.t172 Copy 1 002 824 818 BULLETIN OF THE No. 166 Contribution from the Bureau of Animal Industry, A. D. Melvin, Chief January 22. 1915. (PROFESSIONAL PAPER.) OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF GLANDERS. By John R. Mohlek, Assistant Chief of Bureau, and Adolph ^ichhorn, Senior Bacteriologist, Pathological Division. INTRODUCTION. It is no longer doubted that in tlie work of controlling glanders the destruction of the infected animals should be given prompt con- sideration, and, if possible, the infection should be traced to its origm. Unfortunately, the nature of the disease is such that only a compara- tively small proportion of the cases can be recognized by the ordmary clinical examination, and as long as we limit our efforts to the destruc- tion of these cases the disease will contmue to spread. An effective c'ontrol can be accompUshed only by the eUmmation of all centers of infection of glanders. Therefore it is essential primarily to have a means of diagnosing accurately all forms of the disease. Numerous publications have been issued on the various methods of diagnosis, and it seems that while some favor a certain method or methods, others appear to produce sufficient evidence to point out the inadequacy of these methods. There is no question that m the last decade important progress has been made in the diagnosis of this disease. Smce the discovery of mallem, competent investigators have fruitfully studied this phase of the question of the control of glanders, and at the present tune we possess several methods by which we are reasonably sure of diagnosmg practically all cases of glanders. A minimum percentage of failures will probably always have to be con- tended with, as a good many factors enter into the execution of any test. In judging a method which would be the most satisfactory for the diagnosis of glanders various thmgs have to be taken into considera- NoTE.— This bulletin points out the advantageous and satisfactory use of the ophthalmic mallein test in the diagnosis of glanders and the necessity for prompt action on reactors to this test in eradicating this disease. Of interest to veterinarians and State !ive-stock,sanitary authorities. 68247°-15 j^iJ':^"'^^- C^^-H 3 2 BULLETIN 1(J6, U. S. DEPARTMENT OF AGRICULTURE. Jm ' tion, but especially the reliability of the test. It should bo conven- ient, the results should be manifested as early as possible, the reaction should be distmct and well marked, and, probably the most important of all, it should be possible for the practicing veterinarian to apply the test. The last condition must be seriously considered, since the standing of the veterinarian m the community and the confidence of the pubhc in his work wordd be mor(> manifest if m suspected cases he could personally decide on the diagnosis instead of having to depend entirely on the results of serum tests made at some distant laboratory. VARIOUS METHODS FOR DIAGNOSING GLANDERS. It would require a great amount of space to enter into the liistory of the various methods of diagnosis and to enumerate the data we j)ossess on the different tests. The advantages and disadvantages of the various methods, es])ecially of the subcutaneous mallem tests, have been repeatedly published and are accessible to all those who are interested in the subject. There is no question that the sub- cutaneous mallem test is one of the valuable diagnostic agents for glanders, but no one can any longer deny that failures from this test are more numerous than are desu-able. As a matter of fact, the uncertainty of the results from this test caused numerous investi- gators to seek some other method which might replace the sub- cutaneous mallein test. Besides the failures resulting from it, the technic of executing the test, together with the time required for its determmation, make it unpopular with many veterinarians and sani- tary officers. Of the other tests which from time to time hav(> been devised for the diagnosis of glanders, the precipitation, the opsonic, and the con- glutination tests will not be considered, since the results obtained from them are not encouraging. For laboratory tests the combmed agglutination and complement- fixation test will no doubt remam the most satisfactory, and should always be applied in cases where doubt arises as to the results of other tests carried out by the practicmg veterinarian. These latter should be considered as accessory tests and provision should be made everywhere so that m case of doubt the serum could be subjected to the laboratory test mentioned, and the final decision should rest on its outcome. THE OPHTHALMIC MALLEIN TEST. During the last few years the ophthalmic mulU>iu test has gained great favor in the diagnosis of glanders. The popularity of the test is rapidly gauuiig wherever it has been applied, and among its sup- porters we find at the present time the greatest authorities on the d; op c OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF CLANDERS. 