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Some Further Researches on Johne's Disease
(Beneral Brticles. SOME FURTHER RESEARCHES ON JOHNE'S DISEASE. By F. W. TWORT, M. R.C.S., L.R .C. P. , A ND G. L. Y. I NGRAM, M.R.C.V.S. The B,"own 1nslillition, University of Lo"don .
THE PREPARATION OF AN EFFICIENT VACCINE.
A~D
SPECIFIC DIAGNOSTIC
IN our last paper (Royal Soc., B., vol. 84, 1912) we di scussed various curative and diagnostic vaccines for J ohne 's disease, and pointed out that one of our vacci ne s, prepared by growing J ohne's bacillus on broth containing I per cent. of human tubercle bacilli, g ave a marked reaction with an animal su ffering from Johne's disease plus tuberculosis and with tho se animals suffering from t ubercular disease only. This and other experiments proved that t he medium used was un suitable for the p reparation of a specific diagnostic vaccine. From further experiments we were led to recommend Bacillus phlei as the most suitable for the preparation of media, and we prepared some vaccines on these lines, but the only animal we had the opportunity of testing gave a negative reaction, probably due either to the di sease being very far advanced or the d ose of vaccine being too small. HaIth, of Copenhagen, has publi shed r esults of some experiments with a vaccine prepared by growin g J ohne's bacillus on a medi um containing a g lycerine extract of the t ubercle bacillus. A series of calves were inoculated with pure cultures of J ohne 's baci llus, tested some months later with ordi nary tuberculin with negative re sults, and then with the special vaccine. They all showed some rise of temperature, whilst one gave a typical reaction. The a uthor does not, however, give the ri se of temperwture obtained , but his re sult , in an a nimal apparently free from tuberculosis, is interesting, but as we have al ready indicated, to be specific the vaccine mu st be free from t ubercle bacilli or their extracts. We ourse lves haye teste d a vaccine prepared from a pure culture of Bacillus phlei only, as also ha s Ban g,* with negative r esults in animal s suffering from J ohn e's di sease. We have recently te sted a vaccine prepared from a nin e months' old culture of Johne's bacillus grown on ordinary g ly-ce rine peptone beef broth contain ing glycerine salin e extract of • 66d< Beterning, &c., p. 41.
570 This wa s fir st te sted on three full g rown bovines and on five calves about ,seven months old, and was inoculated intrav enou.sly in do ses varying, accordin g to the size of the animal, from 5 to 10 c. c . No . I, a Jersey bull, and Nos. 2 and 3, Jersey cows, all showed typical clinical manifestations of advanced Johne 's disease which they had contracted naturally . All these animal s had seve ral time s been te sted with ordinary diagnostic tuberculin with negatiye re sult s. Each received IO c.c. of the special vaccine, t he temp erature bein g normal at the time of ino culation : No . I ga ve max imum temp. of I05 .00 F . in I hour. NO. 2 I06.Io F . in 5 hours. " I04·8° F. in 4 NO·3 " " )J os. 2 and 3 developed violent diarrhrea foll owing the inoculatioll. 'vVe consider all the se reaction s po siti ve. The five calves, six month s before t he t estin g of this vaccine, had been inocula ted with living cultures of Johne' s bacillus . isolated from the intestine of a calf in which we had produced the disease . by the inoculati on o f a pure g rowth o f bacilli . Of these five calves No s. I and 2 were injected intrann otl s!y, Nos . 3 and 4 intraperitoneally. a nd No . 5 subcutaneously. Of the special vaccine, calves 2 a nd 3 rec eived 3 c .c ., a nd ca lves I, 4 a nd 5, each 5 c. c. Calves 2 and 3 showed 11 0 ri se of t empe rature in 6 hours. Calf I rose to I04. 6°F . in 3 hours. ,, 4 " 105 .001'. in 6 " " 5 showed no ri se in ten hours, but twenty-four hours after inoculation the t emperature was I05-4°F. and slowly declined. Through an unfortunate error the temperature s of calve s 2 and 3 were not taken after the sixth hour. A few days a ft er the se te sts the fi ve calves were killed and post-mortem examination made with the followin g results : Calf No. I.-Typical lesions of Johne' s di sease and bacilli in the intestines and me senteric g lands . No tuberculo sis . Calf No . 2.-Slight lesions of J ohne' s disease in intestines; several acid-fast bacilli in lesio ns. No tuberculo sis. Calf No. 3.-Typical tubercula r bronchial g lands . No Johne 's disease. C alf No . 4.-Typical tubercul ar bron chial g lands, and Johne 's Ba cillus pltlei.
