U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY— BULLETIN No. 67. D. E. SALMON, D. V. M., Chuf of Bureau. SF J\^7 NECROTIC STOMATITIS. WITH SPECIAL RKFERENCE TO ITS OCCURRENCE IN CALVES (CALF DIPHTHERIA) AXI) PIGS (SORE MOUTH). JOHN R. MOHLER, A. M., V. M. D., Onef of Paitiolo^ical Divisi'iti ^ Bureau of Auintal Industry, GEO. BYRON MORSE, M. D., V. S., Assistaut in Patho/ooiral Division , Bureau of Anivuil tndusiry WASHINGTON: (iOVKKNMRNT I'KINTINC oPFICR. 1905. Class ^} t" ^ ^ — Book ^liX^ ro U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY— BULLETIN No. o7. ' /^ C^ D. E. SALMON, D. V. M., Chief ok Bureau. ^f ^ NECROTIC STOMATITIS. WITH SPECIAL REFERENCK TO ITS OCCURRENCE IN CALVES (CALF DIPHTHERIA) AND PIGS (SORE MOUTH). BY JOHN R. MOHLER, A. M., V. M. D., Chief of Paiholoi^ical Division, Bureau of Animal Industry. AND GEO. BYRON MORSE, M. D., V. S., Assistant in Pathological Division, Bureau of Animal Industry. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1905. "^ ^onccn0k MAY 19 1905 O.otO. LETTER OF TRANSMITTAL U. S. Department of Agriculture. Bureau of Animal Industry, Wasliingtori, I). 61, Fehruary i, 1905. Sir: I have the lionor to transmit herewith a manuscript on "Necro- tic stomatitis, with special reference to its occurrence in calv^es (calf diphtheria) and pigs (sore mouth),*' l)v -Dr. John R, Mohler and Dr. Geo. Byron Morse, both of the Pathological Division of this Bureau. It is definitely known that this disease has been in this country during the last three or four years, and it is probable that it has been pres- ent a much longer time. It affects calves, pigs, adult cattle, lambs, asses, kangaroos, rabbits, dogs, and chickens, but in this countr}' it has so far been reported for calves and pigs only. The percentage of losses varies greatly, but it is always large. I recommend that this manuscript be published as a bulletin of the Bureau series. Respectfully, D. E. Salmon, Chief of Bureau. Hon. James Wilson, Secretary of Agriculture. CONTENTS. Page Introduction 9 Name and synonyms 9 Definition 10 Historical review and geographical distribution 10 Etiology 12 A. Predisposing conditions. 12 B. Active agent {Bacillus necrophorm^) 12 Bacteriology 12 Name 12 History 12 Morphology 13 Motility 13 Staining 14 Biology 15 Plate cultures 15 Shake cultures 16 Stab cultures 17 Cultures in fluid media 18 Action of germicides . IS Chemical activities 18 Pigment production 18 Odor production 18 Production of substances that liquefy 18 ludol formation 19 Production of curdling ferments 19 Production of toxins , 19 Immunity 20 Occurrence in nature 20 Pathogenesis 21 Under natural conditions 21 Under artificial conditions 22 Experiments on rabbits 22 Subcutaneous method 22 Intravenous method 24 Experiments on mice 25 Subcutaneous inoculation 25 . Experiments on guinea pigs 26 Experiments on chickens 26 Experiments on pigeons 26 Experiments on calves 26 Subcutaneous inoculations 26 Sul)cutaneous and intravenous inoi'ulation 28 Intravenous inoculations 28 Experiments on sheep.. 29 Subcutaneous inoculations 29 Experiments on pigs 29 Subcutaneous inoculations 29 CONTENTS. Pathology 30 Symptoms 32 Course and termination 34 Infective character 34 Susceptibility 36 Economic importance 37 Differential diagnosis 39 Prognosis 46 Treatment 40 Prophylaxis , 40 Therapeutics 41 Relation of animal diphtherias to man 42 Disposition of meat of affected carcasses 42 Conclusion 43 Bibliography 44 ILLUSTRATIONS. Plate 1. Necrotic stomatitis with lesions involving tongue and cheek 16 2. Fig. 1. — Section of upper lip of calf showing necrosis bacilli. Fig. 2. — Section of liver of rabbit showing a peripheral area of caseation. 16 3. Section of lung of rabbit showing metastatic foci 24 4. Section of lung of rabbit showing felted network of Bacilhn^ necrophorus 24 5. Fig. 1. — Bacillus necrophorus from metastatic focus in rabbit's heart. Fig. 2. — Petri dish containing characteristic colonies of necrosis bacilli 24 NECROTIC STOMATITIS. WITH SPECIAL EEFERENGE TO ITS OOOUKRENOE IN CALVES (CALF DIPHTHERIA) AND PIGS (SOEE MOUTH). By Jonx 11. Mohlkk, A. M., V. M. D., Chief of Pathological Division, Bureau of Anhnal Industry, Geo. Byron Morse, M. D., V. S., Assistant in Pathological Division, Bureau of Animal Industry. Introduction. The vestibular character of the mouth in its relation to the general organism makes an}- disease of the oral cavit}' an important matter. Mechanic, chemic, and thermic influences ma}' each play a part in the production of various disturbances of its mucous membrane, but the most frec|uentl y operative of all causes — and most serious, too — are the microorganismal. It is these last which render any form of stoma- titis serious and constitute the real source of danger in the grave forms. Not the least interesting in this last group — and, as we shall show, of considerable economic importance, although until recent years not recognized in this country — is the affection to which we have given the name '"necrotic stomatitis/' Owning similar predisposing condi- tions and recognizing a common etiologic agent, its occurrence has been noted in calves, pigs, adult cattle, lambs, asses, kangaroos, rabbits, dogs, and chickens. In this countr}- it has been reported up to the present time onl}^ among calves and young pigs, being probabl}' more prevalent among the latter. For this reason and because our investi- gations in this disease have been limited to cases occurring in these two species only, the following pages will deal with necrotic stomatitis of calves and of pigs. Name and Synonyms. Necrotic stomatitis of calves has been known generally under the unfortunate term of "calf diphtheria.'" Diphtheria has a specitic cause (Klebs-Loffler bacillus) and a definite histologic picture, both of which nre lacking in necrotic stomatitis. The name " calf diphtheria" 18489— No. 67—05 2 9 10 BUREAU OF ANIMAL INDUSTRY. is therefore a. misnomer. The putholooic process in necrotic stoma- titis alwa3's presents the picture of a diphtheric inflammation plus casea- tion — that is to sa\% an inflammation characterized bj^ the production of a membrane, which, as an essential feature of the disease process, undergoes cheesy degeneration. Since this progressive necrosis is caused by the necrosis bacillus, the origin of the term " necrotic stoma- titis" is self-evident. This disease in calves has also been termed gangrenous stomatitis, ulcerative stomatitis, malignant stomatitis, tubercular stomatitis, diphtheric patches of the oral mucous mem- brane, necrosis diphtherica caseosa, stomatitis diphtherica multiplex, and stomatitis membranacea diphtherica. In pigs the affection has been designated ulcerative stomatitis, sore mouth, and canker of the mouth. Definition. Necrotic stomatitis is an acute, specitic, highly contagious inflamma- tion of the mouth, occurring enzooticalh^ in many species of animals and characterized locall}^ by the formation of ulcers and caseo-necrotic patches and by constitutional s3^mptoms, chiefly toxic. The disease is in no Avay related to diphtheria of man. Historical Review and Geographical Distribution. During the last few years farmers and cattlemen of this country have noted the increasing occurrence of sore mouth among the 3'oung animals of their herds. Instead of healing of themselves, as the usual forms do, these cases, if untreated, die. Careful study of some of them has resulted in their identification with cases first reported by Dammann**" in 1876 from the shore of the Baltic. His model clinical investigations, making the description of the disease suitable for to-day, and his accurate postmortem findings were marred by subjec- tion to the then dominating assertion of Eberth, "without micrococci no diphtheria." Finding micrococci everywhere present in the diseased tissues of the mouth, and failing to recognize the fatal results in his experimental inoculations as due to septicemia, Dammann pronounced the disease identical with diphtheria in man. To what extent the malady occurred before his time it is difficult to determine, but from the fact that his clinical observations were soon confirmed b}^ a number of contemporary veterinarians, it is probable that at the time of his writing it was not very uncommon. ^'^ In 1878, Blazekovit^^ gave an extensive report of sixteen cases (fourteen fatal) of calf diphtheria in Slavonia, and, in 1879, Vollers,^** adopting Dammann's theory, recorded four cases of the same disease in Holstein. Near the close of 1880, Lenglen" described quite accurately the local manifestation and gen- «The figures refer to the bibliography at the end of this bulletin. NECROTIC STOMATITIS. 11 eral sjMiiptoms of the disease a^ noted by him in a series of ten cases in the northern part of France. Under the title of gangrene of the mouth in young calves, he identified the disease with gangrenous stomatitis, or noma, in children, and, as predisposing causes, associated the eruption of the teeth with bad sanitary and dietetic conditions. His article attracted the attention of MacGillivray,*" •'" of Banff, Scot- land, who published several articles during 1881-82, describing his experiences with the same disease, and asserting that it was a tuber- cular stomatitis rather than diphtheritis, or gangrenous stomatitis. During these two years there entered into the controversy thus started in the Veterinary Journal and the Veterinarian, the following persons wnth reports of their own cases: Campbell,*^ Cole,** Fleming,^® Gunn,^' James,-** Metherell,'' Smith,*''' and Steel."* In 1884, Loffler*' isolated and described the bacillus of human diphtheria, thus destroying all previous theories as to the cause and relation of micrococci with that disease. Pursuing similar investig-ations, he the same year isolated from diseased tissues in cases of so-called calf diphtheria a long slender thread-like bacterium, which he described and demonstrated as the cause of this affection. Loffler thus proved diphtheria of man absolutely distinct etiologically from that disease, so called, in calves. In 1903, Mettam""^ observed this affection in Ireland in the calves of the prov- ince of Munster and in DuVjlin, while McFadyean*'* has mentioned his familiarity with its prevalence for some years in various sections of England. We have no exact knowledge Of the existence of the disease in this countr}^ prior to 1897, when it was recognized by one of us in southwestern Texas. It was later observed in rather serious outbreaks in 1902 and 1903 in Colorado, Wyoming, and South Dakota. Bang,^ in his notable work on the necrosis bacillus, was the first to announce this parasite's causative relation to the deep necroses often affecting the mouths of hogs. However, Johne,^^ in his pathological reports for 1890, seems to have recognized the disease in pigs, although it was not until 1893 that Schlegel,^** working in con- junction with Johne,^'* proved the relationship of the Bacillus necroph- OTus to diphtheric inflammation of the tonsils, phar3mx, and larynx in hogs. Kitt^^ was also able to recover the causative agent of this disease from the diphtheric lesions of the larynx and phar3'nx of calves and pigs in 1893. In 1903, Lauritsen*** described a necrotic inflam- mation of the mouth in j^oung pigs, believing, though not demonstra- ting, BaciUus iiecrophorus to be the cause. The outbreaks of this disease in pigs, which we were onaljled to stud}^, appeared in an enzootic form in western Tennessee, and, at the Athenia quarantine station, among a herd of young Berkshire hogs recently imported, from which cases the Bacillus necropliorus was recovered. 