An Epizootic of Bovine Tuberculosis in Barbados, West Indies T. M. Wilson and M. Howes*
ABSTRACT A 57 cow dairy herd, believed to be in excellent condition and producing well, was found to contain 30 tuberculous cattle after cervical skin testing employing bovine PPD tuberculin with the single intradermal comparative test. Testing was initiated after two cows from this herd were found at meat inspection to have tuberculosis. The reliability of bovine PPD tuberculin for testing as well as significant epidemiological, laboratory and meat inspection factors are presented.
RESUMe L'epreuve cutanee cervicale, a l'aide d'une seule injection intra-dermique comparative de tuberculine bovine PPD, revela qu'un troupeau de 57 vaches laitieres qui semblait en excellente condition et dont la production etait satisfaisante, recelait neanmoins 30 sujets tuberculeux. On decida de soumettre ce troupeau a l'epreuve de la tuberculine apres que les inspecteurs d'un abattoir eurent diagnostique la tuberculose chez deux de ses sujets. Les auteurs commentent l'efficacite de la tuberculine bovine PPD et certains facteurs epidemiologiques de la tuberculose, ainsi que le role du laboratoire et de l'inspection des viandes dans la lutte contre cette maladie.
INTRODUCTION During meat inspection and follow up studies in 1977 gross and microscopic *Pan American Health Organization/World Health Organization, Bridgetown, Barbados (Wilson) and Tuberculosis Laboratory, Central Laboratories, Ontario Ministry of Health, P.O. Box 9000, Terminal A, Toronto, Ontario M5W 1R5 (Howes).
Submitted June 14, 1978.
Volume 43 -April, 1979
lesions consistent with tuberculosis were detected in lymph nodes and abdominal and thoracic organs from two dairy cows. These mature cows originated from a 57 cow dairy herd which was composed of 39 Holstein-Friesian, 14 Guernsey, one Zebu and three Ayrshire animals. The Ayrshire cattle were imported from Canada while the remainder of the herd was of local origin. Th herd had been maintained on sugarcane tops, pangola grass hay, concentrate, molasses and mineral salts and was permanently housed in a 60' x 40' corral which included a sunshade over approximately one third of the corral. Improper drainage under the sunshade caused that area to be extremely wet. The herd was considered by the owner and attending veterinarian to be in excellent condition and producing well. This report describes the pathological, epidemiological and testing data relevant to this epizootic. Microbiological information regarding the first isolation of Mycobacterium bovis from Barbados is included as well as pertinent public health considerations. Included also are comments regarding the reliability of the recently introduced bovine PPD tuberculin (purified protein derivative tuberculin prepared from Mycobacterium bovis) for cattle tuberculosis skin testing.
MATERIALS AND METHODS Pooled four-quarter milk samples were obtained from 18 randomly selected cows on the farm and prepared for microscopic examination for acid fast bacilli (40). During the interval between initial diagnosis of the disease and testing, six cows were removed from the herd including the two index animals and one cow with suspected tubercular mastitis. Three cows
151
died of unknown causes and were burned. Fifty mature cows and one bull were skin tested in the cervical area employing the single intradermal comparative test. Tuberculin used was PPD, mammalian (bovine), 1 mg/ml (B1/77) and avian, 0.5 mg/ml (A1/77) prepared by the Ministry of Agriculture, Central Veterinary Laboratory, Weybridge, England. The standard interpretation was applied to the first test while subsequent tests were severely interpreted (3). Animals that failed the test and those which had inconclusive results were field slaughtered and necropsied under veterinary supervision. Complete necropsies were performed with particular attention being given to lymph nodes. Selected specimens were placed in 10% buffered formialin, routinely prepared for histological study and stained with haematoxylin and eosin and Zeihl-Neelsen stains (19). Nonencapsulated or poorly encapsulated noncalcified pulmonary lesions that had actual or potential access to the external environment via the airways were classified as open lesions. Selected microbiological specimens obtained at necropsy were refrigerated or frozen and airshipped to Canada. Thirteen samples comprising four lungs, two livers and seven lymph nodes from six bovine carcasses were ground up and treated with 3% hydrochloric acid for one and one-half hours. Smears were made from this sediment and stained with fluorochrome and Zeihl-Neelsen and examined for the presence of acid fast bacilli (37, 40). Treated samples were then inoculated to four different egg media: LowensteinJensen (Jensen, 1932), American Trudeau Society mediump modified LowensteinJensen with pyruvic acid instead of glycerol, and Wooley's medium containing potato water, eggs and no glycerol (1, 13). Biochemical tests performed were niacin production (31), nitrate reduction (41) and amidase activity (12, 34). Also, Tween 80 hydrolysis, arylsulphatase and catalase activity were performed according to standard methods (40). Susceptibility was measured to thiophene-2-carboxylic acid hydrazide and thiosemicarbazone (39) and sensitivity tests to first-line antituberculosis drugs were conducted using the proportion method and Middlebrook 7H 10 agar medium (6, 40). 152
Five, two month old guinea pigs weighing between 250 to 350 g were injected intramuscularly in the right thigh with untreated separate lymph node material. Palpation of the animals at regular intervals for the presence of enlarged lymph nodes was carried out. The animals were euthanized after four weeks on test and macroscopic and microscopic examinations for evidence of tuberculosis conducted (32, 40).
