with vaccines by different observers using methods which are very diverse, and there is no doubt that vaccine therapy can alleviate or cure the condition in a considerable proportion of the cases. Bacteriology. Many attempts have been made to cultivate bacteria from the joints in cases of rheumatoid arthritis. Most of these have been unsuccessful and there must be a very large number of bacteriologists who have made cultivation from small numbers of such joints and who have not troubled to publish their negative findings. Successful cultivations have, however, been recorded from joints, from lymphatic glands draining affected joints, and also from the circulating blood. In most cases the organism isolated has been a streptococcus of the viridans type, but occasionally haemolytic streptococci, diphtheroid bacilli and staphylococcus have been isolated. In considering the significance of these results it must be remembered that staphylococci and diphtheroid bacilli are the most common inhabitants of the skin and are the most likely contaminants of the cultures. Cultures from Joints. In cultures from 54 cases of chronic arthritis Richards (1920) was able to isolate Streptococcus viridans on four occasions. Certain other workers have isolated this organism from small numbers of cases. More recently Forkner, Shands and Poston (1928) obtained Streptococcus viridans from the joints of 11 out of 63 cases examined. They also obtained Staphylococcus aureus once and Gonococeus twice. Shands (1928) reports a further series in which 30 per cent. of positive cultures were obtained, most of these being streptococcus viridans. Cultures from Lymphatic Glands. A number of workers have isolated bacteria from the lymph glands draining the affected joints in chronic arthritis. These have generally been Strep- tococcus viridans. Shands (1928) records having on five occasions isolated the same microbe from the joint and from the glands draining it. The most striking results are those of Baer (1928), 352 CHRONIC RHEUMATIC CONDITIONS who obtained 75 per cent. of positive cultures from the glands. These were usually Streptococcus viridans, but other types of streptococci were occasionally isolated. He attributed his success to the fact that he incubated his cultures for a long time and states that frequently these streptococci take several weeks to show appreciable growth. Allison (1928) by the same procedure obtained positive cultures in six out of ten cases examined. Blood Cultures. Attempts to isolate organisms from the cir- culating blood in cases of rheumatoid arthritis have frequently been made and generally without success. Some successful results have been reported, and probably the most interesting is the report of Cecil, Nicholls and Stainsby (1929). Seventy-eight cases were cultivated and from 48 of these streptococci were isolated, whereas in 54 control cases no streptococci were found. The majority of the streptococci isolated were of one type —a type intermediate between Streptococcus viridans and Streptococcus haemolyticus. Cecil and his collaborators, like Baer and Allison, used pro- longed incubation of their cultures. These were not discarded as sterile until more than a month had elapsed. It seems possible that if this prolonged incubation method had been more widely used more positive cultures would have been obtained. There can be no doubt, however, that by such methods contamination of the cultures with extraneous bacteria may take place unless the most rigid precautions are taken. Dawson, Olmstead and Boots (1932) emphasize this in the report of a series of observations in which, although they followed with the greatest care the tech- nique described by Cecil, Nicholls and Stainsby, they were unable to discover any organisms which could be considered of etiological significance. Septic Foci in Connection with Chronic Rheumatic Conditions. While most observers have failed to find organisms in the affected joint, it is generally held that chronic rheumatic manifestations have their origin in some septic focus in the body. The common sites of this septic focus are the teeth, tonsils, nasal sinuses, genito-urinary and intestinal tracts. The literature on focal sepsis is enormous and there is no need to recapitulate it here. SEPTIC FOCI 353 A good summary of the recent work on this subject as far as it concerns chronic rheumatic conditions is given by Poynton and Schlesinger in Recent Advances in the Study of Rheumatism ” (London, 1931). The following table compiled by Pemberton and Pierce (1927) analyses 945 cases of chronic rheumatism with regard to focal infection :— Civilians. Army. Men. Women. Per No. Por Per cent. No. cent. No. cent. No foci found . é . ¢ 37 23 125 33 107 27 Foci found : 2 ’ . | 126 id 257 67 293 | 73 Tonsillar foci . ‘ ' ' 58 36 110 29 208 | 52 Dental ALS v v 93 57 202 53 134 33-5 Tonsillar and dental. ’ 20 12 49 13 78 19-5 Combination of foci other than tonsillar or dental . . 41 25 57 15 38 9-5 Genito-urinary foci . . . 23 14 8 2 50 | 125 Combination of 3 or more foci . 16 10 22 6 — — Cecil and Archer (1926) in 379 cases of chronic infective arthritis found infected tonsils in 61 per cent. and dental sepsis in 33 per cent. Various observers have pointed out the importance of the nasal sinuses (Willcox, 1926) and the genito-urinary tract as a source of focal sepsis. The intestinal tract has always been looked upon as a frequent source of infection. In connection with all this work on focal sepsis as applied to chronic rheumatism one great difficulty has been to define what exactly is a septic focus. In connection with the teeth, tonsils and intestine this is especially difficult, and the figures given by different workers must be read in relation to their definition of a septic focus. Moreover, it is sometimes very difficult to discover a focus especially in the intestinal tract and more especially in relation to the gall bladder. Some workers accept a great pre- R.A. VACCINES. 12 354 CHRONIC RHEUMATIC CONDITIONS ponderance of streptococci in the faeces as evidence of intestinal sepsis, while others disregard it. A common history of a rheumatic patient who comes for vaccine treatment is something as follows : The patient suffers from pain and possibly swelling of certain joints for a considerable time before he seeks medical advice. His practitioner probably treats him for some time with the appropriate drugs. If the condition persists the teeth are examined and attempts are made to eliminate dental foci possibly by extraction of all the teeth. If the condi- tion still persists attempts are made to locate other possible foci and appropriate surgical measures are taken. In many cases it is only then that vaccine therapy is contemplated. Although the dental and tonsillar foci have been dealt with surgically, it will usually be found that on examination of the faeces there is a great preponderance of streptococci. These streptococci are frequently of the viridans types usually found in the mouth, and it may be supposed that as a result of the swallow- ing of saliva these cocci become established in the intestine, so that although the upper foci are removed the cocci, which are often present in the intestine in enormous numbers, are capable of maintaining the rheumatic condition. This streptococcal flora in the intestine differs from focal sepsis elsewhere in that, as a rule, no definite point of infection can be demonstrated. There is no doubt, however, that most patients with chronic rheumatic conditions have an increased streptococcal flora in the gut, and that this is likely to have great significance in relation to the rheumatic condition. There is evidence to support the view that the streptococci of the ordinary faecal type (S. fwecalis or entero- coccus) may be concerned in chronic rheumatic conditions. It has been shown that the injection into rabbits of some strains of enterococci is followed by an arthritis closely resembling that occurring in man. Crowe (1932) holds that while in fibrositis and osteo-arthritis non-hemolytic streptococci constitute the chief infection, in rheumatoid arthritis the primary infection is a white staphylo- coccus akin to the ordinary saprophytic staphylococcus of the skin. This he has called the ‘ micrococcus deformans.” THe TYPE OF VACCINE EMPLOYED 355 regards all varieties of chronic rheumatism as being associated either primarily or secondarily with both streptococci and staphylo- cocci, and he insists that in the treatment of such cases vaccines of both organisms must be used. Crowe's views as to the associa- tion of staphylococei with chronic rheumatism have not met with general acceptance. Vaccine Treatment of Chronic Rheumatic Conditions It may be premised here that the general opinion is that vaccine therapy should be ancillary to and not a substitute for any radical measures which are desirable for the removal of an infected focus. At the same time, the author does not wish to appear to recommend indiscriminate operative treatment of suspected foci. A discussion of this question is, however, outside the scope of this work. Type of Vaccine Employed. In most cases autogenous vaccines are used. These are prepared from cultures made from an infected focus if such is found. In addition to the examination of any obvious foci in the mouth, tonsils, ete., cultures of the feces should be made, and although this is often omitted it is probable that it is the most important examination of all. Streptococci of the viridans or indifferent types are usually present in abundance and the vaccine is made from one or more types of these cocci. Stock vaccines are also employed. Some workers have made their stock vaccines only from cul- tures from dental or tonsillar sources (Congdon, 1932), but it seems unlikely that a good stock vaccine can be made from streptococci from these regions only. A stock vaccine should contain a large number of strains from the mouth, tonsils and intestine and a lesser number from the genito-urinary tract. The strains selected should only be from cases of rheumatism where by the purity of the culture and by the obviousness of the infected focus there is a reasonable inference that the streptococcus selected has definite relation to the patient’s clinical condition. The merits and demerits of stock and autogenous vaccines are discussed elsewhere (p. 252). Few will object to the generaliza- tion that an autogenous vaccine is at least equal to a stock vaccine 12—2 356 CHRONIC RHEUMATIC CONDITIONS when such autogenous vaccine is made from the actual infecting organism. In rheumatic conditions, however, the vaccine is made from a selection of streptococci from various parts of the body which may be the site of a septic focus, but which in normal conditions contain many types of streptococci apparently leading a saprophytic existence. In such cases the real infecting strep- tococcus may be missed and the * autogenous” vaccine may consist merely of saprophytic streptococci which have no relation to the rheumatic condition. There is no certain method of dis- tinguishing in all patients between the causal strains and those which are merely saprophytic, and the quality of the autogenous vaccine will largely depend on the skill of the bacteriologist in collecting his specimens from the focus and in selecting the streptococci likely to be associated with the clinical condition. If the bacteriologist is unskilful or unlucky and misses the strep- - tococcus which is the actual infecting agent, it can easily be seen that an autogenous vaccine may be definitely inferior to a stock one. The recent report of the Arthritis Committee of the British Medical Association (1933) contains the following statement : “ While stock streptococcal vaccines are sometimes inferior to autogenous vaecines, they are superior to imperfectly made autogenous vaccines or to no vaccine.” Dosage of the Vaccine. In the literature on vaccine therapy of rheumatic conditions the question of dosage is a very confused one. Various authors have expressed very definite opinions on dosage, but unfortunately there is no general agreement between different workers. In most cases an initial dose of less than 5 million streptococci is recommended, but in some reports enormously large doses were employed. Rosenow and Nickel (1928) commenced treatment with a dose of 200 millions ; Wetherby and Clawson (1932) commence with 100 millions intravenously, while some French workers use even larger doses. The best practice in the author’s opinion is, in the average case where there is no reason to suspect excessive hypersensitiveness to streptococci, to commence with a dose of 1 million streptococci, DOSAGE OF VACCINE 357 and if there is no sign of hypersensitiveness to double this dose three or four times every three or four days. If there is no reaction at all to these doses the quantity may be increased by about 50 per cent. each time, and after the dose reaches about 50 million the interval may be lengthened to one week. So long as there is no reaction, the increases can be persisted in up to a dose of 500 to 1,000 million streptococci. In some cases, however, there is very considerable hypersensi- tiveness to streptococci, and it is found that there is a definite reaction, with increase of pain, and increased swelling of joints or malaise following quite a small dose of the vaccine. In such cases the dose must be cut down to a quarter or less, and subsequent increases must be very gradual. The underlying principle is to give as large a dose as possible without inducing a focal or general reaction. In practice it is better to err on the side of too small rather than too large doses, as in this way general reactions can be avoided except in rare instances. When, in a sensitive patient, only very small doses can be given and the improvement in the condition only lasts for a day or two, the injection may be repeated every two, three or four days. It is a good rule when there is a definite improve- ment following a particular dose to repeat the same dose and to keep on repeating it while the improvement is maintained rather than to increase the quantity. Some workers recommend even smaller doses. Willcox (1928) starts with half a million and gradually increases to 50 millions. Crowe (1922) commences with half a million or less, and in bedridden patients the initial dose is as low as 10,000 cocci. Crowe (1932) has now reduced his initial dose to 100,000 cocci. Graham Bonallie, Gallagher and Claxton (1931) commenced treatment with a dose of 100,000 cocci, and in some cases the dose had to be reduced to hundreds of organisms. In the report of the Arthritis Committee of the British Medical Association (1933) practitioners are recommended to commence treatment with a small safe initial dose of from 20,000 to 50.000 streptococci when a septic focus is suspected or half a million when it is fairly certain that no septic focus exists. Increasing doses 358 CHRONIC RHEUMATIC CONDITIONS are suggested every five or six days, and it is stated that it is rare for a dose of more than 10 millions to be required. A very interesting report has been published by Wetherby and Clawson (1932). They used the intravenous method of injection of streptococci for the treatment of rheumatoid arthritis. The reasons they give for this are :— 1. Non-specific chronic arthritis in most cases appears to be due to a streptococcal infection. Streptococci have been recovered in a higher percentage by a greater number of workers from the blood, joints, lymph nodes, and subcutaneous nodules in cases of chronic arthritis than any other organism. 2. Subcutaneous streptococal vaccination does not desensitize the hypersensitive chronic arthritic patient, but tends to increase the hypersensitiveness. The subcutaneous method of giving a streptococcus vaccine increases protective immunity only slightly. 3. Intravenous streptococcic vaccination desensitizes the hyper- sensitive patient and also brings about a high degree of immunity. 4. The desensitizing and protective phenomena do not seem to be strictly type-specific, but are species-specific. They are not in the category of a reaction to non-specific protein. They also state that the agglutination titre of the serum is a reliable indicator of the progress of immunity during treatment. They used throughout one strain of streptococcus which had been isolated from the blood of a patient with rheumatic fever. The vaccine was given at weekly intervals for eight weeks, and then at longer intervals. The agglutination titre of the serum was used as a guide, and they tried in every case to raise it to at least 1 in 6,400. The initial dose was 100 millions, and this was increased by 100 millions each week up to 800 millions. The injections were all intravenous and all the patients were treated as out-patients. Reactions followed about 50 per cent. of the injections in from two to ten hours. These reactions usually consisted of chills of varying degree and duration followed by a rise of temperature to 100° F., 101° F., or occasionally higher for one or two hours. Sometimes there was nausea, vomiting, and diarrhcea. There was apparently no correlation between the reaction and any improvement in the clinical condition. RESULTS OF VACCINE TREATMENT 359 The intravenous injections were followed by a pronounced rise in the agglutination titre of the serum (in one case up to 1 in 100,000), and in the bactericidal power of the blood. The rise in the agglutinating power of the serum appeared to have some relation to the clinical improvement of the patient. Whether we may agree or not with the reasons given for adopting the intravenous method of administering the vaccine, the results of this series of 865 patients treated in this way are interesting. They were immunized in much the same way as, in a laboratory, rabbits are immunized to produce serum containing the usual antibodies. The chief drawback to this method seems to be the reactions with fever, etc., which occur after about half the injec- tions. These reactions might be very unpleasant for patients who are not confined to bed. Results of Vaccine Treatment A survey of the literature would reveal to a statistician nothing on which he could base a judgment. Chronic rheumatic condi- tions vary in their signs and symptoms, and the thing which is most prominent in the patient’s mind—pain—is a symptom in regard to which there is no evidence beyond the patient’s word. The progress of the disease, independently of treatment, is notori- ously erratic, and it is easy for the sceptic to argue that any particular case would have done as well without any special treatment. In most cases, moreover, vaccines have been used in addition to other forms of treatment, and this has increased the difficulty of appraising the value of the vaccines. A further difficulty is that in some cases the most prominent symptoms may be due to tissue destruction or malformation, conditions which it is impossible to influence with a vaccine. In the literature phrases like “ much improved, improved,” and the like, are usual in describing the results. They are in- definite, but unfortunately they are frequently the only ones possible. Most authors state that vaccines are definitely useful in the 3% 360 CHRONIC RHEUMATIC CONDITIONS treatment of chronic rheumatic conditions. There are, however, some reports in which vaccine treatment has apparently conferred no benefit, but what has been said regarding the difficulty of isolating the infecting organism makes negative results inevitable. Of the many reports published regarding the results of vaccine treatment of rheumatic conditions, only a few can be given. Wetherby and Clawson (1932) treated 301 cases of rheumatoid arthritis by intravenous inoculation of a stock streptococcal vaccine. They obtained definite clinical improvement ” in 77-4 per cent. and no improvement in 16-3 per cent. These were out-patients and received no other form of treatment. A table showing the criteria of improvement is reproduced. No. of cases. Percentage. Joint pain (282 cases)— Decreased pain . v . . 233 82-6 Unchanged ‘ ‘ ‘ . 49 17-4 Joint swelling (197 cases) Decreased swelling : : 160 81-2 Unchanged . x ‘ ‘ 37 18-8 Joint motion— Increased motion “ F i 206 84-7 Unchanged : } g 3 37 15-3 Crowe (1926), in a large series treated with small doses of staphylococcus and streptococcus vaccines, obtained good results in 68 per cent. Crowe emphasized the fact that only very small doses should be given, and that harm may follow the administra- tion of large doses. The most recent report of cases treated with this mixed vaccine is summarized in the following table :— ! Ortone Rheuma- | Mixed and Total. itt toid unclassi- | Fibrositis, | Neuritis. | Others. arthritis. | 4 ¢hritis, a, | Symptom free 7 18 Al 13 25 6 4 Much improved | 305 80 23 88 83 20 11 Improved . | 328 112 21 91 86 12 6 Worse or | [ Q am RTH oF S— — No change | . 94 35 10 26 23 Total | 804 245 65 218 217 38 | 21 RESULTS OF VACCINE TREATMENT 361 These figures include all cases treated for over five weeks. The patients were all treated as out-patients with weekly injections of vaccine, and the majority had no other form of treatment. This record might be compared with the latest results from the British Red Cross Society’s Clinic for Rheumatism (1932), where vaccine therapy is a much less important factor in the treatment. Total number of cases discharged during 1932 with treatment completed, 2,133 ; cured, 18-6 per cent. ; improved, 54-8 per cent. ; unchanged, 24-3 per cent. ; worse, 1-3 per cent. Rosenow and Nickel (1928-1929) in a series of 109 cases of arthritis treated with streptococcal vaccines and removal of infective foci concluded that the administration of the vaccine improved the result. They used large doses subcutaneously. Reactions due to hypersensitiveness occurred. Congdon (1932) obtained only an insignificant improvement in a considerable series of cases. The treatment consisted of subcutaneous injection of autogenous and stock streptococcal vaccines from dental and tonsillar sources only. In the Out-patient Clinic attached to the Inoculation Depart- ment, St. Mary’s Hospital, vaccine therapy has, for the last 20 years, been carried out on large numbers of patients suffering from chronic rheumatic conditions. Most of these have been treated with stock vaccines made from a large number of strains of streptococci isolated from the teeth, tonsils, intestine and genito-urinary tract, but frequently autogenous vaccines have been used either from the beginning of treatment or after it was found that the stock vaccine was not giving much benefit. Most of these patients, especially in the earlier years, consisted of those who had failed to obtain benefit with the ordinary medicinal or other treatment in the medical out-patient departments. Except for an interval during the war, the author has been in charge of such a clinic for some 20 years, and has arrived at certain conclusions. In cases where a vaccine is of benefit the first result is a feeling of well-being in the patients. They insist that they are much better, in spite of the fact that there is no obvious physical sign of improvement. It might be said that this was merely a psycho- logical result of the injection treatment, but against this hypothesis 362 CHRONIC RHEUMATIC CONDITIONS is the fact that the effect is manifest in individuals who have had various other injections (and, indeed, other vaccine injections). This feeling of well-being is generally felt after the first two or three inoculations. It is only later that there is any diminution of pain and swelling of the joints. In many cases these results can be obtained by a stock streptococcal vaccine. In some cases, however, the stock vaccine may fail altogether or give only temporary relief. Some of those in whom the stock vaccine fails rapidly improve when an autogenous vaccine is used. This improvement may be uninterrupted, but sometimes after two or three months’ treatment there is a relapse—the joints swell and the pain increases. If another bacteriological examination is made and another vaccine is prepared, some of these patients immediately improve again—the joint swellings diminish and the pain ceases. The streptococci isolated on the second occasion do not apparently differ from those previously isolated. In some of these cases the patient has had no knowledge as to the nature of the vaccine, so that the psychological element was eliminated as far as possible. It may be that the first vaccine had degenerated in the few months since it was made, but this is against all the evidence as to the stability of vaccines. Other explanations suggest themselves. It may have been that more than one streptococcus was concerned in the infection and the first vaccine was not representative of the infection as a whole. It is possible, but this is speculative, that many non-hemolytic streptococci may, at the same time that they have certain specific antigens, have an antigen in common, so that any of them could, up to a point. function as a vaccine in a non-hezemolytic streptococcal infection, Beyond that point, however, only a vaccine of the actual infecting streptococcus would be able to confer benefit. Or it may be that there is some quite non-specific effect of these vaccines, and that if the dosage is correctly adjusted any vaccine can confer some benefit in such cases. This conception is not beyond the bounds of possibility, and indeed certain authorities believe that in vaccine treatment (as distinct from prophylaxis) the non-specific element is very important. Thus by non-specific vaccination the patient may have improved up to a certain point, and it is only when to RESULTS OF VACCINE TREATMENT 363 this is added specific immunization with the actual infecting streptococcus that the improvement is carried further. Most of the results cited are related to arthritic conditions, but the same methods of treatment apply to the other chronic rheu- matic manifestations such as fibrositis and perineuritis. The same group of organisms are apparently involved in these manifestations as in the arthritic cases, and the methods of procedure as regards the preparation and use of vaccines are the same. In many of these patients pain is the chief feature, and there may be little or no physical sign of disease. It is hardly necessary to point out here the importance of accurate clinical diagnosis before vaccine therapy is embarked on. The beneficial results of vaccine therapy are more manifest in these non-arthritic types of rheumatism. In favourable cases there is a distinct improvement, especially as regards the pain, after three or four injections. Treatment must, however, be persisted in for some considerable time. In old-standing arthritic cases treatment has to be persisted with for many months, and usually the condition is not more than relieved. The patients, however, very frequently consider that the improvement is very much greater than a medical examination would warrant owing to the diminution of pain and the improve- ment in their general feeling of health. While in the majority of patients suffering from chronic arthritis the use of a vaccine definitely improves the condition, there is a not inconsiderable percentage in whom no alleviation of the condition occurs. This may be due to our insufficient knowledge of the disease or to lack of knowledge as to the best way in which to employ a vaccine, but the many gaps in our information are (slowly) being closed, and it may reasonably be hoped that the results will gradually improve. The treatment of chronic rheumatic conditions with intravenous non-specific vaccine therapy (Protein Shock) is alluded to in Chapter XXII. REFERENCES ALLISON, N. 1928. J. Bone and Joint Surgery, 10, 756. Baer, W. S. 1928. J. Bone and Joint Surgery, 10, 753. 364 CHRONIC RHEUMATIC CONDITIONS CeciL, R. L., and Arcuer, B. H. 1926. J. Am. Med. Assn., 87, 741. Cecin, R. L., Nicnorts, E. E., and Stainssy, W. J. 1929. Arch. Int. Med., 43, 571. CoNGDON, P. M. 1932. Lancet, 1, 178. Crowe, H. W. 1926 and 1932. * Treatment of Chronic Arthritis and Rheumatism,” London. Dawson, M. H., OLmsteEap, M., and Boots, R. H. 1932. Arch. Int. Med., 49, 173. ForkNER, C. E., Suanps, A. R., and Poston, M. A. 1928. Arch. Int. Med., 42, 675 GramaM BoNaLLIE, F. E., GALLAGHER, E. J. M., and Craxrox, V. E. 1931. Lancet, 1, 615. PemBErTON, R., and Pierce, E. G. 1927. 4m. Jour. Med. Sc., 173, 31. Poynton, F. J., and ScHLESINGER, B. ‘ Recent Advances in Rheumatism,” London. Ricuarps, J. R. 1920. J. Bact., 5, 511. RosuNow, E. C., and NickerL, A. C. 1928-1929. J. Lab. and Clin. Med., 14, 504. WernerBy, M., and CrawsonN, B. J. 1932. J. Am. Med. Assn., 98, 1975. Wirrcox, W. H. 1928. Proc. of Conference on Rheum. Dis., Bath, 266. Wirncox, W. H. 1926. ** A Text-book of the Practice of Medicine,” p- 1212, Edited by F. W. Price. London. ** Report of the Arthritis Committee of the British Medical Association.” 