12 bureau of animal industry. Etiology. Necrotic stomatitis is due to a specitic infection. It is an inocula- tion disease and occurs as a result of the already abraded lining- mem- brane of some part of the oral caA'ity being invaded by the BactUm necroplioTim. \. — PREDISPOSING CONDITIONS. These prepare the nidus for the active ag'ent. The necrosis bacillus will not develop on a normal mucous membrane; its invasion is always secondary. Opportunity for infection of the mouth is afforded by a catarrhal inflammation of its lining-, by a break in the integrity of its protective covering-, or by the role played by the bacterial flora of the mouth in damaging its cells and tissues. Here may be men- tioned, also, irritant foods, rough forage, veiy hot or very cold drink- ing water, chronic and debilitating diseases, and damp, unsanitary pens. Furthermore, as will be pointed out later, some predisposition appears necessary for the development of necrotic stomatitis. In part, this is individual, for whereas the disease is distinctly one of young animals, some of these are more hig-hly susceptible than others. B. — ACTIVE AGENT (BACILLUS NECROPHOKUS). Bacteriology." NAME. This organism has been variously known as Bacillus der Kdlher- dijjhther ie (Lo^er),^^ 188-i; BaciUux diphtlier'tx vitulorwin (Lofl[ler) ^'; Bacillus necrojpliorus {J^\\\^g^^^^ 1880 ; B(iciUus Jiliforrnis (Schiitz)," 1887; N'ekrosebacillus ij&2ing)^^ 1890; Streptothrlx cu^i/ci/Z/ (Schmorl),'*'' 1891; Actinomyces cunicidi {^2k^}Qv'\n\)^^'^ 1892; Bacillus necroseos (Salo- monsen),®^ 1894; Bacillus des Kalhernoma (Ritter)," 1895; and Strep- tothrix necropJiora (Kitt),^* 1899. Until authors are more agreed as to the possession by this organism of the character of branching, a feature not observed during the course of our investigations, it will be well to consider it as belonging to the Bacteriacete and allow priority to designate it Bacillus necrophorus (Fliigge). HISTORY. First observed by Koch, ^'^ it was not isolated and described until Lotfler, in 1884, demonstrated it as the cause of the disease erroneously called by Dammann^ calf diphtheria. It remained for Bang to recog- " It is with pleasure that we acknowledge our inde1)tedness to Dr. B. Bang and Dr. W. Ernst for sending us cultures of Bacillns vecrophorvs with which to compare our organism. '' Without doubt Dammann, on page 12 of his article, in his description of the tangled network of fibrin threads in the deeper layers of the pseudomembranous deposits, really refers to tlie filaments of Bacillvs necropliorus. NECROTIC STOMATITIS. 13 nize its varied activities in the production of coagulation necrosis and describe it as the NekrosebaciUus. A 3^ear later Schmorl made public his careful investigations of a spontaneous rabbit disease in his labora- tory, resulting in the recovery of this same organism which he, either not knowing of Loffler's and Bang's labors or failing to identify his organism with theirs, called Streptothrix cunicuU. Since then numer- ous investigators have confirmed the work of the foregoing scientists and demonstrated a still wider sphere of pathogenic relationship, the latest being the proof furnished by this laboratory of the etiologic con- nection of BdciUu.s necropliorus with foot-rot in sheep. The morpho- logic and biologic characters have been quite thoroughly worked out by Bang, Schmorl, and Ernst. MORPHOLOGY. Bacillus necroj)horus is essentially a pleomorphic organism. It varies, according to nutrient soil and age of culture, from coccoid forms to filaments over 100;< in length and from 0.T.5 to 1.5/^ in width. The longer forms appear as slender, undulating, beaded filaments. Generalh', in the tissues these threads are matted together into an intricate network, like a mass of hair or even the more compact felt. The same appearance may be found in colonies. Frequently the fila- mentous forms present one wide oi" clubbed extremity, with the other extremity tapering. On the other hand, the older cultures — either animal tissues or artificial media — exhibit almost exclusively bacillary forms of various lengths, some so short as to be easily mistaken for cocci. Involution forms may be present in an}^ culture, but certain media, notably that composed of a mixture of agar, gelatin, bouillon, peptone, and salt are particularly favorable to their development. Motility. — Motion has not been observed in our experiments; in fact, it has been reported by Schmorl only. He examined the pleural exudate in hanging drop. He says: '\ The shorter bacillus-shaped forms are mostly motile; they travel slowly through the visual field with a serpentine, or crawling, motion, but generally come very quickly to rest. The longer threads are for the most part absolutely motionless; only in the entirely detached forms does one notice pendulous movements being slowly executed. The signs of motility can be demonstrated only when the material for observation has been taken from an animal just killed or just dead. If it has been dead a longer time, one finds rods and threads altogether in the resting condition, from Avhich they can sometimes be aroused by making the observation on a warm stage in a vacuum. Most of the rather thin and pale threads possess a character- istically stiff appearance. They fioat along sometimes completely extended; at other times they exhibit slight winding motion. But very seldom does one find specimens which appear spirally twisted. Over against this statement of Schmorl ma}' be placed that of Ernst:" The merely feeble, oscillating, molecular movements of the rods and the pendulous, serpentine movement of long, undulating threads in changes of position, according to 14 BUREAU OF ANIMAL INDUSTRY. the law of gravity, permit no positive decision as to motility. In order to examins the bacteria as to their motility I employed cultures ; and since young, 36-hour, cul- tures in the hanging drop showed no active changeof place beside the ordinary molec- ular motion, and furthermore, since in the employment of material from cultures whose growth was just becoming visible (24-36 hours) , flagella were not demonstrated by mordants which gave good results in control experiments with edema and cadaver bacilli as well as colon forms, a denial of motility would appear perfectly just. In this laboratory the examination for motility was made with fresh cultures and with tissue from animals within one hour after death, both b}^ means of hanging drop and by the application of flagella stains. In no case was it possible to claim motion for these Ijacilli. Staining. — The necrosis bacillus stains readily with the ordinary aniline dyes, Loffler's methylene blue, and Ziehrs carbol-f uchsin, pro- ducing particularly good effects. Alkaline toluidine blue (1 per cent solution), while not giving the brilliant effects of fuchsin, makes perhaps the best reagent for routine use. The slide, or cover slip, dipped in the stain, immediateh^ washed in water and mounted, is a very rapid and satisfactory method of bringing out the beaded appear- ance of the organism. In the study of fresh tissue smears, it is usually sufficient to make a film on a slide with a teased particle of the suspected tissue, and, after the usual preliminaries, stain with one of the ordinary dyes mentioned, preferably methylene blue or toluidine blue. Whenever it was desired to employ differential staining, we found the following procedure to answer all requirements: The stains are kept ready for use in wide- mouthed bottles. Prepare the film on the slide in the usual manner, fix in the flame, dip it from two to five seconds in a 1 per cent alka- line toluidine blue, wash it thoroughly in water, counterstain it in a 0.2 per cent Neisser's Vesuvian brown, wash it in water, dry, and then mount it in balsam. An excellent method of double staining was devised for Bacillus necrophoriis by Bang. Bits of the ne(!rosed tissue were hardened in Mailer's fluid, thoroughly washed, and further hardened in alcohol. (Alcohol alone he did not find suitable.) The sections are placed a few minutes in toluidin-safranin (produced like aniline-gentian violet), dehydrated by means of an alcoholic solution of safranin; after this, fluorescin clove oil, pure clove oil, alcohol, watery methyl green, alcohol, xylene, balsam. The bacilli are stained a beautiful red, while the tissue is stained green; no Qther investigated bacteria can stain in this manner. Mention has been made of the beaded appearance of the Bacillus necrophorus in stained preparations. This is noticeable equally in tissue smears or sections and in films from cultures. The longer rods and threads particularly exhibit this characteristic. It is due to the occur- rence in the filaments of unstained spaces which were at first thought to be spores. Spore-staining methods, however, do not alter them. NECROTIC STOMATITIS. 15 Careful study of this peculiarit}- reveals several phases of it. Some- times a thread will be most regularly marked off into alternate sections of stained and unstained material; again, decided irregularity charac- terizes the arrangement — long vacuole-like inclusions alternating with shorter stained squares or Imcillus-like spaces of stained material may alternate with shorter colorless portions; again, the vacuoles may appear like a chain of colorless rods lying on a ribbon of blue or what- ever color may be used for the stain. Sometimes the stained material is so little in quantitv that the thread seems like a string of spores, oval or rod shaped, with thin, deeply stained partitions between them. On the other hand, the filament presents itself as an unstained tube with a regular succession of deeply stained coccus-like granules much resembling streptococci, or these granules may be alternately arranged alono- the sides of the tube. Cultivation of the Bacillus neerophmnis is not easy. It is an abso- lute anaerobe. Investigators differ concerning its requirements as to temperature. Nocard and Leclainche^^ give 30° to 40'^ C. as the limits of growth, with the optimum at 87° C. ; Jensen adopts the same extremes, but places the optimum at 34° C. ; whereas, according to Ernst, development occurs only between 86" and 40° C, and the opti- mum is 89°. Our own investigations have shown that 30° to 40° C. represent the extremes of temperature at which the ordinar}^ work of the laborator}" may be satisfactorily carried on; nevertheless, we have on different occasions been able at 28° C. to grow in agar-bouillon •shakes typical colonies, which responded to the usual tests of mor- phology, odor, and pathogenesis. With us the optimum was 35° C. The usual culture media of the laboratory are either unsatisfactory or altogether inimical to the development of the necrosis bacillus. Agar-agar was often employed with only passable results, but more satisfaction was obtained from the following combinations: Agar- bouillon (A-B); agar-gelatin (A-G); serum-agar (S-A); serum-agar- gelatin (S-A-G); and two suggested by Ernst — 1..5 per cent agar in a peptone-salt-bouillon (A-B-P-S) and 0.7 per cent agar and 7 per cent gelatin in bouillon with .5 per cent peptone and 2.5 per cent salt (A-G-B-P-S). The first four mixtures were usually prepared in the proportions of equal parts, although other proportions were adopted for the purpose of var3'ing the consistence of the medium. Fluid blood serum, milk, rabbit bouillon, and Martin's bouillon were also employed. Plate Cultures. Bouillon agar. — Great difiicult}^ was experienced in getting the organism to develop colonies in Petri dishes. Numerous attempts were made by displacing air with hydrogen in a hydrogen jar, and by 16 BUEEAU OF ANIMAL INDUSTRY. the formation of a vacuum hj withdrawing the air under a bell jar by means of a vacuum pump, but success was not attained in any instance. Recent!