RESULTS Microscopic examination of milk sediment smears from 18 cows on the farm revealed short, plump, acid fast bacteria in one sample. This cow was removed from the herd prior to testing and at necropsy gross and microscopic lesions consistent with tuberculosis were found in the liver and retropharyngeal lymph nodes. Twenty-three cows failed the first test and seven animals yielded inconclusive results. Retesting employing the same testing procedures in 60 days detected seven additional failures totalling 30 test failures and seven inconclusive animals (Table I), all of which were removed from the herd and euthanized. Necropsies were conducted on 28 test failure cows and six test inconclusive cows and tabulated (Table I and II) (Fig. 1). Macroscopic lesions were observed in various lymph nodes, lungs and livers and open lesions in the lungs were detected in 18 cows (Table II). The macroscopic lesions were very variable in size and ranged from focal lesions 3-4 mm in diameter to tubercular mediastinal lymph nodes 20-25 cm TABLE I. Tuberculosis Skin Test and Necropsy Results of 51 Dairy Cattle, Barbados, West Indies
Skin Test + Number Skin Test + Necropsy + Animals Necropsied for Tuberculosis 28 25(89%) 30 Number Skin Test Necropsy + Skin Test Inconclusive Inconclusive Animals for Tuberculosis Necropsied 6 7 3(50%) 34 28(82%) Total 37
Can. J. comp. Med.
TABLE II. The Distribution of Gross Tubercular Lesions in 27 Mature Milk Cows and One Bull, Barbados, West Indies
z hi
'.
. .
z
z .
z . ,..
z ..
.
i..
.
'-.
'.
-
.-
..
..
0. hi
0
..
..
.0
hi
.
.
.
'.
..
e.
.i
hi
hi
.
G.
.
iihi
.
.
2
15
28
0
2
28
28
28
18
16
14
28
28
3
2
28
28
,.
0
28
0
2
27
28
0 28
18 28
28
Fig. 1. Guernsey cow displaying enlarged tubercular parotid and submandibular lymph nodes, Barbados, W.I. (Arrows).
Fig. 2. Very enlarged, calcified tubercular mediastinal lymph nodes from a mature dairy cow, Barbados, W.I. (Arrows).
long and 7-10 cm in diameter (Figs. 2, 3 and 4). The majority of the encapsulated lesions were yellow, firm and calcified while some were caseous and only slightly calcified. No macroscopic lesions were observed in any mammary glands or supramammary lymph nodes. Histologically, lesions in the lung, liver and lymph nodes were characterised by
All smears contained acid-fast bacilli but in very small numbers, usually less than ten bacilli per slide. Initial cultural isolation of 11 mycobacterial strains was achieved in two to four weeks on the egg media with best growth on media devoid of glycerol (Lowenstein-Jensen with pyruvic acid, and Woolley's). The strains were dysgonic on primary isolation, becoming luxuriant on subculture. They grew at 36°C, not at 22°C or 450C and colonies were nonpigmented and rough. All isolates were negative for niacin production, nitrate reduction and Tween 80 hydrolysis. They exhibited positive arylsulphatase activity in two weeks, only slight catalase activity and only a positive urease in the test for amidase activity. They were
necrotic centers, often calcified, surrounded by an admixture of reticuloendothelial cells, lymphocytes and Langhans' giant cells. Acid fast organisms were rarely found. Interalveolar septa in the mammary gland were thickened and infiltrated by reticuloendothelial cells, lymphocytes and the occasional Langhans' giant cell. Acid-fast bacilli were observed in some giant cells.