Some Further Researches on Johue's Diseases
57 I
disease in a moderately advanced stage, the bacilli yery numerous near ileo-crecal valve. Calf No . 5.- Typical t ubercular bronchial glands and tubercular lesion apex right lung. No macr oscopic evidence of Johne's disease, but a few baci lli found beneath mucous membrane of ileo-crecal va lve and several ill a me senteric g land. Cultures were made from beneath ,the mU CO ll S membrane of the intestine of all the cases, and the bacilli gre,,' in all except Calf 3. G RO WTH
OF J OH.:m 's
BACILLCS OX ,",
ORDIX,\ RY
LABORATORY
:NIEDIU)L
After repeated subculturin g, and after living about 18 months outside the animal body, vve succeeded in inducing J ohne's bacillus to grow on ordinary g lycerine liYer broth. From thi s culture we prepared a vaccine and tested it in do ses of 10 c.c , subcutaneously on t\',·o of the Jersey animals above-mentioned. ten a nd twenty-one days respectively having elapsed since the tests with the above-mentioned \'accine . In the latter case (twenty-one day s interval) the temperature r ose from 102.2° F. to 104° F., sufficient to show that the bacillus retained its power of producing a good vaccine in spite of growth having been only moderate in amount and of the slight variation or mutation which has taken place as evidenced by the bacillus gro\\'ing on a medium free from acid-fast bacilli or their extracts. Thi s strain now grows on ordinary g lycerine beef broth. SUCCESSFUL INOCULATION OF GOAT S '\'I TH
JOH NE' S
DISEASE, AND
REACTIONS WITH VACCINES I N TI-IESE ANIMALS.
In July, 19II , we inoculated two young goats with living cultures of Johne 's bacillus . Goat I intravenously, and Goat 2 with a much smaller quantity of bacilli intraperitoneally. Eleven months later Goat I was thin , but otherwise there was no clinical manifestaJtion of the disease. They were te sted with the first vaccine as used on the eight bovines de scribed above, each animal receiving 3 c.c. s ~t bcutan eousl)l. Goat I.-Showed temperature of 106-40 F. in nme hours. reached 106.6° F., and remained above J06° F. for eight hours. the reaction being accompanied by some diarrhcea. Goat 2.-Temperature reached JOSo F . in nine hours and 105.4° F . in ten hour s, but there was no diarrhcea.
57 2
The Veterinary Journal.
Both animals were killed, and on post-mortem, examination both showed lesions of typical John's disease and typical J ohne's bacilli, Goat I being affected in a greater degree than Goat 2. Johne' s bacillus was isolated from both cases. CONCLUSIONS.
From the experiments noted above the following conclusions may be drawn:(I) A diagnostic vaccine has been prepared which is specific for Johne's disease , and which will enable any owner of stock to eradicate the condition from his herd and to prevent its reintroduction by newly bought animals. (2) The disease ha s been shown to be capable of affecting goats, and temperature reaotions have been obtained in these animals with the specific vaccine, and it is probable that the condition demonstrated in deer by McFadyean in 1907, and by S. Stockman in sheep ill 1909, is caused by the same bacillus that affects bovines. (3) A slight variation or mutation of the bacillus has enabled one strain to be grown on ordinary laboratory media. (4) The disease being thus demonstrable to an owner in its earliest stages, on the use of such a vaccine as is here described becoming general, State legislation should be adopted, making slaughter compulsory. No compensation would be called for, the carcase of an affected animal being saleable, and it being to the interest of the owner to remove a source of infection from his herd. AUTOTHERAPY-ITS APPLICATION IN THE TREATMENT OF SEPTIC DISEASES IN THE HORSE.* By D.
J. MANGAN, D.V.S. New York, N. Y.
I HOPE I will be pardoned for attempting to handle such a formidable subject as vaccine therapy; and if my efforts to elucidate the action of autogenous pus when administered internally in septic diseases in the horse will interest you sufficiently to give this treatment a fair trial , I will feel amply repaid for any criticism which my lack of completeness is justly entitled to. According to Wright, when an individual recovers spontane• From the American Veteri11ary R eview. Read before the June Veterinary Medica l Assoc iation of New York City.
meetin~.