}' it was endeavored to grow the organism in Petri dishes placed in a closed jar containing a solution of pyrogallic acid rendered alka- line by sodium hydrate. This method, which permitted the presence of only the inert nitrogen gas, finally resulted in characteristic colo- nies occurring throughout the medium with the formation of numerous gas bubbles. Several of these colonies in the dish of the second dilu- tion (pi. 5, iig. 2) grew so close to the surface that some filaments extended to the upper stratum, and could be removed by means of a platinum needle. In about forty-eight hours after exposing the plates to this method, small, pinhead-sized, dirty-white, opaque, round colo- nies, possessing no distinctive features, were visible to the unaided eye below the surface. Man}^ small round or oval gas bubbles could also be observed. By means of a small magnifying glass these colonies were seen to possess a yellowish-brown center surrounded by a thin, light, almost translucent border^ which, under the mici'oscope, appeared lioccose. After three daj^s the colony presented to the naked eye a woolly appearance, and the microscope now revealed the central struc- ture as a felted maze of threads and the floccose character of the border as long, wav}' filaments. Shake Cultures. Aga/'-hoi(lllon. — In eighteen to twenty-four hours after inoculating' a tube either from necrosed tissue or from a colony in another tube, or with a loopful from the depths of a pure Martin's bouillon culture, Description of Plate 1. Tongue and cheek of calf showing lesions of necrotic stomatitis as a result of natu- ral infection. Notice the zone of thickened granulated tissue surrounding the dry, cheesy, grayish-yellow areas of necrosis both in the tongue and cheek. Description of Plate 2. Fict. 1. — Section of the upper lip showing the necrosis bacilli in their special role of producing a deeply penetrating necrosis. The cuticular layer has suffered com- plete coagulation necrosis and caseation. The drawing depicts the bacilli in the act of carrying the necrosis down between the papillae. Lodged in the interpapillary epithelium they have completely destroyed the portion where they lie and have exerted their toxic influence on the cells to the left. To the right is a section of a papilla whose suprapapillary layer of epithelium has been destroyed next the bacil- lary mass preparatory to the attack on the connective tissue framework of the jjapilla. Stained with methylene blue and eosin. Magnified about 640 diameters. Fig. 2. — Section of liver of rabbit No. 1183, inoculated with an emulsion of necrosed tissue obtained from rabbit No. 1166, which had been inoculated with material from a pig affected with necrotic stomatitis. The periphereal area of caseation delimited bj' the well-defined line of demarcation is the result of the extension of the necrosis by contiguity of structure as the abdominal wall at this point was adherent to the capsule of Glisson by a plastic exudate. No other areas of necrosis were observed in the liver. Stained by hematoxvlin and eosin, and magnified about 22 diameters. Bulletin No. 67, B. A. I. Plate 1. NECROTIC STOMATITIS WITH LESIONS INVOLVING TONGUE AND CHEEK. Haines del Bulletin No. 67, B. A. I. Plate 2. y •\-:t' \'' •'■ %^ii .M Fig. 1 "Section of Upper Lip of Calf Showing Necrosis Bacilli. . ^^f(t4^''^0^-% \ rm Fig. 2. -Section of Liver of Rabbit Showing a Peripheral Area of Caseation. Haines del :^rECROTIC STOMATITIS. 17 the tube is studded with small oval gas bubbles. At this time, also, rarely with the A-B medium, frequently with the softer forms, as S-A-G, A-G (2:1), A-B-P-S, and A-G-B-P-S, the column of culture medium will be transversely ruptured in one, two, or more places by the pressure of the gas. In fortj^-eight hours these sections will often be separated 2 to 5 mm. and even more from each other. We ha\e sometimes seen this gas formation go on for the next two days, suffi- ciently strong to raise the upper portion of the medium 2 cm. The dilution from the above-described tube — tube 2— would often follow tube 1 quite closely in the quantity of gas bubbles formed, though not in the breaking up of the medium. Tubes 3 and 4 would usually show a great diminution in the quantity of gas bubbles and no break- ing up of the medium. In the development of the growth, our experience tallied quite closely in a few notable points with that of Ernst. For instance, shakes sown with necrosed material would often show, after thirty-six to forty -eight hours, a fine gra3'ish white mist of cloudiness at the lower portion of the tube. In our experience, even with slight magnifica- tion, it was possible to detect no particular structure. A film made from this portion of the culture medium would always show beaded forms. Again, when the medium used was jelly-like in consistence, the unabsorbed gas, instead of remaining as bubbles at the point formed, would gradually float upward toward the surface. The original seat of these bubbles and the pathwaj's along which they had risen v\'ould be coated with a fine bacterial growth. Thus would be formed numerous filmy ribboHS, extending from near the surface down into the depths of the tu])e, where they would be anchored bj^ a crescent- shaped bod}'. The time and rate of growth and appearance of colonies in the tube are sufficiently described in the description of the plates. Stab Cultures, Agm-'-agaf. — Near the close of the second day a few grayish-white colonies make their appearance at the bottom of the needle track. Gradually these increase from below upward to within 1-1. .5 cm. of the top of the stab canal. Thus is formed a thin, narrow, opacjue, yellowish or grayish white line of growth surrounded by a thin whit- ish cloud, wdiich on slight magnification is seen to be composed of minute wav}^ threads. Along the needle track, and, if the medium is not too hard when inoculated, throughout the culture may be seen numerous round and oval gas bubbles. Serum-agar. — Time and height of bacterial growth and gas forma- tion are like the preceding. At times the needle track is the center of a whitish film or merely a thin line of cloudiness of the medium; 18489— No. H7— 05 3 18 BUREAU OF ANIMAL INDUSTRY. again, the growth may be denser, similar to that described for agar- agar. The serum is never liquefied, although in very old cultures it will be natural that the zone of cloudiness referred to has spread nearl}^ to the walls of the tube. Cultures in Fluid Media. Bouillon.— rThe organism can be grown in ordinar}" peptonized beef broth, rabbit bouillon, and in Martin's broth, the maximum development occurring at the temperature of 35^ C in a hydrogen jar. The bouillon becomes turbid with the formation of some gas which is noticeable by the surface bubbles. Later the bacillary masses sink to the bottom in the form of whitish viscid Hakes, causing the fluid to become clearer. The cultures have a peculiar odor, very char- acteristic, which will be referred to later. There is no film formation, but a tendency to develop a ring around the border of the medium has been observed. (See "^Milk" and "Fluid serum," on next page.) Action of Germicides. In determining the germicidal power of disinfectants a measured volume of a forty-eight-hour bouillon culture of the necrosis bacillus was intimatel}' mixed with an equal volume of the disinfecting solution, thereby reducing the strength of the germicide to one-half. Three platinum-wire loopfuls were then transferred to fresh rabbit bouillon tubes after varying periods of exposure. After an exposure of 1 minute in a 2 per cent solution of carbolic acid the bouillon tubes showed growth, but in the tube representing a two-minute exposure no development occurred. With bichloride of mercur}- an exposure of nine minutes to a 20S ,t solution prevented growth. Formalin in the strength of 2^ per cent solution (1 per cent formaldehyde) killed the organism in thirteen minutes. Chemical Activities. PIGMENT production. Chromoofenesis is wantine-. odor production. All cultures develop a substance or substances which evolve an odor well described by Ernst as between the odor of cheese and that of glue. The stench is so characteristic that the presence of the bacillus is recognized at once in the tissues of either natural or experimental infection as well as in cultures on artificial media. production of SUBSTANCES THAT LIQITEFY. Gelatin is not liquefied. The growth of the bacillus is likewise without effect on hardened blood serum. NECKOTIC STOMATITIS. 19 INDOI, FORMATION. Indol is formed and ina}' be demonstrated in three-days-old cultures made in Martin's bouillon. PRonrrxioN OF ctrdlixg ferments. Milk is not coagulated nor is acid produced. Fluid serum is coao'ulated. PRODrCTIOX OF TOXI.VS. That the necrosis bacillus produces a toxin is evidenced, not by the isolation of the same from artificial cultures, but by (1) the character of death in the disease, (2) the quality of the rigor mortis, and (3) the study of the pathologic histology. The toxic character of death is not particularly noticed in animals suffering from stomatitis when inappetency and inabilit}' to take nourishment have produced an enfeebled condition. Nor, again, is it noticeable in those animals which die with embolic foci in liver or lungs, the symptoms arising from the diseased organs often masking the signs of intoxication. However, rabbits inoculated subcutaneously in the back will persist, without an}' other sign of the disease except the abscess, for about five or six days. Suddenh% on the sixth or seventh da}", without any premonitory signs, the rabbit will be thrown into convulsions, corning out of one to lie with its head turned side- ways and buried in the bottom of the cage until another attack, dying usually in a few hours after the first convulsion. Quite often in these cases the local lesions will not be sufficient to directly produce death, not being very extensive, and not involving any important organ. Such a course as this points unmistakably to a toxinemia which has attacked the nervous system. The limits of this article do not permit a discussion of the factors entering into the production of cadaveric rigidity. For our present purpose it is sufficient to call attention