Volume 43 - April, 1979
153
TABLE III. Biochemical, Enzymatic and Drug Susceptibility Patterns of 13 Mycobacterium Strains Isolated from Barbados Cattle, West Indies
Fig. 3. Very eniarged calcied-caseous tubercular mediastinal lymph nodes from a mature dairy cow, Barbados, W.I. (Arrows).
Biochemical and Enzyme Results Niacin Negative Nitrate reduction Negative Arylsulphatase Negative three day; positive two weeks Tween 80 hydrolysis Negative 12 days Catalase activity Very slight Amidase activity Positive urease Drug Susceptibility Growth inhibited by 10 pLg/ml Thiosemicarbazone 10 iug/ml Thiophenecarboxylic acid hydrazide 2 ag/ml Streptomycin 2 wg/ml PAS 0.2 ,ug/ml Isoniazid 5 ILg/ml Ethambutol 1 ;Lg/ml Rifampin
tenuated variant of M. bovis, by the more luxuriant growth of BCG and absence of virulence of BCG for rabbits and guinea pigs (7, 11, 38).
DISCUSSION Fic. geal
4. Ver
enlarged caseous tubercular Barbados, W.I. lymph node,
retropharyn-
susceptible to 10 /g/ml thiophene-2-carboxylic acid hydrazide (TCH), 10 /Lg/ml thiosemicarbazone, and also fully susceptible to 2 glg/ml streptomycin, 2 ug/ml PAS, 0.2 pug/ml isoniazid, 5 ,ug/ml ethambutol and 1 ylg/ml rifampicin (Table III). Postmortem examination of all five guinea pigs showed typical tuberculous infection, with the presence of macroscopic, caseating lesions in the inguinal and lumbar lymph glands. The spleen was also grossly affected in two cases. Microscopically, the lesions revealed the presence of numerous, short, acid-fast bacilli. Based on the above results all strains were identified as M. bovis. This organism, recognised as a distinct species (17), is distinguished from Mycobacterium tuberculosis on the basis of its slower growth rate, inhibition by glycerol, inhibition by TCH, negative nitrate reduction, negative niacin production and differences in the amidase patterns. Mycobacterium bovis is differentiated from M. bovis BCG, the at154
In 1966 Barbados initiated a cattle tuberculosis testing program which revealed a very low reactor rate to mammalian tuberculin of 0.172% (14). Some further testing was conducted. However, due to staff and financial constraints island wide coverage was never achieved. Because of the very low reactor rate and the lack of confirmed tuberculosis cases at the slaughterhouse the island was declared free of bovine tuberculosis. This epizootic clearly demonstrates that bovine tuberculosis control/ eradication schemes must rely on continuous testing programs and thorough slaughterhouse inspection programs (2,4,8,16,30,33). Additionally, as depicted in this outbreak and others reported (5,8,16,25), bovine tuberculosis can be a chronic disease, often present for many months or years in a productive herd and only being detected by skin test or at slaughter after the majority of the herd has becomie infected. Significant improvements in materials and methods relevant to bovine tuberculosis testing have taken place. Recently there has been considerable research conducted
Can. J. comp. Med.