3 subject of glanders and on clinical diagnosis. This method of testing is at present officially recognized in Austria, and tlie indications are that ere long it will constitute the official test in other countries. The results obtained m Austria, where the test has been employed for several years, are very gratifying, and Prof. Sclmui'er, of that coimtry, one of oui" greatest authorities on glanders, claims that the control of the disease can be very satisfactorily carried out by the application of the eye test, supplemented in doubtful cases by the agglutination test. Bavaria has recently adopted tliis method of diagnosis for official testmg. In Germany the method is also gaining m favor, and cuiTent veterinary literature contams expressions of satisfaction with this test from many German authorities. In the United States the Bureau of Animal Industry, m consideration of the favorable results obtained, has recognized this method of diagnosis for interstate shipments of equmes. The test has also been officially recognized by the Canadian authorities, and thus far no sanitary offi- cial connected with any of the States in this countiy has decMned to approve this test. The favorable results which have been obtained with this diagnostic method can no longer be denied. Its practicability is apparent, and its use in the control of glanders appears to be now an absolute necessity. — SIMPLICITY OF PROCEDURE. The ophthalmic test has a great advantage over others because of its very simple application. It may be readily executed by any veterinarian, and its other advantages are that the results are obtained in a comparatively short time and are, as a rule, distinct and defoiite. The simplicity of its application is plamly manifest when compared to the subcutaneous test, as it is only necessary to drop two to thi-ee drops of concentrated mallei n mto one of the eyes of the annual to be tested, or, by a still simpler procedure, to dij) a camel's-hair brush into mallem and introduce this mto the conjmictival sac of the anunal. The reaction usually commences in five to six hours after the intro- duction of the mallem and lasts from 24 to 30 hom's. A positive reac- tion is manifested by a purulent secretion from the tested eye. This may be very profuse or slight, somethnes associated with a severe conjunctivitis and edema of the lids, and at other times without any inflammatory symptoms bemg present. At times only a very small quantity of pus may be present in the iimer canthus of the eye. At other times the reaction may manifest a true pyorrhea. The reaction manifests itself ui varymg degrees in the animals, but the intensity of the reaction has no relation to the extent of the dis- ease in the reactor. 4 BULLETIN Itilj, U. S. DEPAKTMEiS'T OF AGKICULTUflE. RELIABILITY OF THE TEST. The available data on the ophthaknic mallem test are suiRcient to draw conclusions as to the rehability of the method, and m Austria alone it has been applied on many thousands of cases with uniformly good results. In considering the good results obtained and the advantages of this method of testing, a concentrated mallein has been prepared for this purpose by the Bureau of Animal Industry, and this was made available to a number of practicing veterinarians who desired to give this method of testing a thorough trial. It has also been employed by inspectors of the Bureau of Animal Industry in their field work, and reports are accessible regarding its action for diagnostic pur- poses on more than 18,000 cases. The results from all sources were uniformly satisfactory. Practicing veterinarians who have given this method a trial have reported very favorably on the results, and the tests conducted by the bureau inspectors on several thousand animals were also satisfactory. The method has been applied here in Washington whenever possible, and recently in some immunizing tests of glanders conducted by the Bureau of Animal Industry there was a good opportunity to repeatedly employ this test. Jn all these instances the results were uniformly good. In cases of glanders there appeared a marked purulent conjunctivitis, and the reaction at times was so severe that the animal could not open its tested eye. BEST RESULTS WITH RAW MALLEIN. The essential factor in obtaining satisfactory results from the test appears to be in the use of the right kind of mallein. It must be by aU means a concentrated mallein, and apparently the best results follow the use of raw mallein, wliich, as a rule, represents the mallein obtained after the concentration of the filtrate from the bouiUon cul- tures of the glanders baciUi. The ordinary mallein used for subcu- taneous testing is not adaptable, and the failures wliich have been reported in the literature were without doubt, in the majority of cases, due to the fact that the mallein employed was not sufficiently concentrated. Marioth ^ correctly asserts that the reaction does not depend as much on the quality and quantity of the mallein as on its concentration. Our experiments in preserving such mallein with the ordinary quantity of 0.5 per cent carbolic acid showed that it does not interfere with the results of the test, although the lacrimation which follows immediately after the introduction of such mallein is more profuse than when carbolic acid has not been added, but this disappears within one or two hours after the application of the test. 1 Monatsh. f. prakt. UerheUk., bd. 24, htt. 7/8, p. 340-373; htt. 9/10, p. 426-456. Stuttgart, 1913. OPHTHALMIC MALLEIN FOE THE DIAGNOSIS OF GLANDERS. 