1933. Brit. Med. Jour., 1, 1044. CHAPTER XV ACNE VULGARIS Tue chronicity of this disease and the disfigurement which it produces at a sensitive age make it an important factor in the lives of many young people. It was one of the first diseases to be treated by vaccine therapy. Bacteriology Unna in his classical work on the histopathology of the skin has shown that in the early acne lesion (the comedo) the deeper portion contains a mass of bacilli (acne bacilli), while in the super- ficial portion these are mixed with staphylococci and sometimes other organisms. Sabouraud (1897) first isolated the acne bacillus in pure culture and this was later done by Gilchrist (1900) and Fleming (1909). These observers used aerobic methods and could only obtain growth when a drop of pus was placed on a suitable medium without spreading. It was, however, shown that the bacillus was essentially an anaerobe, and now by anaerobic methods there is no difficulty in isolating the bacillus from all acne lesions. Craddock (unpublished) made use of the fact that staphylococci are more easily inhibited by penicillin than is the acne bacillus, and by adding penicillin to glucose broth in a concentration just enough to inhibit staphylococci he was able to obtain pure primary cultures of the acne bacillus from every one of fifty consecutive acne lesions. There is no doubt that acne bacilli are present in every lesion whether suppurative or not and there is little doubt that this bacillus constitutes the primary infection. Craddock has shown that there are two types of acne bacillus which can be differentiated by cultural and serological methods. Both of these types may occur in the same patient. A good stock vaccine should include both of these types. 365 366 ACNE VULGARIS The relation of staphylococci to acne vulgaris has been a matter of dispute. Staphylococcus was established as a well-known pyogenic organism, especially in connection with inflammations of the skin, long before the acne bacillus was isolated, consequently when by ordinary aerobic cultural methods it was the only microbe which grew from acne lesions it easily became accepted as the cause of the suppuration in this disease. It is still the current teaching in many places that in pustular acne staphylococcus constitutes the really important infection. Some writers have even doubted the pathogenicity of the acne bacillus. This teaching loses sight of two important findings. In Unna’s descrip- tion of the comedo, acne bacilli are shown to be present in large numbers in the deeper portions, while staphylococci were invariably present in the superficial parts where they would naturally cecur if they were saprophytes. Unna’s findings have subsequently been confirmed by cultural experiments. Gilchrist (1900) showed that acne bacilli could sometimes be isolated in pure culture from pustules. This was confirmed by Fleming (1909) and a very large number of subsequent examinations has substantiated these results. There is a very appreciable number of acne pustules —and many of these are small acute pustules—from which no staphylococci can be grown, but which give pure cultures of acne bacilli. The acne bacillus, therefore, is the only organism con- stantly present in all types of lesions, while staphylococci are more consistently present in the non-pustular lesions than they are after there has been a local reaction to the infection with the forma- tion of a pustule. There are some cases of pustular acne, however, in which a staphylococcal infection certainly plays a part. These are especially the cases in which there is, around the pustules, a very considerable inflammation making the lesions resemble small furuncles. Acne bacilli and staphylococci are normal inhabitants of the skin and there is no certain knowledge as to the conditions which must obtain in the body to allow of their flourishing and becoming pathogenic. It has long been recognized by clinical observation that acne is frequently associated with intestinal stasis. Bac- VACCINE THERAPY 367 teriological examination of the fwces in cases of acne shows that in most patients the faeces contain a great excess of streptococci. It may be that the change in the intestinal flora is the result of the same disturbance which initiates acne or it is possible that the streptococci in the gut produce some change in the body which in certain individuals causes the disease just as a similar condition in other patients is considered to be responsible for rheumatic conditions. However this may be, the correlation exists. If the staphylococcus were the chief factor in the production of acne it might be expected that acne patients would be more liable to suffer from the more typical staphylococcal skin lesion—a furuncle— but this is not the case. Persons suffering from acne are not more susceptible to furunculosis than are other people. Vaccine Therapy of Acne Vulgaris It may be stated at once that anyone who expects, other than in exceptional cases, that the lesions of acne vulgaris will dis- appear in one or two weeks as a result of vaccine therapy is doomed to disappointment. That great benefit can be obtained by the use of vaccines is, in the opinion of the author, indubitable, but the beneficial change is not in the nature of a miracle. That no rapid benefit can accrue is obvious when it is remembered that acne pustules are miniature replicas of abscesses where the bacteria. are flourishing in a fluid from which antibacterial power has disappeared—in what Wright has called a * non-bacterio- tropic nidus ” or ‘ ecphylactic focus.” In a large abscess it would not be expected that any general treatment would be rapidly effective if the abscess were not opened, but it is often expected that vaccine therapy will cause the rapid disappearance of small abscesses which no attempt is made to evacuate or drain. Vaccine therapy of acne was first carried out by Wright (1902), who used staphylococcal vaccines and showed that a marked alleviation of the condition followed. Fleming (1909) was the first to use a vaccine of the acne bacillus and showed that with the aid of a vaccine of this bacillus, either alone or combined with staphylococcus vaccine, the disease could be successfully treated. 368 ACNE VULGARIS He found that better results were obtained by autogenous than by stock vaccines of the acne bacillus. Since that time the usual treatment has been by means of a mixed vaccine of the two organisms. The mixed vaccine is frequently used merely as a matter of convenience or because in many cases it is difficult to decide whether or not there is a staphylococcal infection. In many cases, however, equally good results have been obtained by the use of simple acne bacillus vaccines, especially autogenous ones, as by the use of mixed vaccines. This opinion does not agree with that expressed by Dudgeon (1927) that acne bacillus vaccines are always disappointing unless combined with staphylococcus. Dudgeon’s conception of the bacteriology of acne, however, appears from his monograph to be rather different from that which has been set out above and which is certainly correct. Dosage of Vaccines. The scheme of dosage here given is that of the author after many years’ experience in the treatment of acne. The dose of staphylococcus varies from 200 millions to 2,000 millions. This vaccine should not be given by itself, but always combined with a vaccine of the acne bacillus. It is wise to start with a dose of something like 200 millions and gradually to increase the dose to 1,000 or 2,000 millions. The dosage of acne bacillus is more difficult. This vaccine is very little toxic and practically never gives rise to a general reaction. An unsuitable dose may be followed by a focal reaction which is evidenced by the appearance of fresh pustules. On purely clinical grounds the author has arrived at the conclusion that there are two quite different systems of dosage, either of which may suit an individual patient. Some patients improve very markedly with doses of from 5 to 20 millions, and when somewhat larger doses (say 50 millions) are given focal reactions occur as shown by an exacerbation of the pustular condition. On the other hand, these small doses seem to be quite useless to other patients, but if doses of from 100 to 2,000 millions are employed a rapid improvement is seen without any focal reaction. A good plan is to commence the treatment with a dose of 5 millions and to follow at weekly intervals with 74 and 10 millions. If there is obvious improvement these small doses should be per- DOSAGE OF VACCINE 369 sisted in, but if there is no change the dose should be increased suddenly to 100 millions and then increased by 100 millions or 200 millions up to perhaps 2,000 millions. The association of an excess of intestinal streptococci with acne has led to a vaccine of these streptococci being combined with the acne vaccine. In most cases an autogenous vaccine has been used and definite benefit is sometimes obtained by this procedure. The streptococcus vaccine may be given in doses similar to those of the acne bacillus. Treatment has always to be persisted in for several months and should be combined with a systematic evacuation of the contents of the acne pustules and a removal of the comedones. After several doses of vaccine such treatment of the local lesions is not usually followed by an inflammatory reaction as severe as before the commencement of vaccine treatment. By these procedures in the majority of cases a very distinct improvement can be obtained amounting in many cases to a “cure.” There are, however, refractory cases, especially the type in which the pustules are large and very indolent, and which contain few bacteria. No mention has been made here of treatment other than vaccine therapy but, naturally, this may be supplemented by suitable medicinal, dietetic or physical treatment ; such thera- peutic measures are outside the scope of this work. REFERENCES Dubcron, L. S. 1927. * Bacterial Vaccines and their Position in Therapeutics.” London. FLEMING, A. 1909. Lancet, 1, 1035. Grneurist, T. C. 1900. Johns Hopkins Hosp. Rep., 9, 409. SABOURAUD, R. 1897. Ann. Inst. Past., 11, 134. SupmeRrsEN and TrnovpsoN. 1909. J. Path. and Bact., 14, 224, Wericnr, A. E. 1902. Lancet, 1, 874. CHAPTER XVI FURUNCULOSIS Tunis disease is one of the first in which Wright used vaccines for therapeutic purposes in 1902, and for many years now vaccine therapy has been a standard treatment. Although there have not been any striking advances recently in the vaccine treatment of this disease, it would be well to give some account of the methods which are being used some 30 years after the treatment was initiated. Bacteriology. The furuncle has been regarded as the typical acute staphylococcal lesion of the skin. Cultures from boils always show staphylococci, usually of the aureus variety, and generally nothing else unless special precautions are taken. If, however, cultures are made in glucose broth containing 1 in 500,000 gentian violet from any boil which has been opened for more than 24 hours, it is usual to obtain a pure culture of heemo- lytic streptococci (Maclean, unpublished). The gentian violet inhibits the staphylococci, but allows the streptococci to grow out. In some unopened boils the same result was obtained. These results show that although the staphylococcus is the primary infection these lesions very frequently become secondarily infected with hemolytic streptococci. In the feces of patients suffering from furunculosis it is usual to find a great excess of streptococci especially of the non-haemolytic types. There is no direct connection to be traced between these fecal streptococci and the actual infection of the furuncle, but clinically there is often some relation between the onset of an attack of boils and an intestinal disturbance and again on purely clinical grounds it has been found that better results are obtained in some cases when a vaccine of these intestinal streptococci is combined with the staphylococcal vaccine, 370 VACCINE TREATMENT 371 Normal Resistance to Staphylococcal Infections. Everyone during life has had many minor staphylococcal infections and a certain amount of immunity has been established. We have all, therefore, received a * primary stimulus” and are in a position to respond readily to a secondary stimulus (see p. 424). That we have developed a considerable resistance to staphylococci can be shown by testing the bactericidal power of the blood to this organism. If 3,000 or 4,000 staphylococci are incubated with 1 c.cm. of normal human defibrinated blood some 90 to 95 per cent. of the cocci are destroyed. Everyone has many staphy- lococei on his skin and has frequently come into contact with virulent staphylococci, but the incidence of furunculosis is com- paratively small and is apparently dependent upon some under- lying condition. It is known, of course, that diabetics are especially susceptible to boils, but what it is that enables the disease to occur in an otherwise healthy individual is quite obscure. Vaccine Treatment There are two objects in view in the vaccine treatment of furunculosis, firstly to cause the disappearance of existing lesions and secondly to prevent the appearance of fresh lesions. It is, therefore, a very good example of a combination of therapeutic and prophylactic vaccination. The staphylococci which are isolated from boils are generally of the same serological group, so that theoretically this is a disease which might be treated with stock vaccines and in practice it has been found that the results obtained with stock vaccines differ little from those obtained with autogenous vaccines. There is little danger of a serious mishap if an overdose is given, and indeed it is recommended that in cases where there are semi-indolent boils a large dose should be given with a view to inducing a focal reaction and so hastening the breaking down of the lesions. In the unopened boil staphylococci are growing in a mass of necrotic tissue well removed from the blood stream, so that it is not to be expected that any immunizing process will cause rapid disappearance of the lesion. All that can be done by vaccination is to stop the spread of the inflammatory 372 FURUNCULOSIS process after which the actual boil either breaks down and dis- charges its contents or resolves. Thus following the administration of a staphylococcal vaccine there may be a focal reaction which in some cases is beneficial. With some individuals the local reaction at the site of inoculation is marked and results in a considerable swelling sometimes even resembling a cellulitis, but which seldom lasts more than two days. More usually the local reaction is not severe. Only seldom does a general reaction follow the administration of this vaccine and even when injected intravenously doses of 100 millions may give no reaction. In furunculosis the dosage is from 100 to 2,000 million staphy- lococei. In a severe case the treatment may be commenced with 100 millions ; if less severe, 200 or 300 millions may be the initial dose. The amount is increased at intervals of five to seven days up te 1,000 or 2,000 millions. Usually six to eight injections suffice for patients even when they have been suffering from attacks of furunculosis for years. Dudgeon advocates 25 millions to commence with, then after five days 50 millions and then at weekly intervals doses increasing so that after eight injections the dose is 1,500 millions. Sometimes, however, the simple staphylococcus raccine fails to benefit the patients and in some such cases a “cure 7 is effected by combining with the staphylococcus vaccine one made of streptococci isolated from the faeces or from the boils. There are some obstinate cases in which the lesions persist in spite of this treatment. It seems possible that some of these patients are incapable of breaking up the cocci contained in the vaccine and so making use of the antigens contained in them. Gratia (1930) has used a staphylococcus vaccine in which the cocci were dis- solved by certain streptothrices and moulds. This he has called a mycolysate and he states that it is very efficacious in chronic cases of furunculosis. The author has confirmed Gratia’s finding that in the preparation of staphylococcal mycolysates the antigen is preserved and a fairly extensive use of staphylococcal mycolysate has shown that it is clinically effective in the treatment of furun- culosis. When injected into animals the mycolysate cannot be shown to give a greater yield of antibodies than a similar dose of TREATMENT BY STAPHYLOCOCCAL TOXOID 373 the ordinary staphylococcal vaccine, but the normal animal is capable of breaking down the cocci and liberating the antigen, a function which may be lacking in some patients. Further work on this subject is necessary before it can definitely be said that the mycolysate is superior to the ordinary vaccine. Staphylococcal antigens in solution can be obtained in other ways. notably by the use of a bacteriophage. Staphylococci are planted into broth and when the culture is about four hours old an active bacteriophage is added in quantity sufficient to clear it. The culture is now filtered and the filtrate contains the products of solution of the staphylococci together with the bacteriophage. This has been used extensively especially as a local application with the intention of destroying the infecting staphylococci by means of the parasitic action of the bacteriophage. This local treatment has met with very indifferent success especially as regards recurrence of the furuncles. On the other hand sub- cutaneous injections of staphylococci dissolved by bacteriophage give similar results to ordinary staphylococcus vaccine, but it is quite likely that further work will show that there are some cases in which it will be effective when the ordinary vaccine does not succeed. This method of using bacteriophage is quite different from * bacteriophage treatment,” as here the bacteriophage proper is not a therapeutic agent, but merely a means of bringing the staphylococci into solution. If it is accepted that the important therapeutic element of a staphylococcal “ bacteriophage ” is the dissolved staphylococcal antigen, then the effective method of administration is by injection and not by local application. Furthermore, as it would be immaterial whether the bacteriophage were active or inactive, it would be possible to add an anti- septic to the filtrate, thus rendering very remote the possibility of disaster resulting from contamination of the preparation. Treatment of Furunculosis by means of Staphylococcal ‘‘ Toxoid ’ Many cultures of staphylococci have been shown to produce in culture under certain conditions a powerful exotoxin. Against 374 FURUNCULOSIS this an antitoxin has been prepared and is now used in the treat- ment of staphylococcal infections (see Chapter 373.). Staphy- lococeal toxin can be converted into toxoid by the use of formalin in exactly the same way as is diphtheria toxin (see p. 423). This staphylococcal toxoid has been used by Dolman (1933) for the active immunization of patients suffering from various staphy- lococcal infections and he has recorded very favourable results. Dolman has shown that the antitoxic content of the blood of patients suffering from recurrent boils is not higher than that of the normal man, but that it can be raised very considerably by a few subcutaneous injections of staphylococcus toxoid. It appears, therefore, that repeated superficial infections of this type give no antigenic stimulus capable of evoking antitoxin production. The toxoid was prepared by incubating a very powerful toxin with 0-3 per cent. formalin for two days, after which time its toxicity was reduced to such an extent that 0-1 c.cm, was incapable of causing necrosis when injected intradermally into a rabbit. The toxoid was administered to the patients subcutaneously, commencing with 0-05 c.cm. and increasing the dose every five to seven days by 0:05 c.em. In every case after four such doses there was an increased antitoxin content in the patient’s blood. Dolman treated 16 cases of recurrent boils. An average of 2 c.cm. of staphylococcus toxoid was given in eight doses over a period of six weeks. Two patients received only four and two others as much as 20 doses. In all the lesions disappeared, and at the time of publication all the patients had been free from recurrence for from two to eight months and the antitoxic content of their blood still remained on the average tenfold higher than it was initially. In addition to these cases of furunculosis Dolman treated a number of patients suffering from other staphylococcal lesions and reports complete relief in every case. These results following the use of staphylococcus toxoid are of great interest. It is true that the toxoid must contain a certain amount of the ordinary staphylococcal antigen from the autolysis of some of the cocci during the growth of the culture, and thus it would function to some extent as a staphylococcal vaccine. It possesses, however, an antigen which stimulates the production REFERENCES 375 of antitoxin and which is absent (or only present to a minor degree) in the ordinary staphylococcal vaccine. Staphylococcal Infections other than Furunculosis. These may be treated by staphylococcus vaccines in the same way and with the same doses as cases of furunculosis. In acute infections, such as suppurative arthritis or osteomyelitis, very small doses sometimes have a striking result. The dose may be five millions or less, and this can be repeated every 24 or 48 hours. REFERENCES Dorman, C. E. 1933. Jour. Am. Med. Assn., 100, 1007. DubceoN, L. S. 1927. Bacterial Vaccines and their Position in Therapeutics.” London. GraTIA, A. 1930. C. R. Soc. Biol., 104, 1058. os 1930. Bull. et Mém. Soc. Nation. Chirurgie, 56, 345. WricHT, A. E. 1902. Lancet, 1, 874. CHAPTER XVII B. COLI INFECTIONS B. coli infections are common in the genito-urinary tract and in inflammatory conditions in connection with the intestine, such as colitis, cholecystitis, appendicitis, ete. In many of these inflam- matory conditions radical surgical operation is the treatment of choice, but in urinary infections and in the more catarrhal inflam- mations of the intestine vaccine therapy has been extensively employed. Infections of the Genito-urinary Tract Here B. coli infection is very common ; indeed, Dudgeon states that this is < one of the commonest infections to which the human body is liable, and the amount of ill-health caused in men, women and children by these infections is a much more serious question than is usually recognized.” The infection may be acute, with high fever and much pain, and in this case there is little danger of its being overlooked, but frequently it is chronic, giving rise only to minor local symptoms accompanied by a certain amount of ill-health, and in such cases the infection may not be detected for a long time. After the acute symptoms have subsided the bacteria, in many cases, persist in the urine for months or years, and the patient is subject to relapses. Dudgeon, Bawtree and Wordley (1921) made some observa- tions on the types of B. coli associated with urinary infections, and showed that haemolytic types were particularly important in this connection. In males haemolytic types were the commoner, and although in females the non-hemolytic types occurred more frequently in infections taken as a whole, yet in “ Acute Coli Fever” in women hemolytic types predominated. These observers showed also that the hemolytic types of B. coli 376 INFECTIONS OF GENITO-URINARY TRACT 377 fell into only a few serological types, whereas among the non- haemolytic strains there was an extreme divergence of antigenic characters. The heterogeneity of the B. coli group had long been known, but the work of Dudgeon and his colleagues is of great value in showing that the haemolytic strains, which are responsible for a large part of acute urinary infections, have close antigenic relationships one with the other. B. coli vaccines are used for prophylactic and for therapeutic purposes. For prophylaxis the vaccine is administered to patients suffering from a B. coli infection of the urinary tract to immunize them preparatory to a major operation of the genito- urinary tract. For this purpose Dudgeon uses an autogenous vaccine, and if possible administers three doses (100, 500 and 1,000 millions) at weekly intervals. THe points out that in cases where there is obstruction to the outflow of urine acute symptoms of * coli-fever > may follow the initial dose and that harm can result from these prophylactic doses being used indiscriminately : in other words, the cases chosen for prophylactic treatment must be carefully selected. For treatment Dudgeon recommends B. coli vaccine for cases of bacilluria and pyuria, which have resisted the effects of ordinary treatment, and in whom no exciting cause can be found. In children, he states, the symptoms may rapidly disappear and the urine return to normal. It is recommended that the initial dose for children over five should be 20 million, and that this should be increased at weekly intervals to 500 millions. In adult patients with chronic pyuria and bacilluria, Dudgeon states, the symptoms may be rapidly relieved by vaccine treatment, but it is rare for the B. coli to disappear from the urine after any system of vaccine treatment. The initial dose in adults is 25 millions, and this is increased gradually at weekly intervals up to 2,000 million if there is no focal or general reaction. Dudgeon (1924) has drawn attention to a group of cases of acute infection of the urinary tract caused by a late-lactose-fermenting coliform bacillus. These cases are very acute and are frequently wrongly diagnosed as paratyphoid fever. In such cases vaccine treatment is recommended, commencing with a dose of 25 millions 378 B. COLI INFECTIONS about five days after the temperature has returned to normal, the dose being increased gradually to 500 or 600 millions at weekly intervals. The majority of these patients recover completely and the urine becomes sterile. During the last year or two less attention has been paid to vaccine therapy of B. coli infections of the urinary tract owing to the introduction of the * Ketogenic Diet ” treatment for these infections. This treatment, which has been used with great success in many cases, is outside the scope of this work. Infections of the Intestinal Tract Many cases of chronic diarrhea and * intestinal toxemia > have been successfully treated by vaccine therapy. The infecting agent can only be recognized by bacteriological examination of the intestinal contents and sometimes this has to be supplemented by serological tests. Even then the nature of the infection is some- times obscure. Autogenous vaccines are usually employed and the scheme of dosage is essentially the same as in urinary infections. Dudgeon states that the diarrhea and toxemia may rapidly subside after the administration of vaccines, although other forms of treatment have failed to relieve the condition. Horder’s advice is that in chronic cases of colitis vaccines should always be given a good trial. “‘ Sometimes the good effects are very striking, at others little or nothing is achieved.” The author has seen many patients suffering from chronic B. coli infections of the intestine rapidly improve following the administration of appropriate B. coli vaccines. In this chapter much attention has been given to the opinions expressed by Dudgeon in his monograph on ** Vaccine Therapy,” as this is the most authoritative publication which has recently appeared on the vaccine therapy of B. coli infections. With the opinions expressed by Dudgeon the author is in practical agree- ment, except that he would suggest a smaller initial dose of B. coli vaccine (not more than 5 million). It is certain that many B. coli infections of the urinary and intestinal tract can be successfully treated by vaccine therapy. In urinary infections there is INFECTIONS OF INTESTINAL TRACT 379 usually no doubt as to the infecting agent, as it is found in pure culture, but in intestinal cases it is frequently very difficult, and, indeed, at present sometimes impossible, to distinguish with certainty the infecting B. coli from those which are living sapro- phytically in the intestine. The opsonic index is very useful but very laborious, and agglutination tests are frequently helpful ; but there is as yet no certainty that in all cases the infecting bacteria can be recognized. It seems likely that B. coli is respon- sible to a much greater degree than is generally recognized for minor intestinal ailments, which, although generally not dangerous to life, militate seriously against the capacity of the infected individual. B. coli infections of other regions may be treated with vaccines on the same lines as the urinary and intestinal infections. Frequently the infection is a mixed one and in such cases the B. coli vaccine may be combined with vaccines of the other infecting organisms. REFERENCES DubcreoN, L. S. 1927. * Bacterial Vaccines and their Position in Therapeutics.” London. oN 1924. J. Hyg. 22, 348. DuUpGEON, L. S., WorDLEY, E., and BaAwTree, F. 1921. J. Hyg.,20, 137. 