w SF 796 Glass. Book. Mz BULLETIN OF THE fl No. 70 Contribution from the Bureau of Animal Industry, A. D. Melvin, Chief April 15, 1914. (PROFESSIONAL PAPER.) IMMUNIZATION TESTS WITH GLANDERS VACCINE. By John R. Mohleh and Adolph Eichhorn, Pathological Division, Bureau of Animal Industry INTRODUCTORY. Among the diseases of horses with which the vetermary authori- ties arc concerned glanders is probably the most important, and unless strict measures for its control are enforced the tendency of the disease is to spread more or less rapidly. This fact is due to the character of the disease, to the prevailing methods of carmg for horses, and, probably more hnportant than all, to the frequent latent existence of the disease in apparently healthy animals. The destruction of all infected animals has been accepted as a matter of course in all civilized countries, and owing to the dangerous character of the disease and the possibility of transmission to man, this action appears to be the sanest and most reasonable procedure m its control. On the other hand, the possibility of a method of immunization of healthy animals is worthy of consideration and would be of great advantage. Ever since the discovery of maUein as a diagnostic agent for glan- ders, experunents have been conducted by various mvestigators relative to its immunizing and curative value. Many favorable reports have been made by veterinarians of the results obtained. On the contrary, others appear to have had no satisfaction from its use. [Since it has been proved that cases of glanders may recover it is rather difficult to establish the value of the immunizing agents as to [theu* action on the disease. Fortunately, we now possess a means ! by which the presence of immune bodies can be demonstrated m the animal upon which attempts at immunization are made. With the serological tests at our command we may control to some extent the action of an immunizing substance and observe how long the immune bodies are present in an animal receiving immunization treatment. 24133°— 14 .. Morwgra^ 2 BULLETIN 70, U. S. DEPARTMENT OF AGRICULTURE. Cq> \ V ^ It is unfortunate, however, that the demonstration of immune bodies does not indicate the degree of immunity in the animals. We may obtain in glanders immunization an agglutination value of 1 to 5,000 or over or a complement fixation with 0.02 of a cubic centimeter of serum which may contmue for a period of several months, yet this same animal, which apparently is supphed with a great amount of immune bodies, can be readily infected with glanders bacilli. Therefore, in tests undertaken for establislung the degree of immunity against glanders in the horse, it is necessary to expose the injected animals to an hifection such as occurs under natural conditions. Observations of such animals as to the clinical appear- ance of the disease and periodical ophthahnic tests with maUein are the methods by which the most accurate results of the immunization tests can be obtamed. Serum tests in these cases are of little value, as they invariably demonstrate immune bodies or antibodies in the immunized animals, and since even small quantities of mallein injected mto a horse are sufficient to produce antibodies which remain for 3 or 4 weeks. PREVIOUS RESULTS WITH VARIOUS IMMUNIZING AGENTS. Curative results from maUem were reported by Leclainche, Hueppe, Nocard, Johne, and Wladimiroff, while its immunizing value against glanders was studied by Schindelka, McFadyean, and Semmer, but the results were unsatisfactory. Taking into consideration the litera- ture at our command and drawing conclusions from the results ob- tained, it appears that mallein possesses very little immunizing value and no great benefit can be expected from its use as a curative agent. Other investigators attempted to immunize horses and other animals against glanders with the use of killed glanders bacilli and the literature contains some favorable results from this method of immunization. The preparations which were employed for this pur- pose were in most instances suspensions of glanders bacilli killed by heat. Of the various products which have been prepared and are at the present time used to a limited extent for the immunization of glanders, ''farase," so