to the fact that the intensity and long duration of the rigor mortis observed in the experiment ani- mals and described later in this paper comports perfectly with the well-known fact that the presence of toxins in the blood promotes muscular rigidity. It may be stated with positiveness that Bacillus necvopliorus does not enter an unimpaired tissue. Most, if not all, of its infections with which we are acquainted require for their inception a break in the con- tinuity either of mucous meml)rane or skin. A histologic study of an affected area, elsewhere examined in greater detail, reveals a center of completely destroyed tissue marked by an entire absence of the spe- cific bacteria in question. The boundary of this dead area is formed by great bundles of filaments of Bacillmi necrophorus, large numbei's of leucocytes, and a fair sprinkling of tissue cells whose nuclei still 20 BUREAU OF ANIMAL INDUSTRY. respond to stains. The immediately adjoining- border of surrounding- health}" tissue is seen when carefulh" examined to possess numerous cells whose protoplasm has been more or less destroj^ed, and in among- these dying cells a few scattered filaments have advanced like skirmish- ing parties before the main army. It is a true picture of a bacillary invasion of tissue begun by means of the noxious effects of a soluble toxin. Thus far all attempts to recover the toxic substance either from cul- tures or the bacilli themselves have failed. From this, Jensen,'^ whose assistant, L. Bahr, has made the onl}^ experiments thus far recorded, assumes that either the necrosis bacillus forms these substances only in the living animal or they are of such volatile character that they are destroved as quickly as they are formed. IMMUNITY. The literature of Bacillufi necrophorus has contained no word on acquired immunity until the article by C. O. Jensen ^^ referred to above. This eminent investigator and early worker with the necrosis bacillus states that his assistant, Bahr, has demonstrated by experiments not 3"et published that intravenous injections of cultures of the Bacillus necrophorus carefully given to goats protect them from quite large quantities of the same given subcutaneously. Jensen further states that Bahr has produced in the same manner an immunit}" in guinea pigs from intraperitoneal injections. In view of the fact that most investigators pronounce the guinea pig almost if not quite absolutely immune, the statement needs further elucidation. On the contrary, while we are not willing yet to build any hypothesis upon it, we find that our reinoculation experiments have given us the impression that susceptibilit}^ is increased thereby rather than diminished. OCCUKRENCE IN NATURE. There is hardly room to doubt that the Bacillus necrophcrus is a normal inhabitant of the healthy intestine of at least one species of our domestic animals — hogs — and possibly of the cow and horse. It is also found in the manure, and therefore in soil contaminated with the latter. Bang's"^ discovery of the association of the organism with the necrotic processes in the intestine in hog cholera, and also as cause of an intestinal diphtheritis in calves secondary to an intestinal catarrh ' seemed to require the intestine as the normal habitat of the necrosis bacillus. Could this be demonstrated we should then have an explana- tion of the remarkably ubiquitous character of the organism as exhibited in the wide diversity of diseases caused b}' it. In this manner could be explained its relation not only to the necrotic inflammations occur- ring in the vagina and uterus, but also to all the external necrotic processes. This Bang succeeded in doing. He twice made inocula- NECROTIC STOMATITIS. 21 tions of the intestinal contents of health}' hogs with the result of demonstrating the presence of Baenius neci'Oj>lu)ni><. An analogous investigation by him of the intestinal canal of a cow was not so successful. Pathogenesis. Very few organisms exhibit a wider range of pathogenesis. Accord- ing to clinical observation the necrosis bacillus has so far been found pathogenic for cattle, sheep, goats, antelope, reindeer, red deer, roe deer, horses, asses, hogs, kangaroos, rabbits, dogs, and chickens. Experimental work adds to the foregoing list mice and, under special conditions, guinea pigs and pigeons. It is probably safe to assert that no tissue of the bod}- of a suscep- tible animal is safe from its destructive influence. UXDEK NATURAL CONDITIONS. Natural infections induced by BaciUus ni^crophorus may be consid- ered as follows: Necroses of the skin, hoof, muscle, cartilage, mucous membranes (mouth and uppor air passages, digestive tract, genital tract), navel, and viscera. Necrosis of tJie skin in horses has occurred enzootically on a large breeding farm, the necrosis appearing on portions of the skin exposed to pressure of the harness. A necrotic form of scratches has also been shown to be a local infection of Bacillus necrophorus. Gangrenous pocks in cows have the same origin, and consist of an acute necrotic inflammation of the skin and soft parts of the teats. In hogs such a necrotic process has occurred in the skin of the muzzle, of the outside of the lips, of the feet, and, in sows, of the udder. Necroses of the ho(f include in horses a progressive necrosis of the soft parts of the hoof, often involving the bones and cartilages; in cows, reindeer, and roe deer, panaritium, or so-called foot-rot, involv- ing tendon sheaths, tendons, bones, and joints; in sheep, foot-rot, recently shown to be due to the necrosis bacillus. Necroses of muscle have been reported in connection with a granu- lating wound of the inner side of the hind leg of a cow, also as a result of the passage of a foreign body from the stomach into the heart muscle, and also in a case where a cow's heart, kidney, and some of the voluntary muscles were affected. Necroses of cartilage are represented l)y those already included under hoof necrosis, b}^ quittor, or fistula of the lateral cartilages, and by the laryngeal cartilages in manj'^ cases of necrotic stomatitis. Necroses of hone have been reported in association with the hoof necrosis of various animals, in the vertebrae in the course of some cases of necrotic stomatitis, and in the turbinated bone of the horse. Nec7'oses of mucous membranes may ho, regarded as quite frequent. Those of the mouth and upper air passages are fully discussed in this 22 BUREAU OF ANIMAL INDUSTRY. paper, and have been recoo-nized in the calf, adult cattle, pigs, sheep, kangaroos, rabbits, asses, dogs, and chickens. No less sensitive are the mucous membranes of the digestive tract. In pigs the upper portion of the esophagus, b}^ extension from the mouth, and in calves the esophagus in its entire length, have been afi'ected. In hogs these "necrophorus"' patches have been found in the stomach, small intes- tine, cecum, colon, and rectum. In horses they have been demon- strated in the colon; in cattle, in the rumen, in the reticulum, and in the small intestine; and in the deer and antelope, in the stomach. The mucous membranes of the genital tract of cows that have recently calved are often invaded by the Bacillus necrojjhorus^ result- ing in a necrotic vaginitis and a necrotic metritis. Navel disease in calves and lambs ''^ is dependent in a considerable number of cases on infection of the wound b}" the necrosis bacillus. Bacillary necrosis of the heart, lungs, liver, kidney, and spleen, also of the udder, have all been reported as the result of metastasis from the original seat of the disease, in necrotic stomatitis, necrotic scratches, the various forms of foot-rot, and infection of the genital passage. The classic example of Bacillus necrophorm in mixed infections is hog cholera. Here it has been proved unequivocalh^ that the deep necroses and so-called diphtheric processes occurring in the body of a pig affected with hog cholera are due in some cases to the invasion of the necrosis bacillus. It has also been found as a secondary invader in the necrotic patches in so-called fowl diphtheria. UNDER ARTIFICIAL COXDITIOXiS. The pathogenicity of Bacillus necrophorus under artificial conditions w^as tested upon rabbits, mice, guinea pigs, chickens, pigeons, calves, sheep, and pigs, in order to establish its causal relation to the disease in question. Experiments on Rabbits. Subcutaneous method. — These animals are highh^ susceptible to the action of the Bacillus necroplwrus., and to this susceptibility is due the ease with which the presence of the germ in diseased tissue may be demonstrated. A bit of tissue adjacent to the border of the necrosed area is emulsified in a normal saline solution. The subcutaneous inoculation of a rabbit with 0.5 c. c. of this emulsion will result in the death of the animal within one week. In order to free the tissues of the experiment animal from other forms of microorganisms naturally present in material taken from such an exposed place as the mouth, it is advisable to use a second or even a third rabbit. The first rabbit will, however, in spite of the contam- inated material used in its inoculation, show very characteristic lesions. NECROTIC STOMATITIS. 23 which can be referred to the action of no organism other than the bacillus of necrosis. The chief of these typical changes will be noted at the point of inoculation, where, lying beneath the skin and extend- ing down for a greater or less depth into the muscular tissues, is found an irregular area about 3 to 5 cm. in diameter. This area offers to the naked eye much the appearance of a flattened mass of soft, fresh putty, and to the nose the penetrating odor alread}" referred to as between the smell of cheese and that of glue. This pulpy, yellowish white, malodorous mass is the detritus of muscular, fatt}^ and vascular tissues which have l)een attacked and destro^^ed through the presence of the necrosis bacillus as well as by its effective poisons. Reaching out into the surrounding tissues for about 4 cm. in all directions is found a zone of inflammatory tissue, and the sul)cutaneous and muscular tissues of the abdominal regfion are inflamed and edematous through extension by gravitation of the disease process from the local lesion caused l)y inoculation. Not seldom in these cases is there to be observed a marked development of gas bul)})les. Frequently that portion of the large colon adjacent to the diseased abdominal wall is greatly injected and adherent to the parietal peritoneum by a plastic exudate containing numerous short and long forms of the l)acillus of necrosis. This part of the peritoneum is also inflamed and presents petechial hemorrhages. Examination of the soft mass found in the necrosed area at the point of inoculation shows that it is penetrated in every direction b}^ long thread-like bacilli, and the subcutaneous inoculation of a second rabbit with a small scraping from this mass serves to eliminate a large proportion of the contami- nating organisms. Because of the greater purity of the material now used, the inoculation will not result fatally until a period usually from eight to fourteen days, although occasionally death has l)een prolonged until nineteen, and in one case twenty-three, days. In many instances it will be found that this longer period of time has proved sufficient to allow the circulation to take up a few of the bacilli and deposit them in the plexuses of the lungs, liver, or kidnej's, where small, yellowish white spots of necrosis will result, which may be readily peeled out as if in a capsule. From these secondary visceral necroses, pure cultures of the Bacillus necrophorus may now be obtained, and its further development secured by the utilization of anaerobic methods of culture. The injection of 0.5 c. c. of these pure cultures under the skin of the back produced the same characteristic yellowish white area of muscular necrosis about the point of inoculation and the peculiar penetrating odor so constant with this bacillus. The course of the disease is about the same as when an emulsion of the fresh tissue has been injected, and, in those cases in which life was prolonged several weeks, metastatic areas of focal necrosis were always noted. The approach of death is usually indicated by convulsions; the animal 24 BUREAU OF ANIMAL INDUSTRY. comes out of one to be seized with anotlier, death general!