3 ” ” 1922. J.Hyg., 21,168. Horper, T. 1926. “A Textbook of the Practice of Medicine,” edited by F. W. Price. London, p. 84. CHAPTER XVIII ULCERATIVE COLITIS BarGEN (1924) has associated a certain type of streptococcus with this condition. He was able to isolate this coccus from 189 cases (80 per cent. of the total number examined) of chronic ulcerative colitis in the Mayo Clinic over a period of several years. The organism was obtained by swabbing the ulcers and planting the material into dextrose brain broth. In some ways this coccus resembles the enterococcus, but, according to Bargen, it differs from it in its failure to grow well on agar, in its inability to ferment mannite, in its tendency to form chains, and in its power to produce the * viridans” change on blood agar. Houston, however, has found that some enterococci can change blood in the same way, can in certain circumstances form long chains and have no mannite- fermenting properties. When this coccus is injected into rabbits it gives rise in many :ases to disease of the large intestine with ulceration (Bargen, 1924). Rosenow and his colleagues found that 21 out of 25 dogs injected with this organism developed lesions of the colon. Bargen’s findings have been confirmed by a number of observers (Hogan and Hogan (1929), Zadkin and Gray (1930), ete.). Buttiaux and Sevin (1931) isolated Bargen’s coccus from cases of ulcerative colitis together with another possibly related diplococcus, and both of these, when injected intravenously into mice, reproduced the disease. Bargen prepared vaccines from freshly isolated cultures of the coccus. The organism was grown for 48 hours in broth after which 0-4 per cent. of tricresol was added to sterilize the culture and the concentration was adjusted to 2,000 million per c.cm. He also used sterile filtrates of similar cultures. He states that he always commences with small doses (200 millions), and if there is 380 ULCERATIVE COLITIS 381 no reaction he rapidly increases the dose every third day. His maximum dose for the vaccine is about 2,000 millions, and of the filtrate about 1:5 c.cm. (Some vaccine therapists would not regard these doses as being small.) These vaccines have been used for the treatment of ulcerative colitis. Bargen has found them of little use in the small group of cases in which the disease develops rapidly with fever, leucocytosis, tenesmus, purulent rectal evacuation, and rapid wasting. In such cases he uses passive immunization with an antibacterial serum and only later does he administer vaccines. He has reported that by this method there is a greatly reduced need for ileostomy which formerly was the treatment of choice. At the Mayo Clinic 250 cases of ulcerative colitis were treated with vaccines and bacterial filtrates during a three-year period (1924-1926). Bargen, in 1928, obtained reports from these patients with the following results :—— , Entirely free from symptoms . . ’ . 108 Sufficiently well to resume normal activity . «|| OF Definitely improved . ‘ : . ; Lo 34 No improvement . ‘ . 2 . Lo 11 Dead ‘ » ‘ ‘ : " ' Did not answer inquiry ~~. ‘ : : eli 3 Thus some 70 per cent. of this series of cases had been restored to normal life. Hogan and Hogan (1929) in five cases used no other treatment but vaccines and bacterial filtrates, which were administered alternately every three or four days. In every case improvement was manifest in a few weeks, and this improvement was progressive. Zadkin and Gray (1930) obtained Bargen’s coccus from 12 out of 15 cases, and treated these 12 cases with vaccines prepared from this organism. The initial dose was 660 million cocci, and this was increased by 200 million at each successive inoculation (the vaccine was given three times a week for eight weeks). A rapid improve- ment was noticed in 10 patients—tenesmus was relieved and the number of evacuations was diminished, while at the same time the amount of haemoglobin and the erythrocyte count in the blood 382 ULCERATIVE COLITIS increased markedly. Three of the patients had relapses after three or four months, but cleared up with a further course of vaccine. It would appear from these reports that the diplococcus described by Bargen occurs in the ulcers in cases of ulcerative colitis, and that much benefit is to be derived from the administration of vaccines of this organism, either alone or combined with an antibacterial serum. Bargen, however, emphasizes that this treatment should be combined with strict attention to diet and with removal of septic foci. REFERENCES Barcen, J. A. 1924. J. Amer. Med. Ass., 83, 332. » 1927. Trans. Amer. Proct. Soc., p. 93. 5 1928. Collected Papers of the Mayo Clinic, 20, 230. Burriavx, R., and SevIN, A. 1931. Ann. Inst. Past., 47, 178. Hogan, E., and Hocan, J. 1929. J. Amer. Med. Ass., 93, 263. Rankin, F. M., BArRGEN, J. A., and Bui, L. A. 1932. * The Colon, Rectum, and Anus.” Philadelphia and London. ZADKIN, W. Z., and Gravy, I. 1930. J. Amer. Med. Ass., 94, 849. CHAPTER XIX GONOCOCCAL INFECTIONS GoxococcAL vaccines have been in use for almost thirty years, and an enormous number of publications have been made on their value in the treatment of gonorrhea and other manifestations of gonococeal infections. Unfortunately, most of these publications have little value, and opinions have been expressed on the merits or otherwise of gonococcal vaccine treatment on the most trifling evidence. Preparation of the Vaccine. In the earlier work a simple" vaccine was used, prepared from cultures of gonococcus grown on serum agar. Such a vaccine is probably still the one most commonly employed. It has been emphasized by most workers in this field that the cultures from which the vaccine is made should be recently isolated and that vaccines made from old stock cultures should not be used. The evidence in favour of this procedure in the case of the gonococcus is not so clear cut as it is with some other bacteria (B. typhosus, B. pertussis, ete.), but from what is now known regarding bacterial variation there is little doubt that this recommendation is valuable. Some workers, e.g., Eyre (1925), have laid great stress on using autogenous vaccines wherever possible, but the evidence in favour of this procedure is not very strong. It is true that Torrey found that by agglutina- tion tests he could split up gonococei into several groups, and from this it might be argued that autogenous vaccines would have a distinct advantage, but, on the other hand, it has been found that there is little difference between one strain of gonococcus and another when the complement fixation test is used showing that the antigenic differences between the different strains are slight. A great many attempts have been made to modify gonococcal vaccines with a view to increasing their cfficiency or reducing their 883 384 GONOCOCCAL INFECTIONS toxicity. For a time sensitized vaccines (p. 245) had a consider- able vogue and numbers of observers reported favourably on their use (McDonagh and Klein, 1912-1913, Cruvheilier (1913), Dopter and Panron (1913), ete.). Sensitized vaccines are now, however, little used. Hirschfelder (1913) used as a vaccine a filtrate of a gonococcal suspension which had been digested by 2 per cent. pancreatin. It has been shown in connection with other bacteria that digestion does not destroy the antigen, and bacterial digests have been used as vaccines in other infections. Thomson (1919) dissolved the cocci in caustic soda and subsequently neutralized the solution with acid, thus obtaining a preparation which could be injected into patients in large amounts without causing a reaction. He later modified the method by breaking up the cocci mechanically and then dissolving them in very weak alkali (1/50 normal caustic soda), and vaccines prepared in this way have been extensively advertised, and largely used in practice. It has been said that in the process of manufacture the antigen is largely destroyed and Eyre (1925) has expressed the opinion— on what grounds is not stated—that he has come to regard * vaccines of this kind merely in the light of indifferent vegetable proteins, and the effects they produce to depend on the little understood protein shock.” That the antigen is not completely destroyed follows from Thomson's observations that after the injection of such vaccines complement fixing antibodies can be detected in the serum. Dimond prepared a gonococcal vaccine by growing the cocci on a medium rich in animal nucleo-protein on which some strains are said to produce large numbers of polar bodies similar in their staining properties to the well-known polar bodies of the diphtheria bacillus. The culture is suspended in 2 per cent. saline in which the polar bodies can easily be separated from the cocci. The suspension is then centrifuged and the supernatant fluid, containing the polar bodies, constitutes the vaccine, which is standardised by estimating the number of gonococei present in the original suspen- sion. Oliver (1933) has modified the method in certain particulars and only uses strains of gonococei which have been frequently subcultured so that they will grow well on ordinary agar. This DOSAGE OF GONOCOCCAL VACCINE 385 procedure seems to be in opposition to the results of recent research on the antigenic characters of bacteria which have shown that the most effective protective vaccines are to be obtained only from freshly isolated or virulent cultures (see Part II., Chapter II.) There is, apparently, considerable difficulty in preparing different batches of vaccine of equal potency. This type of vaccine has received commendation from Lambkin and Dimond (1927), Harrison (1933), Oliver (1933) and Clements (1933). It has been recommended that it should be injected intradermally into the penis or the region of the groins, and this may be combined with installation of the vaccine into the urethra. The dosage has to be carefully watched (Clements) and local, focal, and general reactions may follow its administration. The difficulty of preparing a standard vaccine of this type will militate against its general acceptance unless it can be shown to have a very great advantage over the ordinary vaccine. A certain number of workers have used vaccines of living gonococei, but on general grounds these are not to be recommended. Mixed vaccines have frequently been used, this practice being based on the fact that in the later stages of gonorrhoea secondary infections frequently supervene. These secondary infections usually consist of staphylococci, streptococci, or diphtheroid bacilli, and the mixed vaccines contain one or more of these organisms in addition to the gonococcus. Dosage of Gonococcal Vaccine. The doses of vaccine recom- mended by various workers have been extraordinarily varied. Wright's school have used small doses—from 4 to 10 millions in acute cases and in those complicated with arthritis, and up to 500 millions in chronic cases. Eyre, in acute cases, starts with half a million and, at intervals of four or five days, increases the dose gradually up to 5 millions at intervals of four or five days, then, while the dose is between 5 and 10 millions the interval is seven days and when the dose is over 10 millions the interval is lengthened to twelve or fourteen days. The experimental evidence on which this system is based is not, however, stated. In some papers on the vaccine treatment of gonococeal infections little attention seems to be paid to dosage ; for instance, Barbellion R.A, VACCINES. 13 386 GONOCOCCAL INFECTIONS (1929) in an article of 20 pages makes no mention of the doses he used. Generally speaking, in the French literature the quantity administered appears to be regarded as a subsidiary matter and results, good or bad, are discussed with little reference to dosage. It appears, however, that it is the custom in France to administer large doses of vaccine. Bourdelles and Sédallian (1930) mention a number of stock gonococcal vaccines made in France with the doses recommended :— Pasteur Institute Vaccine. 1,000, 2,000 and 4,000 millions at two-day intervals. (If complications exist the initial dose should not be greater than 400 millions.) Vaccine of Lebeuf (Bact. Inst. of Lyons). 2,000, 4,000, 6,000 and 8,000 millions at intervals of two or three days. Iodized vaccine of Ranque and Senex. 250 to 1,000 millions. Vaccine of Demonchy. 100,000 to 200,000 millions. Lipo-vaccine of Le Moignac, Sezary and Demonchy. 7,500 millions and upwards every two or three days. Audebart and Giscard (1929) use doses up to 25,000 millions of autogenous gonococcus vaccine even in pregnant women and in arthritic cases. Thomson commences with 5,000 millions of his detoxicated vaccine and increases the dose up to 80,000 or 100,000 millions. In this case, however, the number merely represents the original number of cocci before the process of *“ detoxication > was com- menced. There are no intact cocei in the final product. Pelouze (1929) is very emphatic as to the danger of large doses ol vaccine, which, he states, undoubtedly do harm. He has no doubt as to the value of gonococcal vaccine in the treatment of gonorrheeal urethritis and its complications. His system is to give four “small” doses, 150, 200, 250 and 300 millions at intervals of two, three and four days respectively. The word ‘small is here a purely relative one and a large number of workers would not regard the above doses as coming within that category. The great variation in the doses which have been used makes the whole subject very confusing. Large doses are often followed by focal and general reactions, and in some cases the benefit resulting from the vaccine may be the result of the febrile reaction and only RESULTS OBTAINED BY VACCINE THERAPY 387 an indirect result of the vaccine. In the author’s opinion the most satisfactory system is to use doses so small that reactions are avoided. With the simple vaccine this involves the use of doses of from } to 10 millions in acute and complicated cases. In chronic. cases the initial dose should still be small, but the amount administered may rapidly be increased up to 500 millions or more so long as there is no reaction. Results obtained by Vaccine Therapy in Gonococcal Infections Just as there is a great diversity in the dosage of gonococcal vaccine, so there is considerable difference of opinion as to the value of this vaccine in the various gonorrheeal manifestations. Thomson, in his book on * Gonorrheea ** (1923), cites about 250 articles on the results obtained by vaccine therapy up to the year 1921. The following figures are extracted from a table constructed by Thomson, summarizing the opinions expressed by the various authors. | No. of Phase of the Disease. Action Opinion Expressed on Vaccine Therapy. : ; 34 Good results The disease in general . . 35 : = g 1 No effect. f { ( 17 Good results. { Acute urethritis . 9 . 33 16 Little or no elfedt. Chronic urethritis : : 12 12 Good results. Complications in general : 4 4 Good results. .. . | 66 Excellent results. Arthritis. . : : 68 | 2 No effect. Epididymitis : 2 . 35 35 Very valuable. 11 Good results (especially in Prostatitis ‘ . : 14 | chronic follicular). | 3 No effect. Eye complications : Conjunctivitis, Iritis, and 11 Curative. {10 Choroiditis. I 1 No effect. x " e ( 23 Good results. Female gonorrhcea y . 28 |) 75 No effect. aminiite § i o (16 Good results. Vulvo-vaginitis in children . 20 | 4 No effect. 388 GONOCOCCAL INFECTIONS Thomson argues from the fact that nearly all the observers cited have obtained favourable results that there can be no doubt whatsoever that gonococcal vaccine is of therapeutic value in practically every phase of gonorrhoea. While not disagreeing with the conclusion, it must be pointed out that the figures given in a table such as this need not necessarily be taken at their face value, for certain obvious reasons. There is always a tendency to publish positive rather than negative results and also many opinions have been expressed for and against on evidence which is unsatisfactory and untrustworthy. These figures have, however, a certain interest in that, if we except acute urethritis, there is an enormous preponderance of opinion that vaccine therapy is definitely useful in all gonococcal infections. Acute Urethritis. Among the older observers, as shown in the above table, opinion was equally divided as to the value of vaccine therapy and the literature in recent years leaves us in very much the same position. Maclachlan (1923) makes a useful comment on the attitude of many surgeons in regard to vaccine treatment. * Most medical men who have treated gonorrhoea know that it will take weeks or months to clear up a case with irrigations and medicines, even under the best conditions, and although they expect to find gonococci in the discharge and in the gleet which follows they do not doubt the efficacy of this form of treatment even after months of it, while gonococeal vaccine is condemned if it does not work a wonder in a week or two.” Harrison (1931), after enormous experience in the treatment of gonorrheea, states that the observation of a series of parallel cases has convinced him that in the vaccine-treated case the disease pursues altogether a milder course than in cases from which vaccines are withheld. He quotes the figures obtained by Thomson and by Lees in a military hospital, showing that patients treated with vaccines were cured in a substantially less time than were the control patients in whom the same local treatment was carried out, but who received no vaccine. The results obtained by Boas and Thomsen (1919) are worth quoting. They used a simple vaccine made from 24-hour cultures URETHRITIS, ACUTE AND CHRONIC 389 of gonococel, and showed that among 126 cases of recent uncom- plicated gonorrhoea in men only 19 per cent. developed complica- tions as against 45 per cent. of 202 cases not treated with vaccine. The complications which did occur were mild and there were only three cases of prostatitis among the vaccine-treated patients, while in the control group 45 developed this complication. Chronic Urethritis. In this condition there is more unanimity of opinion as to the value of vaccine therapy. Generally speaking, larger doses are used here than in the acute manifestations and as very frequently secondary infections with staphylococei, streptococei, or diphtheroid bacilli have supervened these organisms are frequently incorporated into mixed vaccines with the gonococcus. In these cases the injection of vaccine is some- times accompanied by a mild focal reaction which is beneficial rather than otherwise, but care should be taken to adjust the dose so that severe focal reactions er general reactions are avoided. Vulvo-vaginitis of Children. Most of the writers on this subject have concluded that vaccine therapy was definitely beneficial, but there have been many exceptions. References to these are included in Thomson’s book on ¢ Gonorrheea” (1923) and in a paper by Reith Fraser (1925-1926). Hamilton (1910) treated two comparable series of cases, one with irrigation alone and the other with vaccine alone, with the following results :— n : b Thang Percentage Average Length of Time Treatment. No. of Cases. Cured. Under Active Treatment, Irrigation ‘ ‘ 260 60 10-1 months Vaccine : . 84 90 17 vi In this series stock vaccine was used in doses commencing with 50 millions and increasing by 10 millions every five days up to 100 millions, after which 100 millions was given every ten days. Most of the acute cases were cured after six injections of vaccine. Terwilliger (1931) has published an interesting report on the use of gonococcal vaccine for the prophylaxis and treatment of vulvo-vaginitis in two institutions in which the disease had existed 390 GONOCOCCAL INFECTIONS for a long time. The doses administered were small-——200,000- 4,000,000 cocci. It was noticed that in a number of cases after the second or third injection the infection temporarily flared up, and this was followed by a marked improvement. In some such patients gonococei were found in the discharge during this period of focal reaction although it had been impossible to demonstrate their presence earlier. In these institutions it had been noticed that in a patient suffering from a chronic vulvo-vaginitis the infection flared up by contact with a fresh case, but this did not happen in the children who had received gonococcal vaccine. In all, 42 children were treated with vaccine—of these 19 were infected—and it is stated that 16 were cured. The uninfected children who were inoculated remained well, and one institution became free from infection for the first time in five years. Gonorrheeal Arthritis. There is a considerable measure of agreement among the different observers that vaccine therapy is beneficial. The procedure adopted has, however, in the main followed two distinct lines. The early workers, following the teaching of Wright and his school, used subcutaneous inoculation of small doses (4 million to 10 millions) at intervals of three to seven days. When this system of dosage is employed it is unusual to have any focal or general reaction and the arthritic condition usually clears up after a longer or shorter period. This system of dosage is still the one most commonly used in practice. Lees (1932) has studied 388 cases of arthritis which occurred in a series of over 13,000 gonorrheeal patients, and expresses a very definite opinion that vaccine therapy has given consistently good results and that it assists materially in the relief of pain and swell- ing, in cutting short the disease, and in lessening the liability to complications. This worker has a preference for *“ detoxicated ” vaccines in doses commencing with 2,000 to 5,000 millions and increasing progressively up to 100,000 million. Bruck and Sommer (1912-1913) used intravenous injections of gonococcal vaccine and reported excellent results in gonorrheeal arthritis. To a certain extent the benefit derived from an injec- tion depended on the severity of the general reaction. This GONORRH(EAL ARTHRITIS 391 general reaction consisted in fever, malaise, and sometimes nausea and vomiting, and it was usually accompanied by a focal reaction evidenced by increased pain and swelling of the affected joints. These reactions disappeared in 24 hours or less, and were followed by a feeling of relief and a reduction of pain and swelling of the joints. Culver (1916) confirmed Bruck and Sommer’s work, but showed that the same results could be obtained by the intravenous injections of other vaccines, and even by non-bacterial protein. This system of treatment falls, therefore, into the category of * protein shock ”* (see p. 412). Some workers have reported good results with very large doses of vaccine administered subcutaneously. It seems likely that in these cases the result is obtained by the same process as in the intravenous method, namely, protein shock.” Gonorrheeal Epididymitis. What has been said of arthritis applies equally to epididymitis. Culver (1916) has reported that by protein shock methods excellent results have been obtained in this condition (see p. 417). Ophthalmic Complications. Various reports have appeared claiming that vaccine therapy is of benefit in conjunctivitis, iritis “and choroiditis. These all refer to small numbers of patients, and the results would in no way satisfy a statistician, but must be accepted merely as the opinion of the observer. The author has seen gonorrheeal iritis apparently very much benefited by the administration of gonococcal vaccine in doses of from half to five millions, but the results are not so dramatic that it is possible to ascribe the improvement wholly to the vaccine. However, in all these cases vaccine therapy can be recommended if small doses are employed. Larger doses may give rise to unpleasant focal reactions. i Summary of the Position of Vaccine Therapy in Gonococcal Infections. Provided that care be taken to avoid overdosage vaccines may be administered safely in all phases of gonorrheea ; there is some evidence that in vaccine-treated cases the duration of the primary infection is reduced ; the evidence is stronger that the administration of vaccine diminishes the number and severity of the complications ; and there is no doubt that in complications 392 GONOCOCCAL INFECTIONS such as arthritis vaccine therapy is beneficial. It seems clear, also, that mn gonorrhwal complications, such as arthritis and epididy- mitis, vaccines may have a definitely beneficial non-specific action. It is probable that the results obtained depend more on the dosage than on the way in which the vaccine is prepared (assuming the vaccine to be made from recently isolated cultures). The author strongly recommends the use of small doses (up to 5 or 10 millions) in acute and in complicated cases, although in chronic cases doses up to 500 millions or more can be administered with safety. REFERENCES BarsrLuioN. 1929. J. Urologie., 28, 8. Boas. H., and Tnousex, O. 1919. Hospitalstidende, Copenhagen, 42, 1185. Le BourpeLLEs, B., and SEpaALLiaN, P. 1930. ** Precis d’Immuno- logie.”” Paris, p. 709. Bruck and Sommer. 1913. Miinch. med. Wschr., 60, 1185. CLEMENTS, P. A. 1933. Brit. J. Ven. Dis., 9, 147. CrUVEILHIER, L. 1913. Lancet, 2, 1311. 5 1919. J. Med. Francais, 8, 124. CuLver, H. 1916-1917. Proc. Inst. Med. Chicago, 1, 63. 1920. J. Am. Med. Ass., 76, 311. Eyre, J. W. H. 1925. Brit. J. Ven. Dis., 1, 149. Fraser, A. R. 1925. Brit. J. Ven. Dis., 1, 268. " 1926. Ibid. 2,1. HavivroN, B. W. 1910. J. Am. Med. Ass., 54, 1196. Harrison, L. W. 1931. * Diagnosis and Treatment of Venereal Diseases.” London, p. 278. Lamkin, E. C., and DimoNp, L. 1927. Brit. Med. J., 2, 302. Lees, D. 1932. Brit. J. Ven. Dis., 8, 79, 192. Macracuran, K. 1923. In‘ Gonorrhea,” by D. Thomson. London, p. 343. OLIVER, J. O. 1933. Brit. J. Ven. Dis., 9, 159. Prrouze, P. S. 1929. * Gonococcal Urethritis.” * Philadelphia and London. TERWILLIGER, W. G. 1931. Can. Med. Ass. J., 25, 294. Tromson, D. 1923. * Gonorrhea.” London. (Incorporating a complete bibliography up to 1921.) CHAPTER XX HAY FEVER AND OTHER IDIOSYNCRASIES Hypersensitiveness of an individual to some protein may manifest itself as Hay Fever, Asthma, Urticaria, Angio-neurotic (Edema, Migraine, ete., and to this group of symptoms Freeman has given the convenient name ** Toxic-idiopathy.” With regard to these an enormous literature has collected in recent years, and it is impossible here to touch on many of the interesting and important discoveries which have been made, but as these ail- ments are now treated by injections of an antigen a consideration of the practical therapeutic question of desensitization by means of an antigen is not out of place in a work on Vaccine Therapy. It has been clearly established that there is a very definite hereditary element in these disorders, but the proteins to which an individual is sensitive may not be the same in the children as in the parent nor may the symptoms exhibited be the same.! A typical family chart (Freeman 1930) shows the extraordinary changes which may occur in the allergic manifestations in the same family. Father. Epilepsy and food idiosyncrasy. Mother. Asthma and Hay fever. Children. 1. Hay fever, food idiosynecrasy. angio-neurotic cedema. 2. Asthma. 3. Asthma, horse idiosyncrasy. 4. Migraine. 5. Hay fever. 6. ? Nil. 7. Hay fever, asthma. 1 There is often, however, a strong tendency to heredity sensitiveness to the actual protein irritant. 393 394 HAY FEVER AND OTHER IDIOSYNCRASIES 8. Migraine. 9. Hay fever. 10. ? Nil. Hay Fever For a consideration of the process of desensitization it is con- venient to take hay fever as an example, as this is a common ail- ment in which the exciting cause is well known and in connection with which many of the problems of desensitization have been worked out. The methods which are used in hay fever can, with obvious modifications, be used in the case of other protein sen- sitizations. Hay fever has been shown to be due to a hypersensitiveness to plant pollen. A person may be sensitive to the pollen of many plants, but for a particular pollen to be important in the causation of the disease it must fulfil two conditions :— 1. The plant must be common for pollen to get into the air in quantity sufficient to excite a reaction in a sensitive individual. 