termed by Levy, Blumenthal, and Marxer, gives apparently the best results. It is prepared by kiUing glanders bacilli with 80 per cent glycerin or 10 per cent urea. The baciUi are then dried and the substance is used in that condition for the immu- nization. It does not contain living bacteria. Favorable results were obtained with farase by Bautz and Machodin, and by Dediuhn. The results of Dediuhn are probably the most remarkable, since he reports that on an estate where previous to immunization 276 glandered ani- mals had been destroyed, he injected 303 animals and after one year and V), OF Q. APR 2? .914 IMMUNIZATION TESTS WITH GLANDEES VACCINE. 3 four months not a single case of glanders developed, although in the meanwhile 14 cases of glanders developed among 300 nonimmunized animals. Bautz and Machodin subjected farase to various tests to establish its immunizing value. Their results on guinea pigs, cats, and horses were very satisfactory. Guinea pigs which were given two injections of farase resisted six weeks later an intraperitoneal infection with 1/2500 and 1/5000 mg. of glanders bacilli. Of six horses which received two immunizing injections of farase, two were given 1/2500 mg. of glanders bacilli subcutaneously, two received 1/500 mg. of glanders baciUi per os, and two were exposed with the other animals 45 days after the second injection. For each of the groups one check was used. Post-mortem examination of the check animals four to five weeks after the infection showed typical glanders, while the two immunized animals which received subcutaneous injections of glanders bacilli failed to show any lesions of the disease. No record was obtained of the four remaining immunized animals, as they were turned over to another laboratory for study of the duration of immu- nity in these horses. One of the recent works on the immunization of glanders was pub- lished by Zurkan, who studied the formation of specific antibodies in the blood of horses under the action of glanders antigens. He con- cludes that of various antigens such as farase, killed glanders bacilli, mallein, and malleo-aggressin, the fu'st and the last (farase and malleo- aggressin) proved most active in the production of immune bodies. The degree of immunity in the animals was estabhshed by Zurkan from the comparative results of the serological reactions he obtained with the complement-fixation, agglutination, precipitation, and opsonic tests. Since there were no practical tests made on these animals, his statement that malleo-aggressin may be used for the immunization of horses against glanders can not be accepted as conclusive. At the meeting of the American Veterinary Medical Association in Indianapolis, MacKellar presented his conclusions on the protective effect of glanders vaccine. The proportion of infections in the stables where these outbreaks occurred, as indicated by the agglutination test, is astonishing. As there is no mention made in the article of the time the agglutmation tests were applied subsequent to the mallein test, it suggests that the large proportion of reactors to the agglutina- tion test were the result of the mallein injection and not due to the presence of the infection. If this be true, then the effect of the vaccine remains indefinite and the control of the disease must be accredited to the other precautions which were observed. At best it will require several years before the value of any method of immu- nization can be satisfactorily estabhsl^ed. 4 BULl.ETIN '70, V. S. DEPARTMENT OF AGRICULTURE. EXPERIMENTS WITH DRIED GLANDERS BACILLI. The New York City board of health has been conducting immu- nizing experiments with a vaccine prepared in their laboratory, con- sisting of a suspension of dried glanders bacilli. Each cubic centi- meter of the suspension contains 2 mg. of dried bacUli. Through the courtesy of Dr. William H. Park, director of the laboratory, a suffi- cient quantity of such vaccine was obtained for conducting a series of investigations relative to the possibility of conferring immunity to animals injected wdth this vaccine. GUINEA-PIG EXPERIMENTS. The experiments were made on guinea pigs and on horses. Twenty guinea pigs, about 600 grams in weight, were divided into 4 groups, 4 pigs of each