5 PREPARATION OF THE MALLEIN. The concentrated mallein which has been. used for our work and which gave such satisfactory results was prepared at the request of the authors by and in cooperation with Mr. A. M. West, of the Biochemic Division, as follows: The media consists of bouillon containing 5 per cent glycerin, 1 per cent peptone, and 5 per cent NaCl. The reaction is that of the natural acidity of the meat, no acid or alkali being added. The flasks of media are inoculated with virulent cultures of Bacillus mallei and placed in the incubator at 37.5° C. for a period of two months or more. The stock cultures of B. mallei are kept on agar, and their \'irulence is re- newed when necessary by passage through a series of guinea pigs. The well-grown cultures show a heavy mass of organisms, which generally sinks to the bottom of the flask. This growth is of a whitish color splotched with brown. The cultures are then removed from the incubator and heated for one hour in the Arnold sterilizer. Then they are stored for two weeks in ai dark closet to settle. The clear liquid is then carefully decanted and the growth proper is discarded. A measured amount of the decanted licjuid is concentrated over a steam bath to one-third its volume. It is then filled into flasks and sterilized and again filtered while hot, first through one then through three paper filters. Next the clear liquid is passed through a Berkefeld filter. This is followed by a concentration to one-tenth its original volume and by sterilization. To the raw mallein, concentrated to one-tenth its original volume, is added 0.5 per cent carbolic acid and 20 per cent glycerin. Then the liquid is again concentrated to one-tenth its original volume, filtered while hot through filter paper, and sterilized. It is kept in a dark place for a week, and if upon inspection a precipitate is foimd the mallein is again passed through paper filters and sterilized. The finished product is a clear, sirupy, dark-brown liquid,' with a disagreeable odor. The mallein is then bottled, under aseptic conditions, in small ^•ials and is ready for use. It is advisable to provide the mallein for the tests in small vials, each containing about 1.5 c. c. of mallein, which is sufficient for testing 15 horses. After the vial has been opened and part of the contents used for testing, especially if the mallein has been taken out with a camel's-hair brush, the remainder should not be used for tests applied on subsequent days, but should be discarded. THE USE OF DRY MALLEIN. Another form of mallein which has been used quite extensively for the eye test is the mallein siccum, or dry mallein. This represents an alcoholic precipitate of mallein. It is a fine gray powder and must be dissolved in water before it is used. The solution loses its effective- ness in a very short time and must be prepared fresh on the day of the test. Dr. K. F. Meyer, formerly of the University of Pennsyl- vania and now of the University of California, has used the dry maUein extensively, and at the present time this preparation is em- ployed in Pennsylvania for the application of the ophthalmic test. For this purpose two vials are sent out from the laboratories of the Pennsylvania Livestock Sanitary Board, one containing the pow- 6 BULLETIN 166, U. 8. DEPARTMENT OF AGRICULTURE. ciered maUeiii and the other sterile or sahne water in quantities which will make a 5 per cent solution of mallein. The content of the bottle containing the fluid is poured into the bottle containing the mallein powder and the test solution is thus prepared. The results with this form of testing in Pennsylvania appear to be highly satisfactory, as may be seen from a publication by Dr. Meyer on the " Conjunctival reaction for glanders," in the May, 1913, mmiber of the Journal of Infectious Diseases. The advantages of the use of one as compared with the other of these forms of mallein for the eye test are not marked, as equally good ' results were obtained from the application of both forms of this ])roduct. The fact that the preparation of the raw mallein is less laborious and expensive than the mallein siccum and that it is ready for use on opening the vial would probably give this product a greater popularity. It is only natural, however, that in the event subsequent extensive testings show the superiority of the dry mallein, it will be given preference over the raw product. METHOD OF APPLYING THE TEST. Before the application of the ophthalmic test the animals should be carefuUy examined to ascertain whether the eye shows conjunctivitis or other changes which are associated with suppuration. Should such be present the test should not be applied. The test consists in introducing into the conjunctival sac of the ^ye several drops of either undiluted raw mallein or a solution of pre- cipitated mallein (0.1 to 0.2 c. c. per horse). This may be introduced either with the aid of a cameFs-hair brush or with an eyedropper. Only one eye is treated ; the other serves as a control for comparison of tlie reaction. For the testing of horses in the same stable the same dropper or camel's-hair brush may be used for all the animals. The results of the test should be recorded as follows: N=Negative — eye uuchauged. S=Suspicious — seromiicoue discharge. P-|-=Positive — seromucous discharge with purulent flakes. P4-+=Po8itive — distinct purulent discharge. P-f4- 4- = Positive — purulent discharge with swelling of the eyelids. P-f + + +=Positive — strong purulent discharge with swelling and gluing together of both lids. OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF GLANDERS. 