2. The pollen must be sufficiently light to blow about in the air for some time. Two types of plants are especially concerned in hay fever. namely the grasses and certain of the composite. In Europe the disease is only common during the pollinating season of the grasses, but in America there is also an autumnal hay fever due to the pollen of some of the composite. Preparation of the Pollen Extract. Noon (1911) who was the first to use pollen extracts for the prevention of hay fever prepared his extracts by repeated freezing and thawing of the pollen in distilled water. Clowes (1913) made a watery extract after pre- cipitating with acetone. Goodale (1915) made extracts in 15 per cent. alcohol. Clock (1917) compared extracts made in different ways for antigenic capacity. Rabbits were immunized with pollen extracts and the antibodies produced were estimated by the complement fixing reaction using different types of extract as antigen. He concluded that the most potent antigen is made by extracting pollen with 2 parts of glycerin and 1 part of saturated salt solution. There is no certainty, however, that the test used TESTS FOR HYPERSENSITIVENESS 395 by Clock gives any real indication of the potency of the extract for the purpose of desensitizing a hay fever patient. Armstrong and Harrison (1925) modified Clock’s method by substituting a buffered salt solution (0-27 per cent. NaHCO3 + 0-5 per cent. NaCl) for the saturated salt solution and Brown (1932) has extracted pollen with equal parts of glycerin and this buffered salt solution. Brown has found these extracts less irritating to the tissues than those prepared with the saturated salt solution. In England the pollen extracts are usually made by repeatedly freezing and thawing the pollen in normal salt solution. The extract is sterilized by filtration through a Seitz filter and for use it is diluted in normal saline containing 0-5 per cent. phenol. By this procedure a very potent extract can be prepared. Standardization of the Extract. Noon (1911) established as a pollen “unit” the amount of extract corresponding to one- millionth of a gram of pollen. This unit has been fairly generally accepted. It is obvious that the potency of the extract must vary with the pollen used and the method of extraction, and the only accurate method at present of comparing one extract with another is by testing serial dilutions of both extracts on the same sensitive patient either by the ophthalmic or one of the dermal tests. When fresh batches of pollen extract are prepared they should be compared in this way with previously tested batches so that the number of units recommended for administration may be greater or less according to the strength of the extract. Tests for Hypersensitiveness in Hay Fever. The hypersensitive condition can be detected and the degree of hypersensitiveness can be measured with some degree of accuracy by methods similar to those which have been used in the past for the diagnosis of tuberculosis by means of tuberculin. The first attempt to estimate the sensitiveness of hay fever patients to pollen extracts was that of Noon (1911), who used an ophthalmo-reaction similar to the Calmette tuberculin reaction except that in this case the reaction was quantitative as well as qualitative. A drop of weak pollen extract (5 units) is introduced into the conjunctival sac. If in three minutes there is no reaction a stronger extract (15 units) is used on the other eye. If this gives 396 HAY FEVER AND OTHER IDIOSYNCRASIES no reaction the test is continued with progressively increasing concentrations of pollen extract up to 500 units until a reaction is obtained. The reaction consists of a slight but definite reddening of the caruncle. This estimation of the degree of hypersensitive- ness was used by Noon to give an indication of the initial dose of pollen extract for desensitization. He found that a safe initial dose was one-third of the number of units contained in 1 c.cm. of the concentration of pollen extract which just caused a reaction. The test can also be done on the skin and a dermal test is now the most popular largely owing to the reluctance of patients to have materials instilled into the conjunctival sac. Drops of various concentrations of pollen extract are placed on the skin of the forearm or thigh and then with a needle the skin is scratched through the drops commencing with a control drop of salt solution and proceeding from the weakest to the strongest concentration of pollen extract. After quarter of an hour wheals appear in the sensitive individual surrounding some of the scratches, and the degree of hypersensitiveness of the patient determines the size of the wheal as well as the concentration of pollen extract which is necessary to produce it. The pollen extract may be injected intradermally. This method has been especially favoured by American workers, but it appears that it is not very accurate in that duplicate tests on the same individual with the same pollen extract do not always give the same quantitative results. A very easily performed and satisfactory method of performing the dermal test is by the * prick ”” method used by Lewis in con- nection with his work on histamine. Drops of various concentra- tions of pollen extract are placed on the skin and then with a fine hypodermic needle the skin is pricked through the drops (begin- ning, of course, with the control drop of saline and proceeding from weaker to stronger pollen extract). The drops can then be wiped off. After 7 to 12 minutes a wheal appears varying in size from a split pea to a shilling (Freeman, 1933). The area of the wheal can be traced on a microscope slide for further reference. The prick test is very easy to perform and is accurate in that duplicate tests give practically identical results. Freeman uses DESENSITIZATION WITH POLLEN EXTRACTS 397 in his hay fever clinic two concentrations of pollen extract, 5,000 and 50,000 units per c.em., which is sufficient to give a rough estimate of the degree of sensitiveness of the patients. The difference in reactivity to pollen extract between the hay fever patient and the normal individual is, as a rule, enormous. In hay fever conjunctival reactions may be detected in very sensitive patients with a concentration of as low as 4 units per c.cm. (Noon, 1911), and practically all react with less than 500 units per c.em. With the “prick ” dermal reaction hay fever patients almost all react to 5,000 units or less. The normal indi- vidual gives no reaction to the strongest pollen extracts. Specificity of the Pollen of Different Grasses. It has frequently been recommended that a pollen extract for use as a desensitizing agent in hay fever should contain the pollen of a number of different grasses. in other words, it should be * polyvalent.” Freeman (1933) in a recent article has dealt with this problem. He quotes the results of three separate investigations, the first “in 1908 in conjunction with Noon and two later ones in 1920 and 1932. The results of all three investigations were essentially the same. A patient who was sensitive to one grass pollen was sensi- tive to all. The grass pollen to which hay fever patients were most sensitive was that of the common timothy grass, Phleum pratense, and desensitization with this pollen desensitized to all the grass pollens. He concludes that * extracts from all the grass pollens furnish one and the same antigen for purposes of desen- sitization to hay fever.” This being so. it is quite unnecessary to employ in hay fever patients a *“ polyvalent” pollen extract ; a simple extract of Phleum pratense suffices, and as this pollen is more potent than that of the other grasses it is even more satis- factory than a mixed pollen extract for the prevention of summer hay fever. Desensitization with Pollen Extracts A very large number of publications have been made giving the individual views of the various observers. The literature up to 1930 has been collected by Rackemann and is to be found in the bibliography included in his book on ¢ Clinical Allergy ” (1931). 898 HAY FEVER AND OTHER IDIOSYNCRASIES It would not be profitable here to consider these various publica- tions in detail, as there is a considerable measure of agreement on the general outlines of the treatment. The methods employed may be briefly summed up. Doses of pollen extract are given, usually subcutaneously, and gradually increasing in quantity, but so graduated that little or no reaction is produced. The interval between the doses is determined largely by the time available for the desensitization process to be carried out before the onset of the hay fever season, and it may be varied considerably to suit the convenience of the patient. The physician aims at being able to increase the tolerance to pollen extract to such an extent that before the onset of the hay fever season a large dose can be injected without disturbing the patient. Coincidentally with this tolerance to subcutaneous injection of pollen extract there is a diminution in the sensitivity of the skin and conjunctiva as shown by the dermal and ophthalmic reactions. The details of desensitization are given by Freeman in a recent article (1933). He first determines the degree of sensitiveness by the dermal or ophthalmic reactions in order to arrive at a safe initial dose. The rate of increase of the doses and the intervals at which they are given depend largely on the time at the disposal of the patient. If treatment is applied for some months before the hay fever season is due to begin a leisurely ”” method of weekly injections may be employed ; if the desensitization has to be carried out within a few weeks a “rapid ” method of injec- tions every one or two days is used ; but if the patient is seen for the first time during or only just before the hay fever season then the * rush” method described by Freeman (1930) must be used. ’ There is, however, no need to adhere strictly to one method. It may suit a patient some time before the hay fever season to give up two or three days to a “rush” treatment, and when this is completed and desensitization has been accomplished he can carry on with weekly injections of the pollen extract till the end of the season. The actual details of the treatment in any particular case may be governed largely by the convenience of the patient and the doctor, and the same result may be achieved DESENSITIZATION WITH POLLEN EXTRACTS 399 with doses of pollen extract administered at widely different intervals. Freeman (1933) has given detailed instructions as regards dosage. The initial dose ranges from 20 to 100 units, as may be determined from the results of the dermal or ophthalmic reaction. If the initial dose is under 100 units the amount administered is increased by 20 units each time up to 200 units and then by 15 per cent. If the initial dose is over 100 units the increases may be bolder until a dose of 300 or 400 units is reached, when the routine increase of 15 per cent. is adopted. Frequently by these methods the patient can be desensitized without any reaction occurring, but sometimes in the course of the treatment a * sticking point is found where the patient will not tolerate the usual rate of increase. When this occurs the rate of increase in the dose has to be reduced or the same dose may have to be repeated several times. If the amount administered is too large the patient suffers from a ‘reaction ” which may show itself as urticaria, angio- neurotic cedema, migraine or asthma, but Freeman points out that unless the dose has been very badly estimated nothing more will be noticed than a transient urticaria which is easily controlled by adrenaline. The interval between the inoculations may, as has been stated above, be varied considerably to suit the circumstances of the particular case. Freeman prefers the interval not to be longer than one week, and (unless the ‘‘ rush” method is adopted) not shorter than four hours. If the rush method is used then the patient is best treated in a nursing home and doses are given every two or three hours during the day. The treatment is continued until the patient is able to tolerate doses of 10,000 or 20,000 units. A similar method was used by Alexander (1917) to desen- sitize a patient to horse serum. Here gradually increasing doses of horse serum were administered at intervals of from 20 minutes to two hours and eventually 40 c.cm. of horse serum could be injected intravenously without causing any symptoms. Freeman points out that patients differ very much in the amount of clinical improvement they get from small amounts of 400 HAY FEVER AND OTHER I1DIOSYNCRASIES treatment. Doses up to 500 units, or even only up to 300 units, improve many and ‘‘ cure” some patients. The larger the dose of pollen extract which can be tolerated the greater is the chance of the patient being completely relieved of symptoms of hay fever. Freeman concludes that * The practitioner may justifiably tell his hay fever patient that he can be cured for certain ; but should add in the next breath that unless symptoms are really exceptionally severe such complete treatment may be hardly worth while. Far less treatment than this usually gives satisfactory or even complete relief, but it is impossible at present to guarantee how much less will do sufficiently well.” Brown (1932) describes methods of administration of pollen extract which do not differ essentially from those described by Freeman. He regulates his dose by the local reaction at the site of injection. The initial dose is 60 units. If no local reaction occurs the dose is trebled ; if the local reaction is transient (dis- appears in 24 hours) the next dose is increased two and a half times ; after he reaches a dose of 6,000 units the increases are much smaller (10 to 20 per cent.). The intervals between the doses are arranged according to how much time is available before the commencement of the hay fever season. If the patient applies for treatment six or more months before the season, weekly doses are given ; if only five months are svailable the interval is six days; and so on till if desensitization has to be carried out in one month doses are given every two days. These times are apparently purely empirical, but the underlying principle is the same as in Freeman's *“ leisurely,” * rapid ” and * rush >> methods of desensitization. Brown aims at being able to attain a dosage of 60,000 to 100,000 pollen units before the hay fever season commences and this dose is repeated weekly throughout the season. He states that *“ by gradually working up to large enough pre-seasonal doses, namely 60,000 to 100,000 units, failures are eliminated and perfect results practically assured.” . . . “ Fur- thermore the evidence at hand would seem to indicate that the administration of such massive doses leads to complete and permanent desensitization, with a disappearance of positive skin reactions.” REFERENCES 101 It is quite clear from the results obtained by Freeman and by Brown cited above that individuals sensitive to pollen can be desensitized by subcutaneous injections of pollen extract to such an extent that they do not suffer from hay fever. Rackemann and a large number of observers quoted in his book on ** Clinical Allergy > support this view. Desensitization to Substances other than Pollen Many people are sensitive to various products of animal or vegetable life, and contact with these substances give rise to symptoms which have already been alluded to under the term “ toxic-idiopathies.” In most cases all that is necessary is to discover the particular substance which is the exciting agent and then the patient can avoid it, but in certain circumstances it is desirable to desensitize the patient. This can be done exactly as has been described in the case of hay fever by injecting gradually increasing amounts of sterile extracts of the exciting substance until a tolerance is established. REFERENCES ALEXANDER, H. L. 1917. Arch. Int. Med., 20, 636. ARMSTRONG, C., and Harrison, W. T. 1925. Public Health Rep., 40, 1466. Brown, G. T. 1932. Jour. Allergy., 3, 2. Crock, R. O. 1917. Jour. Inf. Dis., 21, 523. Crowes, G. H. A. 1913. Proc. Soc. Exp. Biol. and Med., 10, 70. FREEMAN. J. 1911. Lancet, 2, 814. 1930. Ibid. 1, 228. 1930. Ibid. 1, 744. 1933. 1Ibid., 1, 573, 630. GoopaLe, J. L. 1915. Boston Med. and Surg. Jour., 178, 751. Noon, L. 1911. Lancet, 1, 1572. RACKEMANN, F. M. 1931. * Clinical Allergy.” New York. CHAPTER XXI ASTHMA Patients frequently complain of * asthma > when they are merely suffering from dyspneea associated with bronchitis. So far as vaccine therapy is concerned, such patients come under the heading of chronic bronchitis (see Chapter X.). True asthma often results from a hypersensitiveness to some foreign protein which may be ingested with the food or inhaled from the air. A great variety of food-stuffs have been incriminated in different patients; the proximity of certain animals such as the cat, dog or horse is responsible for a certain number of cases ; others are sensitive to mould spores which are inhaled and, of course, some attacks of asthma are associated with hay fever, the treatment of which has already been discussed. There is no need here to enter into details of the specific treatment of such cases. It is necessary to discover the exciting substance and the patient must either avoid this or be desensitized on the same lines as those reviewed in connection with hay fever. There are, however, many cases of true asthma in which this is associated with a bacterial infection, and it is to this type that special reference is here made. Vaccine therapy has been used by many physicians in the treatment of such cases. and many reports of * cures” have appeared. Bray (1931) in “ Recent Advances in Allergy ” quotes results which have been obtained, and these results are condensed in the Table on p. 403. From this Table it will be seen that in all the reports there was improvement in the clinical condition in a considerable proportion of the cases treated. The number in which the symptoms were entirely relieved does not appear in the Table, but some of the reports state that over 50 per cent. fell into this category. Autogenous or stock vaccines made from bacteria isolated from the sputum have chiefly been used. It is a common observation 402 RESULTS OBTAINED BY VACCINE THERAPY 403 Table showing the Results obtained by Vaccine Therapy in Asthma No. of No. of Observer. Date. | Type of Vaccine. cases cases treated. |improved. Pirie . | 1913 | Autogenous 9 % Montgomery and Sicard . | 1917 16 12 Hutcheson and Budd 1918 »s > v . . 20 17 Walker 1919 .. (when patient showed no | 150 609%, hypersensitiveness to bacterial protein). Stock (made from bacteria to 28 969%, which the patient was sensitive). Rogers . 1921 | Autogenous from sputum 40 529, Rackemann and Graham. | 1923 | Autogenous 90 48 Veitch . 1924 | Peptone - vaceine 24 23 Thomas and Touart 1924 | Autogenous from sputum. 62 87% Brown . 1926 ” 144 909, Voorsanger and Firestone.| 1929 | Autogenous 66 64.9, Thomas 1929 % . 300 609%, that in many cases of asthma an examination of the sputum does not reveal a great preponderance of any well-known pathogenic bacterium, but shows merely small numbers of the microbes normally found in the respiratory tract. The following figures (Knott, 1930) clearly bring this out :— Bacteria found in the Sputum of 132 cases of Asthma Sputum collected during the Paroxysm Fosinophilic Sputum. Non-eosinophilic Sputum. Gram-negative bacilli 42 | Pus + catarrhal micrococci Micrococei 8 .. Gram-negative bacilli Moulds . 4 . Moulds . No bacteria 31 | No abnormality Total 85 Total wo } J | Sw 404 ASTHMA In view of this, it is extremely unlikely that the most efficient bacterial vaccine could be prepared from the sputum if we assume that the action of the vaccine is a specific one. Of course, if the vaceine has no specific immunizatory or desensitizing action in asthma but merely acts as so much bacterial protein it would matter little from which region the bacteria composing the vaccine were derived, and indeed it would be reasonable to assume that some bacteria from the air or the soil would make just as good vaccines as bacteria from human sources. .A certain number of observers hold the view that bacterial vaccines have a purely non-specific action in asthma. Bray (1931) states that as auto- genous and stock vaccines appear to be of equal value the effect must be a non-specific one due to the bacterial protein. This argument is not, of necessity, correct. Rackemann (1931) expresses the opinion that the value of a vaccine in asthma goes hand in hand with the production of a local reaction at the site of inoculation and that this local reaction is essential if a good result is to be obtained. He selects for use the vaccine which, when injected intradermally, is followed after 12 or 24 hours by the greatest local reaction. There is no doubt that in asthma benefit can be obtained by non-specific vaccines ; Mackenzie (quoted by Rackemann) was successful with typhoid vaccine, and van Leeuwen (1922), Maxwell (1932) and others have used minute doses of tuberculin with considerable success in cases where there was no suspicion of tuberculosis. Moreover, quite non-specific substances, such as peptone, milk, ete., have been used successfully in many cases. The specific immunizing or desensitizing effect of a vaccine may, however, be of great Importance, and if the vaccine is intended to act as a specific immunizing agent care must be taken in the selection of the bacterium used in its preparation. In some cases of asthma there is a definite chronic respiratory infection by B. influenze, pneumococcus, streptococcus, or Friedlander’s bacillus, and in such cases the infecting organism should be incorporated in the vaccine. In many patients, however, there is no definite respiratory infection. and in these a search must be made elsewhere than in the sputum. Tt is an old observation RESULTS OBTAINRD BY VACCINE THERAPY 405 that attacks of asthma are frequently associated with alimentary disturbances and for many years past it has been our custom at St. Mary’s Hospital to pay special attention to the intestinal tract. The importance of intestinal infections in asthma is brought out by the following case. The patient, a physician practising in one of the western States of America, commenced to have typical and severe asthma at the age of 45 following an attack of influenza (during the 1919 pan- demic). Medicinal and climatic treatment was unavailing ; operative measures on the nasal passages failed to improve matters, and a vaccine made from the sputum gave no relief. After suffering for about two years, he crossed to England, where he had several severe asthmatic attacks. The sputum did not show any definite infection, but when his feces were examined an enormous preponderance of enterococei was discovered. A ‘accine was prepared of these enterococci, and doses were given every three or four days commencing with 0-3 million and increas- ing at first very gradually up to 5 millions and then by larger increments until finally a dose of 3,000 millions was easily tolerated. Apart from one asthmatic attack soon after the vaccine treatment commenced, the condition was entirely relieved and the patient has remained well for some 10 years. In this patient vaccine therapy had been a failure when the vaccine used was made from the sputum, but it was a brilliant success when an autogenous enterococcal vaccine was adminis- tered. This is by no means an isolated case, and it emphasizes the importance of looking elsewhere than the sputum for the bacteria wherewith to prepare vaccines for the treatment of asthma. Freeman, who has had a very large experience in the treatment of asthma with vaccines, has stated in a personal communication to the author that in from one-third to one-half of all his asthma cases intestinal organisms play a part. This is particularly so in the common type of patient who suffers from early morning attacks. In the preparation of vaccines for the treatment of asthma, examinations should be made of the sputum and feces as well 406 ASTHMA as examinations of any region where there is reason to suspect a septic focus. In many cases in which a duodenal or gall-bladder infection has been suspected, examination of material from the duodenum has revealed the infecting organism. While streptococci are probably the most common organisms associated with asthma a great many other pathogenic bacteria have been incriminated in different cases. The sensitiveness of the patient to the various bacteria isolated can be determined by means of the dermal reaction (see p. 396), and if definite hypersensitiveness is discovered to any organisms they should be included in the vaccine. The dermal reaction in bacterial asthma usually takes much longer to appear than it does when protein solutions are pricked into the skin of a hyper- sensitive patient. In the latter case the reaction is at its height in about half an hour, but with bacteria this is unusual and more commonly the reaction appears in 12 or 24 hours. The reactions obtained are comparable with the cutaneous ttiberculin reaction or with the reactions to toxins as shown in the Schick and Dick tests. Stock-mixed vaccines have frequently been used with success. A satisfactory vaccine of this type may be prepared by diluting tenfold the mixed vaccine which is made for the prevention of “colds ” and adding 2 millions of mixed intestinal streptococci. Treatment may be commenced with 0-1 to 0-2 c.em, Dosage of Vaccines in Asthma. It is necessary to commence with very small doses otherwise there is danger of precipitating an attack. A suitable initial dose of a streptococcal vaccine is 0-1 to 0-5 million, and this may be increased by 0-1 million up to 1 million and then by gradually increasing increments up to as large a dose as can be tolerated—it may be several thousand million. If an overdose is given and the patient develops urti- caria, angio-neurotic cedema, headache, or asthma, the next dose must be reduced and the subsequent increases must be very gradual. To avoid the risk of reactions following an overdose many physicians now follow the practice introduced by Freeman some years ago of mixing 0-5 c.cm, or less of adrenalin with the vaccine MULTIPLE SENSITIZATIONS 107 in the syringe and injecting the mixture. This applies not only to asthma, but to all similar desensitization processes. Multiple Sensitizations. Frequently asthmatic patients are sensitive to several different proteins, all of which may contribute to the attacks. In such cases desensitization to one protein may be sufficient to stop the attacks if the protein selected happens to be the most important factor. Freeman has stopped cat asthma > by vaccine treatment of the accompanying bronchitis, and, on the other hand, asthma associated with bronchitis has been cured by desensitization with horse dandruff in a horse- sensitive patient. He considers bronchial infections as important for two reasons :— (a) The infecting bacteria may act as one of the protein irritants. (b) They may act as a local traumatic agency which determines the general allergic tendency of the patient in the direction of the lungs. If the local trauma is removed the asthma may disappear and the allergic manifestation may change to eczema or migraine. A single desensitization may have little or no clinical effect, and in those cases in which desensitization to a protein fails to give relief it is wise to search for a bacterial cause. Some cases are apparently so complicated that all attempts to treat them by specific desensitization methods fail. REFERENCES *Bray, C. W. 1931. ° Recent Advances in Allergy.” London. Knorr. 1930. Guy's Hosp. Rep., 80, 421. van Leeuwen, W. S. and Varexkamp, H. 1922. Miinchen. med. Wschr., 69, 849. MAXWELL. 