group receiving three immunizing injections of a definite amount of vaccine at intervals of one week. The size of the doses and other details are presented in Table 1. After the conclusion of these vaccinations one pig from each group was subjected to infection with suspensions of glanders bacilli. These injections with infectious material were administered at various intervals. In aU instances the same strain of glanders baciUi was used for the infections. The fifth pig in each group was not vaccinated, but served as a check, receiving only a corresponding quantity of glanders bacilh. The results of these guinea-pig tests showed that there was not a sufiicient increased resistance among the vaccinated guinea pigs to warrant any hopes of successful immunization by this method. It is to be regretted, however, that in the infection of these pigs probably too large a quantity of glanders baciUi was used. On the other hand, it would appear that if there had been any appreciable immunity present in the vaccinated guinea pigs they would have manifested it by a greater resistance against the infection. IMMUNIZATION TESTS WITH GLANDEKS VACCINE. ^ , a iS a a a r" 3 HI '-' -H -< _-o 3 3 3^ 3 3i->'^ 5 j?-j;'3 3 3 ■atj oj o m >>ti3 >>bi a a 3 d : a '■ 3 c o —1 a :^ a ^ • :3 s 3 3 1 3 : 3 • 3 1 o : o : •'^ xi .o : ^ ^ o o o o o ■ o o o «^ 1 Cj" o ; o ■ o o o o o C a 3 .3 1 3 ■ 3 3 H c — 3 : 3 • 3 3 <= c _ o • _ c ^ _ o _ c . 3 _o •- o _o ~ _c •■ « :q M CO M CC CO 05 0^0 010 o © a> ►», o 3 3 3^-3 3 3 3 3 3 COCOCOCO CO CO CO CO CO cocococo OiOOOS OiCSOiO^O C^CiCjCi i-H rt N CM ^T-HIN 333—3 333^ 33333 3333 . CO CO CO CO lO lO ic >o 5 o o >^ i!; o o o m" i-KNCO-fO -HtNCO- i-HiMco'^o ^Mco-ri . Pi o p. o S " is .B M 03 » 6 BULLETIN 70, U. S. DEPARTMENT OF AGEICULTURE. EXPERIMENTS ON HORSES. In the experiments conducted on horses, 17 animals were used, which were purchased on the open market. Most of the animals were aged, but otherwise in fair condition. AH the horses w^ere subjected to the agglutination, complement-fixation, and the ophthal- mic mallein tests, prior to the vaccination. All of them proved free from glanders on all the tests. Since the amount of vaccine to be injected for immunizing purposes has not been established, it was deemed advisable to employ varying quantities for the injections, and in order to determine the resistance of the animals against infection durmg and after the vaccination they were subjected to exposure at different times during the investigation. The smallest amount of the suspension used for the vaccination was the quantity recommended by the New York City Board of Health, viz, 1, 3, and 5 c. c. per injection, while the largest amount any of the horses received was 4, 8, and 12 c. c, respectively. Two of the vaccinated horses received an infection on the nasal mucosa with glanders bacilli, taken up on the end of a platinum loop, one week after the last vaccination. Both of these horses promptly developed glanders and one of them. No. 102, died of an acute form of the disease 21 days after the infection. Thus, there appeared to be no resistance, or at least no increased resistance, against artificial infection. To establish the resistance of the vaccinated animals against contact infection a corral was built where all the animals, including two artificially infected glanders cases, were kept. They were fed in common feed boxes and were watered from a common trough. Only one hayrack was used for all animals. Simultaneously with this exposure a stable with three stalls was likewise used for exposing the horses. The construction of the stalls in this stable was such that the animal in the center could reach to the feed boxes of either of the horses in the side stalls. The horse placed in the center was a good discharging case of cHnical glanders, whereas the horses placed in the side stalls were either two immimized animals or two controls, all of wliich were given one week's exposure with this infected horse. This was accomplished by changing the horses in the two side stalls every week, and bringing in two others from the corral, so as to make the exposure as uniform as possible in all ani- mals, including the checks. The conditions of exposure were appar- ently severe, yet they did not exceed the exposure wliich occurs in the stables of large cities where the sanitary conditions are very poor and where poor light and ventilation afford a splendid oppor- tunity for the propagation of the disease. In fact, the exposure in the corral was rather slight, since the sunlight no doubt had a destructive influence on the infection. IMMUNIZATION TESTS WITH GLANDEES VACCINE. 