7 The following is a copy of Q. D. Form 69, Recortl of Ophthalmic Mallein Test, which is used by the Bureau of Animal Industry to record all official tests: [Obverse.] RECORD OF OPHTHALMIC MALLEIN TEST. No. of ani- Breed or markings. Sex (stal- lion, geld- ing, or mare). Age. Weiglit. Time of in- stilla- tion. Time of observation. Temperatures, if taken. Ante-mortem symptoms. Re- sults of test; de- (Give date and hour.) Be- fore. After. ci- sion. 1 1 ! 1 (Decision should be recorded in accordance with results obtained, by use of: [N]= Negative, eye un- changed. [S]= Suspicious, seromucous discharge. [P+]= Positive, seromucous discharge with purulent flakes. (P-t--{-]= Positive, distinct purulent discharge. [P +-!--(-]= Positive, purulent discharge with swelling of the eyelids. [P-l--f -l--f ]= Positive, strong purulent discharge with swelling and gluing together of the lids.) (Identify each animal by complete description; if necessary use two lines for an animal.) BULLETIN 166, U. S. DEPAETMENT OF AGRICULTURE. [Reverse.) RECORD OF REACTORS. No. of Disposition of reac- tors (slaughtered or quarantined). Date: Place of slaughter: . . . Post-mortem by: , (Q. D. Form 69.) U. S. Department of Agriculture, BUREAU OF ANIMAL INDUSTRY. Record of ophthalmic mallein test. Name of person making test Address: Dates: Owner: Address: Number passed: Number reacted: Number suspicious: Total number tested: EFFECT OF THE TEST ON GLANDERED AND HEALTHY ANIMALS. As soon as the mallein is introduced into the eye practically all animals show a lacrimation, increased reddening of the conjunctiva, and slight photophobia. No significance should be given to these symptoms. They disappear in one to two hours. Glandered animals are hy])ersensitive to mallein in a way that the administration of small quantities of mallein produces local inflam- matory processes. In larger quantities it produces a febrile general reaction. The hypersensitiveness appears as a rule during the third week after the infection, and reaches its height in the first few month.s after the infection. In the subsequent course it may subside in retrogressive cases even to the degree observed in healthy animals, but even in these cases various conditions may bring on an increased sensibility. The characteristic manifestations of the reaction for glanders commence as a rule in from 5 to 6 hours and last 24 to .36 hours, some- Bui. 1 66, U. S. Dept. of Agricultur Plate I. Fig. 1.— P + = Seromueous discharge with purulent flakes. Fig. 2.— P-f + = Distinct purulent discharge. Fig. 3.— P + + + = Purulent disehurge with swelling of the eyelids. Fig. 4.— P + + + + = Strong purulent dis- charge with .swelling and gluing together of both lids. Varying Degrees of Reactions in the Ophthalmic Mallein Test for Glanders. OPHTHALMIC MALLEIN FOE THE DIAGNOSIS OF GLANDERS. 9 times longer. The reaction consists of a purulent discharge from the conjunctival sac which is typical, as well as swelling and gluing of the eyelids. It is advisable to examine the tested animals in a good light from 12 to 24 hours after the application of the test. Varymg degrees of reactions are illustrated in Plate I, figures 1 to 4. A suppurative discharge of varying quantities is considered a positive reaction. The conjunctiva and the eyeball should also be included in the examination after examining the discharge. A pseudo-reaction can be produced b}^ artificial or accidental irrita- tion of the eye. On the other hand the purulent discharge maj have been removed (either by the stable attendant or by the animals licking each other, etc.), and the positive result thus obliterated. In such cases dried pus may be frequently found on the ])arts around the eye. Generally the positive ophthalmic reactions are not accompanied by fever or systemic disturbances. Occasionally, however, affected horses are hypersensitive to such a degree that even the few drops of mallein placed in the eye may enter the circulation and produce fever. Therefore it is advisable, when possible, to accompany the ophthalmic reaction with temperature readings. For this purpose the temperature should be taken twice, the first time when the eye test is being made and the second time when it is judged. In a doubtful eye reaction, where there is an increased temperature of IJ degrees F., the test should be considered positive if the animal had a normal temperature at the time the test was made. As stated before, it should be remembered that the intensity of the reaction has no relation to the extent of the disease in the animal tested. In the absence of any secretion the test should be considered nega- tive. When there is a mucous secretion or lacrimation dui^g the period of reaction the test must be considered as atypical, and in such cases it may be repeated the same day, when, as a rule, the results are more confirming. The application of the ophthalmic test should not be repeated more than three times on the same animal within three months, as experiments show that the reaction after the third application within this short period usually loses its intensity in positive cases, and on subsequent tests may be entirely absent. In cases where the results of the second test immediately following the first test are atypical, the blood of such animal may be drawn and forwarded to a labora- tory for the serum diagnosis. From experience gained with the eye test such a procedure would become necessary only in a compara- tively few cases. In the control of glanders, animals may be retested every six months with satisfactory results. 