THE PREPARATION OF AN EFFICIENT VACCINE.
A~D
SPECIFIC DIAGNOSTIC
IN our last paper (Royal Soc., B., vol. 84, 1912) we di scussed various curative and diagnostic vaccines for J ohne 's disease, and pointed out that one of our vacci ne s, prepared by growing J ohne's bacillus on broth containing I per cent. of human tubercle bacilli, g ave a marked reaction with an animal su ffering from Johne's disease plus tuberculosis and with tho se animals suffering from t ubercular disease only. This and other experiments proved that t he medium used was un suitable for the p reparation of a specific diagnostic vaccine. From further experiments we were led to recommend Bacillus phlei as the most suitable for the preparation of media, and we prepared some vaccines on these lines, but the only animal we had the opportunity of testing gave a negative reaction, probably due either to the di sease being very far advanced or the d ose of vaccine being too small. HaIth, of Copenhagen, has publi shed r esults of some experiments with a vaccine prepared by growin g J ohne's bacillus on a medi um containing a g lycerine extract of the t ubercle bacillus. A series of calves were inoculated with pure cultures of J ohne 's baci llus, tested some months later with ordi nary tuberculin with negative re sults, and then with the special vaccine. They all showed some rise of temperature, whilst one gave a typical reaction. The a uthor does not, however, give the ri se of temperwture obtained , but his re sult , in an a nimal apparently free from tuberculosis, is interesting, but as we have al ready indicated, to be specific the vaccine mu st be free from t ubercle bacilli or their extracts. We ourse lves haye teste d a vaccine prepared from a pure culture of Bacillus phlei only, as also ha s Ban g,* with negative r esults in animal s suffering from J ohn e's di sease. We have recently te sted a vaccine prepared from a nin e months' old culture of Johne's bacillus grown on ordinary g ly-ce rine peptone beef broth contain ing glycerine salin e extract of • 66d< Beterning, &c., p. 41.
570 This wa s fir st te sted on three full g rown bovines and on five calves about ,seven months old, and was inoculated intrav enou.sly in do ses varying, accordin g to the size of the animal, from 5 to 10 c. c . No . I, a Jersey bull, and Nos. 2 and 3, Jersey cows, all showed typical clinical manifestations of advanced Johne 's disease which they had contracted naturally . All these animal s had seve ral time s been te sted with ordinary diagnostic tuberculin with negatiye re sult s. Each received IO c.c. of the special vaccine, t he temp erature bein g normal at the time of ino culation : No . I ga ve max imum temp. of I05 .00 F . in I hour. NO. 2 I06.Io F . in 5 hours. " I04·8° F. in 4 NO·3 " " )J os. 2 and 3 developed violent diarrhrea foll owing the inoculatioll. 'vVe consider all the se reaction s po siti ve. The five calves, six month s before t he t estin g of this vaccine, had been inocula ted with living cultures of Johne' s bacillus . isolated from the intestine of a calf in which we had produced the disease . by the inoculati on o f a pure g rowth o f bacilli . Of these five calves No s. I and 2 were injected intrann otl s!y, Nos . 3 and 4 intraperitoneally. a nd No . 5 subcutaneously. Of the special vaccine, calves 2 a nd 3 rec eived 3 c .c ., a nd ca lves I, 4 a nd 5, each 5 c. c. Calves 2 and 3 showed 11 0 ri se of t empe rature in 6 hours. Calf I rose to I04. 6°F . in 3 hours. ,, 4 " 105 .001'. in 6 " " 5 showed no ri se in ten hours, but twenty-four hours after inoculation the t emperature was I05-4°F. and slowly declined. Through an unfortunate error the temperature s of calve s 2 and 3 were not taken after the sixth hour. A few days a ft er the se te sts the fi ve calves were killed and post-mortem examination made with the followin g results : Calf No. I.-Typical lesions of Johne' s di sease and bacilli in the intestines and me senteric g lands . No tuberculo sis . Calf No . 2.-Slight lesions of J ohne' s disease in intestines; several acid-fast bacilli in lesio ns. No tuberculo sis. Calf No. 3.-Typical tubercula r bronchial g lands . No Johne 's disease. C alf No . 4.-Typical tubercul ar bron chial g lands, and Johne 's Ba cillus pltlei.