7 All animals were subjected periodically to clinical examinations and only four of the vaccinated horses developed signs of the disease up to the conclusion of this experiment, although some of them were exposed since May 16. Horse No. 99, which received 4 immunizing injections and was exposed to a discharging case of glanders in the stable, died 15 days after the exposure from acute broncho-pneu- monia rhalleosa. In order to determine whether any of the vaccinated horses were infected with the latent form of the disease, all were subjected July 23 to the ophthalmic test. This gave sm-prising results. Two of the vaccinated animals gave a marked reaction (P + -h + ) . A similar reaction was also obtamed in the affected horses used for exposui'e, while of the two check animals wliich were not vaccinated but had been exposed to a similar extent as the vaccinated animals, only one responded to the test; the other check animal failed to give any reaction. One month later all horses in the experiment were again subjected to the ophthalmic test. The results were the same as on the previous test, but it was noted that the intensity of the reaction was not as pronounced as in the first test. The inflammation and amount of purulent discharge were somewhat less than in the previous test. This observation coincides with that of Meyer, who states that after several eye tests in positive cases of glanders the degree of the reaction becomes less distinct. The detailed account of the results of the immunizing tests in horses is given in Table 2. BULLETIN 70, U. S. DEPARTMENT OF AGEICULTUEE. — «> ^ H 3^ 2 2 2 <^ "^S " g " 'a -W o ^ o S 22S Ol Ci ^H CO-:; O) t-H OS f-t ^ '-' _. S f5 °S ^ o^"""! jS siM ■3 5 Ho , i3 S .« .'^ Ol 0> ^H ^ ^ 05 ^ -• 2 2 S 1 p< H -a T3 -c -a . CJ e-'a n o o ffl OJ K O ,T^ cS _g o " o •=• 2 2 3p " rt ° MM p "M q'°S53m' c3 o g O j3 2 3 a .^:^ "2-2 2?^ a £3 ..2 ^o ° o © ; O g d g : o o e o ■ o~ . O « O 0) ; f5 "2 c "2 ; ac I2 M _^~3 Ma, «g§a §5B •zo < o < ^■,'zo O B3 • d *- gas •o S"; ^a : + : + : + + I + I III + : + :+ ' + + I I U"^ ^ CO -J c C3T3 155 ::§ s coo •rl C o o o o 2 p,-0 -3 x) X3 <- • ■ • IS a> 1^ CD cvi .-H Oi O 00 O --* ^O i-H ^H r-^ C^l C^ C<1 Oi J -^ Tf ^^ C^ 5-a 00 00 3 3 9 o T3 0-3T3 C^ J-. -J IMMUNIZATION TESTS WITH GLANDERS VACCINE. 9 AGGLUTINATION AND COMPLEMENT-FIXATION TESTS. Ill order to study the effect of tlie immuiiizing injections on the serum tests, the blood of the horses in this experiment was subjected to the agglutination and complement-fixation tests from the time of the first injection until the conclusion of the work. It was found that the agglutination value of the serum of the vaccinated horses as a rule increased from the tliird day after the first vaccination and continued to rise for a time. A decrease was again noted from two to fom' Aveeks after the last vaccination and appeared practically normal after six weeks to two months. A complement fixation with the sera of the A^accinated horses Avas obtained from the seventh to the ninth day after the fii'st vaccination and they continued to giA^e positiA'e fixations from two to three months after the last vaccination. These negatiA-e serological results Avhich folloAved the positiA^e reac- tions due to the injected vaccine, appeared only in the animals Avhich gaA^e no reaction to the ophthalmic test, Avhile the blood of those A'acci- nated horses wliich gave a positive reaction to the eye test continued to give a positive fixation until they had been destroyed and proA-^ed to be affected Avitli the disease. The same condition was observed in the animals wliich had been artificially infected with glanders. The serological results from these investigations appear to have a great significance AA-ith reference to the immunity produced by the injection of dead glanders bacilU. The fact that the demonstration of the presence of immune bodies