1932. Brit. Med. Jour., 2, 1182. *RACKEMANN, F. M. 1931. © Clinical Allergy.” New York. * These contain references to the important publications on Asthma. CHAPTER XXII NON-SPECIFIC VACCINE THERAPY It has been a common assumption that the action of a vaccine is strictly specific and that to obtain good results by vaccine therapy it is absolutely necessary that the vaccine used should correspond with the infecting bacterium. There is no doubt whatever that when a vaccine is injected antibodies are produced which are highly specific, and it is now a commonplace in bac- teriology to classify bacteria by their antigen-antibody reactions. In recent years, however, the idea has spread that in addition to the specific response to vaccines there is also a non-specific effect, and some authorities hold that this non-specific response is of the greatest importance in vaccine treatment. The ordinary vaccine is far from being a simple antigen, indeed, recent work has shown that the antigenic structure of bacteria is very complicated. Some portion of the antigenic complex is known to be common to several bacteria, and an extension of this work will doubtless show further common factors among the antigens contained in different microbes. Much has yet to be learnt also as to the factors which bring about the cure of a bac- terial infection. It may be that it is an increase in the antitoxic content of the body fluids, an enhancement of the bactericidal power of the blood, or an increase in the opsonic power of the serum whereby the leucocytes are enabled to phagocyte and destroy the infecting bacteria. It may also be that some change in the environment acts on the infecting organism and, without actually killing it, alters its invasive properties. There are. however, many gaps in our knowledge, and until these are} filled up the complete explanation of the beneficial action of a bacterial vaccine must wait. The injection of a vaccine is known to increase the antitryptic 408 NON-SPECIFIC VACCINE THERAPY 409 power of the blood and also to induce some leucocytosis. Wright (1917) showed that an increase in the antitryptic power of the blood had an inhibitory power on the growth of bacteria, and Fleming (1926) has shown that the bactericidal power of whole blood—other things being equal-—depends directly on the number of leucocytes. Here, then, are two quite non-specific effects of a vaccine which may well have a bearing on the treatment of infections with vaccines. Sir Almroth Wright, who was a strong believer in the strict specificity of vaccines, has more recently emphasized the non- specific aspects of vaccine therapy. He has said (1919) © I confess to having shared the conviction that immunization is always strictly specific. Twenty years ago, when it was alleged before the Indian Plague Commission that antiplague inoculation had cured eczema, gonorrhea, and other miscellaneous infections, 1 thought the matter undeserving of examination. I took the same view when it was reported in connection with antityphoid inocula- tion that it rendered the patients much less susceptible to malaria. Again, seven years ago, when applying anti-pneumococeus inocula- tion as a preventative against pneumonia in the Transvaal mines I nourished exactly the same prejudices. «But here the statistical results which were obtained in the Premier Mine demonstrated that the pneumococcus had, in addi- tion to bringing down the mortality from pneumonia by 85 per cent., reduced also the mortality from other diseases by 50 per cent. From that on we had to take up into our categories the fact that inoculation produces in addition to ° direct * also © col- lateral > immunization.” Calmette has likewise claimed that in children immunized against tuberculosis with BCG vaccine the death-rate from diseases other than tuberculosis is materially reduced. The data which he has given are not, however, convincing. Ledingham (1926) lays great stress on the non-specific effects of vaccines for therapeutic purposes, but adheres to the idea of strict specificity for prophylaxis. He reiterates the view that vaccine therapy as ordinarily practised has in view not only the cure of an existing infection, but the prevention of fresh lesions, 110 NON-SPECIFIC VACCINE THERAPY thus being a combination of therapeutic and prophylactic vac- cination. This is notably so in patients suffering from recurrent infections such as furunculosis or acne vulgaris. In such cases while he would attribute the rapid improvement observed in existing lesions to the non-specific action of the vaccine, Ledingham still advocates the specific vaccine with a view to preventing fresh outbreaks. Wright has, during the last 10 years, published results which he has obtained by adding vaccines to human blood in vitro. With carefully graduated doses of some vaccines he has been able to show that the blood very rapidly acquires an enhanced bac- tericidal power and an increased opsonic content. This response is quite non-specific. For instance, Wright has found that small doses of tubercle vaccine added to blood evoke a greater anti- bacterial response against staphylococci than does staphylococcus vaccine itself. This non-specific response in vitro has been shown by Wright to take place almost immediately. Immuno-transfusion As a result of his work on non-specific immunization in vitro Wright has advocated the method of treatment known as immuno- transfusion, which has attained some popularity in connection with acute septic conditions. This method was further elaborated by Colebrook and Storer (1923). Immuno-transfusion may be carried out by *“ immunization of the blood after it is withdrawn from the donor or by non-specific or specific “ immunization ** of the donor before the blood is taken. Blood is withdrawn from the donor. This is defibrinated or citrated and a suitable dose of staphylococcus vaccine (6,000 cocci per c.cm.) is added. Wright showed that the immunizing change occurred almost immediately so that the blood could be administered to the patient without delay. The immunizing change was manifested by an increase in the opsonic content of the blood serum and by an increased bactericidal power of the whole blood. The action is non-specific, so that the same vaccine can be used whatever the nature of the infection, IMMUNO-TRANSFUSION 411 A dose of 800 million staphylococcus vaccine may be adminis- tered to the donor and then after four to six hours, when the non- specific response is at its height, blood is taken from the donor and administered to the patient. When specific immunization of the donor is aimed at, this indi- vidual receives a series of doses of a vaccine made from the particular organism with which the patient is infected. After a week or more, when sufficient time has elapsed for the donor to have developed specific antibodies, the transfusion is performed. These immuno-transfusions have been performed mainly in cases of acute streptococcal infections and a number of favourable reports have appeared (Colebrook and Storer, 1923. Linser, 1925. Brody and Crocker, 1932. Dalsace, 1932, ctc.). Barach (1931) reports that he obtained no benefit in eight cases of lobar pneumonia when transfusions were performed from donors who had been immunized over long periods. In many cases the simple transfusion of blood from a healthy donor appears to be beneficial, and it is sometimes difficult to assess the special value of the “ immunization *’ of the donor or his blood. Among most of those who have practised vaccine therapy for a long time there is a strong opinion that the specific element is important in treatment as well as in prophylaxis. It is largely on this assumption that autogenous vaccines have been used in preference to stock ones. Frequently the preference for auto- genous vaccines rests on no very solid foundation. Many prac- titioners treat all of certain types of case with autogenous vaccines, so that they have no controls treated with other vaccines with which to compare their results. In the Out-patient Department at St. Mary’s Hospital the writer has on many occasions treated patients in the first instance with stock vaccines and, when the improvement was not marked, has used autogenous vaccines with the result that a very definite improvement in the condition was manifest. Of course, this did not happen in every case, but it was sufficiently frequent to be significant. Occasionally, also, a wrong bacteriological diagnosis has been made and a wrong vaccine given without appreciable benefit, but later when the bacteriology was further investigated and a vaccine was pre- 412 NON-SPECIFIC VACCINE THERAPY pared of the infecting organism a prompt amelioration of the infection occurred. There can be few people who have practised vaccine therapy seriously over a period of years who have not convinced themselves that vaccines for therapeutic purposes in chronic diseases have a definite specific action, and that in most cases better results are to be obtained by using a vaccine of the infecting organism than by using a vaccine made of some quite different microbe. This does not, however, oppose the hypothesis that vaccines have also a non-specific effect. There are some types of case where a non-specific vaccine seems of value. This is especially seen in the case of tubercle vaccine, and it is of interest that it is with tubercle vaccine that Wright (1931) gets his most striking results in the * immunization > of human blood in vitro. In cases of phlyctenular conjunctivitis and some cases of iritis (non-tuberculous) tubercle vaccines in very small doses (144-556 Mg- or less) have had marked success, and many cases of asthma have been successfully treated with tuberculin (van Leeuwen and Varekamp, 1921). In a recent report Maxwell (1932) has shown that in some 60 per cent. of cases of asthma treated with small doses of tuberculin there was considerable improvement or complete cessation of the attacks. There was no suggestion that a tuberculous infection played any part in the asthma in these cases. NON-SPECIFIC INTRAVENOUS VACCINATION (PROTEIN THERAPY) It has long been known that an acute febrile attack may result in the disappearance of a chronic infection. McIntosh, Fildes and Dearden (1912) made some observations on the non-specific influence of the intravenous administration of dead bacteria on chronic infections. These observations were the outcome of work on salvarsan fever, and it was shown that intravenous injection of saline solutions which had been made up with distilled water contaminated with bacteria and subsequently sterilized, was followed by a febrile reaction, and that syphilitic and other ulcers could by this method be made to heal. PROTEIN THERAPY 413 During the last 15 or 20 years an enormous literature has accumulated on this so-called * protein shock ” or * protein therapy,” which is essentially the same as the phenomenon described by McIntosh, Fildes and Dearden. In addition to bacterial vaccines a great variety of substances have been injected for the purpose of inducing fever and non-specific immunity. These are dealt with by Petersen (1922) in his monograph on “Protein Therapy,” which includes a fairly complete bibliography. In this chapter attention will be confined to the intravenous use of bacterial vaccines. Much work has been done in attempting to elucidate the changes which occur in the body during the * shock ”” which follows the intravenous injection of a vaccine. It has been shown that the numbers of leucocytes are at first reduced and later increased. Changes have been noted in respect of the protein content of the serum, in the ferments and antiferments present, and in the coagula- tion time, and it has been shown that in many other ways the blood and the tissues are altered. Stress has been laid by a number of authors on the increased capillary permeability, especially in the splanchnic region. Some evidence has recently been adduced (Boak, Carpenter, and Warren, 1932) that in certain infections, for example, syphilis, continued high fever has a directly lethal action on the infecting organism. It is impossible here to enter into details regarding all the work which has been done on these subjects. Readers who are interested may refer to Petersen’s monograph on Protein Therapy (1922) or to his more recent article in Jordan and Falk’s “ Newer Knowledge in Bacteriology (1928). Choice of a Vaccine for Protein Shock. A great variety of vaccines have been used and very different results have been obtained with different vaccines. The object of the injection is to cause a febrile reaction, and this is readily obtained by the use of vaccines of the coli-typhoid group of bacteria. Typhoid vaccines probably do not differ much in their *“ protein shocking * qualities, but, as a large number of bacteria with widely different characters are classed as * B. coli,” vaccines made of these cer- tainly vary considerably. An appreciable number of the popula- tion, also, have at some time or other suffered from minor infec- 414 NON-SPECIFIC VACCINE THERAPY tions with B. coli, so that in some cases the reactivity of the body is altered to this organism. Likewise some patients at the time of treatment may have an unsuspected focus of B. coli infection, and there is a danger of an exacerbation of such infection as a result of an intravenous injection of B. coli vaccine. For these reasons typhoid (or typhoid-paratyphoid) vaccine is to be pre- ferred to B. coli vaccine as a means of inducing protein shock. Petersen (1922) quotes reports indicating that the clinical results in different conditions may vary with the vaccine used, so that while one vaccine may be more useful than another in some particular infection, it may not give as good results in another infection. This question, however, is not yet fully worked out. Reaction to the Intravenous Injection of a Bacterial Vaccine. This varies enormously with the nature of the vaccine, being much more severe with some than with others. Vaccines of the Gram-negative intestinal bacteria are in general toxic, whereas those of the pyogenic cocci in the same doses are followed by little reaction. It has already been noted in connection with the treatment of rheumatic conditions (p. 858) that intravenous injections of considerable doses of streptococci frequently cause no reaction. Following the intravenous injection of T.A.B. or B. coli vaccine (in a dose of say 25 to 50 million bacilli) the temperature rises in from one to four hours to from 101° F. to 103° F. The degree of fever varies in different patients, in some being hardly perceptible, while in others the temperature may be as high as 105°. One or more rigors occur ; there may be nausea and headache, and the pain in any infected area may be increased. This lasts for a few hours, then the temperature falls, there is profuse perspiration, the pain disappears, and there is a feeling of well-being. In arthritic cases joints often move much more freely at this stage. Blood tests at this stage have shown that there is considerable leucocytosis and the bactericidal power of the blood is increased. (This increased bactericidal power is non-specific, that is, it affects not only the type of bacterium injected, but all other microbes to which it can be tested.) PROTEIN THERAPY 415 Sometimes the temperature persists for a day or two, but this is unusual. Dosage of Vaccine for producing ‘‘ Protein Shock.” When T.A.B. or B. coli vaccine is used a suitable initial dose is 25 million organ- isms. It is not wise to start with a larger dose unless an excessive reaction is desired. The injection may be repeated in about four days and the quantity injected will depend on the first reaction. If this was mild then 50 millions may be given, if severe 25 mil- lions should again be given. Proceeding on these lines the dose may be increased every four days up to 500 millions (by incre- ments of 100 millions). These suggested doses are not absolute and should be varied to suit the patient. The first dose may fail to produce a satisfactory reaction and in such a case double the amount may be given next day. Digests of B. typhosus (either by trypsin or by ferments ela- borated by moulds) serve excellently for intravenous injection to induce protein shock. They have the slight advantage that no particulate matter is introduced into the circulation. They may be used in the same doses as the intact bacteria. Clinical Applications of Protein Shock It has already been mentioned that McIntosh, Fildes and Dearden were able to induce healing of syphilitic and other ulcers by the intravenous injection of salt solution containing the dead bodies of various contaminating bacteria. In 1912 Ichikawa published the results which he had obtained in the treatment of typhoid fever by intravenous injections of typhoid vaccine. In some cases the fever terminated by crisis, in others by lysis shortly after the injection. Roughly one-third of the cases recovered by crisis, one-third were greatly improved and one-third were not benefited by the treatment. These results were confirmed by an Argentine group of clinicians (Penna, Torres and others). Kraus and Mazza (1914) showed that the same results could be obtained by the injection of vaccines of B. coli, thus showing that the treatment was not specific. 416 NON-SPECIFIC VACCINE THERAPY Protein shock has been used in other acute diseases, such as pneumonia, puerperal fever, endocarditis and septicaemia, and favourable reports have appeared, but the benefit conferred is not of the same degree as in the case of typhoid fever. Gow (1918-1919) recommends protein therapy for a great many different conditions. (a) Acute diseases, e.g., typhoid and paratyphoid fevers, coli- form infections of the urinary tract, etc. (b) Infective diseases of uncertain origin. Rheumatic fever, toxic arthritis, ete. (¢) Chronic diseases of uncertain origin. Psoriasis, lupus erythematosus, ete. In the rheumatic diseases protein therapy has had a great vogue since Miller and Lusk issued their first reports on the subject. The results obtained by these observers in acute and subacute arthritis were very good in that more than half * recovered promptly,” although most of them had previously been having the usual drug treatment without much success. In the chronic cases the success was not so great, although in some the pain diminished and the patients weregmore comfortable after a few intravenous injections of typhoid vaccine. Similar results have been published by Scully (1917), Cecil (1917), and others. Snyder (1918) reported excellent results in 110 cases of arthritis following the use of very small doses (5 to 10 million) of typhoid vaccine. Petersen, referring to acute arthritis, concludes that “in perhaps forty per cent. of the cases one or two injections completely terminate the disease, in another thirty per cent. the improvement is marked and recovery made complete on further injections, while in the balance there may be either a transient improvement with a relapse later or no marked clinical improvement.” He points out that the methods used are not pleasant for the patient. This last remark is certainly true, but in certain cases the discomfort of the treatment is much more than compensated for by the almost immediate benefit which follows an intravenous injection of typhoid vaccine. Bruck and Sommer (1912-1913) used intravenous injections of a polyvalent gonococcal vaccine for the treatment of the com- PROTEIN THERAPY 417 plications of gonorrhecea with remarkable results. This they attributed to a specific immunizatory effect. Culver (1916) investigated the effect of protein shock on the complications of gonorrhoea. A series of patients were divided into four groups and these received intravenously every fourth or fifth day one of the following :— (a) Gonococcus vaccine 100 millions. (b) Meningococcus vaccine 100 millions. (¢) B. coli vaccine 25 to 100 millions. (d) Deutero albumose, 2 c.em. of a 4 per cent. solution. A curious phenomenon was observed with the meningococcus vaccine in that more than half of the patients developed herpes labialis after the first injection. Whatever preparation was used, the injections were followed by the usual symptoms of protein shock. The therapeutic results were the same in each case. In all 31 cases of gonorrheeal arthritis were treated. Most of these were acute or subacute cases, but some were over five months’ duration and many had lasted over 10 weeks. The length of treatment varied from two days to one month and all but three were completely cured or decidedly improved. Three of these patients with acute arthritis felt so well after one dose that they insisted on getting up and leaving the hospital in three days. Of these, two had effusions into the knee joint which completely disappeared before leaving the hospital. Twelve patients were treated for gonorrheeal epididymitis. Invariably the pain sub- sided after the first injection and usually not more than two injections were necessary. Since Culver’s work a considerable literature has accumulated and there is agreement that protein therapy gives very good results in the complications of gonor- rheoea, but the primary urethritis is not much influenced. To give a list of all the diseases in which protein shock has been used would be to enumerate practically all the more or less chronic diseases of obscure origin together with many of quite well-known wtiology. Some favourable results have been observed in an enormous variety of conditions, but at the same time there are many failures. The treatment is very unpleasant for the patient and it may be dangerous when administered indiscriminately. R.A. VACCINES, 14 418 NON-SPECIFIC VACCINE THERAPY Cardiac weakness, pregnancy, and alcoholism are regarded as contra-indications to protein therapy, and Petersen has recorded several patients who developed delirium tremens after such treatment, one ending fatally. The primary stimulation of an infected focus may in the cases of tuberculosis assist in the spread of the infection. At the same time the benefit obtained in typhoid fever seems to be definite and it is striking in some cases of rheu- matic arthritis where, within a few hours of the injection, the pre- viously swollen and painful joints are quite comfortable. This form of treatment is without doubt useful in certain cases, but it is not to be recommended indiscriminately in every chronic infec- tion which resists ordinary treatment. It differs from vaccine therapy as ordinarily practised in that the best results are seen when the injection is followed by a short sharp febrile attack, whereas in vaccine therapy it is customary to obtain the maximum benefit without any clinical reaction what- ever. REFERENCES Baraca, A. L. 1931. Amer. J. Med. Sci., 182, 811. Boaxk, R. A., CARPENTER, C. M., and WARREN, S. L. 1932. J. Exp. Med., 56, 741. Bropoy, W., and CrockER, W. J. 1932. J. Amer. Med. Ass., 98, 2191. Bruck and SommEeR. 1913. Miinch. med. Wschr., 60, 1185. CeciL, R. L. 1917. Arch. Int. Med., 20, 951. COLEBROOK, L., and Storer, E. J. 1923. Lancet, 2, 1394. CuLveERr, H. 1916-1917. Proc. Inst. Med. Chicago, 1, 93. >3 1920. Jour. Amer. Med. Ass., 76, 311. Darsace, J. 1932. Bull. Soc. Obstet. Gyne., 21, 382. FLEMING, A. 1926. Brit. Jour. Exp. Path., 7, 281. Gow, A. E. 1919. Quart. Jour. Med., 13, 82. % 1918-1919. St. Bart's. Hosp. Jour., 26, 75. Icnikawa, S. 1914-1915. Ztschr. Immun. Forsch., 23, 32. Kraus, R., and Mazza, S. 1914. Deut. med. Wechschr., 40, 1556. vAN LEEUWEN, W. S., and Varekame, H. 1921. Lancet, 2, 1366. LepiNneuaMm, J. C. G. 1926. Brit. Med. Jour., 1, 815. LINSER, P. 1925. Miinch. med. Wchnschr., 72, 1281. McI~rosH, J., FiLpes, P., and DearpeN, H. 1912. Zeit. Immun. Forsch., 12, 164. MAXWELL. 1932. Brit. Med. J., 2, 1182. MivLLER, J. L., and Lusk, F. B. 1916. Jour. Amer. Med. Ass., 6, 1756. PererseN, W. F. 1922. “ Protein Therapy and Non-specific Resist- ance.” New York. (With bibliography.) REFERENCES 419 PrrerseN, W. F. 1928. ** Newer Knowledge in Bacteriology.” Jordan & Falk, Chicago, p. 1086. Scurry, F. T. 1917. J. Amer. Med. Ass., 69, 20. SNYDER, R. G. 1918. Arch. Int. Med., 22, 224. WricHrt, A. E. 1917. Lancet, 1, 939. 5s 1919. Lancet, 1, 489. 5 1931. Lancet, 2, 225, 277, 333. » 1931. Ann. Inst. Past., 46, 639. 14—2 CHAPTER XXIII ACTIVE IMMUNIZATION AGAINST DIPHTHERIA The immunity following an attack of diphtheria is very lasting, and it has been the aim of many workers actively to immunize children against this disease. In the last decade great success has been obtained in this direction, and when the public has been sufficiently educated to appreciate the benefit which can be conferred by prophylactic inoculation there is reason to hope that they will consent to have their children immunized and that the incidence of the disease will be markedly reduced. The diphtheria bacillus has very limited invasive powers and in diphtheria the infected area is usually small and superficial ; serious or fatal symptoms develop as a result of the absorption of the very powerful toxin which the bacillus produces. Hence efforts in the prophylaxis of diphtheria have differed from those directed against other bacterial diseases such as pneumonia or typhoid fever in that the aim has been to establish an antitoxic rather than an antibacterial immunity. The vaccine therefore consists of some modification of diphtheria toxin and does not contain the actual bacterial bodies, whereas the ordinary vaccine contains the bacterial bodies and is usually heated to 60° C., a temperature which destroys most exotoxins. It may be that in the future diphtheria vaccines will be used containing the antigens of the bacterial bodies as well as the antigen of the toxin, but the results which have already been obtained by the induction of a purely antitoxic immunity have been extra- ordinarily good. Schick Test as a Guide to Immunity. It is unnecessary to enter into the practical details regarding the performance of the Schick test; they can be found in most bacteriological text-books. The test consists in the intradermal injection of a very small 420 SCHICK TEST 421 quantity of diphtheria toxin. If there is no antitoxin in the circulating blood the toxin injected induces a local inflammation which is clearly visible, but if there is an appreciable amount of antitoxin in the blood (3'; unit per c.cm.) the toxin is neutralized and no inflammatory reaction occurs. There is a large amount of evidence showing that individuals who are Schick-negative, that is, who have diphtheria antitoxin in the blood, are relatively immune to diphtheria, while Schick-positive individuals are susceptible. The Schick test, therefore, has been valuable in that it has provided a ready method of determining whether an indi- vidual is immune. Such a test has been very badly wanted in antibacterial immunity and in its absence recourse has had to be made to the cumbersome method of statistical evaluation of the protection in animals or man. If it could be established that the estimation of any particular antibody in the blood was a certain index of immunity, research on bacterial vaccines would be enormously simplified, as it has been in diphtheria prophylaxis by the Schick test. The Schick test has been used to determine the susceptibility of the population to diphtheria. New-born infants are immune ; this immunity disappears during the first year of life, and in the majority of cases returns after a longer or shorter interval either following an attack of diphtheria or as a result of minimal infec- tion with the diphtheria bacillus. The age at which immunity becomes re-established varies greatly in different communities, being less in circles where the children mix freely one with another, as in such cases the risk of exposure to infection is greater than in circles in which the children are more protected (Dudley, 1928). Active Immunization by Mixtures of Toxin and Antitoxin As long ago as 1892 von Behring and Wernicke showed that it was possible, with graduated doses of diphtheria bacillus cultures, actively to immunize animals which had been temporarily passively protected by the administration of antitoxic serum. In 1913 von Behring immunized man with toxin-antitoxin mix- tures. Since the introduction of the Schick test in 1913 immuniza- 422 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA tion of children with accurately titrated mixtures of toxin and antitoxin has been very extensively practised all over the world, but especially by Park and Zingher in America. Although the exact nature of the mixture has varied at different times, it has been clearly established that by inoculation of these toxin- antitoxin mixtures individuals can in the great majority of cases be rendered Schick-negative and therefore presumably immune to diphtheria. A certain number of disasters have occurred with these toxin- antitoxin mixtures. One (the Bundaberg disaster) was due to contamination with staphylococci, an accident which might happen in the careless use of any kind of vaccine containing no antiseptic. The other disasters had a definite relation to the diphtheria toxin contained in the mixture. In one the manu- facturer miscalculated the quantity of toxin so that the mixture was too toxic, in another the mixture, containing a small amount of carbolic acid, was kept frozen and on thawing it was found to be too toxic owing to some of the antitoxin having been destroyed by freezing and thawing in the presence of carbolic acid ; in yet another the mixture was made by adding the requisite amount of toxin slowly to the antitoxin, in which circumstances the antitoxin is neutralized by a smaller quantity of toxin than when the mixture is made rapidly (the Danysz phenomenon), so that the final mixture contained an excess of free toxin. These toxin-antitoxin mixtures, containing as they do free toxin, are not now to be recommended. They have been of immense service, but it is now possible to use a toxin which has been so modified that while as an immunizing agent its activity is not diminished it has lost its toxic properties. Toxoid Lowenstein showed that when tetanus toxin was treated with formalin it lost its toxic properties but retained its antigenic efficiency. Glenny and Siiddmersen (1921) showed that the same thing happened with diphtheria toxin. Ramon (1928) confirmed these findings. Ramon gave the name ‘ anatoxine ” to this TOXOID 423 detoxicated toxin, and it is by this name that it is generally known in France, but elsewhere Ehrlich’s old name ““ toxoid * is generally applied. The rate of * toxoiding ”’ of diphtheria toxin with formalin varies with the temperature and the strength of the formalin, as is shown from the following figures taken from Glenny, Hopkins, and Pope (1924). Effect of temperature. Toxin +4 0-3 per cent. formaldehyde for 24 hours. Temperature ‘ . 30° C. 33° C. 36° C. 39°C. Strength of toxin (m.r.d. per c.c.) ‘ ‘ v 1,250 500 250 25 Effect of strength of formalin. Toxin - formalin at 37° C. for 24 hours. Strength of formal- dehyde per cent. . | 0-01 | 0:03 | 0-1 0-2 0-3 0-4 0-5 Toxicity compared with original toxin 4 1/3 | 1/20 | 1/100 | 1/400 |1/10,000 [not toxic 3 The rate of * toxoiding »” with formalin varies to some extent with the nature of the broth in which the bacteria have been grown and also with the reaction, being more rapid in alkaline solutions. Moloney and Weld (1922-1925) have shown that it occurs more rapidly with the whole culture than with the filtered toxin. Ramon uses 0-3 to 0-4 per cent. of commercial formalin for the purpose of *“toxoiding™ his cultures, and he leaves the formo- lized cultures for one month at 37° C. before filtering. The rate of detoxication is shown by the following figures (Ramon, 1924) : Toxin + formalin (0-35 per cent.) at 37° C. 1 3 50 10 10 1 Time of exposure (days) . : 0 30 20 Number of M.L.D. per c.c. . | 800 1 The toxoid is more stable than the original toxin. Ramon (1924) showed that heating for one hour at 65° C. had no effect on its combining power. 