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An Assessment of the Single Intradermal Comparative Test in the Diagnosis of Bovine Tuberculosis in Cattle Previously Vaccinated Against Johne's Disease
Brit. vet. J. (1962), liB, 15
AN ASSESSMENT OF THE SINGLE INTRADERMAL COMPARATIVE TEST IN THE DIAGNOSIS OF BOVINE TUBERCULOSIS IN CATTLE PREVIOUSLY VACCINATED AGAINST jOHNE'S DISEASE By
J.
S. S.
INGLIS
and
MARY WEIPERS
Department of Animal Husbandry, University of Glasgow
With the eradication of bovine tuberculosis it has now become apparent that Johne's disease is one of the greatest sources of economic loss remaining in cattle in this country. The recent Ministry of Agriculture publication, "Disease Wastage and Husbandry" (Leech, Davis, Macrae & Withers, 1960), would seem to confirm that this is true, at least in the dairy herds. This survey showed that Johne's disease was one of the two disease conditions causing the greatest total loss through depreciation in market value. The difficulty in making an early diagnosis and the ( fact that many animals are affected without showing clinical signs makes eradication by slaughter impracticable, and the most promising approach to controlling this disease would appear to be by vaccination. One of the main difficulties in recommending the widespread use of the vaccine to date has been the assumption that vaccination interferes radically with the diagnosis of tuberculosis by means of the tuberculin test. This point has been emphasized by Hebert, Doyle & Paterson (1959) in their work with attested cattle vaccinated against Johne's disease, and by Chandler, Macdonald, Caldwell, Griffiths & Isaacs working in New Zealand with untested cattle. The only precise experimental attempt to clarify the position with tuberculosis-infected cattle was by Ritchie, Robertson & Muir (1950). They concluded "that on methods used for interpreting the single intradermal comparative test it is impossible to distinguish bovine type infection in animals previously vaccinated ; with Johne's disease vaccine" and "that it is inadvisable to adopt widespread :' use of this vaccine against J ohne's disease in view of the efforts presently being: made to eliminate bovine tuberculosis from cattle." There is, however, more recent evidence from the field trials conducted by the Ministry of Agriculture and reported by Stuart in 1960 which suggests that the vaccine may not interfere as seriously as was thought earlier. In view of the limited nature of the previous work we considered that a reappraisal of the relationship of Johne's disease vaccination to the interpretation of the single intradermal comparative test might be valuable, and consequently we decided to initiate an experiment on a limited scale. We infected animals vaccinated against Johne's disease with bovine tuberculosis and later subjected them to the single
16
BRITISH VETERINARY JOURNAL, 118,
1
intradermal comparative tuberculin test. At the same time a johnin test was applied with a view to obtaining some indication of its possible value in this connection. PROCEDURE
Twenty-four cattle of mixed breeds aged between 12 months and 21 years which had been vaccinated against Johne's disease at eight days of age were used. Nodules were present in all these animals at the site ofinoculation (brisket). The vaccines used were Johne's disease vaccine as supplied by the Ministry of Agriculture's Central Veterinary Laboratory, Weybridge (21 animals), and Sigurdson's vaccine as used in Iceland for sheep vaccination (3 animals). These animals had all been negative to the single intradermal comparative tuberculin test carried out at six-monthly intervals. The animals were divided into two groups, as follows:Group I: vaccinated only. Group 2: vaccinated and infected. These animals were infected on 28th October, 1960, with bovine tuberculosis. Five drops of suspension of bovine culture were instilled into each eye; each millilitre of suspension contained 2·5 mg. of organisms suspended in normal saline. The bovine culture used was obtained from the Ministry of Agriculture's Central Veterinary Laboratory, Weybridge. A portion of the material was subcultured and the final inoculum consisted of equal parts of the original Weybridge culture and a subculture prepared by ourselves. The material was also tested for viability and virulence by animal inoculation prior to use. Both groups were subjected to the comparative tuberculin test and the johnin test prior to the commencement of the experiment (23rd September, 1960) and again on 2nd February, 1961, approximately three months after the date of infection. RESULTS
The results of the tuberculin and johnin tests are shown in Tables I and II. It will be seen that, according to the interpretation of the tuberculin test as laid down by the Ministry of Agriculture, all animals in Group 1 (Table I) would be retained on the reading of both tests. On the other hand, although all animals in Group 2 (Table II) would be retained as a result of the test in September, 1960, they would all be removed as a result of the test in February, 1961, as every animal in Group 2 at the February test gave a mammalian reaction more than 6 mm. greater than the avian reaction. It will be seen that the reaction to the johnin test was generally greater than that to the avian tuberculin test, with the result that, when the avian mammalian interpretation criteria were used, certain infected animals would not have been removed if the johnin test had been used instead of the avian tuberculin test.