in the vaccinated horses ceased entirely in two or three months from the last A^accination would indicate that after the lapse of such a time the animals have A^ery little or no immunity against the disease. This is further substan- tiated also by the agglutmation value of the sera returning to the normal level. As a matter of fact, preAaous investigations carried out by Dr. Buck, of this laboratory, showed that one or two sub- cutaneous injections of mallein Avill give a complement fixation which may last from one to tAvo months. The agglutination A^alue of the serum of such animals is also markedly influenced by subcutaneous malleinization. The serum reaction of horses folloAving the sub- cutaneous injections of mallein is giA^en in detail in Table 4. From this it seems that a mallein injection has almost the same action on the production of immune bodies in a horse as killed glanders bacilH. Table 3 indicates the results obtained with the agglutination and com- plement-fixation tests in the animals used in tliis uivestigation. On August 20 two A^accinated horses and one check animal wliich gave positiA^e results to the eye test Avere destroyed, and in ail three animals marked pulmonary glanders was observed. Horse No. 105 showed the presence of glanders nodules in the lungs in very great numbers, some of which were of the size of a walnut. In the two other cases, A\-hile the nodules Avere A^ery numerous and from their 10 BULLETIN 70, U. S. DEPAETMENT OF AGRICULTURE. appearance appeared to be active, they were of smaller sizes, ranging from a pinhead to the size of a pea. Horses Nos. 110, 120, and 124 were killed on the same day, although they had failed to show any indication of glanders by the eye test, which was also substantiated by the complement-fixation test with the blood of these animals. Post-mortem examination showed no signs of glanders in these animals. The final results are quite striking relative to the deficiency of immunization against glanders by killed bacteria. Of the remaining horses which were kept under observation, as indicated in Table 2, Nos. 117 and 119 died on October 17 and 25, respectively, of acute glanders after developing the cHnical form of the disease. No. 8G also showed indications of the disease in the early part of October. The final test on the remaining horses, namely, Nos. 86, 111, 121, and 123, was undertaken in the early part of January, 1914, when they were subjected to the ophthahnic and subcutaneous mallein tests and also to the complement-fixation and agglutination tests. All horses reacted to these different tests with the exception of No. 86, which reacted to the fixation, agglutina- tion, and ophthalmic tests but failed to react to the subcutaneous mallein test. Two days following the tests all the animals were destroyed and careful post-mortem examinations were made. The results showed glanders lesions in all animals, including No. 86, in varying degrees. The lungs in all cases contained numerous glanders nodules most of which were in an active stage, and in horse No, 86 the apex of the left lung showed a typical glanderous pneumonia with the characteristic gelatinous infiltration and numerous nodules of various sizes throughout the remainder of the pulmonary tissue. It is interesting to note that all these vaccinated horses returned to the normal serum reaction of a negative case on or before the twelfth week after the vaccination, as may be seen from Table 4. The exposure in the corral was continued the same as during the entire course of the experiments and the weekly changes of stable exposure were also carried out. The appearance of the disease in these remaining animals seems to offer a more substantial basis for drawing conclusions as to the unsatisfactory results of these vaccina- tion tests. From our experience with outbreaks of glanders in stables, it appears that these experimental horses did not develop chnical manifestations of the disease in greater proportion than is the case with the average exposed horse. It is true that the exposure of the horses in the experiments was continuous although not unusually or unreasonably severe. Whether the horses which were included in this final test possessed a certain degree of immunity as a residt of the vaccination, during the period including the time between the first exposure and the last negative eye test, it is impossible to say; but IMMUNIZATION TESTS WITH OLANDERS VACCINE. 