}^ resulting in a few hours after the onset of the first convulsion. In the majority of rabluts that succumbed to this disease, a marked rigor mortis was observed, especially noticeable in the hind quarters, causing the back to be arched and the legs contracted as if in a tetanic spasm. Micro- scopic examination of tissue taken from the necrosed area or from metastatic lesions of the liver or lungs shows the presence of typical necrosis bacilli in great numbers. In the case of organic lesions these filaments are seen to be arranged along the border of the area of necrosis, whereas the central portion is amorphous and does not reveal the presence of any microorganisms. Intravenous metliod. — The intravenous method of inoculation was adopted in nine cases, four of which resulted in the death of the animal. Each rabbit received in the posterior auricular vein 0.3 c. c. of an emulsion of the tissue filtered through cotton, the filtrate con- taining numerous bacilli. The course of the disease ranged from seven to twelve days. The symptoms exhibited in all cases were the same as those induced by subcutaneous inoculation and consisted of gradual emaciation followed by the loss of appetite and by convulsions, paralysis, and death. The postmortem examination showed the principal lesions to be located in the thoracic cavit3\ The lungs contained several caseous nodules the size of peas surrounded by a hemorrhagic zone, and in one rabbit the left principal lobe was adherent to the costal pleura by a thick, purulent exudate. In each case the costal pleura contained several metastatic foci and the chest muscles in two instances were the seat of Description of Plate 3. Section of lung of rabbit No. 1197, inoculated subcutaneously with a bouillon culture of Bacillus necrophorus, showing four metastatic foci. Stained with Lo filer's methylene blue. Magnified al)out 25 diameters. Description of Plate 4. Section of lung of rabbit No. 1197, showing the superior focus in plate 3 greatly amplified. Notice the felted network of wavy filaments and smaller forms of Bacillus necrophorus. Stained with Loffler's methylene blue. Magnified about 450 diameters. Description of Plate 5. Fig. 1. ^Cover-glass preparation from a metastatic focus in the wall of the left ventricle of rabbit No. 1219, which died on the fourteenth day following a subcutaneous injection of a Martin's bouillon culture of the necrosis bacillus. Stained with Ziehl's carbol-fuchsin. Composite drawing made with camera lucida at base of stand with Zeiss No. 6 compensating ocular, 2 mm. oil immersion objective and 160 mm. tube length. Fig. 2. — Photograph of Petri dish containing numerous characteristic colonies of necrosis bacilli forty-eight hours after their inoculation into bouillon agar and fol- lowing their inculcation at 39° C. in a closed jar containing pyrogallic acid. Notice the gas formation and the fluffy outlines of the colonies, with their dark dense centers. Bulletin No. 67, B. A. I. Plate 3. ^.\0 ^' Section of Lung of Rabbit Showing Metastatic Foci. Haines del Bulletin No. 67, B. A. I. Plate 4. r ^'^ %, V^ * •!,. 4/* ^j'' * / / t,^wr Section of Lung of Rabbit Showing Felted Network of Bacillus necrophorus. Haines del Builstin No. 67, B. A. I. Plate 5. FiG. 1.— Bacillus necrophorus from Metastatic Focus in Rabbit's Heart. Fig. 2.— Petri Dish Containing Characteristic Colonies of Necrosis Bacilli. X 2. NECROTIC STOMATITIS. 25 one or two localized areas of coagulation necrosis. In another the caseous process had included the entire surface of both lungs, the pericardium, and the heart itself until all were superticially fused into one chees,y mass. The liver of one animal contained eight superticial areas of caseation varying in size from a pea to a hazelnut and was adherent to the diaphragm and abdominal muscles. All other organs were normal. No lesion was observed about the point of inoculation in three cases; the fourth showed a narrow strip of necrosis along the line of the vein for about half an inch. Cover-glass preparations made from the lesions contained numerous characteristic specimens of Bacillus n ecrophor m. " Experiments on Mice. Sithcutaneous ?V?oci/ia^/w?.— The remarkable susceptibility of a white mouse to "necrophorus infection'' makes this animal an excellent medium for the preservation of the virulence of that microorganism, as well as a means of obtaining it in pure culture. Unfortunately, this animal's extreme susceptibility to many forms of the bacterial flora of the mouth disqualities it for use in the early stages of an investigation. The inoculation experiments may be carried on In- placing a small bit of necrosed tissue into a pocket in the skin of the back, or by injecting into the same region 0.1 c. c. of a fluid culture or emulsion containing the germ in a pure state. In two to four days the point of inoculation is covered with a rather thick, blackish, or dark-brown dry scab around which is a zone of redness. Beneath this scab proceeds a coagulation necrosis spreading through the subcutane- ous tissues until the mouse is completely mummifled, shriveled up. and covered with a dry parchment-like coating; or. penetrating into the body cavities in its course, successively caseates muscle, cartilage, bone, and viscera. In some of these cases so general becomes the caseous process that it is difficult to decide whether the viscera have suffered embolic necrosis or have become involved in the progressive caseation through extension by contiguity. In cases in which necrotic action was less widespread, small focal necroses containing the Bacillm o^ecrophorus in pure culture were found in the lungs, liver, and spleen. In our experiments mice have died as early as five, and as late as twenty-one days. « While these pages were in press an interesting experiment in natural infection was completed. Rabbit 1262 died, presenting a complete picture of Schmorl's dis- ease. It had been associated with an inoculated rabbit affected with a " necropho- rus " ulcer on the right ear, and was repeatedly noticed licking this sore spot. The necropsy revealed a necrotic stomatitis involving the gums, dental alveoli, body of the inferior maxillary, floor of the mouth, sublingual and submaxillary glands, and the tissues of the face and neck. There were no internal necroses. Cover-slip preparations and inoculations, both artificial and animal, demonstrated the presence of Bacillux necrophorus. 26 BUREAU OF ANIMAL INDUSTKY. ExPERUrEXTS ON GUINEA PiGS. Guinea pigs were inoculated with pure cultures of necrosis bacilli by both the subcutaneous and intraabdominal methods, but these were followed by negative results. Nine animals were used in the experiments, and doses ranging from 0.25 to 1 c.c. of a forty-eight-hour bouillon culture were injected without producing any unfavorable effects. However, one positive result was obtained with guinea pig No. 3181, which was injected intraabdominally with 0.5 c.c. of an emulsion of the necrosed tissue taken from the mouth of calf No. 3. Death fol- lowed on the tenth day. On autopsy the carcass appeared emaciated. The point of inoculation in the prepubic region was surrounded by an abscess several millimeters in diameter containing a rich yellowish pus. In the linea alba several inches above this abscess was a metastatic nodule 1.5 cm. in diameter, involving the muscular tissue and the peritoneal lining. It contained the same character of pus. On the right side of the linea alba the peritoneum was adherent to the contiguous loop of the colon opposite the last rib. The right and left lobes of the liver were almost fused into one by abscess formation. The right lobe particularly was the seat of several abscesses 1^ cm. long ])y 1 cm. wide, with a line of demarcation plain and straight between the part abso- lutely necrosed and the invaded portion. The spleen appeared mot- tled and slightly enlarged, and the kidneys were deeply congested. The lungs were engorged on the right side and showed one or two foci of hepatization. The presence of Bacillus necrophorm in the liver abscesses was demonstrated by microscopic preparations and by animal inoculations. Experiments on Chickens. Four fowls were injected— two l^y intravenous and two by intra- muscular inoculations- with 0.75 c.c. of a forty-eight-hour Martin's bouillon culture, but in no case was the result fatal. Nothing was observed that would indicate that the birds had been injected, and after a period of two months they were chloroformed. The postmortem examination revealed absolutely healthy birds. e;xperiments on Pigeons. The inoculation of two pigeons indicated that the bacillus is not pathogenic for these birds. One pigeon was inoculated intravenously with 0.3 c.c. of a forty-eight-hour culture in Martin's bouillon, the other bird with 0.5 c.c. of a similar culture into the pectoral muscles. This experiment was concluded six weeks after its inauguration by chloroforming the pigeons, but in neither case was any abnormal condition apparent. EXPERIiMENTS ON CaLVES. Suheutaneous inoculations. — Heifer calf No. 351, 7 months old, was injected October 5, 1904, on the inside of. the upper lip with 1 c.c. NECROTIC STOMATITIS. 