THE SINGLE INTRADERMAL COMPARATIVE TEST CLINICAL EXAMINATION
At the time of the February test most of the animals in Group 2 showed varying degrees of lachrymation and conjunctivitis of both eyes. Three cases showed a granulomatous thickening of the conjunctiva. One or both parotid lymph nodes and one or both submaxillary nodes were enlarged in all the animals. The prescapular glands were enlarged in one case. POST-MORTEM EXAMINATION
A detailed post-mortem examination was carried out on all animals in Group 2. One or both eyes of all animals showed a tuberculous conjunctivitis. Macroscopic tuberculous lesions were found in one or more of the lymph nodes of the heads of all the animals in this group. In addition macroscopic lesions were found in the bronchial lymphatic nodes in four cases, in the mediastinal glands in three, in the prepectorals in one, and in the lung substances in four animals. No macroscopic lesions were found in any other part of the carcasses or organs. 23·9 ·60
,.
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23..9-60
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TABLE II
Results of comparative tuberculin tests and johnin tests on two groups of animals. DI SCUSSION
In this experiment all the animals used had been vaccinated against Johne's disease with either the Ministry of Agriculture vaccine or Sigurdson's vaccine. Even though interpretation was based on the assumption that non-specific infection was present there was no difficulty in detecting the tuberculous animals by means of the single intradermal comparative test. This finding would appear to be contradictory to the view generally held. The difference may have arisen as a result of the longer interval between vaccination and superimposed tubercle infection; in addition previous workers used much younger animals and, as pointed out by Hebert et at. (1959), the general level of reactivity to both the avian and mammalian tuberculin tests is high in calves after vaccination. D
18
BRITISH VETERINARY JOURNAL, 118, I
Although the present results were unequivocal as far as identification of tuberculous animals was concerned, the number of animals used was small and it would be desirable to repeat the work with larger numbers of cattle of different ages and also to test at varying intervals after infection up to a period of about two years. Furthermore, this was an artificial infection and results might possibly be different with natural infection. We hope, however, to clarify this point in the near future with work already in progress. The reaction to johnin in vaccinated animals was generally greater than the reaction to avian tuberculin, although a few animals gave reactions to the johnin test which were equal to or lower than those to the avian test. The usefulness of the johnin test in testing vaccinated herds cannot be properly evaluated until its response in vaccinated cattle affected with tuberculosis is known. The results with our group of infected vaccinated cattle would certainly indicate that the johnin/mammalian comparative test would be much more liable to lead to error and to the retention of tuberculous animals than the avian/mammalian test. SUMMARY
An experiment with 24 cattle, 12 of which served as controls, was carried out to study the single intradermal comparative test in tuberculous animals that had been vaccinated with Johne's disease vaccine 1 year-2! years prior to experimentally produced tuberculosis tubercle infection. When the criteria applied by the Animal Health Division of the Ministry of Agriculture were used, it was possible to identify all the infected animals although these animals had previously been vaccinated against Johne's disease. The findings also suggested that an avian/mammalian comparative test is more satisfactory than a johnin/mammalian comparative test in the detection of tuberculosis in vaccinated animals. ACKNOWLEDGEMENTS
We wish to thank the Veterinary Field Officers of the Department of Agriculture, Stirling area, for their co-operation; the staff of the Ministry of Agriculture's Central Veterinary Laboratory, Weybridge, for supplying the Johne's disease vaccine, johnin, and the culture of Mycobacterium tuberculosis; Dr. G. B. S. Roberts, Ruchill Hospital, Glasgow, for preparing cultures; and also Mr. R. M. Peacock for his supervision and care of the infected animals. REFERENCES CHANDLER, R. L., MACDONALD,j. H., CALDWELL, D. W., GRIFFITHS, C. V. W. & IsAACS, C. E. (1957)· N.Z· vet. ]·,5, 24· HEBERT, C. N., DOYLE, T. M. & PATERSON, A. B. (1959). Vet. &c., 71, 108. LEECH, F. B., DAVIS, MURIEL E., MACRAE, W. D., WITHERS, F. W. (1960). Disease, Wastage, and Husbandry in the British Dairy Herd, p. 60. Ministry of Agriculture, Fisheries and Food, London: H.M.S.O. RiTCHIB,j. N., ROBERTSON, A. & MUIR, R. O. (1952). Personal communication. STUART, P. (1960). Personal communication. (&ceivedfor publication, 4 October 1961)
AN ASSESSMENT OF THE SINGLE INTRADERMAL COMPARATIVE TEST IN THE DIAGNOSIS OF BOVINE TUBERCULOSIS IN CATTLE PREVIOUSLY VACCINATED AGAINST jOHNE'S DISEASE By
J.