424 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA “~ Primary and Secondary Stimulus Immunization with diphtheria toxin or toxoid preparations has permitted a more exact detailed study of certain problems in active immunity than is possible in the case of antibacterial immunization. When a bacterial vaccine is inoculated into an animal various antibodies are produced, but there is no certainty that the recognizable antibodies (agglutinins, precipitins, comple- ment-fixing substances, opsonins, ete.) represent the sum total of the immunizing response. When, however, the antigen is a 301 nN wo L » Ss > wv 2 Ss Antitoxin Units == Injection <—/njection 0 20 40 69 60 10 20 140 160 Time, in Days Fig. 1. Antitoxin production in horse, showing difference between primary and secondary response. (From Topley and Wilson after Glenny and Siidmersen.) toxin, it is recognized that antitoxin production represents the complete immunizing process. The antitoxin content can be accurately estimated in the blood of the immunized animal by direct titration with a standard toxin, so that the process of immunization can be closely followed. Glenny and Sidmersen (1921) examined a large number of guinea-pigs which had survived the routine injections for the titration of toxin. They all showed the presence of antitoxin (not to a high titre) in the blood. When these guinea-pigs were injected with a further dose of toxin, the antitoxin response was much PRIMARY AND SECONDARY STIMULUS 425 greater than it was in the normal animal. The first injection of toxin, therefore, in addi- tion to evoking 0400 ] x {jection S300 5 3200 k: GS 700 & Qo J 1 1 T 10 30 40 eR SS 0 5 10 5 Time, in Days (b) Fic. 2.-— Precipitin production. Response of rabbit to a single injection of horse serum. (a) Normal rabbit. (b) Previously sensitized rabbit. (From Topley and Wilson after Dean and Webb.) 20 Time, in Days (a) the production of some antitoxin, had altered the animal in some way so that it gave a greater response to a second in- jection of toxin, This question has been fully dis- cussed by Glenny and his co-workers (Glenny and Siidmersen, 1921, Glenny 1925, 1931). The first response of the animal to the antigen Glenny has called the “Primary Stimulus” and the subsequent response the Secon- dary Stimulus.” Fig. 1 shows the antitoxin response of a horse to two injections of toxin-antitoxin mixture. Following the first dose there was, after an interval of about 14 days, a slight rise in the antitoxic content of the blood, which persisted until the second injection was given 14} weeks later. The response to this second injec- tion was quite different. After a latent period of only three days, the antitoxic content of the blood rose very rapidly, so that in 10 days the titre was 20 times the maximum titre attained after the first injec- tion. 426 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA Fig. 2 shows a similar result obtained by Dean and Webb when working with precipitins formed as a result of the injection of horse serum into rabbits. The response to a bacterial vaccine administered to the non- immune and the partially immune animal is somewhat similar, as can be seen from Fig. 3. A glance at these charts will show that typhoid vaccine, given to a man who had been previously inoculated with this vaccine, was followed by a much more satisfactory response of antibodies (agglutinins) than was the same dose of vaccine given to an uninoculated individual. In the previously inoculated man, also, the high concentration of antibodies persisted much longer. Interesting work on this increased reactivity of an animal previously immunized with a bacterial vaccine is that of Harvey and Iyengar (1928). Pigeons were immunized with two doses of B. avisepticus (the bacillus of fowl cholera) after which they were found to be resistant to infection with this organism for several months. After that time the resistance began to disappear, but it was shown that the falling immunity could be restored to the original level by a single dose of the vaccine, which was only a small fraction of the original immunizing dose. This restored immunity lasted as long as the original immunity following the two large doses. The importance of these observations in con- nection with the prophylactic and therapeutic use of vaccines is obvious. It seems likely that the increased reactivity of the body following a primary stimulus with an antigen is a general phenomenon. The obvious benefit of prophylactic inoculation may not rest entirely on the antibacterial or antitoxic content of the blood, but may largely depend on the altered state of the immunizing mechanism whereby the inoculated individual is enabled to respond to a subsequent infection by a prompt and copious production of antibodies. In the treatment of chronic bacterial infection with vaccines this question is likely to be of extreme importance. Here the primary stimulus will have been administered by the infection itself, and the patient will be in a position to respond satisfactorily =Injection 1600 | 800 + 400 Q C 3 200 + 700 + 04d I 1 v 1 1 0 40 80 120 160 200 Time, in Days (@) 3200+ |" Injection 1600+ 800 hi S 4001 ~ 2004 1004 0 0 40 _80 120 160 200 Time, in Days (b) Response to a single injection of typhoid IF16. 3.—Agglutinin production. (b) Previously vaccine. (a) Previously uninoculated person. inoculated person. (From Topley and Wilson.) 128 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA and rapidly to doses of vaccine so small that they would evoke little response in the normal individual. Glenny and Allen (1921) showed that following the injection of an antigen this increased state of reactivity could be demonstrated before any antitoxin could be detected in the blood, and also that it persisted after the antitoxic response to the primary stimulus had disappeared. This “actively immune state 7 in the absence of demonstrable antitoxin was demonstrated by a shortening of the latent period after the injection and by the large amount of antitoxin produced. Glenny (1931) states that “the increased power of response conferred by a primary stimulus lasts for a long period, and there is no recorded case of its diminution.” He instances a series of guinea-pigs which had a secondary stimulus of toxin nine months after the primary stimulus, and whose pooled serum yielded 30 units of antitoxin per c.cm. This titre of anti- toxin could only be passively attained in man by the administra- tion of about 75.000 units of diphtheria antitoxin. Different Forms of Diphtheria Prophylactic Toxin-antitoxin Mixtures. The various combinations which have been used are discussed in the Medical Research Council's System of Bacteriology * (1931, Vol. 6) by Glenny, whose article may be read with profit by anyone interested in the subject. The objections to active toxin in any mixture used for human immunization have already been mentioned, and the use of these mixtures is rapidly becoming less common. Toxoid-antitoxin Mixtures. The toxoid is prepared by treating diphtheria cultures or toxin with formalin. Glenny, Hopkins and Pope (1924) showed that the presence ol antitoxin within a wide range of concentration did not affect the immunizing power of the toxoid. The most extensively used diphtheria prophylactic in England since 1924 has been a toxoid-antitoxin mixture con- taining either completely * toxoided ” toxin or toxin so altered by formalin that one human dose even without antitoxin contains less than one guinea-pig fatal dose. Glenny (1931) suggests that the addition of antitoxin may be regarded as a concession to FORMS OF DIPHTHERI.{ PROPHYLACTIC 429 British conservatism, as any type of toxin-antitoxin mixture was accepted freely, while toxoid alone had little support. Toxoid (Anatoxine). Although Glenny and Hopkins (1923) first suggested the use of toxoid alone as an immunizing agent, it is Ramon and his co-workers in France who have used the method most extensively. The toxin is completely toxoided ” by incubation with formalin at 37° C. The ease with which toxoid can be prepared and the good results which have followed its use, make it very suitable for human immunization. There is a somewhat greater local reaction following its use than there is with toxin-antitoxin mixtures or with the floccules alluded to below, but this reaction is not so severe as to militate seriously against its use in man. Toxin-antitoxin Floccules. Ramon showed that toxin and antitoxin, when mixed in equivalent amounts, produce a precipi- tate. This precipitate contains all the specific antigen and antibody in the mixture with relatively little non-specific material (Hartley, 1925; Sordella and Serpa, 1925). The floccules are collected and well washed before final suspension. By the use of these floccules a large part of the non-specific matter contained in a toxin-antitoxin mixture is eliminated and the reaction following the injection of the floccules is less than with toxin-antitoxin mixtures. This preparation makes an excellent prophylactic, but it has no advantages over the next, and the use of active toxin in its preparation makes it less suitable for human immunization. Toxoid-antitoxin Floccules. This preparation is made in the same way as the last, except that toxoid is substituted for toxin. This renders it absolutely safe for the immunization of children, and Glenny and Pope (1927) have shown that the * toxoiding process does not interfere with the antigenic capacity of the floccules. They have further shown that the immunity following the injection of the floccules comes on rapidly and that the antigenic efficiency can be enhanced by partial destruction by heat of the antitoxin contained in the floccules. The floceules give practically no reaction, so that in special cases, where it is important that no reaction should occur, they may be 430 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA used in preference to toxoid. They are much more difficult to prepare and the antigenic power is not quite so great as that of the best toxoid, but a satisfactory immunity can be obtained by their use, especially if an extra dose is given. There is no reason why toxin-antitoxin mixtures should now be used for immunization against diphtheria. The presence in such mixtures of active toxin makes it always possible for accidents to happen owing to carelessness in the preparation, storage or use of the mixture, and as toxin has no greater immunizing value than has the non-poisonous toxoid, there is no justification for its use. The only thing which has militated against the use of toxoid has been the occurrence of reactions in a small number of the treated individuals. This reaction depends on a state of hyper- sensitiveness of certain individuals to the proteins contained in the culture medium which is used in the preparation of the toxoid. The reactions are unpleasant, but are harmless to healthy indivi- duals. Children under the ages of four or five rarely give any reaction with undiluted toxoid, and for this reason there is no contra-indication to its use in such young children. For older children Moloney has devised an intradermal test which shows whether the individual is sensitive to the toxoid. Using this test, O’Brien and Parish (1932) showed that it was possible to immunize children rapidly without reactions. Those insensitive to toxoid were inoculated with potent unconcentrated toxoid, while the sensitive ones received toxoid-antitoxin floccules (T.A.F.).. Ramon, in France, has made light of the reaction following toxoid, but it is likely that in England, where there is a deep-rooted prejudice against inoculation, it is wise to take precautions against the occurrence of such reactions so that the immunization procedure may be more widely used. Although Ramon has stated that thousands of tuberculous children have been inoculated with toxoid without any serious effect, it is recorded that in some children inoculations of toxoid have been followed by a flare up of tuberculous foci (Aubertin and Boudou, 1932). The addition of alum (2 per cent.) to toxoid increases its efficiency as an antigen (Glenny, 1930), but it increases the reaction, and so PROCEDURE 431 it has attained no popularity for human immunization, however useful it may be in the immunization of horses for the production of antisera. Ramon and his confréres have used the method of *“ vaccins associés,” which consists of mixing anatoxine (toxoid) with typhoid-paratyphoid (T.A.B.) vaccine and injecting the two simultaneously. In this way the immunity produced to diphtheria was increased and the anatoxine did not interfere with the immunity to the enteric organisms. In communities where it is desirable to immunize against both diphtheria and the enteric fevers the advantage of this procedure is obvious. The non-toxic character of anatoxine or toxoid has been amply demonstrated in a case recently described by Aubertin and Boudou (1932). A patient suffering from diphtheria inadvertently had 100 cc. of anatoxine injected subcutaneously, and after 24 hours another 50 c.c. These injections were followed by a certain degree of * protein shock,” but this was transient and the patient made an uninterrupted recovery. It would appear, therefore, that even when an individual is suffering from diphtheria one hundred times the ordinary dose of toxoid can be injected without serious symptoms. Procedure in the Active Immunization against Diphtheria A very important memorandum on this subject has been issued by the Ministry of Health in November, 1932. This accords with the findings of the conference of experts from different countries held in London in June, 1931, under the auspices of the Health Committee of the League of Nations. The eight recommendations made in this memorandum are here reproduced. 1. Among children in this country a proportion which varies in different communities, but may be as high as 90 per cent. or as low as 10 per cent., is susceptible to diphtheria. In order definitely to ascertain whether a child is susceptible or not, a preliminary (Schick) test should be made. This consists of an injection into the superficial layers of the skin (intradermally) of a forearm of 0-2 c.c. of standardized diluted diphtheria toxin (Schick Test Toxin). For control purposes a second and precisely similar 432 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA injection is made into the skin of the other forearm, but with toxin which has been previously rendered inactive (Schick Control). 2. At the same sitting a third injection is given, also intra- dermally into the skin of the forearm, of a very small dose of formol toxoid (0-2 c.c. of a 1/100 to 1/200 dilution) with a view to determining whether there is any hypersensitiveness which requires to be taken into consideration if immunization is to be proceeded with as a result of the Schick test. 3. If the Schick reaction when read on or after the third day proves to be negative the child, if over one year old, can for all practical purposes be considered naturally immune to diphtheria. 4. If the Schick reaction is positive, as indicated by the appear- ance of a flush of § to 1 inch or more in diameter (which may not appear until the third day), lasting for some days and gradually fading into a brown discoloration, the child should be artificially immunized. The observer should have the necessary experience in reading the results of the test and should record the diameter of the reaction. 5. The method of immunization should be modified in accordance with the reaction to the small dose of formol toxoid which consti- tuted the third injection. If there is no local response to this within 24 to 48 hours, or if there is a local reaction of not more than 4 inch in diameter with no induration, immunization should be undertaken by the subcutaneous injection of formol toxoid of 25 to 35 LI, or of even greater strength, in doses of 1 c.c. on three occasions with fortnightly intervals. 6. If the local reaction to the test dose of formol toxoid is strong, however, (as occurs in approximately 5 per cent. of children of all ages), i.e., if there is a vivid flush of more than } inch in diameter with central induration, toxoid-antitoxin floccules should be used as the immunizing agent, instead of formol toxoid. 7. Not less than two months after the last immunizing dose has been given the Schick test should be repeated. In the great majority of cases it will be found to be negative, indicating that immunity has been established. If the Schick test is positive, however, or doubtfully positive, then one additional dose of the prophylactic, equal to the last one given, should be administered PROCEDURE 433 and the test again applied not less than two weeks later. If the test proves again positive, then the whole immunizing course should be repeated, as should the subsequent retesting. 8. Immunization should not be attempted in children under the age of one year ; with this proviso the procedure is applicable to all healthy children found to be susceptible.” It is laid down that immunization should be preceded by a Schick test to ascertain whether the individual is already immune. This is. of course, the ideal procedure, but in dealing with young children the preliminary performance of a Schick test may in certain circumstances definitely interfere with the ease with which the consent of the parents is obtained to the immunizing process. In such cases and in cases where there may be lack of facilities for performing the Schick test, there is no reason why immunization should not be proceeded with. The great majority of children under, say, 6 years of age, are susceptible, and the inoculation of all such children would merely mean that a certain amount of prophylactic material was to all intents and purposes wasted in the * immunization > of already immune individuals. Although the preliminary Schick test may be dispensed with in certain circumstances, it is important that the Ministry’s second recommendation should be carried out in order to detect the children who are hypersensitive to the toxoid and so avoid undue reactions which would militate against the general acceptance of diphtheria prophylaxis. It is recommended that some months after immunization another Schick test should be done to ascertain whether sufficient immunity has developed. It has been shown, as we shall see later. that a course of three doses of toxoid renders the Schick test negative in something like 98 per cent. of individuals. It is likely that, with the improvements which have taken place in the preparation and use of toxoid, in the near future 100 per cent. of the children inoculated will be immunized. When that stage is reached the post-immunizatory Schick test will be quite un- necessary. Typhoid vaccine has been successfully employed without applying any subsequent test for the purpose of ascertaining 434 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA whether immunity had been established in the inoculated indi- viduals. Had such tests been demanded, it is certain that the spread of typhoid prophylaxis would have been definitely hindered. It may be the same with diphtheria prophylaxis. When it becomes known that a certain type of antigen confers immunity in 98 to 100 per cent. of the cases, insistence on a Schick test may definitely interfere with the spread of immunization and thus be a danger from the public health point of view, although in an individual case it may be satisfying as a demonstration that immunity has developed. Results of Immunization There is ample evidence that immunization with toxin-antitoxin mixtures causes a rise in the antitoxin content of the blood, as shown by direct titration (Romer, 1914) or by the Schick test (Park, Zingher, etc.). In New York, Park (1922) has collected figures concerning the incidence of diphtheria in immunized children. Among 90,000 children who had been Schick tested and, where necessary, immunized with toxin-antitoxin mixtures, there were 14 cases of diphtheria, whereas in the same number of children who were neither tested nor immunized there were 56 cases. The very small rate of incidence of the disease, however, lessens the value of these figures, and more obvious benefit is noticed in small communities of people immunized in conditions where the risk of infection is greater. Woods (1928) gives figures for certain fever hospitals where the staff were immunized from 1922 onwards. In the Little Bromwich Hospital, among 90 nurses, the incidence of diphtheria in the three years previous to 1922 was 17-8 per cent., whereas in the three years following the introduction of active immunization it fell to 3:7 per cent. Similar results were obtained at the City Hospital in Edinburgh. In the pre-immunization period (1919-1922) the attack rate was 9:5 per cent., and this was reduced to 3:5 per cent. in the post-immunization period (1923-1925). In a more recent paper, Park and Schroder (1932) state that RESULTS 435 during 15 years up to 1931 some 500,000 school children were treated, and since 1929 some 250,000 children of pre-school age had been inoculated. The deaths from diphtheria in New York in 1920 were 880, in 1929 there were 416 deaths, and only 198 in 1930. Results of Immunization with Toxoid (Anatoxine) Most of the earlier results come from Ramon and other French workers. Ramon (1928) has published a full description of ana- toxine and its applications. He prescribes three subcutaneous doses, the first of 0-5 c.cm., followed after an interval of three weeks by 1 c.em. and a third dose of 1-5 c.cm. after a further period of 15 days. A Schick test done one or two months after the last dose shows that the reaction is negative in practically all the inoculated persons. Martin, Loiseau and Lafaille (1928) have shown how the immunity, as measured by the Schick test, developed. After the first dose some 37 per cent. of the inoculated children became Schick-negative, and after the second dose 95 to 98 per cent. Records are given from a large number of institu- tions in which inoculations of anatoxine were controlled by the Schick test ; in practically all of them over 97 per cent. of those receiving three doses of anatoxine became Schick-negative. These authors record, also, very favourable results during epi- demics of diphtheria. Fitzgerald (1928) has published results obtained in Canada. From October, 1925, to March, 1927, almost 400,000 persons were inoculated with toxoid (anatoxine) without any untoward result. Fitzgerald shows that there is a marked diminution of the diph- theria morbidity and mortality among the inoculated individuals. The immunization against diphtheria in Canada has been con- tinued, and in a recent paper Fitzgerald (1932) concludes that in the treated children the incidence of diphtheria has been reduced by 90 per cent. He has shown that in certain communities where inoculation with toxoid has been very thorough, such as Hamilton, with a population of 150,000, diphtheria has been practically eliminated. It is of interest that during 1932 in the city of 46 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA Toronto there was not a single death from diphtheria. Tomesik (1932) reports the results obtained in Hungary where during 1930 and 1931 250,000 children were vaccinated with anatoxine. He has collected the results in many districts where more than 50 per cent. of the children were vaccinated and has found that in 100,000 children fully vaccinated from 1930 to the end of 1931 the incidence of diphtheria was reduced by 90 per cent. as com- pared with the incidence in non-vaccinated children of the same age living in the same localities. The vaccinated children who contracted the disease had, as a rule, only a very light attack, but there were some severe cases and three deaths among the children who had received the three injections. Tomesik states that the rarity of severe cases among the vaccinated justifies the conclusion that the immunizing process reduces the mortality from the disease by considerably more than 90 per cent. An extensive review of the results of diphtheria immunization in many countries has been published by Graham Forbes (1932). This work may with advantage be consulted by anyone requiring further detailed information on the subject. Age when Inoculation with Toxoid should be Carried Out Statistical studies have shown that fatal diphtheria occurs most frequently at early school age, and from this it is obvious that children should be immunized either before or immediately after they go to school. If the procedure is postponed the child is exposed to serious risk of infection in his carly school days and the value to the community of immunization is correspondingly diminished. The time of election for immunization is between the years of one and three. At this time hardly any of the children are sensi- tive to toxoid and the injections are not followed by any reaction. At the same time, the immunity is provided when it is most wanted. Godfrey has shown that there is no sensible decline in the attack rate if 60 to 70 per cent. of the school children are inoculated when the schools are continually recruiting only susceptible children. It is only when about one-third of the children of pre-school age REFERENCES 437 are immunized that a noticeable decline in the prevalence of the disease is observed. There is no reason why every child should not be immunized against diphtheria. When toxoid is used the procedure is quite safe ; there is no danger of poisoning, as there was when active toxin was contained in the mixture—the regulations imposed on the manufacturer by the Ministry of Health quite obviate that ; there is no danger to the general health of the child, and the local reaction is not severe. It is only the prejudice of parents against any inoculation procedure which prevents wholesale immuniza- tion and the practical disappearance of diphtheria as a menace to public health. The saving to the community in time, money and lives would be enormous, and it is to be hoped that in the near future Britain will not lag behind in diphtheria prophylaxis. REFERENCES AuserriN and Boubou. 1932. Paris Med., 393. von BEHRING. 1913. Deut. Med. Wschr., 89, 873. von BEHRING and WERNICKE. 1892. Zeit. f. Hyg., 12, 10. DaNvysz, J. 1902. Ann. Inst. Past., 16, 331. Drax and Wess. 1928. J. Hyg., 24, 301. Duprey, S. F. 1928. Brit. J. Exp. Path., 9, 290. Frrzeerarp, J. G. 1928. Ann. Inst. Past., 42, 1089. FrrzeeraLD, J. G., Derries, R. D., FRASER, D. T., MoLoNEY, P. J., and McKinnon, N. E. 1932. Am. J. Pub. Health, 29. 25. Gren~y, A. T. 1925. J. Hyg., 24, 301. hn! 1930. Brit. Med. Jour., 2, 244. 1931. “A System of Bacteriology,” London, H.M. Stationery Office, 6, 161. GLENNY, A. T., and ALLEN, K. 1921. J. Path. and Bact., 24, 61. Grenny, A. T., and Hopkins, B. EK. 1923. Brit. J. Exp. Path., 4, 283. GLENNY. A. T., and Por, C. G. 1927. J. Path. and Bact., 30, 587. GrLEsNy. A. To. et al. 1925. J. Path. and Bact., 28, 279. v5 "” 1926. J. Path. and Bact., 29, 38. GLENNY. A. T., and Stpmersex, H. J. 1921. J. Hyg, 20, 176. GranavM Fomrses. 1932. * Diphtheria: Its Distribution and Pre- vention.” London. Harmison, W. T. 1932. Amer. Jour. Public Health, a8. 17. HarrtLEy. P. 1925. Brit. Jour. Exp. Path., 6, 112. Harvey, W. F., and IveNcar, K. R. K. 1928. Ind. Jour. Med. Res., 15, 935. LOWENSTEIN, BE. 1909. Zeit. Hyg. InfektKr., 62, 491. Martin, L., Lomseav, G., and LaraiLLe, A. 1928. Ann. Inst Past., 42, 959. 438 ACTIVE IMMUNIZATION AGAINST DIPHTHERIA Ministry of Health Memo. 1932. 170/Med. O’Brien, R. A., and Parisi. 1932. Lancet, 2, 176. Park, W. H. 1922. J. Am. Med. Ass., 79, 1584. Park, W. H., and ScHroDER, M. C. 1932. Amer. J. Public Health, 22,7. Parr, W. H., and Zineuer, A. 1915. Amer. J. Public Health, 65, 2216. Park, W. H., and ZINGHER, A. 1924. Amer. J. Dis. Child., 28, 464. Parisu, H. J., and OkeLL, C. C. 1928. Lancet, 2, 322. PouraiN. 1932. Ann. Inst. Pasteur. 42, 588. Ramon, G. 1923. C. R. Soc. Biol., 86, 661. # 1924. C. R. Acad. Sc., 179, 422. % 1924. C. R. Acad. Sc., 179, 485. v5 1926. Ann. Inst. Past., 40, 1. 3 1928. Ann. Inst. Past., 42, 959. 5 1932. Bull. Inst. Past., 30, 1, 65. SorpELLA and Serra. 