27 of an emulsion prepared from the point of inoculation of rabbit No. 1215, which was the seventh in a series of animals inoculated with the material from a calf affected with necrotic stomatitis. October 7. — There is a slight reddening about the site of injection, with a temperature of 102.6° F. October 10. — The temperature has reached 103.2° F., and a large tumor with a soft center is observed at the point of inoculation. Pus can be seen oozing from the opening made by the needle in the lip. October 11. — There is an enlargement about 1 by 2 by 1 inch in size at seat of injec- tion, with an ulcerating surface surrounding the point of puncture. October 12. — The caseous nodule is about the same size, and is still suppurating. October 14. — The enlargement on lip does not appear to have increased any in size. An opening simulating a rodent ulcer about one-half inch in diameter has formed ( n the inner side of lip which leads to the pus cavity in the center of the necrotic area. Thick creamy yellow pus issues from it. October 17. — The pus has been evacuated from the abscess in lip, and the lesion has begun to heal. October 18. — The lesion in the mouth is healing rapidly, and the animal has been removed from the experiment. On Auoust 1-1, 1904, a fragment of necrosed tissue about one-half the size of a pea was inserted into a pocket made beneath the mucous membrane on the right side of upper lip of calf No. 31S, 4 months old. The tissue w^as taken from the seat of injection of rabbit No. 822, the second in a series of rabbits inoculated with a pure culture of the Bacillus necrophonis obtained from the lip of a Berkshire shoat affected with necrotic stomatitis. August 15. — Temperature of the calf is 103.2° F., and an intiannuatory z(jne is noticed about the punctured mucous membrane. Atigust 17. — At the seat of inoculation there is a hard spheroidal enlargement about 1 inch in diameter. August 18. — The abscess at point of inoculation has not increased any in size. A plug of pus and necrotic material of one-half cubic inch in volume has been extruded from the caseous nodule in the mucous membrane at the seat of injection. August 19. — A small quantity of very thick pus is being secreted from the abscess. Temperature remains about the same as on previous days, 103° F. August 22. — Only a small ulcer with a slight reddening at seat of inoculation remains. Temperature 104.8° F. August 24. — Lesion is healing; appetite is good; temperature 104° F. September 1. — The ulcer at seat of inoculation has about healed. Temperature 102.4° F. September 12. — Animal removed from this experiment. Roan calf No. 350, 2 months old, was inoculated May 24, 1904, on the lower portion of the left cheek with tissue taken from rabbit No. 1015, which was one of a series of rabbits injected with the necrosis bacillus obtained from a case of foot-rot in sheep. May 26. — There is a reddening of the mucous membrane about the seat of injection. May 27. — The animal appears dull and languid and eats sparingly. The mucous membrane is of a grayish color and presents an ulcer about \h inches in diameter. 28 BUREAU OF ANIMAL INDUSTRY. May 28. — A distinct and characteristic ulcer has formed, containing a caseous necrotic center surrounded by a zone of inflammation. From the caseous material scraped from the deeper portion of this ulcer rabbit No. 1117 was injected under the skin of the back. (This rabbit succumbed on the sixth day following, with charac- teristic lesions, from which the Bacillus necrophorm Avas obtained. ) May 31. — Point of inoculation in cheek of calf still shows ulceration, but swelling and inflammation are disappearing. June 11. — Healing is about completed, there being only a small, depressed ulcer remaining. Suhcutcmeo'us and intravenous inoculation. — On November 27, 1903, calf No. 337, 11 months old, was injected on the left side of tongue with 1 c.c. and inoculated in the right jugular vein with 5 c.c. of a culture of Bacillus nec7'02)horus obtained from necrotic stomatitis of calves. December 3. — The animal has remained perfectly well in every respect. No lesions. December 16. — The calf to all appearances continues well. December 26. — General condition remains good. Temperature has been normal throughout the experiment. Owing to the negative result of this experiment it mav be concluded that the animal in question was not susceptible to the injections, proba- bly on account of its age. Intravenous inoculations. — Gray calf, No. 359, was injected intra- venously (right jugular vein) with 10 c. c. of a culture of the Bacillus necrophorus obtained from necrotic stomatitis of calves. November 23. — The only reaction shown from the injection yesterday is the temper- ature of 103° F. November 24- — The temperature reaches 106.8° F. The calf is now very sick, walks with a staggering gait, and hock joints are flexed much more than normal. It trembles, and eats scarcely anything. November 25. — The animal shows about the same symptoms as yesterday, but more marked; pulse, 104. November 26. — It is considerably weaker than on November 25, and has difficulty in getting on its feet. Temperature, 107° F. November 28. — The condition is about the same; very weak, but can stand up; spiritless and refuses food. November 29. — The calf seems somewhat weaker and walks with a staggering gait. Temperature, 104.6° F. November 30. — It is still very weak and more stupid than yesterday; appetite is much improved. Temperature, 104° F. December 1. — It shows no improvement in general appearance, but has a much better appetite. December 2. — The condition is same as yesterday. Animal killed by bleeding. The postmortem examination held shortly after death showed the carcass to be in a rather poor condition and the hair and skin dry and harsh. On opening the thoracic cavity the lungs were found to con- tain a number of encapsulated caseous nodules scattered quite regu- larly in the peripheral and superficial portions of the lobes. There were nine such areas in the right lung and seven in the left lung. Four of these nodules were surrounded b}^ hemorrhagic zones, while NECROTIC STOMATITIS. 29 the remainder bore a striking- resemblance to the early pulmonary lesions of caseous lymphadenitis of sheep, and were circumscribed by normal lung tissue. On sectioning the}^ were seen to contain a yel- lowish white, thick, rather dry mass, from Avhich the necrosis bacillus was obtained in purity. The liver showed three small superticial foci of necrosis the size of a grain of wheat, while the heart, spleen, and kidneys were apparently without lesions. No alteration of any kind was observed at the point where the intravenous injection was made. Culture media were inoculated with the caseous material from the necrotic areas in the lungs, and a rabbit was injected subcutaneously with similar material, with the result that characteristic development in the former case occurred within forty-eight hours, and in the latter case death of the rabbit followed on the eighth day with typical post- mortem lesions. EXPERIiMENTS ON SHEEP. Suhci(t(i7ieoiis hwculatiom. — On August 22, ll>0-i. a small fragment of necrotic tissue, obtained from the same source as that used on calf No. 318, was introduced into a pocket made under the mucous mem- brane of the right cheek of sheep No. 23, about 1 year old. August 24. — At the seat of inoculation in the mouth there is an inflammatory proc- ess which has caused a small ])atch of necrosis in the mucous membrane. Temper- ature 104° F. August ^6'.— The ulcerous area in the mouth has reached one-half inch in diameter and is secreting pus. The inflammation and swelling are somewhat reduced. Tem- perature 103.6° F. August 30. — The lesion at the point of injection is decreasing in size and the tem- perature of the animal is 102.4° F. September 1. — The ulcer has nearly healed. Temperature 102° F. September 9. — No lesion at seat of inoculation. Condition of animal is good. Sheep No. 102, pure Merino, born in the fall of 1902, was inoculated May 9, 1903, on the lower lip with a culture of necrosis bacillus obtained from necrotic stomatitis of calves. Maij 21. — No symptom of the disease has thus far appeared. June 1. — No lesions are to be seen in the mouth, and the sheep seems to be well and gaining flesh. June 5. — Animal is removed from experiment. Experiments on Pigs. Suhcutaneous inoculation. — On October 5, 1904, pig No. 1448, weigh- ing 50 i^ounds, was injected inside of upper lip on the right side with 1 c.c. of the emulsion obtained from the same source as that used on calf No. 351. Following the injection the pig continued in good con- dition, and no alterations were observed at point of inoculation until October 12, when a small inflamed swelling was detected, which showed a small necrotic center. -By October 14 this enlargement had reached the size of a hazelnut, with a small ulcerative surface about 5 cm. in 30 BITREAU OF ANIMAL INDUSTRY. diameter. The lesion remained stationary for several days, when healing started, and by November 2 the ulcer had almost disappeared. On November 12 the animal was in good condition, without any altera- tion at seat of injection. Pathology. The pathologic process which everywhere characterizes Bacillus necfophorus is a necrobiosis, involving coagulation necrosis with sub- sequent caseation, and marked by a progressive invasion of the sur- rounding (particularly the deeper) tissue and a remarkable tendency to metastasis. When this process is applied to a mucous membrane we have presented to us a diphtheric intiammation plus caseation. A discussion of terms at this point is necessary for clearness of definition. Tlie Greek (5zrt's)oi this disease has also been isolated in this laborator}^ from alxscesses iii the livers of cattle slaughtered at Kansas City and Chicago, from the liver of a deer from the National Zoological Park, and from sheep affected with foot-rot. would indicate that the organism is widespread in this country-, only awaiting an opportunity for entering the tissues of a susceptible animal. In reviewing the economic importance of this disease, considera tion must be given to the other infections produced by BadUns- oiecrophoynis, some of which are even more grave than necrotic stomatitis. The presence of the latter disease on a farm would indi- cate the possibility of the causative germ affecting other susceptible animals in tissues already referred to in the chapter on pathogenesis. Thus the importance of this organism is far beyond even its relation to necrotic stomatitis of calves and pigs since foot-rot of cattle and sheep, necrotic inflammation of the uterus and vagina of cows, a simi- lar inflammation of the large intestine, and necrotic scratches of horses, gangrenous processes of the lips and nose, and necrotic stomatitis in sheep, may all follow on premises contaminated with the infectious principle of this disease. Among the letters that have been received 38 BUREAU OF ANIMAL INDUSTRY. requesting information relative to this malad}^ the following will con- vey a general idea of its nature and importance: Within the last two weeks several complaints from different parts of the State, mostly from the mountain regions, have come into this office regarding a disease in calves that were dying from sore month. I have just returned from Gunnison, where I examined several calves that died of this disease. I found that at the base of the tongue and in the larynx there were large sores, some of them as large as a silver dollar. They presented a thickened croupous matter usually white in color. After this matter was scraped off with a knife, a red granulated membrane was left. The owner told me that in some cases these sores appeared on the anterior part of the tongue, but in the cases I saw they were at the base of this organ. The trouble occurs in sucking calves of one to three months old. The first symptoms noticed are that they can not swallow, and soon cease to nurse. In calves that are old enough to eat hay or other forage, large boluses Of food are found between the lips and teeth, giving the apjiearance of a large swelling on the side of the