S. S.
INGLIS
and
MARY WEIPERS
Department of Animal Husbandry, University of Glasgow
With the eradication of bovine tuberculosis it has now become apparent that Johne's disease is one of the greatest sources of economic loss remaining in cattle in this country. The recent Ministry of Agriculture publication, "Disease Wastage and Husbandry" (Leech, Davis, Macrae & Withers, 1960), would seem to confirm that this is true, at least in the dairy herds. This survey showed that Johne's disease was one of the two disease conditions causing the greatest total loss through depreciation in market value. The difficulty in making an early diagnosis and the ( fact that many animals are affected without showing clinical signs makes eradication by slaughter impracticable, and the most promising approach to controlling this disease would appear to be by vaccination. One of the main difficulties in recommending the widespread use of the vaccine to date has been the assumption that vaccination interferes radically with the diagnosis of tuberculosis by means of the tuberculin test. This point has been emphasized by Hebert, Doyle & Paterson (1959) in their work with attested cattle vaccinated against Johne's disease, and by Chandler, Macdonald, Caldwell, Griffiths & Isaacs working in New Zealand with untested cattle. The only precise experimental attempt to clarify the position with tuberculosis-infected cattle was by Ritchie, Robertson & Muir (1950). They concluded "that on methods used for interpreting the single intradermal comparative test it is impossible to distinguish bovine type infection in animals previously vaccinated ; with Johne's disease vaccine" and "that it is inadvisable to adopt widespread :' use of this vaccine against J ohne's disease in view of the efforts presently being: made to eliminate bovine tuberculosis from cattle." There is, however, more recent evidence from the field trials conducted by the Ministry of Agriculture and reported by Stuart in 1960 which suggests that the vaccine may not interfere as seriously as was thought earlier. In view of the limited nature of the previous work we considered that a reappraisal of the relationship of Johne's disease vaccination to the interpretation of the single intradermal comparative test might be valuable, and consequently we decided to initiate an experiment on a limited scale. We infected animals vaccinated against Johne's disease with bovine tuberculosis and later subjected them to the single
16
BRITISH VETERINARY JOURNAL, 118,
1
intradermal comparative tuberculin test. At the same time a johnin test was applied with a view to obtaining some indication of its possible value in this connection. PROCEDURE
Twenty-four cattle of mixed breeds aged between 12 months and 21 years which had been vaccinated against Johne's disease at eight days of age were used. Nodules were present in all these animals at the site ofinoculation (brisket). The vaccines used were Johne's disease vaccine as supplied by the Ministry of Agriculture's Central Veterinary Laboratory, Weybridge (21 animals), and Sigurdson's vaccine as used in Iceland for sheep vaccination (3 animals). These animals had all been negative to the single intradermal comparative tuberculin test carried out at six-monthly intervals. The animals were divided into two groups, as follows:Group I: vaccinated only. Group 2: vaccinated and infected. These animals were infected on 28th October, 1960, with bovine tuberculosis. Five drops of suspension of bovine culture were instilled into each eye; each millilitre of suspension contained 2·5 mg. of organisms suspended in normal saline. The bovine culture used was obtained from the Ministry of Agriculture's Central Veterinary Laboratory, Weybridge. A portion of the material was subcultured and the final inoculum consisted of equal parts of the original Weybridge culture and a subculture prepared by ourselves. The material was also tested for viability and virulence by animal inoculation prior to use. Both groups were subjected to the comparative tuberculin test and the johnin test prior to the commencement of the experiment (23rd September, 1960) and again on 2nd February, 1961, approximately three months after the date of infection. RESULTS
The results of the tuberculin and johnin tests are shown in Tables I and II. It will be seen that, according to the interpretation of the tuberculin test as laid down by the Ministry of Agriculture, all animals in Group 1 (Table I) would be retained on the reading of both tests. On the other hand, although all animals in Group 2 (Table II) would be retained as a result of the test in September, 1960, they would all be removed as a result of the test in February, 1961, as every animal in Group 2 at the February test gave a mammalian reaction more than 6 mm. greater than the avian reaction. It will be seen that the reaction to the johnin test was generally greater than that to the avian tuberculin test, with the result that, when the avian mammalian interpretation criteria were used, certain infected animals would not have been removed if the johnin test had been used instead of the avian tuberculin test.