animals lesions were present only in the lymph nodes of the head while the major pulmonary lymph nodes were lesion free. Additionally, 11 of 28 (39%) cows had gross lesions in the major pulmonary lymph nodes while designated lymph nodes of the head had no lesions. Meat inspection staff must make complete and detailed gross examinations of the major lymph nodes of the head and lungs. This should be particularly stressed in developing countries where oftentimes the mneat inspection service is in its very formative stages. The proper laboratory follow-up of granulomatous lesions is a most important activity in bovine tuberculosis programs and should be strongly encouraged (2,4). One important aspect of this laboratory study of suspect granulomatous lesions is a thorough histological examination of the lesions. However, over reliance on histopathology as a definitive diagnostic tool must be avoided. Acid fast bacteria were rarely observed histologically in a variety of tissues from 28 cattle on this farm and this observation was supported by the rarity of such organisms under fluorescent and acid fast examination of smears from various diseased tissue. One study on bovine tuberculosis (35) completely disregarded acid fast histological staining as it did not aid significantly in the diagnosis of bovine tuberculosis when microbiological culturing was done. Histopathological find(2). Depopulation of the entire herd in ings where possible should be correlated large outbreaks of bovine tuberculosis is with the isolation and identification of the generally considered to be a most appro- bacteria. priate procedure (2,4,8,25,42). Immediate Microbiological identification of the economic consideration, unfortunately, pre- causative agent in bovine tuberculosis is cluded entire herd depopulation on this essential (2,4,29) as it is well recognised farm and eight animals from the original that atypical mycobacterium may cause herd remain. tuberculosis-like lesions in cattle and proAn attempt was made to correlate the duce sensitization to the skin test (28). Adresults of the skin test (dimensions and ditionally, microbiological identification is characteristics of reaction) to the size and most important in those geographical areas number of gross lesions found at necropsy. where little information exists about boNo simple relationship appeared to exist in vine tuberculosis. this situation. Mycobacterium bovis isolates from this Meat inspection is considered to be one herd clearly indicate it to be the etiological of the most important surveillance activi- agent and more importantly, add valuable ties in bovine tuberculosis control pro- information relevant to the reliability of grams and is today responsible for the bovine PPD tuberculin under field testing identification of many tuberculous cattle conditions. Little information (10, 14, 15) herds (2,4,8,16,25,26,29,35,42). Special sig- exists on the epidemiology of bovine tubernificance relevant to the distribution of the culosis in Barbados or the English speakgross lesions and meat inspection is evident ing Caribbean and this report should prowhen one notes that in five of 28 (17%) vide a clearer understanding of this imon the use of bovine PPD tuberculin in cattle tuberculosis testing in place of the widely used human PPD tuberculin and several studies have indicated a high degree of sensitivity and specificity for PPD tuberculin of bovine origin (9,18,2024,27,36). Our results tabulated from a natural field epizootic revealed a high degree of reliability in the use of bovine PPD tuberculin. This herd was gradually built up over a ten-year period by purchasing cattle in small numbers from around the island. The tuberculosis testing history of the major herd and the animals added to the herd was not clear. Investigations concerning cattle movement in and out of this herd and trace back tuberculosis testing have failed to reveal the source of infection. Three cows found to be infected were imported from Canada. However, because of Canadian testing programs this is an unlikely source of infection (42). Lack of proper records of many of the small farmers considerably complicated the epidemiological follow-up. It is quite clear, however, that after introduction the disease may have spread very rapidly and diffusely. The close confinement of the cattle under very wet conditions would have allowed opportunity for repeated exposure to the infectious agent which may have caused the development of the very large lesions noted, especially in the pulmonary lymph nodes
Volume 43 - April, 1979
155
portant zoonosis. Tuberculosis of the bovine udder may take several forms including disseminated miliary, lobular infiltrating and caseous mastitis (2,33). Lobular infiltrating tubercular mastitis is reported to be the most common form and is unique in that the macroscopic changes in the udder and regional lymph nodes are very minimal. Examination of milk samples from a portion of this herd revealed one cow that was shedding acid fast bacilli resembling M. bovis in the milk. Necropsy of this cow revealed gross tubercular lesions in several sites, excluding the udder. However, histological lesions consistent with lobular infiltrating tuberculosis of the udder were observed. Although no macroscopic changes were detected in the udder or suprammamary lymph nodes of 27 cows, histological lesions consistent with lobular infiltrating tuberculosis were detected in five cattle. Microbiological tests were not conducted on mammary tissue. Bovine tuberculosis, in particular the shedding of M. bovis in the milk, has very important livestock and public health implications (2). In 1977 fresh milk production in Barbados amounted to approximately ten million pounds of which six million pounds was pasteurised prior to public sale. Four million pounds or 40% of the total milk supply was available to the public as a raw product. Because of the well recognised threat of M. bovis to human health an epidemiological study was initiated by health officials to trace any persons in close contact with the farm animals or farm milk products. Additionally health officials have advised all those consuming raw milk to boil it before use. In preparation of an island wide cattle tuberculosis testing program health officials have com,pleted a cattle census detailing all owners who sell raw milk to the public. Bovine tuberculosis occurs throughout the world and can have a serious impact on cattle development in both developed and developing countries. It is clear that through proper implementation of both testing and slaughterhouse surveillance this important zoonosis can be efficiently controlled. It is particularly important for those countries embarking on cattle development schemes to plan and implement modern tuberculosis control programs and avoid unnecessary livestock losses.