face. Practically speak- ing, 90 per cent of the affected calves have died. One party lost 21, another 10, with no cases recovering on their fainis. Another correspondent from Salida, Colo., writes: We have lost 33 calves with throat and mouth disease and will lose more. A stock owner, writing from Lithia, S. Dak., states: There is a disease attacking my calves and which is new to the cattlemen of this vicinity. The calves stop eating; about three days after they begin to foam at the mouth, but continue to move their mouths. The throat swells very hard and the jaws are set tight, so that they can not open. A foam}^ pus discharges from the nose two days before they die, and they seem to strangle to death from accumulation of this pus in the nose and throat. They have high temperature and stand up and lie down alternately during the last two days. The bowels move freely and there is a tendency to urinate frecjuently. Death occurs in four or five days. Upon cutting into the throat or larynx and nasal cavities, they were found completely filled with thick pus. The muscles around the larynx and throat and back and under the tongue seemed to reseml)le the contents of an abscess. The heart and lungs seemed to be in their natural condition. I now have 7 head that have this disease. From Aladdin, Wyo., the following was received: A disease has broken out among my calves. I have 117 head and I believe half of them have sore or ulcerated tongues. I can not see that they are affected other- wise. The sore is generally located on the top of the tongue about the center and in some cases has eaten the tongue nearly in two. Two head have died, and in one of them nearly the entire surface of the back half of the tongue had rotted awaj' and contained numbers of cavities filled with pus, but I could not find any foreign sub- stance in them. A letter from Belle Fourche, S. Dak. , indicates that the disease is amenable to treatment. There are a number of herds (calves) affected in this country, and I know of 1 cow. I have been using boracic-acid wash for mine, and they seem to be improving. A ranchman sends the following record of his observations from Bixby, S. Dak.: All of my calves and some of my yearlings have some kind of a disease. Their jaws swell and sometimes their throats, and when I come to examine them I find NECROTIC STOMATITIS. 39 that their jaws are sore on the outside of the grinders and inside the cheek. In some it rots holes in their tongues, mostly at the back part. The breath smells terribly and the calves get dumpy and poor. A letter from Mason ville, Colo., sng-g'ests the possibility of the disease originating from foot-rot in cattle. I should like your opinion of what might be called an epidemic among my calves. Three of them have been attacked with swellings in the lower jaw on both sides. Yesterday one of the calves died, and on examining the jaw^ I found it apparently rotten on the inside from the grinders down. There was no pus. Last summer I noticed a calf of this same herd similarly afflicted, but it came out all right. Last summer about 10 per cent of my cattle were troubled with their feet cracking between the hoofs. They became very lame. Mr. G., of Wheatland, Wyo., requested— information regarding some young calves I have. There are 2 calves less than a month old running with their mothers. One of them has his jaw swollen up on one side. The other calf has both sides of the jaw and under the jaw swollen. I exam- ined their mouths and found "canker" alongside and behind their teeth, and it was awfully offensive. A few weeks ago I lost one six-months-old calf with a similar disease. Many cases of sore mouth in pigs are reported from various sec- tions of this country, and, assuming that these cases were similar to those investigated by us, it would seem that this disease causes the death of man}^ young animals and also stunts the growth of many that survive. Almost ever}^ pig in the litter suffers, and litter after litter becomes affected. The same lack of recognition of the disease as in calves prevents any accurate description of the importance of the affection in pigs, but in a few outbreaks observed it was noted that from 60 to 90 per cent of the animals in the herd died if left untreated. The disease in pigs, while usuall}^ affecting the young, has been reported at all ages, from sucking pigs one week old to hogs two years of age. One writer reports a loss of 33 out of 50, another 33 out of 37, while a third states that of 12 four-months-old shoats 11 succumbed to the disease. Differential Diagnosis. Necrotic stomatitis mav be differentiated from foot-and-mouth dis- ease by the fact that in the latter malady there is a rapid infection of the entire herd, as well as of any hogs or sheep that may be on the prem- ises; it is also highly infectious, spreading rapidly to neighboring herds and to cattle of the same herd. The characteristic lesion of foot-and-mouth disease is the appearance of vesicles containing serous fluid in the mouth, upon the udder, teats, heels, and coronary bands of the affected animals. Drooling is profuse, and there is a peculiar smacking sound made by sucking the affected lips. In hogs the mouth is not so likely to be affected as are the feet. Foot-and-mouth 40 BUREAU OF ANIMAL INDUSTRY. disease has appeared in this country on four occasions onl3\ always near a seaport, and does not exist in the United States at the present time. In necrotic stomatitis vesicles are never formed, necrosis occur- ring- from the beginning- and followed by the formation of yellowish patches, principally in the mouth. M^'cotic stomatitis is a sporadic disease which atl'ects cattle of all ages that are on pasture, but more especially adult animals. It is characterized by inflammation and ulceration of the mucous membranes of the mouth, producing salivation and inappetence, and, secondarih', afl'ecting the feet, which become sore and swollen. Superficial erosions of the skin, particularh' of the muzzle, and of the teats and udder of cows may also be present. It occurs onl}" in a few animals of the herd, usually in the earl}' fall after a dry summer; it does not run a reg'ular course and can not be inoculated. Actinomycosis of the tongue ma}" occur as superficial erosions but is usually deep-seated, where, instead of producing ulceration and destruction of the mucous membrane, it causes a difl'used induration of the muscular tissue of the tongue, resulting in an enlargement of that organ. Hence the term '"wooden tongue'' has been applied to this condition. The course of the disease is chronic and the tongue is extensively affected before attracting the attention of the owner. The indurated tissue when incised is found to be hard and gritty and con- tains bright yellow sulphur-like granules of actinomyces, which are the causative agents of the disease. Prognosis. Ordinarily animals affected with necrotic stomatitis show no tend- ency to spontaneous cure. Left to themselves, they either die or become permanently stunted in growth. On the contrary, if taken in hand early, the disease is readily amenable to treatment. In the latter event the prospects of recovery are excellent, and under favorable con- ditions it takes place as a rule in twelve to fifteen days. Treatment. prophylaxis. Prophylaxis should be carried out along three lines: (1) Separation of the sick from the healthy animals; (2) close scrutiny and thorough disinfection once daily for five days of the mouths and nasal passages of those animals that have been exposed by the eruption of the first teeth, by the shedding of the milk teeth, or through association with affected animals; (3) complete disinfection of all stalls, sheds, and NECROTIC STOMATITIS. 41 farrowing pens with a 5 per cent solution of carbolic acid to which has been added sufficient lime to make the disinfected area conspicuous. The disease appears to break out in winter and holds over to spring. It is conceivable that exposure to cold might so disturb the normal circulation of the oral tissues as to make the mucous membrane an excellent location for the development of the causative factor of the disease. There is another possibilit}^ however, which bears on the third line of prophylaxis. The diphtheric inflannnation of the vagina and uterus in cows is caused by the same organism that induces necrotic stomatitis. Ellinger ^^ has recently pointed out the almost constant relation of such attacks to previous occurrences of foul-foot or foot-rot in the same or other cattle on the place. In all likelihood, the stalls and sheds are the harborers, in such cases, of this germ. Wh}' may it not be that many of these outbreaks of necrotic stomatitis in calves have some relation to preceding cases of the above-mentioned diseases and the greater use in winter of stalls and sheds harl)oring the Bacillus necrophorus; and in pigs to the fact of their following cattle affected with one of the above diseases'^ THERAPEUTICS. The treatment consists almost solely in careful and extensive cleansing and disinfection of the mouth and other affected surfaces. The mucous membrane of the mouth should be copiously irrigated with a 2 per cent creolin solution in warm water. This should be performed at least twice daily, but care should be used with this as with all other antisep- tics, to prevent swallowing of any considerable quantity of the solution. Since exposure to oxygen kills the bacilli one need have no fear al)out disturbing or tearing off the caseous patches or necrotic tissue during irrigation. The irrigation of the sores should be followed by the application with a brush or rag on a stick of a paste made with salicylic acid 1 part and glycerin 10 parts; or the affected spots may be painted with Lugol's solution of iodine (iodine 1, potassium iodide 5, water 200). Frequent injections into the mouth of a 1 per cent car- bolic-acid solution make an excellent treatment. In calves the inter- nal administration of 2 grams of salicylic acid and 3 grams of chlorate of potash three times a day has also proved very benelicial. A very simple and efficacious method of treating pigs is to catch them and hold their heads for a few moments in a solution containing in each gallon of water 2 ounces of potassium permanganate, or in a solution of creolin of like strength. This treatment ma}' be repeated twice daily for six daj's. If the predisposed animals are sucking, it would also be advisable to batbe the udder and teats of the cows or sows with a 5 per cent solu- 42 BUREAU OF ANIMAL INDUSTRY. tion of carbolic acid in order to prevent infection from tlie probably contaminated manure on these parts. In animals that have been weaned it will be necessar}" to feed soft, nutritious food, such as whole milk, bran mashes, ground feed, and gruels, and to keep clean, cool water constantly within reach. Kelation of Animal Diphtheeias to Man. We have already shown that the cause of necrotic stomatitis and the cause of human diphtheria are entirely distinct microorganisms. We have also shown that the disease process in necrotic stomatitis of animals is markedly difl'erent from that in human diphtheria. A superficial resemblance in the local manifestations of the two diseases and a