11 S£ ^B3 O o .2 § §3 5 So eoES =: . o a Ok2 •5c ••£"'' BC O "^ O K Q Ph ft p.^ I O yi '-' ' c j; o O l^ -n c Wl ^,■3 3.5?© P^ ■gT6T 'n "PO ■:>jDnAV •J[09AV •3(8aM •JJOSAV •JfOO.W P -I m X + I + + + + + + + + I t/i ■jjaeAV O O c3-r- > s tcQt a •g ft • 5(99 A PUOO 9 S •UOIJBU -lOOBA + + + + + + + + + + I I •3[90AV •3(99AV •3[D8M •^a9Ai •jjeaM puooas I I ■J(99AV •J(99AV pUOO 9 S •UOIJ'Bn -TOOBA •CTJ 13X5 C != P-" c3 u a; cs u.a ►> q;* . t" © ci Co—" o'ja t3 iD ■0>I 8SI0H o 12 BULLETIN 70, U. S. 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IT — ' -^ >-!« '^ c cs C3 C3 K X X X X ^ c c c ^ « c «= n tC n i 1 1 1 a i S § s C3 a ft = ft = ft = a = ft 3 * S "bJ S "S s "^ S "S 3 M ^ ) 6- 1 ' > M a < C/ < c < < <1 5i^ H-t ' ^ ,^ ^ ,^ (O ;^ ^ ^! 1 1 ^ 1 1 ^11' S'o ^£ '"' ■^ 1 £J ? ^ i 1 '^.+ 1 '^+ 1 -*^ rH+^ -'i.'^ ss ^ ^■"w ". 1 1 =^+8 ■^.+8 ° 8 rt-^O (tH-CO ^+;; ^+^- s^ .-1 2"e "^ 1 1 ■=^+8 "^+8 p^H-OO ^ ^+:^ ^4-^ §^ "1 '^. 1 1 ■=■^+8 =^+g i?^ ^ |§ ^+;h- -,+.1; ^5! ^ "^"b '=~^ 1 1 ^+8 ^+8 ^_j_^ -1+^ rt '-^>, ^ 1 1 ■^+8 "^+8 >>o3 (S'O 1+'^ '^.+^ ^^ .-1 yS'^ ^ 1 1 "=^+8 =^-f g K^ '-:+'^ -l+Tl -.-rt ^+f^ '^. +7l <;',:: •72 tn ^+8 ^.+8 "^ +8 Or^OO £,'=' W+;.; '".+"rf ^+Tf -— a; i^ ■^ o ^ -+-. -+-:1 S ^„ ^ , ^ -H , ^ >1 S o s •3^5 C-M IMMUNIZATION TESTS WITH GLANDERS VACCINE. 13 from the sanitarian's standpoint this would be of theoretical impor- tance only, since even if such should be the case an immunit}^ of from two to four months could not be considered sufhcient for practical vaccination purposes. Furthermore, it should be remembered that some of these horses developed a latent form of the disease in less than three months from the last vaccination during the period in which the blood still contained the so-caUed immune bodies. CONCLUSIONS. The results obtained by these investigations appear to be sufficient to demonstrate the unsatisfactory results of this method of immuni- zation. Of the 13 immunized animals, 9 contracted the disease from natural exposure, which is a large proportion when it is consid- ered that all animals were aged and kept most of the time during the exposure out of doors. Of the 4 remaining immunized horses, 1 died of impaction after the second vaccination, wliile the other 3 animals were IdUed August 20, 1913, in order to ascertain by post mortem examination the possibiUty of glanders existing in these animals which had given positiA'C serum reaction, but which had returned to normal. In artificial infections of the vaccinated ani- mals they showed no resistance whatsoever, as both vaccinated horses promptly developed an acute form of the disease from touch- ing the Schneiderian membrane with a platinum loop v/hich had been touched to a growth of glanders bacilU. For the present, therefore, it seems ad^^sable to abstain from immunizing horses by this method, as a practice of this kind may do more harm than good. Owners ha^dng horses which are supposedly immunized would naturally become careless, thinking their animals were resistant to the disease, and thus even a better opportunity would be offered for the propagation of the disease than if the horses were not vac- cinated. Furthermore, the fact that the blood of vaccinated ani- mals can not be utihzed for serum tests for two or three months after the injections is also a great disadvantage in the eradication of the disease. As a result of this preUminary work it appears that the control and eradication of glanders must still be dependent upon the con- centration of our efforts in eliminating infected horses and the adop- tion of proper precautions against the introduction of infected ani- mals into stables free from the disease. The results achieved in Germany, Austria, and Canada by these methods have proved very encouraging, and no doubt if executed in the same spirit in this country a marked reduction in the cases of glanders would result. ADDITIONAL COPIES OF THIS PUBLICATION MAY BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE ■WASUINGTON, P. C. AT CENTS PEK COPY V LIBRARY OF CONGRESS 002824 814 3 j:„,;r.T;i,i,ii lii i 11 mmm N 11 s