156
ACKNOWLEDGMENTS Field work and testing was assisted in part by Doctors D. Armstrong, W. Huey, M. Proverbs, T. King and S. St. John. We thank them for their assistance. The authors are particularly indebted to Dr. W. J. McCabe, Regional Veterinary Director, Health of Animals Branch, Toronto and staff for their assistance in shipment and entry of tissue specimens. Special thanks to Dr. 0. W. Kelton, Contagious Diseases Division, for his assistance. Funds were provided by the United Nations Development Program (UNDP). REFERENCES 1. ANONYMOUS. Trudeau Mycobacterial Culture Collection. Trudeau Institute Incorporated. Saranac, N.Y. 1972. 2. ANONYMOUS. First International Seminar on Bovine Tuberculosis for the Americas. Scientific Publication no. 258, Pan American Health Organization, Washington, D.C. 1972. 3. ANONYMOUS. The Single Intradermal Comparative Tuberculin Test. Ministry of Agriculture, Fisheries and Food, Government Buildings, Garrison Lane,
Chessington, Surrey, England. 1975. 4. ANONYMOUS. Proceedings of the National Bovine Tuberculosis Conference. pp. 1-41. Animal and Plant Health Insp. Service, U.S.D.A. 1976. 5. BALDWIN, J.H. Pulmonary bovine tuberculosis in an owner and in his dairy herd. Cornell Vet. 58: 81-87. 1968. 6. CANETTI, G., S. FROMAN, J. GROSSET, P. HAUDUROY, M. LANGEROVA, H.T. MAHLER, G. MEISSNER. D.A. MITCHISON and L. SULA. Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull. Wld Hlth Org. 29: 565-578. 1963.
7. DAMSKER, B., E.J. BOTTONE and S. SCHNEIRSON. Human infections with Mycybacterium bovis. Am. Rev. resp. Dis. 110: 446-449. 1974. 8. DIEHL, K.E. An epizootic of bovine tuberculosis traced from slaughter. J. Am. vet. med. Ass. 159: 1534-1537. 1971. 9. FRANCIS, J., C. L. CHOI and A.J. FROST. The diagnosis of tuberculosis in cattle with special reference to bovine PPD tuberculin. Aust. vet. J. 49: 246-251. 1973. 10. GUILBRIDGE, P.D.L. Veterinary public health. The importance of animal disease to public health in the Caribbean with special reference to Jamaica. W.I. med. J. 105: 1-21. 1952. 11. HARRINGTON, R. and A. G. KARLSON. Differentiation between M. tuberculosis and M. bovis by in vitro procedures. Am. J. vet. Res. 27: 1193-1196. 1966. 12. HELLBECQUE, D.M., V. HANDZEL and L. EIDUS. Simple amidase test for identification of mycobacteria. J. clin. Microbiol. 1: 50-53. 1975. 13. HUGHES, M.H. Pyruvic acid egg medium for tubercle bacilli. J. Clin. Path. 19: 73-75. 1966. 14. HUTSON, L.R. A review of tuberculin testing of cattle in Barbados. Can. vet. J. 8: 228-230. 1967. 15. HUTSON, L.R. Clinical tuberculosis in a goat. Can. J. comp. Med. 5: 259-262. 1941. 16. JOHNSON. D.C., A.N. ROGERS, J.F. ANDREWS. J.A. DOWNARD and C.O. THOEN. An epizootic of bovine tuberculosis in Georgia. J. Am. vet. med. Ass. 167: 833-837. 1975. 17. KARLSON, A.G. and E.F. LESSEL. Mycobacterium bovis nom - nov. Int. J. syst. Batc. 20: 273-282. 1970. 18. KONYHA, L.D. Replacement of mammalian tuberculin with an M. bovis PPD tuberculin for field testing for tuberculosis. pp). 345-350. 19th Annual Proc. Am. Ass. Vet. Lab. Diag. 1976. 19. LUNA, L.G. Manual of Histologic Staining Methodq of the Armed Forces Institute of Pathology, 3rd Edition. New York: McGraw-Hill. 1968.
Can. J. comp. Med.