1925. C. R. Soc. Biol., 92, 824. Tomesik, J. 1932. Ann. Inst. Past., 49, 574. Torrey, W. W., and WirLson, G. S. 1929. * Principles of Bacteriology and Immunity,” 2, 737. Woops, H. M. 1928. .J. Hyg., 28, 147. CHAPTER XXIV ACTIVE IMMUNIZATION AGAINST TETANUS Our knowledge of this subject is mainly due to the work of Ramon and his co-workers in France. They have used tetanus anatoxine prepared in exactly the same way as diphtheria ana- toxine by the action of formalin on the toxin at 38° C. The pre- paration and mode of employment of tetanus anatoxine is described by Ramon and Zoeller (1927). They have shown that by the injection into man of three doses of anatoxine, spaced in the same manner as diphtheria anatoxine already described, antitoxin is produced in amounts such that 1 c.cm. of serum is capable of neutralizing 300 to 1,000 fatal doses (for a guinea-pig) of toxin. This is a large yield, and the serum of such an actively immunized man is considerably more antitoxic than is that of a man passively immunized by the injection of the ordinary prophylactic dose of antitoxine. Ramon and Zoeller found that no antitoxin appeared in the blood after anatoxine administered by the mouth, but when the antigen was applied to the nasal mucosa the yield of antitoxin was comparable with that following subcutaneous injection. Nattau Larrier, Ramon and Grasset (1927) immunized mothers in the late stage of pregnancy with three doses of anatoxine and they found that the new-born infant had in its serum antitoxin in quantity such that 1 c.cm. would neutralize 100 to 300 guinea pig lethal doses of toxin. (The mothers’ serum had roughly three times this quantity.) This method of protecting both mother and child can be practised where there is danger of tetanus following childbirth. Of course, as Ramon and his colleagues point out, this risk is negligible in Western countries, but there are parts of the world where the danger is real. Ramon and Zoeller have shown that the action of tetanus 430 440 ACTIVE IMMUNIZATION AGAINST TETANUS anatoxin is markedly increased by the method of * vaccins associé¢s.” The anatoxine is injected together with a bacterial vaccine, e.g., typhoid-paratyphoid (T.A.B.) vaccine. The first two doses of anatoxine are combined with the first and second doses of T.A.B. vaccine respectively, and the third dose of anatoxine is given alone. In this way there is simultaneous immunization against tetanus and the enteric fevers and at the same time the immunity to tetanus is enhanced. This is an important practical observation, as in most of the Western countries the only urgency for tetanus immunization occurs in time of war and it is in just such a time that it is imperative to immunize against enteric fever. Combined Active and Passive Immunization. When a man has been wounded and it is feared that tetanus may develop, any immunizing process designed to prevent the disease must be aarried out promptly and the patient’s blood must acquire a considerable antitoxic power before there is time for the tetanus bacilli to multiply and produce toxin. The toxin, as is well known, is absorbed from the blood by the motor nerve endings and reaches the central nervous system by means of the motor nerves. The greater part of the latent period between the occurrence of the wound and the onset of tetanus is due to the time occupied by the toxin travelling up the nerves to the cells of the central nervous system. Once the toxin has entered the nervous system it is very inaccessible to antitoxin circulating in the blood. To prevent tetanus, therefore, the blood must contain sufficient antitoxin to neutralize the toxin before it is absorbed by the nerve endings. This urgency makes it imperative to use passive immunity with antitoxin for immediate protection. Ramon and Zoeller, however, have shown that active immuniza- tion with anatoxin can be combined with the passive immuniza- tion. The injection of a prophylactic dose of antitoxin does not prevent the development of active immunization. They suggest the following procedure in such a case : on the first day 10 c.cm. of’ antitoxin is injected and 1 c.cm. anatoxine, on the tenth day another 10 c.em. of antitoxin is given, and on the 20th and 30th days 2 c.cm. of anatoxine. The anatoxin must not be injected REFERENCES 441 into the same site as the antitoxic serum. In this way the patient is passively immunized for 20 days and after that time active immunity develops and he remains immune for a long period, thus preventing any danger of late tetanus occurring. REFERENCES NATTAU LARRIER, L., Rayon, G., and Grasset, EK. 1927. Ann. Inst. Past., 41, 848. RaMoN, G., and ZorLLer, C. 1927. Ann. Inst. Past., 41, 803. RAMON, G., and Descomsry, P. 1927. Ann. Inst. Past., 41, 834. CHAPTER XXV PREVENTION OF CERTAIN ANIMAL DISEASES BY THE ADMINISTRATION OF FORMOL CULTURES Lamb Dysentery Tis is a disease which has been very disastrous to new-born lambs in certain parts of Britain, especially the Border country. The incidence of the disease naturally varies from year to year, but on infected farms it averages about 10 to 20 per cent. of the total number of lambs born, although sometimes it may rise to over 50 per cent. The infection is very deadly and the case mortality approaches 100 per cent. Since 1921 Dalling and his co-workers have been attempting to prevent the disease by immunological methods. After investiga- tion of various strains of B. coli which were at one time thought to be of importance, Dalling succeeded in producing with cultures of a type of B. welchii the characteristic clinical syndrome in new-born lambs born of non-immune mothers. There are slight differences between the standard type of B. welchii and that isolated from lamb dysentery, which Dalling calls B. welchii (type agni),” the most important being that whereas lamb dysen- tery bacillus antitoxin will completely neutralize standard B. welchii toxin or culture, the converse does not hold good. On the assumption that lamb dysentery is due to an infection by this type of B. welchii, Dalling and his colleagues attacked the disease in two ways :— 1. By passive immunization of the new-born lambs with antitoxic serum. 2. By active immunization of the mothers with toxin-antitoxin mixtures or with *toxoided > culture of the lamb dysentery bacillus. 442 LAMB DYSENTERY 443 Passive immunity. This is dealt with in the section on serum therapy. Suffice it to say here that the injection of new-born lambs with antitoxic serum has proved extremely successful in that it has protected almost 100 per cent. Antitoxic serum, however, is expensive, and the inoculation of new-born lambs is troublesome on the northern hills where the lambs may be born far from the fold, so that active immunization of the mothers was tried. Active immunization. At first toxin-antitoxin mixtures were used as in diphtheria prophylaxis. These were given to the mothers shortly before parturition with excellent results. Field experiments were carried out in which some of the ewes on infected farms were inoculated with the toxin-antitoxin mixtures, and others were left uninoculated as controls. It was found that the mortality among the lambs born to inoculated ewes was greatly reduced as compared with the control lambs. Dalling, Mason and Gordon (1928) have published figures showing that by this pro- cedure the mortality was reduced from over 14 per cent. to less than 1 per cent. More recently Dalling and his co-workers have abandoned the toxin-antitoxin mixtures (just as similar mixtures have been given up for diphtheria prophylaxis), and they have used formolized cultures of the lamb dysentery bacillus as their vaccine. Cultures of the bacillus are grown on meat broth. They are filtered through a sand filter to remove the meat, and formalin is added to the filtrate to make a concentration of 0-3 per cent. The formolized cultures are kept at 37° C. until they are detoxicated to such an extent that a dose of 0-5 c.c. just fails to kill a mouse. The anti- genic strength of the formolized culture is tested by its power to immunize guinea pigs against multiple lethal doses of the specific toxin. This method of preparing vaccines from toxic cultures has already been made use of in connection with B. chauver (Lopez y Lopez, 1926, Cordier, 1926, McEwen, 1926, etc.). Injection of the mothers with this formol culture a few weeks before parturition gave results practically as good as those obtained with the toxin- antitoxin mixtures. The following figures are taken from Dalling, Mason and Gordon (1928) :— 444 PREVENTION OF CERTAIN ANIMAL DISEASES 1,224 lambs (mothers inoculated with formol culture), 13 {=1-06 per cent.) died. 1.215 lambs (mothers inoculated with toxin-antitoxin), 10 (= 0-82 per cent.) died. 1,333 lambs (mothers not inoculated), 191 (= 14-33 per cent.) died. These figures clearly show the great value of active immunization with ** formol culture >> in lamb dysentery, and as a result of this procedure the disease has almost disappeared in districts where it was once a scourge. The first injection of vaccine is given in the autumn and the second in the spring, a week or two before parturition. The Immunity conferred on the ewe by this active immunization is a durable one. It is essential to secure a high concentration of antitoxin in the ewe’s blood just before lambing, and Dalling has shown that when the blood of the ewe contains antitoxin there is a very high concentration of this in the colostrum which the lamb sucks immediately after birth, and which it is capable of absorbing in the first few days after birth. The immunity in the lamb lasts for about a week (Dalling, Mason and Gordon, 1927), but this is sufficient to prevent the disease. These observations on the antitoxic immunity of the new-born lamb are to be contrasted with the findings in human diphtheria and tetanus. In the sheep antitoxin does not pass through the placenta from the blood of the ewe to that of the lamb, but the lamb has the power of absorbing antitoxin from the alimentary tract during the first days of life. Exactly the opposite holds in man. As stated above, if ewes are injected in the autumn and again just prior to lambing, a very high degree of protection is conferred on the lambs. Further observations have shown (Dalling, personal communication) that if the same ewes are allowed to lamb in the following year without receiving further vaccine treatment the antitoxin content of their blood has dropped considerably, and so far as can yet be judged from the data the protection conferred on the lambs is much less than before. The ewes, however, by the previous inoculations, have received the * primary stimulus (see p- 424), and a single dose of vaccine administered in the spring BRAXY 445 just before lambing is sufficient to cause a rapid rise in the anti- toxin content, and the lambs are protected. A number of farmers in the north have now made it a routine practice that in the first instance the ewe receives one dose of vaccine in the autumn and another in the spring, but in subsequent years they only give one dose, in the spring just before lambing. Braxy This is a disease of young sheep which is common in Scandinavia and certain parts of North Britain, and causes considerable losses in the autumn and winter. In 1888 Nielsen described a bacillus which he considered to be the cause of braxy. In 1896 Jensen confirmed this work, and, 1922, Gaiger identified the anaerobe found in the tissues of sheep killed when affected with braxy as Vibrion septique. He showed that he could protect animals by inoculation with Vibrion septique toxin. It was later shown by several observers that by inoculation with partially neutralized toxin a very fair measure of protection could be obtained. Bosworth, in a field experiment, showed that only some 2 per cent. of inoculated animals died as compared with 7-7 per cent. in the uninoculated sheep on the same farms. Dalling, Mason and Allen obtained similar results. Of recent years the toxin-antitoxin mixture has been replaced by Vibrion septique formol-culture prepared in the same way as that used in lamb dysentery. Stewart (1929) has recorded results obtained with this formol culture. Of 1,222 sheep inoculated with one dose of formol culture, 17 (or 1-4 per cent.) died from braxy, whereas among 456 uninoculated animals on the same farms, 43 (or 9-4 per cent.) died of the disease. Here, as in lamb dysentery. there is a very striking reduction in the mortality as a result of the use of formol culture. One of the most important things in connection with this work on lamb dysentery and braxy is the great success which has followed active immunization with formol vaccines which contain not only the dead bacterial bodies, but also toxins transformed into toxoids. It seems likely that this method of preparing 446 ~~ PREVENTION OF CERTAIN ANIMAL DISEASES vaccines will in future be used more largely in human medicine than it has been in the past. REFERENCES Lamb Dysentery Corpier, G. 1926. C. R. Soc. Biol., 95, 848. DavriNg, T. 1926. Jour. Comp. Path. and Ther., 39, 148. » 1928. Ann. Cong. Nat. Vet. Med. Assn. of G. B. and 1I., p. 55. DarniNg, T., Mason, J. H., and GorponN, W.S. 1927. Vel Record, 40, 217. 5 » o 1928. Vet. Jour., 84, 640. McEwEeN, A. D. 1926. Jour. Comp. Path. and Ther., 39, 253. Braxy Daring, T., Mason, J. H., and ALLEN, H. R. 1925. Vet. Record, 5, 501, ” 1926. Vet. Jour., 82, 406. ' on 33 1929. Vet. Record, 9. 903. GalGer, S. H. 1922. Jour. Comp. Path. and Ther., 35, 191, 285. Stewart, W. L, 1929. Vet. Jour., 85, 400, INDEX OF Abdominal conditions, treatment of, 55 Acne vulgaris, bacteriology of, 365 vaccine treatment of, 367 dosage, 368 Adrenaline, intracardiac injections of, in serum accidents, 13 solution of, use in serum accidents, 12 African horse sickness, prophylaxis by vaccines, 230 treatment by serum, 230 Agglutinin content of anti- meningococcus serum, 136 Agglutinins, flagellar and somatic, 261 Alimentary disturbance, association of asthma with, 404, 405 Alkalies and acids, detoxication of vaccines by, 245 Allergic shock, risk of, 8, 10, 120 Allergy, 393 in serum treatment, 8, 10, 120 Ana-cultures (formolized vaccines), 247 in treatment of Shiga type of dysentery, 284 Anaerobic infections, treatment of, 60 puerperal infections, serum treatment of, 58 Anaphylaxis in serum treatment, 8 Anatoxine, 246, 422 of Ramon, use of, 2 tetanus, preparation of, 439 Angio-neurotic cedema, 393 Animal diseases, prevention of, by administration of formol cultures, 442 Animals, immunization of, against paratyphoid infections, 281 practical immunization of, 281 Anthrax in animals, prophylaxis by vaccines, 224 treatment by sera, 152, 224 Anti-anthrax serum for human use, 152 preparation of, 154 results of treatment, 154 Sclavo’s, 1563 titration of, 155 acute, serum various, serum SUBJECTS Antibacterial serum, local application of, in treatment of diphtheria, 33 preparation of, 4 use of, in diphtheria, 33 Antibodies following oral administration of vaccines, 270 of pneumococcus vaccine, 300, 308 4 Anti-cholera immunization, 290 dosage of vaccine, 290 results obtained, 291 serum, 162 Anti-cold vaccines, 324 Anti-dysentery inoculation, type, 286 Shiga type, 283 serum, ‘¢ Flexner,” 92 multivalent, in treatment of ulcerative colitis, 97 ¢«¢ Shiga,” 94 method of titration, 94 use of, general considerations on, 91 Flexner Anti-influenza vaccine, 329 Anti-meningococcus serum, 130 agglutinin content of, 136 antitoxic component in, 138 concentration of, 140 desiccation of, 138 dosage of, 143 mode of action of, 142 modes of administration, combined routes, 143 intracisternal route, 144 intrathecal route, 143 intraventricular route, 146 occasional inefficiency of, analysis of reasons for, 131 preparation of, 139 in inter-epidemic periods, 137 reinforcement of, by fresh human serum, 141 titration of, 136 Shwartzman’s method, 141 See also Cerebrospinal fever. 447 448 INDEX OF Anti-plague inoculation, 293 effect of heat on immunizing power of vaccines, 294, 295 results of, 295 serum, 160 Anti-pneumococcus serum, British Provisional Unit for, 127 concentration of, 125 preparation of, 125 production of, 125 titration of, 126 precipitation test, 127 Anti-pneumococcus vaccine, 301 Anti-poliomyelitis serum, 181 mode of action of, 196 titration of, 195 Anti-staphylococcus serum, 83 experimental work, 84 producti and titration of, 85 therapeutic effects of, 86 Anti-streptococcus serum, multivalent, Rosenow’s, use of, in treatment of poliomyelitis, 196 Antitoxic component in anti- meningococcus serum, 138 sera, preparation of, 2 Antitoxin, normal, horses, 3 use of, in diphtheritic paralysis, 32 Antitryptic power of blood, influence of vaccines on, 408, 409 Anti-typhoid inoculation, 273 duration of immunity after, 276 results of, 275 serum, 157 vaccine, preparation of, 273 importance of choosing suitable strain of typhoid bacillus for, 275 which will not interfere with subsequent diagnosis of typhoid fever, possibility of using, 274 Antivenins for bite of spiders, 110 for scorpion sting, 109 for snake-bite, 102 preparation of, mode of, 106 titration of, 106 value of, statistical evidence of, 109 Antiviral sera, preparation of, 5 human, in treatment of poliomyelitis, 181. See also under Immune serum. Antiviruses, 251 Appendicitis, serum treatment of, 57 SUBJECTS Arthritis deformans, 350 gonorrheeal, vaccines in treatment of, 390 non-specific, infective, 350 rheumatic, bacteriology of, 351 blood cultures in cases of, 352 cultures from joints in cases of, 351 cultures from lymphatic glands in cases of, 351 Asthma, 393, 402 association with alimentary disturbance, 404, 405 importance of bronchial infections in, 407 multiple sensitizations in, 407 serum reactions associated with, 8, 10, 11 vaccine treatment in, 402, 403 dosage, 406 preparation of vaccines for, 405, 406 Asylum dysentery, vaccine treatment of, 286 Atopy, 8 Autogenous and stock vaccines, 252 vaccines, preparation of, 252 Autolysates, use of, as vaccines, 250 Avidity of diphtheria antitoxin, 28 Bacillary dysentery, vaccine treatment of, 283, 287 B. coli infections, 376 of genito-urinary, frequency of, vaccines, prophylactic therapeutic, 377 B. pertussis, antigenic structure of, 334 Bacteria found in sputum in asthma, 403 solution of, by means of a bacteriophage, 251 specific affinities of, 268 Bacterieemia in pneumonia, serum treatment of, 115, 117, 119, 124 meningococcal, chronic, serum treatment of, 148 Bacterial enzyme, use of, in treatment of infections of pneumococci (Type 111.), 306 extracts, filtrates and digests, use of, as vaccines, 249 flora in common cold, changes in, 315 vaccines, intravenous injection of, reaction to, 414 and INDEX OF SUBJECTS 449 Bacterial vaccines, response to, in non- | Cerebrospinal fever, fulminating cases, immune and partially immune animals, 425, 426 variation in regard to immunity, 258 “Hand “ O > variation, 261 “R” and *“S” variation, 258, 259 to tests of immunity after use of vaccines, 265 Bactericidal power of blood, enhanced by vaccines, 408, 410 Bacteriophage, solution of bacteria by means of, 251, 293 therapy in prevention and treatment of dysentery, 289 - Bargen’s serum in treatment of ulcerative colitis, 97, 380 Biological products, veterinary, standardization of, 238 Blackleg, prophylaxis by vaccines, 228 treatment by serum, 228 Blood, anti-tryptic power of, influence of vaccines on, 408, 409 cultures in rheumatic arthritis, 352 Bordet’s bacillus in whooping cough, 334 Botulism, serum treatment of, 99 Bovine pleuro-pneumonia, prophylaxis by vaccines, 228 Braxy, prevention of, by cultures, 445 prophylaxis by vaccines, 236 Bronchial infections, importance of, in asthma, 407 Bronchitis, chronic, vaccine treatment of, 325 Bubonic plague, 160, 293, 294, 295 formol Calcium chloride, use of, in preparation of sera, 3 Calmette’s anti-viperine serum, 104 Canine jaundice, prophylaxis by vaccines, 234 treatment by serum, 234 Catarrh, respiratory, prophylactic vaccines against, composition of, 323 Catarrhal respiratory infections and common cold, prevention of, 314 Cattle, hmmorrhagic septicemia of, prophylaxis by vaccines, 223 treatment by serum, 220 plague, prophylaxis by vaccines, 220 treatment by serum, 220 R.A. VACCINES. serum in, 142 in infants, serum treatment of, 146 mode of = action of anti- meningococcus serum in, 142 preparation of serum in inter- epidemic periods, 137 serum treatment of, 130 importance of antigenic quality of immunizing strains, 136 preparation of serum for, 135 variable results of, 134 treatment of, choice of multivalent sera or monotypical sera, 139 spinal drainage in, 133 See also Anti-meningococcus serum. Chemical substances, vaccines killed by, 248 Chicken-pox, serum treatment of, 206 Chill-producing substance in serum, 14 Cholera, fowl, prophylaxis by vaccines, 237 treatment by sera, 237 in man, serum treatment of, 162 antibacterial component in, 163 antitoxic component in, 164 toxin of, V. cholercee, 163 Cold, common, and other catarrhal respiratory infections, prevention of, 314 bacterial flora in, changes in, 315 bacteriology of, 314 division of public into three classes in regard to, 319 filter-passing virus in relation to, 314 immunity to, 317 in hospital practice, results obtained ~~ with prophylactic vaccines, 323 mixed vaccines in, prevention of, 317 season of year for inoculations, 322 Shope’s work on swine influenza in relation to, 320 treatment by vaccines, 324 variability of, 317 ‘¢ Colds *’ associated with pneumococcal infection, vaccine treatment of, 312, 313 Colitis, ulcerative, 380 serum treatment of, 97 treatment with Bargen’s serum, 97 15 450 INDEX OF Colitis, ulcerative, treatment with multivalent anti-dysentery serum, 97 vaccines in treatment of, 380, 381 Common cold and other catarrhal respiratory infections, prevention of, 314. See also under ** Colds,” + Concentration of sera, 140 Conjunctival and intracutaneous tests in serum treatment, 11 Convalescent serum in poliomyelitis, 181 measles serum, organization of supplies of, 169 preparation and storage of, 168 prophylactic value of, 166 use of, in treatment of scarlet fever, 73 Culturai methods of choosing infecting organism in preparation of vaccines, 254 treatment of Defatted vaccines, 247 Dengue fever, serum treatment of, 211 Desensitization, methods of, to avoid serum accidents, 12 Detoxication of vaccines by alkalies and acids, 245 Diaplytes (defatted vaccines), 247 Diarrhea, chronic, vaccines in treatment of, 378 Diphtheria, active immunization against, 420 by mixtures of toxin and antitoxin, 421, 434 by toxoid, 435 procedure in, 431 results of, 434 antitoxin, avidity of, 28 prophylactic use of, 34 bacillus, association of streptococci with, 19 C. diphtherice gravis in, 22 C. diphtheriee mitis in, 22 immunization with toxoid (anatoxine), results of, 435 intraperitoneal method of administration of antitoxin in, 30 malignant, character of strains from, 20 insulin treatment in, 24 intensive treatment of, 24 serum treatment of, 18 use of streptococcus antitoxin, in, 19 SUBJECTS Diphtheria, modes of administration of antitoxin in, 30 prophylactic, different forms of, 428 toxin-antitoxin floccules as, 429 toxin-antitoxin mixtures, 428 toxoid-antitoxin floccules as, 429 toxoid-antitoxin mixtures as, 428 toxoid (anatoxine) as, 429 Schick test as guide to immunity, 420 serum treatment of, scales of dosage in, 26 time of administration of serum in, 31 toxin, production of, 4 “ toxoiding 77 of, 422, 423 toxoid inoculation against, age for, 436 use of antibacterial serum in, 33 Diphtheritic paralysis, antitoxin in, 32 Distemper, dog, prophylaxis by vaccines, 232 treatment by sera, 232 Dosage in desensitization with pollen extracts in hay fever, 398, 399 in vaccine treatment of acne vulgaris, 368 of chronic rheumatic conditions, 356 of furunculosis, 372 of whooping cough, 341 of anti-cholera vaccine, 290 of anti-meningococcus serum, 143 of convalescent serum in prophylaxis of measles, 167 of gonococcal vaccines, 385 of normal adult human serum in prophylaxis of measles, 171 of pneumococcal vaccine for treatment, 311 of T.A.B. vaccine, 282 of vaccines for producing protein shock, 415 . in asthma, 406 Dysentery, asylum. vaccine treatment of, 286 bacillary, vaccine treatment of, 283, 287 bacteriophage therapy in prevention and treatment of, 289 diagnosis of, value of cytological method in, 90 Flexner-group infections, treatment of, 92 therapeutic effects of, 93 serum INDEX OF Dysentery, lamb, passive immunity in, 443 prevention of, by active immunization, 443 prophylaxis by vaccines, 235 treatment by sera, 235 serum treatment of, 88 Shiga” infections, serum treatment of, therapeutic effects of, 95 treatment of, importance of early diagnosis in, 88 vaccine prophylaxis, 283 vaccine treatment, 288 Encephalitis, post-vaccinal, treatment of, 207 Enzyme, bacterial, use of, in treatment of infections of pneumococci (Type I11.), 306 Epidemics, of measles, control of, by human immune-serum, 173 Epididymitis, gonorrheeal, vaccines in treatment of, 391 Erysipelas, serum treatment of, 75 swine, prophylaxis by serum, 220 treatment by sera, 220 European vipers, antivenin for, 103 serum Febrile reactions after intravenous injections of serum, 13 Fibrositis, 350 Filter-passing virus in relation to the common cold, 314 to influenza, 328 virus diseases, vaccine and serum treatment of, 200 Flexner group infections, serum treatment of, 92 vaccine treatment of, 286 Foot-and-Mouth disease, by vaccines, 224 treatment by sera, 224 Formalin, toxoiding of toxin with, 423 Formol cultures, administration of, prevention of certain animal diseases by, 442 Formolized toxins, use of, 2 in diphtheria, 422 in dysentery, 284 in furunculosis, 373 in tetanus, 439 vaccines, 246 Fowl cholera, prophylaxis by vaccines, 237 prophylaxis diphtheria SUBJECTS 451 Fowl cholera, treatment by sera, 237 pox, prophylaxis by vaccines, 237 treatment by sera, 237 Furunculosis, bacteriology of, 370 treatment of, by staphylococcal “toxoid,” 373 vaccine treatment of, 371 dosage, 372 Gas-gangrene antitoxin, and titration of, 61 bacteriology of, 51 serum treatment of, 51 during late war, 52 in civil practice, 54 Genito-urinary tract, B. coli infections of, frequency of, 376 Glenny’s method in antitoxic sera, 3 Goats, anti-poliomyelitis serum from, 193 Gonococcal infections, 383 preparation of vaccines for, 383 vaccine therapy in, summary of, 391 vaccines, dosage of, 385 results obtained with, 387 Gonorrhea, complications of, protein shock therapy in. 417 Gonorrheeal arthritis, treatment of, 390 epididymitis, vaccines in treatment of, 391 ophthalmic complications, vaccines in treatment of, 391 Guinea-pigs, immunization of, 281 preparation production of vaccines in Hemorrhagic septicemia of cattle, prophylaxis by vaccines, 22: treatment by sera, 22: of sheep, prophylaxis by vaccines, 236 treatment by sera, 236 Haffkine’s vaccine, preparation of, 293 Hay-fever and other idiosyncrasies, 8, 393 hereditary element in, 393 desensitization in, 394 preparation of pollen extract, 394 pollen extracts in, 397 dosage and spacing of injections, 398, 399 with substances other than pollen, 401 hypersentiveness in, tests for, 395 156—2 452 INDEX OF Hay-fever, specificity of different grasses, 397 standardization of pollen extract, 395 ‘“H” and ‘‘ 0» variation, 261 importance of, in vaccine therapy, 262 Horse, anti-poliomyelitis serum from, 191 clinical experience with, 194 distribution of protective substances in, 193 ‘¢ Horse-asthmatics,”” 10 Horse sickness, African, prophylaxis by vaccines, 230 treatment by sera, 230 Horses, immunization of, by B. abortus equa, 281 Human immune-serum (measles), con- trol of epidemics by, 173 prophylactic value of, 170, 172 Human serum in treatment of polio- myelitis, 181 experimental! foundations for use of, 181 normal, value of, in treatment of poliomyelitis, 184 Huntoon’s antibody solution in treatment of pneumonia, 115 Hypersensitiveness in hay-fever, tests for, 395 pollen of Immune-serum, from horse, prophylaxis by, in poliomyelitis, 190 human, in treatment of poliomyelitis, 181 critical review of, 186 curative dose and route of administration, 185 general conclusions on, 188 preparation, storage and organization of supplies, 188 prophylaxis by, in poliomyelitis, 190 Immunity, bacterial variation in regard to, 258 duration of, after anti-typhoid inoculation, 276 in diphtheria, development of, 421 rate of appearance of, after pneumococcus vaccines, 300 restoration with minute dose of vaccine, 426 to pneumococcus, species specific, 321 type specific, 299 SUBJECTS Immunity to virus of common cold, 317 Immunization, local, 268 Immuno-transfusion, 410 Infants, cerebrospinal fever in, serum treatment of, 146 Influenza and its complications, vaccines for prevention of, 329 results obtained, 330, 331 bacilli present in health and in disease, distinction between, 316 bacteriology of, 327 preparation of vaccine against, 332 virus, 328 Insulin treatment in diphtheria, 24 Intestinal infection, association of asthma with, 405 obstruction, serum treatment of, 55 tox@mia, vaccines in treatment of, 378 tract, infections of, vaccines in treatment of, 378 Intestine, streptococcal flora in, significance of, in relation to acute rheumatic conditions, 354 Intracardiac injections of adrenaline in serum accidents, 13 Intracutaneous and conjunctival tests in serum treatment, 11 Intrathecal injections of anti- poliomyelitis horse serum, reactions from, 194 Intravenous administration in intensive treatment of malignant diphtheria, 24, 25 injection of bacterial vaccines, reaction to, 414 of scarlet fever antitoxin, 69 inoculation of typhoid vaccine in man, 276 of vaccines fever, 348 vaccination, non-specific (protein shock), 412 in acute rheumatic Jaundice, canine, prophylaxis by vaccines, 234 treatment by sera, 234 Joints, cultures from, in rheumatic arthritis, 351 Lamb dysentery, passive immunity in, 235, 443 prevention of, by active immunization, 443 INDEX OF Lamb dysentery, prophylaxis by vaccines, 235 Leucocytosis due to infection of vaccines, 409 Lipo-vaccines, 248 Lobar pneumonia, 298 in man, prophylactic against, 301 response to vaccine, 300, 308 serum treatment of, 113 vaccine treatment of, 306 dosage, 311 results obtained, 30% Local immunization, 268 Lymphatic glands, cultures rheumatic arthritis, 351 inoculation from, in Manganese chloride, use of, in production of sera, 3 Mares, before or during immunization of, 281 Measles, convalescent serum in, conferment of full protection by, 166 dosage, 167 duration ofl mmunity conferred by, 168 mitigation of attack by, 167 preparation and storage of serum, 168 prophylactic value of, 166 use as diagnostic agent, 168 pregnancy, epidemics of, control by human immune-serum, 173 human immune-serum in, curative value of, 174 prophylactic value of normal adult human serum in, 170, 172 dosage, 171 prophylaxis of, by serum, 165 = sera obtained from animals, use of, 174 Meninges, toxic effect on, of Rosenow’s antistreptoccus serum, 196 Meningococcal bacterieemia, serum treatment of, 148 Migraine, 393 Monotypical sera in treatment of cerebrospinal fever, 139 Mouth, administration of vaccines per, in man, 270 Multivalent anti-streptococcus serum, 78 sera in treatment of cerebrospinal fever, 139 chronic, SUBJECTS 453 Mumps, serum treatment of, 208 Mycolysate, 249 Neurological complications of serum treatment, 14 Non-specific intravenous (protein shock), 412 vaccine therapy, 408 vaccination Official control of sera, 6 Ophthalmic complications of gonorrhoea, vaccines in treatment of, 391 Oral administration of anti-dysentery