Some Further Researches on Johue's Diseases
57 I
disease in a moderately advanced stage, the bacilli yery numerous near ileo-crecal valve. Calf No . 5.- Typical t ubercular bronchial glands and tubercular lesion apex right lung. No macr oscopic evidence of Johne's disease, but a few baci lli found beneath mucous membrane of ileo-crecal va lve and several ill a me senteric g land. Cultures were made from beneath ,the mU CO ll S membrane of the intestine of all the cases, and the bacilli gre,,' in all except Calf 3. G RO WTH
OF J OH.:m 's
BACILLCS OX ,",
ORDIX,\ RY
LABORATORY
:NIEDIU)L
After repeated subculturin g, and after living about 18 months outside the animal body, vve succeeded in inducing J ohne's bacillus to grow on ordinary g lycerine liYer broth. From thi s culture we prepared a vaccine and tested it in do ses of 10 c.c , subcutaneously on t\',·o of the Jersey animals above-mentioned. ten a nd twenty-one days respectively having elapsed since the tests with the above-mentioned \'accine . In the latter case (twenty-one day s interval) the temperature r ose from 102.2° F. to 104° F., sufficient to show that the bacillus retained its power of producing a good vaccine in spite of growth having been only moderate in amount and of the slight variation or mutation which has taken place as evidenced by the bacillus gro\\'ing on a medium free from acid-fast bacilli or their extracts. Thi s strain now grows on ordinary g lycerine beef broth. SUCCESSFUL INOCULATION OF GOAT S '\'I TH
JOH NE' S
DISEASE, AND
REACTIONS WITH VACCINES I N TI-IESE ANIMALS.
In July, 19II , we inoculated two young goats with living cultures of Johne 's bacillus . Goat I intravenously, and Goat 2 with a much smaller quantity of bacilli intraperitoneally. Eleven months later Goat I was thin , but otherwise there was no clinical manifestaJtion of the disease. They were te sted with the first vaccine as used on the eight bovines de scribed above, each animal receiving 3 c.c. s ~t bcutan eousl)l. Goat I.-Showed temperature of 106-40 F. in nme hours. reached 106.6° F., and remained above J06° F. for eight hours. the reaction being accompanied by some diarrhcea. Goat 2.-Temperature reached JOSo F . in nine hours and 105.4° F . in ten hour s, but there was no diarrhcea.
57 2
The Veterinary Journal.
Both animals were killed, and on post-mortem, examination both showed lesions of typical John's disease and typical J ohne's bacilli, Goat I being affected in a greater degree than Goat 2. Johne' s bacillus was isolated from both cases. CONCLUSIONS.
From the experiments noted above the following conclusions may be drawn:(I) A diagnostic vaccine has been prepared which is specific for Johne's disease , and which will enable any owner of stock to eradicate the condition from his herd and to prevent its reintroduction by newly bought animals. (2) The disease ha s been shown to be capable of affecting goats, and temperature reaotions have been obtained in these animals with the specific vaccine, and it is probable that the condition demonstrated in deer by McFadyean in 1907, and by S. Stockman in sheep ill 1909, is caused by the same bacillus that affects bovines. (3) A slight variation or mutation of the bacillus has enabled one strain to be grown on ordinary laboratory media. (4) The disease being thus demonstrable to an owner in its earliest stages, on the use of such a vaccine as is here described becoming general, State legislation should be adopted, making slaughter compulsory. No compensation would be called for, the carcase of an affected animal being saleable, and it being to the interest of the owner to remove a source of infection from his herd. AUTOTHERAPY-ITS APPLICATION IN THE TREATMENT OF SEPTIC DISEASES IN THE HORSE.* By D.
J. MANGAN, D.V.S. New York, N. Y.
I HOPE I will be pardoned for attempting to handle such a formidable subject as vaccine therapy; and if my efforts to elucidate the action of autogenous pus when administered internally in septic diseases in the horse will interest you sufficiently to give this treatment a fair trial , I will feel amply repaid for any criticism which my lack of completeness is justly entitled to. According to Wright, when an individual recovers spontane• From the American Veteri11ary R eview. Read before the June Veterinary Medica l Assoc iation of New York City.
meetin~.