10 BULLETIN 166, U. S. DEPAETMENT OF AGRICULTURE. REPORT OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION ON THE OPHTHALMIC TEST. The special committee on the control of glanders of the American Veterinary Medical Association has issued a most excellent report on the various phases of diagnosis of glanders. The conclusions on the value of the eye test offered by this committee are in perfect accord with our findings, we therefore deem it advisable to include them in this paper, as follows: 1. The ophthalmic test not only meets all the reqiiirements, but is without doubt the most convenient diagnostic method at our command. 2. Its reliability compares favorably with any of the other tests available. 3. The reaction is usually very distinct, and doubtful or atypical reactions are rather infrequent. 4. The ophthalmic test has the advantage that it does not interfere with subsequent serum or other mallein tests if such are deemed necessary. 5. The test may bP repeated within 24 hours on same or control eye. If another retest is necessarys it should not be made in less than three weeks. 6. The ophthalmic test should be recognized by State and Federal authorities, since its reliability can no longer be doubted. 7. In all atypical and doubtful cases of the ophthalmic test, the combined comple- ment-fixation and agglutination or subcutaneous mallein test should be utilized for confirmation. 8uch a procedure would minimize the failure and would assure the l)e8t results in the control of the disease in a single stable or in an entire community . CONCLUSION. The results achieved in Austria with the o})hthalmic test have been remarkably successful and deserve the most earnest considera- tion. The report of Prof. Schnurer on The Residts of the Diagnostic Procedure in Glanders in Austria is a convincing proof as to the value of the eye test in the control of glanders. The senior writer received a communication only a short time ago from Prof. Schnurer, and since it deals principally with the diagnostic value of the eye test, a quotation from the letter will no doubt be permissible: 1 am at the present contemplating collecting the results of the eradication of glanders in Austria during the last three years (19I0-10T2). During this time 60,894 tests were undertaken on 47,973 horses. Of 272 cases which were found on post-mortem to be affected with glanders 240 (8S.2 per cent) were positive, 21 (7.7 per<'ent) gave an atypical reaction, while IJ (4 per cent) \yere negative. Of the 47,701 healthy horses, 189 (0.39 per cent) were positive or atypical, the remaining 47.512 (99.61 per cent) gave a negative reaction. According to these results, therefore, the eradication of glanders is only a question of organization — that is, the malleinization of horses at the border and conscientious following up of all suspected horses. Such procedure would, without doubt, result in a complet<> eradication of glanders. At the Veterinary School of Austria we now have difficulty in showing the student cases of glanders, and for demonstration piu-- poses we are compelled to infect horses artificially, whereas several years ago we had every week at least one case of glanders in our clinics. I use as mallein at the present time a product which I, myself, prepare, which represents a bouillon filtrate from seven different strains of glanders bacilli which has becji concentrated to one-tenth of the original volume OPHTHALMIC MALLEIN FOR THE DIAGNOSIS OF GLANDERS. 11 The Optimistic view of Prof. Schnurer is {'crttiinly justilied from the results he achieved, and clearly shows that with proper organiza- tion in the control work of glanders tte eradication of the disease is onU' a question of time. The eradication of outbreaks of glanders (^an not, of course, be altogether attributed to the eye test, since from the report of Never- mann, veterinar}' councilor of Prussia, glanders has diminished remarkably in that ct)initry, where they employ the combined complement-fixation and agglutination test for the diagnosis, while McGilvray has practically eradicated glanders from the Province of Manitoba by means of the subcutaneous mallein test. The method of testing by means of complement-fixation and agglutination is undoubtedly the most acciu-ate of any available, but since 'it can not be as conveniently applied as the eye test, its disadvantages are apparent. There is no doubt that with the application of either the eye test or the combined complement-fixation and agglutination tests, equally good results may be obtained provided that the work is conscientiously carried out and that all the reactors are destroyed without hesitation. As long as the authorities will limit themselves to the destruction of clinical cases only and will not take immediate action on reactors of the occult and latent character, glanders will not only continue to exist, but it will spread. WASHINGTON : i ;f ivp:r ^•^f KNT PitrNTixr; office : 1915