THE SINGLE INTRADERMAL COMPARATIVE TEST CLINICAL EXAMINATION
At the time of the February test most of the animals in Group 2 showed varying degrees of lachrymation and conjunctivitis of both eyes. Three cases showed a granulomatous thickening of the conjunctiva. One or both parotid lymph nodes and one or both submaxillary nodes were enlarged in all the animals. The prescapular glands were enlarged in one case. POST-MORTEM EXAMINATION
A detailed post-mortem examination was carried out on all animals in Group 2. One or both eyes of all animals showed a tuberculous conjunctivitis. Macroscopic tuberculous lesions were found in one or more of the lymph nodes of the heads of all the animals in this group. In addition macroscopic lesions were found in the bronchial lymphatic nodes in four cases, in the mediastinal glands in three, in the prepectorals in one, and in the lung substances in four animals. No macroscopic lesions were found in any other part of the carcasses or organs. 23·9 ·60
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TABLE II
Results of comparative tuberculin tests and johnin tests on two groups of animals. DI SCUSSION
In this experiment all the animals used had been vaccinated against Johne's disease with either the Ministry of Agriculture vaccine or Sigurdson's vaccine. Even though interpretation was based on the assumption that non-specific infection was present there was no difficulty in detecting the tuberculous animals by means of the single intradermal comparative test. This finding would appear to be contradictory to the view generally held. The difference may have arisen as a result of the longer interval between vaccination and superimposed tubercle infection; in addition previous workers used much younger animals and, as pointed out by Hebert et at. (1959), the general level of reactivity to both the avian and mammalian tuberculin tests is high in calves after vaccination. D
18
BRITISH VETERINARY JOURNAL, 118, I
Although the present results were unequivocal as far as identification of tuberculous animals was concerned, the number of animals used was small and it would be desirable to repeat the work with larger numbers of cattle of different ages and also to test at varying intervals after infection up to a period of about two years. Furthermore, this was an artificial infection and results might possibly be different with natural infection. We hope, however, to clarify this point in the near future with work already in progress. The reaction to johnin in vaccinated animals was generally greater than the reaction to avian tuberculin, although a few animals gave reactions to the johnin test which were equal to or lower than those to the avian test. The usefulness of the johnin test in testing vaccinated herds cannot be properly evaluated until its response in vaccinated cattle affected with tuberculosis is known. The results with our group of infected vaccinated cattle would certainly indicate that the johnin/mammalian comparative test would be much more liable to lead to error and to the retention of tuberculous animals than the avian/mammalian test. SUMMARY
An experiment with 24 cattle, 12 of which served as controls, was carried out to study the single intradermal comparative test in tuberculous animals that had been vaccinated with Johne's disease vaccine 1 year-2! years prior to experimentally produced tuberculosis tubercle infection. When the criteria applied by the Animal Health Division of the Ministry of Agriculture were used, it was possible to identify all the infected animals although these animals had previously been vaccinated against Johne's disease. The findings also suggested that an avian/mammalian comparative test is more satisfactory than a johnin/mammalian comparative test in the detection of tuberculosis in vaccinated animals. ACKNOWLEDGEMENTS
We wish to thank the Veterinary Field Officers of the Department of Agriculture, Stirling area, for their co-operation; the staff of the Ministry of Agriculture's Central Veterinary Laboratory, Weybridge, for supplying the Johne's disease vaccine, johnin, and the culture of Mycobacterium tuberculosis; Dr. G. B. S. Roberts, Ruchill Hospital, Glasgow, for preparing cultures; and also Mr. R. M. Peacock for his supervision and care of the infected animals. REFERENCES CHANDLER, R. L., MACDONALD,j. H., CALDWELL, D. W., GRIFFITHS, C. V. W. & IsAACS, C. E. (1957)· N.Z· vet. ]·,5, 24· HEBERT, C. N., DOYLE, T. M. & PATERSON, A. B. (1959). Vet. &c., 71, 108. LEECH, F. B., DAVIS, MURIEL E., MACRAE, W. D., WITHERS, F. W. (1960). Disease, Wastage, and Husbandry in the British Dairy Herd, p. 60. Ministry of Agriculture, Fisheries and Food, London: H.M.S.O. RiTCHIB,j. N., ROBERTSON, A. & MUIR, R. O. (1952). Personal communication. STUART, P. (1960). Personal communication. (&ceivedfor publication, 4 October 1961)