vaccine, 288 T.A.B. vaccine, 271 vaccines in man, 270 versus subcutaneous administration of vaccines, 271, 272 Organization of supplies of antiviral serum for measles, 169 for poliomyelitis, 188 Osteo-arthritis, 350 human Paratyphoid infections, immunization against, 280 inoculation in man, 281 Perineuritis, 350 Peritonitis, serum treatment of, 55 Pettit’s anti-poliomyelitis horse serum, 193 Plague bacillus, antigens of, effect of heat on, 294, 295 cattle, prophylaxis by vaccines, 220 treatment by sera, 220 in man, serum treatment of, 160 Pleuro-pneumonia, bovine, prophylaxis by vaccines, 228 Pneumococcal infections, minor, vaccine treatment of, 311 vaccine treatment of, 308 Pneumococei (Type III.) infections of, use of a bacterial enzyme in treatment of, 306 Pneumococcus, species specific vaccine, 321 typing of, technique of, 122 vaccine in lobar pneumonia, type- specific, 299 use of, in relation to predisposition to pneumonia, 304 vaccines, rate of appearance of immunity after, 300 route of administration, 299 454 INDEX OF Pneumonia, bacteriology of, changes in, 304, 305 fatality-rate of, influence of serum treatment upon, 118 lobar, 298 in man, prophylactic inoculation against, 301 serum treatment of, 113 vaccine treatment of, 308 dosage, 311 results obtained, 309 rate before and after inoculation, comparison of, 302 et seq. serum treatment of, dosage, 121 mode of action of serum in, 119 of administration, 120 therapeutic effects produced by, 16 technique of typing pneumococcus, 122 treatment of, Huntoon’s antibody solution in, 115 serum preparations used in, 115 Type I, serum treatment of, 114 Type I1., serum treatment of, 124 Poliomyelitis, anterior, acute, antiviral serum from horse, clinical experiences with, 194 general account of, 177 human serum in treatment of 18 critical review of, 186 curative dose and route of administration, 185 experimental foundations for use of, 181 general conclusions, 188 preparation, storage and organization of supplies, 188 prophylaxis by antiviral serum from man or from horse, 190 serum treatment of, 177 importance of early diagnosis in, 179 use of Rosenow’s antistreptococcus treatment of, 196 value of normal human serum in treatment of, 184 Pollen extracts, desensitization with, in hay-fever, 397 dosage and spacing of infections, 398, 399 serum in SUBJECTS Pollen extracts, preparation of, in desensitization in hay-fever, 394 standardization of, 395 of different grasses, specificity of, in hay-fever, 397 Post-vaccinal encephalitis, treatment of, 207 Preparation of anti-pneumococcus serum, 125 of antivenins, 106 of gas-gangrene antitoxin, 61 of human antiviral serum poliomyelitis, 188 of scarlet fever antitoxin, 72 of tetanus antitoxin, 48 Primary stimulus, 424 Production of anti-staphylococcus serum, 85 Prophylactic use of antitoxin, 34 of scarlet fever antitoxin, 71 value of tetanus antitoxin, 38 Protein shock, 412 choice of vaccine for, 413 clinical applications of, 415 dangers of and contraindications to, 417, 418 induction of, by intravenous injection of typhoid vaccine, 277 reaction to intravenous injection of bacterial vaccines, serum for diphtheria 414 vaccines for producing, dosage of, 415 Psittacosis, serum treatment of, 209 Puerperal fever, serum treatment of, 77 treatment of, by scarlet fever antitoxin, 74 infections, anaerobic, serum “treatment of, 58 Pyo-sero-culture, Wright's method of, 254 Rabies, acute immunization against, 201 Ramon and Zoeller method of prophylaxis with tetanus antitoxin, 40 Rashes, recurring, in serum treatment, 10 ‘“ Rawlings >> strains of B. typhosus, unsuitability for preparation of vaccines, 261, 275 INDEX OF Respiratory catarrhs, prophylactic vaccines against, composition of, 323 results obtained in hospital practice, 323 infections, catarrhal, and common cold, prevention of, 314 Rheumatic conditions, chronic, 350 septic foci in connection with, 352 vaccine treatment of, 355 dosage, 356 results of, 359 type of vaccine employed, 355 diseases, protein shock therapy in, 416 fever, acute, 343 bacteriology of, 343 haemolytic streptococci in, 345 intravenous inoculation of vaccines in, 348 nature of infection, 343 non-hemolytic streptococci in, 344 vaccine treatment with haemolytic streptococci, 346 Rheumatoid arthritis, 350 Rigor-producing substance in serum, 14 Rocky mountain fever, immunization against, 201 Rosenow’s antistreptococcus serum, use of, in treatment of poliomyelitis, 196 “RR » and ‘¢8 *’ variation in cultures, 258, 259 Salmonella group of bacilli, immunization against, 280, 281 Scarlet-fever and other streptococcal infections, serum treatment of, 65 antitoxin as prophylactic agent, 71 intravenous injection of, 69 preparation and titration of, 72 therapeutic effects of, 67 use of, in various streptococcal infections, 73 streptococcus, relationship other streptococci, 73 treatment of, by convalescent serum, 7: Schick test as guide to immunity in diphtheria, 420 Sclavo’s serum, 153 dosage of, 156 Scorpions, sting of, sera for, 109 Secondary stimulus, 424 of, to SUBJECTS 455 Sensitiveness, serum, mechanism of, 13 Sensitized vaccines, 245 Septic foci in connection with chronic rheumatic conditions, 352 Septiceemia, haemorrhagic, of cattle, prophylaxis by vaccines, 223 treatment by sera, 223 of sheep, prophylaxis by vaccines, 294 treatment by sera, 236 Sera and vaccines in veterinary practice, 215, 442 antibacterial, preparation of, 4 antitoxic, preparation of, 2 antiviral, preparation of, 5 clinical value of, statistical evidence of, 7 concentration of, 5 official control of, 6 preparation of, 2 titration of, 6 Serologicalmethods of choosinginfecting organism in preparation of vaccines, 256 Serum accidents, 10 conjunctival and intracutancous tests, 11 method of desensitization to avoid, 12 precautionary measures against, 11 Serum, anti-cholera, 162 anti-staphylococcus, 83 disease, 8 for bite of spiders, 110 for sting of scorpions, 109 from animals, use of, in treatment of measles, 174 human, normal adult, prophylactic value of, in measles, 170 intravenous injections of, febrile reactions after, 13 prophylaxis of measles, 165 reactions in treatment of lobar pneumonia, 120 sensitiveness, mechanism of, 13 sickness, incidence of, 8 treatment, complications of, 8 neurological complications of, 14 of acute abdominal conditions, 55 of acute anterior poliomyelitis, 177 of anaerobic infections, 60 of anthrax, 152 of B.-dysenterice (Shiga) infections, 94 (Sonne) infections, 96 456 INDEX OF Serum, treatment of botulism, 99 of cerebrospinal fever, 130 of chicken-pox, 206 of dengue fever, 211 of dysentery, 88 of erysipelas, 75 of Flexner-group infections, 92 of gas gangrene, 51 of lobar pneumonia, 113 of malignant diphtheria, 18 of mumps, 208 of plague, 160 of post-vaccinal encephalitis, 207 of psittacosis, 209 of puerperal fever, 77 of scarlet-fever and other streptococcal infections, 65 of Type I. pneumonia, 114 of Type 11. pneumonia, 124 of typhoid, 157 of typhus fever, 210 of ulcerative colitis, 97 of virus diseases, 206 of yellow fever, 210 specific, for snake-bite, sting of scorpions and bite of spiders, 102 mode of administration, 107 Sheep, anti-poliomyelitis serum from, 193 hemorrhagic septicemia of, prophylaxis by vaccines, 236 treatment by sera, 236 Sheep pox, prophylaxis by vaccines, 2:36 treatment by sera, 236 Shiga infections, serum treatment of, 94 type of dysentery, anacultures (formolized vaccines) in treatment of, 284 vaccine treatment of, 283 Shope’s work on swine influenza in relation to the common cold, 320 Shwartzman’s method of titration of anti-meningococcus serum, 141 Sickness, serum, 8 Snake-bite, antivenins for, 102 general treatment of, 108 serum treatment of, mode of administration, 107 Snakes, poisonous, of Africa, 106 of Australia, 105 of North America, 105 of South America, 105 Sonne infections, serum treatment of, 96 Spiders, sera for bite of, 110 SUBJECTS Spinal drainage in treatment ot cerebrospinal fever, 133 Sputum in asthma, bacteria found in, 403 Statistical evidence of clinical value of sera, 7 Stock and autogenous vaccines, 252 vaccines, preparation of, 255 Storage of human antiviral serum for measles, 168 for poliomyelitis, 188 Staphylococcal infections in acne, 366 normal resistance to, 371 other than furunculosis, vaccines in treatment of, 375 serum treatment, 83 “toxoid,” treatment of furunculosis by, 373 Streptococcal flora in gut, significance of, in relation to chronic rheumatic conditions, 354 infections, various, use of scariet- fever antitoxin in, 73 Streptococci, association of, with diphtheria bacillus, 19 hemolytic, in rheumatic fever, 345 vaccine treatment of acute rheumatic fever with, 346 non-hemolytic, in acute rheumatic fever, 344 Streptococcus antitoxin, severe diphtheria, 19 Subcutaneous versus oral administration of vaccines, 271, 27% Swine erysipelas, prophylaxis by serum, 220 treatment by sera, 220 fever, prophylaxis by serum, 217 treatment by sera, 217 use of, in T.A.B. vaccine, inoculation of, in man, 281, 282 dosage, 282 oral administration of, 271 Tapioca, use of, in production of sera, 2 Tetanus, active immunization against, 439 anatoxine, preparation of, 439 antitoxin, preparation of, 48 methods of titration, 49 prophylactic value of, 38 therapeutic use of, 42 curative effects, 45, 46 mode of administration, 43 scheme of dosage, 42 INDEX OF SUBJECTS Tetanus, cephalic, treatment of, 48 combined, active and passive immunization, 440 delayed, treatment of, 47 early symptoms of, diagnosis of, 40 local, treatment of, 47 prophylaxis by vaccines, 228 recurrent, treatment of, 48 serum treatment of, 38, 220 special forms of, treatment of, 47 Thermal reactions after intravenous injections of serum, 13, 121 Titration of anti-anthrax serum, 155 of anti-dysentery serum (Shiga), 94 of anti-meningococcus serum, 136 Shwartzman’s method, 141 of anti-pneumococcus serum, 126 of anti-poliomyelitis serum, 195 of anti-staphylococcus serum, 85 of antivenins, 106 of gas-gangrene antitoxin, 61 of scarlet-fever antitoxin, 72 . of sera, 6 of tetanus antitoxin, 49 Toxsemia, intestinal, vaccines in treatment of, 378 Toxic-idiopathies, 393 desensitization with substances other than pollen, 401 Toxin-antitoxin floccules as diphtheria prophylactic, 429 Toxin-antitoxin mixtures as diphtheria prophylactic, 428 in active immunization diphtheria, 421, 422 primary and secondary stimuli, 424, 425 Toxin of V. cholere, 163 Toxins, formolized, use of, 2 Toxoid (anatoxine), 246, 422, 423 as diphtheria prophylactic, 429 inoculation against diphtheria, age for, 436 immunization against with, results of, 435 staphylococcal, treatment of furunculosis by, 373 use of, 2 Toxoid-antitoxin floccules in diphtheria prophylaxis, 429 mixtures as diphtheria prophylactic, 428 Trypaflavine in treatment of chronic meningococcal bacterizmia, 149 against diphtheria 457 Typhoid bacillus, suitable strain of, importance of choosing for preparation of anti-typhoid vaccine, 275 fever, serum treatment of, 58 treatment of, by vaccines, 278 vaccine, intravenous inoculation of, in man, 276 oral administration of, 270 Typhus fever, active immunization against, 202 serum treatment of, 210 Typing of pneumococcus, technique of, 122 Ulcerative colitis, 380 serum treatment of, 97 vaccines in treatment of, 380, 381. See also under Colitis. Urethritis, acute, vaccines in treatment of, 388 chronic, vaccines in treatment of, 389 Urticaria, 393 Vaccine and Vaccines, 247 and sera in veterinary practice, 215 anti-cholera, preparation of, 290 anti-plague, preparation of, 293 anti-typhoid, 273 preparation of, 273 autogenous and stock, 252 comparison of results with, 411, 412 by mouth, man, 270 dead, versus living attenuated vaccines, 244 detoxication by alkalies and acids, 245 for prevention of whooping cough, preparation of, 336 for protein shock, choice of, 413 dosage of, 415 formolized, 246 gonococcal, 383 dosage of, 385 large doses recommended in France, 386 results obtained with, 387 in B.-coli infections, prophylactic and therapeutic, 377 influence on bactericidal power of blood, 408, 410 administration of, to 458 INDEX Vaccine and Vaccines, injection of, effect on antitryptic power of blood, 408, 409 leucocytosis due to, 409 intravenous inoculation of, in acute rheumatic fever, 348 intravenous use of, 412, 413 killed by chemical substances, 248 mixed, in prevention of common cold, 317 in prevention of whooping cough, 336 oral wersus subcutaneous administration of, 271, 272 pneumococcal, species specific, 321 type-specific, 299 preparation of, 243 for treatment of asthma, 405, 406 methods of choosing infecting organism, 254 prophylactic, against respiratory catarrh, composition of, 323 reaction after injection of, 245 protective. ** R”’ variant useless as, 260 sensitized, 245 treatment, importance of *“ H” and “0,” variation in, 262 non-specific, 408 of acne vulgaris, 367 of acute rheumatic fever with haemolytic streptococci, 346 urethritis, 388 of asthma, 402, 403 dosage, 406 of chronic bronchitis, 325 of chronic diarrhea, 378 of chronic rheumatic conditions, 350, 3556 results of, 359 of chronic urethritis, 389 of common cold, 324 of furunculosis, 371 of gonococcal infections, summary of, 391 OF SUBJECTS treatment of arthritis, 390 epididymitis, 391 of infection of intestinal tract, 378 of lobar pneumonia, 308 of minor pneumococcal infections, 311 of ophthalmic gonorrhea, 391 of pneumococcal infections, 308 of staphylococcal infections other than furunculosis, 375 of typhoid fever, 278 of ulcerative colitis, 380, 381 of vulvo-vaginitis of children, 389 of whooping cough, 339 dosage, 341 Variola, immunization against, 201 Veterinary biological products, standardization of, 238 practice, vaccines and sera in, 215 Vibrio cholerae, 163 - serological types, 163 Villous arthritis, 350 Vipers, European, antivenin for, 103 Virus diseases, active immunization against, 200 serum treatment of, 206 Vulvo-vaginitis of children, vaccines in treatment of, 389 Vaccine, conorrheeal complications of Whooping cough, 334 prevention of, mixed vaccines for, 336 prophylactic inoculation results of, 337, 338 vaccine for prevention preparation of, 336 vaccines in treatment of, 339 dosage, 341 Wool-sorter’s disease, 152 for, of, Yellow fever, active immunization against, 204 serum treatment of, 210 INDEX OF AUTHORS ABDOUSH, 162, 163 Abramson, 186 Abt, 49 Achard, 248 Acton, 95, 288 ‘Adams, 60 Adamson, 284 Agulnik, 18, 21 Alessandrini, 97 Alexander, 231, 399 Alivisatos, 288 Allen, 14, 15, 238, 428 Allison, 52 Alstead, 2 Amaral, re. 107, 111 Amoss, 187, 195 Anderson, 22, 76, 169, 173 Andrewes, 47, 48, 92, 329 Appelbaum, 140, 148 Aragao, 205 Archer, 353 Arkwright, 139, 15: 210, 258, 259, 260, 2 Armstrong, 118, 12: 300, 308, 395 Aronson, 65 Arthus, 110 Aschoff, 53 Ashhurst, 43 Aubertin, 430, 431 Audebart, 386 Austin, 303, 304, 311 Avari, 203, 294 Avery, 113, 306, 307 Aycock, 179, 180, 182, 184 Ayer, 183 Aznar, 58 BACHER, 28, 106 Badger, 204 Baer, 351, 352 Bain, 12 Baird, 74 Baldridge, 13 Baldwin, 115, 119, 124 Balteano, 73 Banks, 12, 26, 29, 69, 70, 144 Banzhaf, 85, 140, 192, 193 Barach, 300. 308, 300, 411 Barbellion, 385 Barenberg, 171, 209 Bargen, 97, 98, 380, 381, 382 Barlow, 34 Barnett, 48 Barotte, 62 Barsky, 148 Batten, 179 Bauer, 49 Bawtree, 376 Bazgan, 61 Bedson, 209 Behring, 33 Bell, 21, 339 Beltrami, 154 Bengston, 62 Benjamin, 147 Benn, 24 Bennett, 145 Benson, 76, 78 Bensted, 275 Berg, 154 Berry, 209, 210 Besredka, 155, 11 268, 269, 278 Bessa, 287 26 Bie, 2 A Bieling, 85, 96, 131 Bigger, 85, 250 Birkhaug, 73, 75, 343, 344, 346 Bischoff, 284 Blackfan, 134, 144, 206 Blake, 114, 120, 299, 327 Blaxall, 201, 236 Bliss, Bloom, Blum, 172, Boak, 413 Boas, 388 Boehncke, 284, 237 Bogen, 111 Bokenham, 157 Bolton, 286 Bonallie, 287 Boots, 352 Bordet, 334, 336 Bosworth, 228, 235 Botzei, 149 Boudou, 430, 431 Bourdelles, 386 Bowen, 247, 285 Bower, 56, 57, 145 Brain, 144, 187 Branham, 135, 136, 137 Braun, 35 Bray, 13, 402, 404 Brazil, 106, 109, 111 Brebner, 191 Bridré, 236 Brocq-Rousseu, 155 Brodie, 185 Brody, 411 Bronfenbrenner, 100 Broom, 132 Brown, Sh pass 400, 401 Bruce, 38, 39, Bruck, oy ha Brutsaert, 135 Buchanan, 170 Buie, 98, 382 459 . 341 339 Bull, 299 Bullowa, 116, 117, 119, 124 Bunim, 142 Burgers, 284 Burn, 172 Burnell, 34 Burnet, 85, 189 Burt-White, 74, 75 Buttiaux, 380 CAFFEY, 146 Callender, 90 Calmette, 104, 105, 409 Calvin, 43 Cameron, 77 Canon, 19 Cantacuzene, 135 Capon, 74 Carlin, 136, 137 ( ArpgnLoY 328, 413 Carré, (‘astanec a, 203 Cecil, 114, 115,116, 117, 118, Ey 130, 121, 124, (ésari, 79 Chaillot, 281 Chantemesse, 157 Chapin, 35 Charlton, 66 Charteris, 310 Claiborn, 11 Clark, 56, 57, 85 Clausen, 348 Clawson, 356, 358, 360 Claxton, 357 Cleland, 211 Clements, 385 Clock, 394 Clow, Clowes, Cluver, 2 Coburn, 346 Coca, 8,10, 13 Cockshutt, 342 Cohen, 135, 141, 254 Cole, 116 (Colebrook, 75. 410, 41! Collier, 179, 180 Collins, 77 Collis, 346, 347, 348 Comber, 69 Cominotti, 226 Compton, 293 Congdon, 355, 361 (Connor, 339 Conroy, 206 460 Conseil, 166, 208, Cooper, 124 opeman, 169, 170 Sino Cotoni, 79 Courmont, Cowan, 118 Cowie, 72 Craddock, 62, 365 Craighead, 202 Creswell, 13 Crocker, 411 Crowe, 354, 357, 360 Cruickshank, 74, 118 Cravheilier, 334 Culver, 391, 417 Cummins, 38, 47 Cunningham, 202 Curasson, 223 Curson, 997 Cushing, 67, 68, 183, 180 210, 271 270 DALE, 9,13 Dalling, 234, 2 Dalsace, 411 Dauer, 30 Davey, 317 Davide, 178, 190 Davidson, 118 Daw, 270 Dawson, 352 Day, 321 Dean, 33, 44, 426 , 238, 246 160, 284, Dearden, 412, 413, 415 Debré, 33, 167, 168 Decourt, 210 Defoe, 74 Degkwitz, 171, 175 d’Herelle, 251 Deicher, 18, 20, Delarue, 34 de Lavergne, 200 de Schmidt, 293 de Schweinitz, 217 Dible, 327 Dick, 66, 68, 69, 73 Dimond, 384 Dochez, 66, 315, 317, 328 Doerr, 13 Dold, 23 Dolman, 374 Doorenbos, 163 Dopter, 134, 384 Dorset, 217, 218, 219 Douglas, 163, 247 5 21, 148 Du Bois, 186 Dubos, 306 Dudgeon, 97, 284, 289, 368 372, 376, 377, 378 Dudley, 421 Dulaney, 148 Dumas, 247, 285 Dunkin, 232, 234 Dunn, 17, 131, 133 Durand, 247, 985 Du Toit, 231 Duval, 331 Dyer, 204 INDEX OF EBAUGH, 145 Eguchi, 299 Eichhorn, 154, 238, 239 Eley, 76 Elkeles, 32 Elliott, 144 Enders, 136 Erber, 49 Escherich, 66 Eurich, 152, 153 Evler, 41, 42 Eyre, 318, 331, 383, 384 FAGGE, 57 Fairbrother, 178, 185, 193, 197, 290 Fairley, 105, 142 Falk, 2909 Fantus, 12, 144, 146 Farjot, 278 Feierabend, 21 Felix, 62, 135, 203, 210, 262, 263, 264, 265 Fellowes, 72 Felton, 6, 14, 116, 1 127, 233 Ferguson, 317 Ferran, 290 Ferris, 43 Ferry, 138, 174, 249, 250 Fildes, 46, 47, 48, 412, 413, 415 Findlay, 205, 206, 238, 275 25, 126, Finkelstein, 19 Finland, 117, 121 Fisher, 174, 209, 249, 250 Fitzgerald, 435 FitzSimons, 103 Flaum, 61 Fleming, 247, 366, 367, 409 Fletcher, 92 Flexner, 131, 133, 134, 139, 140, 181, 182, 186, 187, 188, 1900, 191, 192, 195, 197 316. 328, 365, Florentin, 209 Florey, 47 Flu, 251, 293, 297 Flueckiger, 238 Foetten, 262 Foix, 248 Forbes, 436 I orkner, 351° Foster, 133, 315 Fourie, 227 Fox, 145 Francis, 122 Fraser, 389 Frasey, 210, 211 Freedlander, 43 Freeman, 168, 172, 339, 303, 396, 397, 398, 399, 400, 401, 405, 406, 407 French, 310 Friedberger. . 24 Friedemann, 32, 134, 133, 148 Frosch, 224 Fuerth, 43 192, AUTHORS ({ABRIEL, 68 Gabritschewsky, 66 Gaessler, 78 raffky, 10 Gaiger, 235 Galeotti, 245 Gallagher, 357 Gardner, 88, 92, 336, 337 Graskell, 133 Gates, 328 Gatewood, 13 Gauthier, 287, 288 Gay, 276, 278 Gebhardt, 193 Geiger, 219 Gengou, 85 Gerlach, 218 Gibson, 284 Gilchrist, 365, 366 Ginsbourg, 49, 52, 101 Giiscard, 336 Githens, 108 Glenny, 3, 49, 246, 285, 422, 423, 424, 425, 428, 4% 29, 430 Glover, 346 Godfrey, 436 Goehle, 30 Goeppert, 133 Gohar, 162, 163 Goldberg, 43 Goldman, 145 Goodale, 394 Goodall, 27 Goodner, 126, 306 Gordon, 13, 15, 76, 132, 135, 136, 138, 130, 184, 206, 207, 208, 209 Gordon-Taylor, 60 Gory, 158 Gotschlich, 162 Gould, 60 Gow, 416 Graham, 95 Granata, 208 Grasset, 158, 439 Gratia, 153, 249, 372 Graves, 148 Gray, 346, 380, 381 Greenfield, 207 Greenwood, 7, 275, 291 Grenet, 34 Griffith, 112, 117, 34 93, 286, 335 119, 140, Grinnell, 260, 261, 266, (ross, 84, 85, 159 Groves, 315 Gunn, 167, 168, 169, 172, 173, 207, 209 Gunther, 85 171 HAAS, 172 Hadden, 15 Haffkine, 244, Hahn, 162, 276 Hamilton, ‘50 Hanger, 9, 11, 13 Happold, 22 Harispe, 154 Harmon, 184 Harries, 27, 31, 70, 72, 171 290, 291 INDEX OF AUTHORS Harris, 331 Harrison, 385, 388, 395 Hartley, 4, 9, 21, 72, 1 223, 429 Harvey, 162, 201, 244, 276, 426 o= 29, Haselbauer, 193, 194 Hiissler, 96 Haughwout, 90, 93 Havens, 21 Hayano, 335 Hecksher, 9, 25, 27 Hector, 24, 183 Heggs, 160, 296 Heidelberger, 113 Hekman, 207 Helsby, 209 Herrick, 143, 144 Herrman, 339 Hervey, 72 Herzog, 264, 281 Hess, 208, 339 Hetsch, 162 Hewlett, 99, 100, 152, 153 Hindle, 204, 205, 211 Hine, 139 Hirschfelder, 384 Hodgson, 153 Hoffstadt, 270 Hogan, 380, 381 Holmes, 224 Holtz, 148 Hooker, 13 Hopkins, 85, 423, 428, 429 Hortopan, 61 Horder, 250 Houston, 380 Howell, 281, 339 Howitt, 193 Hoyle, 316 Hughes, 24 Huntoon, 76, 92 Hurst, 97, 178, 181 Hutchison, 76, 92, 103, 107, 108, 109 Hutyra, 219 Hyman, 116 IBRAHIM, 260, 264 Ichikawa, 278, 415 Inman, 92 Irvine, 331 Ivens, 60 Iversen, 209 Iyengar, 265, 426 JACKSON, 108, 140, 146 James, 72 Janvier, 8 Jenkins, 48 Jenner, 243 Jensen, 230 Jepps, 92 Joannon, 168 Jobling, 140 Joe, 71, 72 Johnson, 118, 122 Jordan, 99, 318, 330 Jung, 293 KANAGARAYAR, 93 Kaplan, 73 Karsner, 13 Kauffmann, 14 Kaupe, 340, 342 Kauntze, 288 Kearney, 222, 223 Kellaway, 105, 106, 111 Kellogg, 186 Kelser, 154 Kennedy, 134 Ker, 131, 133 Kessel, 116 Kingsbury, 93, 169, 172 Kinsella, 78 Kirkbride, 72, 135 Kirkhope, 71 Kitchen, 205 Klein, 65, 384 Kleinschmidt, 9, 33, 35 Knott, 403 Knowles, 95, 288 Knox, 99 Kolle, 22, 162, 244, 290 Kolmer, 142 Konigsberger, 19 Koves, 219 Kramer, 184, 187 Kraus, 28, 29, 104, 106, 107, 108, 154, 159, 162, 415 Krause, 279 Kristensen, Krumbein, 1 Krumwiede, 123, 209, 334 Kruse, 314 Kudrjawzew, 155 ow 9 LADE, 287 Lafaille, 435 Laidlaw, 232, 234, 329 Laigret, 205 Lamb, 104 Lambert, 310 Lambkin, 385 Lamson, 10 Landsteiner, 163, 181 Lantin, 95, 96 Larrier, 439 Larsen, 115, 338 Larson, 54, 300 Lash, 73, 77 Leclainche, 228 Ledingham, 139, 189, 208, 251, 269, 409 Lees, 389, 390 Leishman, 273, 330 Leitner, 149, 340 Le Moignic, 248 Lempriere, 319 Leonard, 76 Leslie, 335, 336, 337 Levine, 163 Lewis, 76, 171, 181, 396 Lichtenstein, 27, 70, 73 Lignieres, 22 Lindsay, 105 Lingelsheim, 78 Linser, 411 Lister, 299, 300, 301, 303, 305, 332 Lloyd, 205 Loeffler, 224, 225, 269 161 Logan, 122 Loiseau, 435 Long, 196, 328 Look, 73 Lowe, 318, 331 Lowenstein, 48, 106, 246, 285, 422 Lucksh, 286 Lusk, 416 Lustig, 245 Lusztig, 155 Luther, 179, 180, 182 Luttinger, 341, 342 Lynch, 281 Lyon, 118, 146 MCCANN, 76 McCartney, 174, 175, 315 McClean, 71 MacConkey, 40, 93, 140, 160 McCoy, 7, 107, 132 McCracken, 26, 29 McDonagh, 384 Macdonald, 308 McEachern, 183 McEwen, 235 Macfadyen, 157 MacFarlane, 27 Mc(irath, 209 McIntosh, 412, 413, 415 Mackay, 146, 147 McKee, 299 McKendrick, 201 Mackenzie, 9, 11, 13, 69, 300 404 McKenzie, 141 McKhann, 147 Mackie, 74, 161, 162, 163 McLachlan, 74, 388 Maclean, 316, 328, 370 McLean, 146 McLeod, 22, 78 Macnamara, 184, 187, 188, 189 : MecNalty, 170 MacWilliam, 74 Madsen, 28, 29, 337, 338 Maegraith, 136 Magalhies, 110 Mair, 29, 66 Malone, 202, 294 Mann, 21 Manninger, 237 Manson, 288 Manson- Bahr, 90, 91, 288 Marchoux, 210 Marlow, 148 Marmorek, 65 Martin, 33, 90, 104, 105, 141, 435 Mason, 238 Massingham, 170 Master, 148 Matsunami 142 Maxwell, 404, 412 Mazza, 415 Meader, 206 Melnotte, 278 Mendelewa, 170, 173 Mennona, 248 Messer, 171 462 Meyer, 19, 85, 96, 99, 100, 131 Michelson, 148 Miessner, 219 Miller, 416 Mills, 315 Minaker, 331 Minett, 238 Mishulow, 334 Mitchell, 206 Mohler, 238, 239 Moloney, 423 Monro, 99 Mooser, 203 Morgan, 106, 186, 192, 193, 208, 309 Moritsc h, 104 Moro, 190 Morrison, 293 Morse, 187 Moser, 65, 66 Murata, 268, 291 Murdick, 141 Murray, 135, 136, 137, 138, 143 Musser, 31 . NABARRO, 96, 170 Naidu, 160, 293, 294 Neal, 137, 140, 143, 144, 146, 186 Neisser, 83 Nelson, 300 Netter, 133, 183, 186, 189, 278 Neufeld, 122 Neustaedter, 192, 193, 194, 196 Newman, 174 Nickel, 356, 361 Nicolas, 269 Nicholls, 343, 344, 352 Nicolle, 166, 208, 210, 2 339 Nicoll, 43, 47 Nijland, 291 Noel, 24 Nolf, 287 Noon, 394, 395, 396, 397 Norton, A 136, 138 Novak, NaS 218 O’BRIEN, 22, 72, 430 O’Connor, 90 Okell, 72, 74, 234, 345 Oldenbusch, 209, 334 Olitsky, 196, 315, 328 Olitzki, 263. 265 Oliver, 384, 385 Olmstead, 352 Ordman, 304 Ornstein, 220 Ostroff, 209 Otto, 104 PAINE, 343 Panron, 384 Panton, 85, 86 INDEX OF 120, 127, 184, ~1 79, 80, 174, Pape, 224 Parish, 22, 62, 72, 127, 345, 430 Park, 10, 12, 13, 21, 25, 27, 47, 66, 67, 68, 116, 119, 124, 166, 168, 170, 172, 174, 187, 192, 193, 194, 318, 330, 422, 434 Parker, 85, 202 Pasteur, 237, 243 Paterson, 44, 339 Paul, 179 Pauli, 346 Pearl, 17 Peet, 147 Pelouze, 386 Pemberton, 353 Penna, 415 Permin, 46 Perry, 275, 288 Petersen, 206, 288, 413, 414, 416, 418 Petrie, 120, 293 74, 85, 127, 136, 208, Petrowitch, 278 Pettit, 193, 194, 210 Pfaundler, 10 Pfeiffer, 244 Phear, 95 Picken, 174 Pierce, 353 Pierret, 338, 339, 340, 341 Pijper, 270 Pinoy, 248 Place, 31 Platou, 29, 77 Plummer, 114, 115, 116, 117, 118, 119, 124 Pope, 4, 266, 423. 428, 420 Popper, 181 Porebski, 3 Poston, 351 Powell, 291 Poynton, 343 Prausnitz, 6, 131 Preisz, 217, 221 Priet, 149 Prigge, 162 Proscher, 84 Pugh, 234 Pulford, 54 Pulvertaft, 72 Purves-Stewart, 144, 146 RACKEMANN, 397, 401, 404 Raia, 114 Rake, 136 Ramon, 2, 4, 8, 28, 33, 2 246, 247, 285, , 423, 429, 430, 431, 435, pi 440 Rankin, 23, 98 Ravenel, 206 Ravina, 167, 168 Reagh, 261 Regan, 208 Reiche, 27 Reinthaler, 159 Reye, 27 Reymann, 49 Reynolds, 211 Rhoades, 124 AUTHORS Rhoads, 192, 193, 195, 196 Richards, 351 Riven, 148 Rivers, 209, 210 Robb, Robertson. 52, 62, 315 Rochaix, 270 Rodet, 158 Rolleston, 8, 14, 25, 32, 68 70 Romanow, 155 Romer, 434 Rosenbliith, 116, 119, 124 Rosenhol, 154 Rosenow, 196, 197, 356, 361, 380 Rosher, 74, 315 Roux, 28 Rowland, 160, 204 Rumpel, 53 Rumreich, 204 Ruppel, 220 Russ, 84 Russell, 17, 271, 292 Ryle, 07 SABIN, 14, 122, 123 Sabolotnyi, 153 Sabouraud, 365 St. John 211 Sallard, 210 Salmon, 243 Sanderson, 74 Sandidge, 35 Savas, 291 Sawchenko, 66 Sawyer, 205 Schiiffer, 262 Schelenz, 284 Schick, 9 Schiff, 264, 281 Schlutz, 77 Schmidt, 2, 20, 21, 23, 29, 315 Schobl, 93 Schottmiiller, 72 Schroder, 434 Schubert, 21 Schultz, 66, 193, 224, 281 Schiitze, 260, 264, 265, 293, 204, 295, 297 Schweinburg, 252 Schwentker, 24 Sclavo, 153 Scott, 13, 68. 108, 135, 327 Scully, 416 Sédallian, 386 Séguin, 53 Seligmann, 35 Sellards, 205 Serpa, 429 Sevin, 380 Seyfarth, 295 Shands, 351 Sharp, 318, 330 Sharpey-Schafer, 12 Shaughnessy, 184 Shearman, 89 Shelden, 346, 347, 348 Sherrington, 46 INDEX OF AUTHORS Shibley, 315 Shiga, 270, 283 Shope, 320, 329 Shultz, 114 Shwartzman, 141 Siebner, 54 Siegl, 148 Signy, 170 Silverman, 168, 170 Simey, 319 Simig, 21 Simmons, 211 Simond, 210 Simpson, 209 Skrotskiy, 208 Sleeswyck, 334 Small, 343, 345 Smeall, 122 Smellie, 339 Smith, 127, 174, 262, 329, 339 Smithburn, 136, 140, 144 Snyder, 416 Sobernheim, 152, 153, 155, 227, 228, 268 Sollgruber, 148 Sommer, 390, 416 Sordella, 429 Sordelli, 154 Spencer, 202 Spicer, 13 Springutt, 264 Starr, 179 Staub, 155 Steele, 138 Steffen, 299, 328 Stainsby, 343, 344, 352 Stefanopoulo. 210 Stein, 278 Stevens, 31 Stevenson, 148 Stewart, 13, 142, 182, 186, 190, 193, 194 Stillman, 299 Stocanne, 281 Stocks, 173 Stoges, 53 Stookey, 144 Storer, 410, 411 Strauss, 144, 187 Stroe, 61 Strong, 293 Stuppy, 299 Stidmersen, 422, 424, 427 Sutherland, 169, 173 Sutliff, 117, 121 Sutton, 310 Suvansa, 45 Swift, 344, 346, 347, 348 Swyer, 71, 72 Symmers, 75, 76 Tart, 136, 137 Takerka, 149 Tapia, 58 243, 261, Tavel, 157 Teachenor, 144 Teissier, 209 Tenbroeck, 49 Terwilliger, 389 Theiler, 205, 23 Theobald, 78 Thibault, 58 Thomas, 84 Thompson, 270 Thomsen, 388 Thomson, 22, 25, 3, 717, 317, 339, 384, , 388, 389 386, * Tidy , 97 Todd, 109, 110, 223, 345, 346 Tomesik, 436 Toomey, , 68 Topley, 33, 47, 163, 260, 264, 280, 315, 317 Torres, 415 Torrey, 383 Toyama, 142 Toyoda, 68 Trask, 179 Trautwein, 225 Troise, 111 Tulloch, 49 Tunnieliff, 174 UDE, 77 Uhlenhuth, 218, 219 Uhry, 33 Unna, 365, 366 Urbain, 155, 281 VALENTINE, 85, 86, 122 Vallée, 228 Van de Velde, 83 van Leeuwen, 404, 412 van Loghem, 162 Varekamp, 412 Vaughan, 248 Vellard, 111 Velu, 269 YX Si 148 Viljoen, 227 Vincent, 48, 286 von Behring, 421 von Bormann, 20, 67 v. Lingelsheim, 83 von Pirquet, 9 von Sholly, 318, 330, 339 WADSWORTH, 72 134, 136 Wainwright, 43 Walbum, 3 77, 132, Wallace, 14, 52, 53, 55 463 Warren, 413 Weaver, 78 Webb, 426 Webster, 270, 315 Wechsberg, 83 Weech, 206 Weigl, 203 Weil, 203, 262, 264 Weinberg, 49, 52, 53, 57, 58, 62, 101 Welsbecker, 7 73 Weiss, To Weld, 423 Wells, 13 Wenyon, 90 Wernicke, 21, 421 Western, 209 Wetherby, 348, 336, 358, 360 Weyer, 192 Whatley, Wheeler, 72 Wherry, 247, 278, 285 Whitby, 60 White, 29 Whitehead, 275 Whitmore, 248 Whittingham, 145 Wies, 142 Willcox, 309, 350, 353, 357 Willems, 229 Williams, 21, 55, 56, 57, 174 Willmore, 89 Wilson, 15, 33, 47, 109, 163, 174, 346, 347 Wolff, 19 Wollstein, 208 Woodcock, 24 Woods, 17, 434 Wordley, 376 Workman, 204 Worster-Drought, 47, 134 Wright, 23, 158, 244, 254, 260, 273, 275, 300, 301, 302, 304, 305, 309, 311, 332, 367, 370, 409, 410, 412 Wrigley, 58, 59 Wynn, 179, 276, 310, 311, 331 YERSIN, 245 Yodh. 44 Yoshioka, 298 Young, 15, 76 Yule, 17, 275, 291 Yunowich, 135 ZADKIN, 380, 381 Zeissler, 52 Zingher, 166, 182, 422, 434 Zinsser, 203 Zoeller, 40, 439, 440 PRINTED IN GREAT BRITAIN BY THE WHITEFRIARS PRESS LTD., LONDON AND TONBRIDGE indi (029400513