total misconception of the cause of each gave rise to an identity of terms which is confusing. As we have elsewhere stated, calf diphtheria is a misnomer, false in its characterization of the disease, and misleading in its effect upon the mind of the public. The same statement holds with reference to so-called chicken diphtheria, or roup, and similar affections in avian species. Many different species of the mammifeis have revealed disease processes in mouth and throat that superticially resemble the diphtheritic process in man, but the weight of evidence at the present time goes to show that the patho- logical alteration in man}^ of these cases is not identical with that in human diphtheria. However, satisfactory proof has been offered of the recover}^ of the bacillus of diphtheria from a few isolated cases, especially in the cat, horse, and dog. At this point we would remind the reader that in the human famih^ diseases of the mouth, throat, and upper air passages frequentl}" arise which closely simulate diph theria, giving rise to almost identical clinical manifestations, but which upon bacteriological examination fail to yield the Klebs-Loffier bacillus, thus proving them to be distinct from diphtheria. In view of the apparent resemblance between the true and false diphtherias of man and animals, and because the former disease appears to be inter- communicable between man and animals, we urge upon the reader the isolation of all animals, particularly pets, affected with mouth and throat diseases, and the most punctilious observance of all details of sanitary precautions. Disposition of Meat of Affected Carcasses. If necrotic stomatitis were transmissible to man, the germicidal action of cooking might be counted on to obviate such danger. Proof that necrotic stomatitis is not transmissible to man does not demon- strate that the meat of animals so affected is fit for food. Other fac- tors enter into this question of the edibility of such meat. The disease NECKOTIC STOMATITIS. 43 ma}' be regarded as at tlie lirst a local affection. Now it is manifest that those portions of the animal body affected with caseonecrotic patches should be condemned for food. The remaining- portions of the carcass, however, if in a good state of nutrition, might be placed on the market. On the other hand, should the disease have progressed beyond the condition of localization to a condition of toxinemia, which would be evidenced by emaciation, enlargement and discoloration of the lymph nodes, and cloudy swelling of the liver, the carcass should be rejected as both innutritions and noxious. Conclusion. We ma}' note that in necrotic stomatitis of animals we have to do with an acute, infectious inflammation of the mouth and upper air passages, of widespread occurrence both geographically and zoologic- alh", caused by the invasion locally of a thread bacterium, the Bacilhts necrophoTux^ a nonmotile, poh^morphic anaerobe, inhabiting normallv the intestinal tract of hogs and, in all probability, of herbiv- ora, and found also in manure. The disease is observed most frequentlv in calves and pigs, and usually in connection with the eruption of the lirst teeth, although other injuries of the mucous membranes may ])e the starting point. The disease process is a clearly defined cheesy degeneration of a pro- gressive coagulation necrosis characterized by the formation of ulcers and caseonecrotic patches, and manifesting itself clinically by saliva- tion, refusal to suck, difficult breathing, and rapid emaciation. The duration of the disease is from five days to five weeks, these animals, if untreated, dying early with a toxinemia. Necrotic stomatitis is not difficult of diagnosis and may quite easily be differentiated from other inflammations of the mouth, is most amenable to treatment, and hence offers a highly favorable prognosis. The exquisitely infectious nature of the cause rendering most easy the rapid development of an enzootic, and favoring the spread of other forms of bacillary necrosis, some of them even more grave than necrotic stomatitis, lifts the disease to a high place in agricultural economics. Evidence is not at hand for pronouncing it transmissible to man, and it is absolutely distinct from human diphtheria. 44 BUREAU OF ANIMAL INDUSTRY. BIBLIOGRAPHY. Ill the bibliography appended all the works referred to in the text have been given and several additional references have been included which bear more or less directly on the subject-matter. (1) Bang, B. Om aarsagen til lokal nekrose. Maanedssk. f. dyrl., bd. 2, p. 235. KJ0ben- havn, 1890-1891. Referat by C. O. Jensen. Ueber die ursache der nekrose. Cent. f. bakt., bd. 13, no. 5-6, pp. 201-203. Jena, Feb. 17, 1893. (2) Banc;, B. De l)akteriologische forhold ved svinepesten. Maanedssk. f. dyrl., bd. 4, p. 190. Kj0benhavn, 1892-1893. Referat by C. O. Jensen. Cent. f. bakt., bd. 13, no. 5-6, pp. 20.3-205. Jena, Feb. 17, 1893. Referat l)y C. O. Jensen. Jahresb. ii. d. fortsehr. in d. lehre v. d. path, mikroorganismen, jahrg. 8, pp. 141-143. Braunschweig, 1892. (3) Berg, V. Gangra^n0s mundbettendelse hos k0er. Maanedssk. f. dyrl., bd. 8, no. 6, pp. 205-207. Kj0benhavn, Sept., 1896. (4) Berndt, K. T. Die lebernekrose bei rindern. Arch. f. wissensch. u. prakt. thierh., bd. 21, no. 2-3, pp. 194-196. Berlin, 1895. (5) Blazekovic, F. Znr kenntniss der kiilber-diphtherie. Deut. ztschr. f. thiermed., bd. 4, no. 1-2, pp. 64-77. Leipzig, Mar. 12, 1878. (6) Campbell, Thomas. Muco-gastro-enteritis in calves. Vet. jrn., v. 14, pp. 404-408. London, June, 1882. (7) Caudwell, William. Bacillary necrosis of the lungs in a calf. Vet. rec, v. 14, no. 713, p. .552. London, Mar. 8, 1902. (8) Cole, R. R. The transmissibility of diphtheria from calves to pigs. Vet. jrn., v. 13, pp. 395-396. London, Dec, 1881. (9) Dammann, Carl. Die diphtherie der kiilber, eine neue auf den menschen iibertragbare zoonose. Deut. ztschr. f. thiermed., bd. 3, heft 1-2, pp. 1-27. Leipzig, Nov. 30, 1876. (10) Dammann, Carl. Die gesundheitspflege der landwirthschaftlichen haussaugethiere. 1318 pp. 23cm. Berlin. Paul Parey, 1886. (Seep. 1007.) (11) Dieckerhoff, Wilhelm. Lehrbuch der speciellen pathologie und therapie fiir thieriirzte. 2 v. 244 cm. Berlin, August Hirschwald, 1891-1894. (See bd. 2, lief. 1, pp. 102-105, 1891.) (12) Diem. Diphtheritis bei liunmern. Woch. f. thierh. u. viehz., jahrg. 41, no. 36, p. .339. Miinchen, Sept. 7, 1897. NECROTIC STOMATITIS. 45 (13) Ellinger. Zur casuistik der iiljertragbaren bosartigen scheideuentziindungen des rindes mit besoiiderer berucksichtigung der wirkungen des necrosebacillus, zu- gleich audi eiu beitrag zur lehre von der puerperalen infection. Berl. thieriirztl. woch., no. 2, pp. 25-29. Berlin, Jan. 8, 1903. (14) Ernst, Wilhel.m. Ueber nekrosen und den nekrosebacillus ( Streptothrix necrophora ) . Monatsh. f. prakt. thierh., 1)d. 14, hft 5, pp. 193-228. Stuttgart, 1902. ( 15) Feldmann. Diphtheritis beini kalbe. AitIi. f. vet. med. Referat. Revue f. tierheilk. u. viehz., bd. 1, no. 6, p. 94. Wien, June 1, 1878. (16) Fleming, George. The transmissibilitj' of diphtheria from animals to mankind. Vet.jrn., v. 13, pp. 153-171. London, Sept., 1881. (17) Fleming, George. Diphtheria in calves. Vet. jrn., v. 13, pp. 311-315. London, Nov., 1881. (18) Flugge, Carl. Die mikroorganisnieu mit beson.lerer beriieksichtigung der iitiologie der infektionskrankheiten. 23 cm. Ed. 2. Leipzig, F. G. W. Vogel, 188H. (See pp. 265-266 (Bacillus diiihtheriie vituloruni, LofHer); p. 276 (Bacillus necrophorus, liotfler).) (19) Flugge, Carl. Idem. Ed. 3, 2 v., 1896. (See p. 61, theil 2, (Streptothrix cuniculi, Schmorl). ) (20) Foulerton, Alexander G. R. Streijtothrix infection in the lower animals. Jrn. of comp. path, and therap., V. 14, pt. 1, pp. 45-59. Edinburgh and London, Mar., 1901. (21) Francke. Der necrose-bacillus als krankheitserreger bei unseren hausthieren. Berl. tiehirztl. woch., no. 25, pp. 299-303. Berlin, June 22, 1899. (22) Gasperini, Gustavo. Ricerche morphologiche e biologiche sul genere actinomyces harz. Ann. dell, ist. d' igiene sperim. dell. r. univ. di Roma, v. 2 (nuova serie), pp. 167-228. Roma, 1892. (23) Grappunder. Die schweinepest in der Neumark. Berl. tieriirztl. woch., no. 4, pj). 39-43. Berlin, Jan. 25, 1894. (24) Gratia. Quelques considerations speciales sur la diphterie. Ann. de med. vet., v. 39, no. 1, pp. 1-11; no. 2, p. 81-87. Bruxelles, 1890. (25) GuNN, Alexander. Tubercular stomatitis in young cattle. Veterinarian, v. 54, no. 639, p. 170. London, Mar., 1881. <^26) Haeren, M. Den s. k. " brandbacillen " ( nekrosbacillen ) sasom orsaken till brandig difteri hos husdjar. (The so-called necrosis bacilli as a cause of gangrenous diph- theria in domestic animals). Eira, v. 16, no. 11, pp. 336-338. Stockholm, June 15, 1892. 46 BUKEAU OF ANIMAL INDUSTRY. (27) Imminger. Einiges iiber den sogenannten klanenkrebs (klauennecrose) beim rinde. Woch. f. thierh. u. viehz., jahrg. 42, no. 41, pp. 377-382, Oct. 11; no. 42, pp. 381-393, Oct. 18. Miinchen, 1898. (28) James, J. C. 'Gangrenous stomatitis. — Tubercular stomatitis. — Diphtheritis? " A disease affecting tiie tongue and mouth of the ox. V^et. jrn., v. 13, pp. 315-316. London, Nov., 1881. (29) James, J. C. "Tubercular stomatitis." Vet. jrn., v. 14, pp. 12-13. London, Jan., 1882. (30) Jensen, C. O. Die vom nekrosebacillus (bacillus necroseos) hervorgerufenen krankheiten. Ergebnisse der allg. path. u. pathol. anat. des menschen und der thiere, jahrg. 2, 1895, pp. 122-129. Wiesbaden, 1897. Referat. Woch. f. tierh. u. viehz., jahrg. 41, no. 15, pp. 144-147. Miinchen, Apr. 13, 1897. (31) Jensen, C. O. Handbuch der pathogenen mikroorganismen; herausgegeben von Prof. Dr. W. Kolle und Prof. Dr. A. Wasserman. bd. 2, lief. 9-12, pp. 693-706. Jena, Gustav Fischer, 1903. (32) JoHNE, Heinrich Albert. Bericht iiber die pathologische anatomic. Bericht iiber das veterinarwesen im konigreiche Sachsen fiir das jahr 1890, jahrg. 35, pp. 24^1. Dresden, 1891. (33) JoHNE, Heinrich Albert. Bericht iiber die pathologische anatomie. Bericht iiber das veterinarwesen im konigreiche Sachsen fiir das jahr 1893, jahrg. 38, pp. 39-73. Dresden, 1894. (34) KiTT, Theodor. Bakterienkunde und pathologische mikroskopie fiir thieriirzte. Dritte, neu- arbeiteteaufiage. 525 i)p. illus. 245cm. Wien, Moritz Perl es, 1899. (See p. 405. ) (35) KiTT, Theodor. Diphtherische erkrankungen bei schweinen und kalbern. Deut. ztschr. f. thiermed., supplementheft 18, pp. 81-88. Leipzig, 1895. (36) Koch, Robert. Zur untersuchung von pathogenen organismen. Mittheilungen aus dem kai- serl. gsndhts., bd. 1, pp. 1-48. Berlin, 1881. (37) Koudelka, Florian. Ueber kalberdiphtherie. Oesterr. monatschr. f. tierheilk., jahrg. 17, 1892, bii. 16, no. 6, pp. 250-253. Wien, June, 1892. Referat. Berl. tienirztl. woch., no. 40, p. 472. Berlin, Sept. 29, 1892. (38) Kruse, W. Systematik der streptothricheen. (In Fliigge, Carl. Die mikroorganismen, mit besonderer beriicksichtigung der jitiologie der infektionskrankheiten. Ed. 3. 2 V. Leipzig, F. C. W. Yogel, 1896. See p. 61.) (39) Larsen, B. Kalvedifteritis. Maanedssk. f. dyrl., bd. 6, p. 50. Kj0benhavn, 1894. NECROTIC STOMATITIS. 47 (40) Lauritsen, L. J. Nekrotiserende nuindbetit-ndelt-e lios smaagrise. Maanedssk. f.