D.A. NEWTON-TABRETT, 20. LEPPER, A.W.D., L.A. CORNER, M.T. CARPENTER, W.A. SCANLAN, O.J. WILLIAMS and D.M. HELWIG. The use of bovine PPD tuberculin in the single caudal fold test to detect tuberculosis in beef cattle. Aust. vet. J. 53: 208-213. 1977. 21. LESSLIE, I.W., C.N. HERBERT, K.J. BURN and B.N. MacCLANCY. Comparison of the specificity of human and bovine tuberculin PPD for testing cattle. 1 - Republic of Ireland. Vet. Rec. 96: 332-334. 1976. 22. LESSLIE, I.W., C.N. HERBERT and D.N. BARNETT. Comparison of the specificity of human and bovine tuberculin PPD for testing cattle. 2 Southeastern England. Vet. Rec. 96: 335-338. 1975. 23. LESSLIE, I.W. and C.N. HERBERT. Comparison of the specificity and sensitivity of human and bovine tuberculin PPD for testing cattle. 3 - National trial in Great Britain. Vet. Rec. 96: 338-341. 1975. 24. LESSLIE, I.W., C.N. HERBERT and G.N. FRERICHS. Practical application of bovine tuberculin PPD in testing cattle in Great Britain. Vet. Rec. 98: 170-172. 1976. 25. McLAUGHLIN, A.R. and A.I. MOYLE. An epizootic of bovine tuberculosis. J. Am. vet. med. Ass. 164: 396-397. 1974. 26. MEDLAR, E.M. Pulmonary tuberculosis in cattle. Am. Rev. Tuber. 41: 282-306. 1940. 27. O'REILLY, L.M. and B.N. MacCLANCY. A comparison of the accuracy of a human and a bovine tuberculin PPD for testing cattle with a comparativecervical test. Irish vet. J. 29: 63-70. 1975. 28. PRITCHARD, D.G., D.A. FRANCIS, R. GRIPP. R.B. HARDING, E.P. JONES, C. MINTERN and P.T. McGOVERN. An abattoir survey of bovine tuberculosis in the Karamoja Region of Uganda. Br. vet. J. 131: 120-127. 1975. 29. ROSWURM, J.D. and A.F. RANNEY. Laboratory responsibility in diagnosis of mycobacterial disease. Hlth Lab. Sci. 9: 139-144. 1972. 30. ROSWURM, J.D. and A.F. RANNEY. Sharpening the attack on bovine tuberculosis. Am. J. publ. Hlth 63: 884-886. 1973.
Volume 43- April, 1979
31. RUNYON, E.H., M.J. SELIN and H.W. HARRIS. Distinguishing mycobacteria by the niacin test. Am. Rev. Tuber. 79: 663-665. 1959. 32. SOLTYS, M.A. Tubercle Bacillus and Laboratory Methods in Tuberculosis. Edinburgh: E. and S. Livinstone Ltd. 1952. 33. STEELE, J.H. and A.F. RANNEY. Animal tuberculosis. Am. Rev. resp. Dis 77: 908-922. 1958. 34. TACQUET, A., F. TISON, P. ROOS et B. DEVULDER. Activite amidasique des mycobacteries. Annls Inst. Pasteur, Paris 3: 876-383. 1967. 35. TAMMEMAGI, L., G.C. SIMMONS, R. KELMAN and W.T.K. HALL. A study of tuberculosis-like lesions in cattle slaughtered in Queensland meatworks. Aust. vet. J. 49: 507-511. 1973. 36. THOEN, C.O., R.D. ANGUS and M. SWANSON. Method for evaluation of Mycobacterium bovis purified protein derivative tuberculin in experimentally infected cattle. Am. J. vet. Res. 38: 1019-1022. 1977. 37. TRUANT, J.P., W.A. BRETT and W. THOMAS, JR. Fluorescence. Microscopy of Tubercle bacilli stained with auramine and rhodamine. Henry Ford Hosp. Bull. 10: 287-296. 1962. 38. TSUKAMURA, M. Niacin-negative Mycobacterium tuberculosis. Am. Rev. resp. Dis. 110: 101-103. 1974. 39. VESTAL, A.L. and G.P. KUBICA. Differential identification of mycobacteria, 111 use of thiacetozone, Thiophene-2-carboxylic acid hydrazide and triphenyltetrazolium chloride. Scand. J. resp. Dis. 48: 142-148. 1967. 40. VESTAL, A.L. Prcedures for the isolation and identification of mycobacteria. National Communicable Disease Center, Atlanta. Hew Publication No. 76-8203. 1976. 41. VIRTANEN, S. A Study of nitrate reduction by mycobacteria. Acta tuber. scand. Supp. 48: 1-119. 1960. 42. WELLS, K.F. The administration of bovine tuberculosis eradication in Canada. V InterAmerican Meeting on Foot and Mouth Disease and Zoonoses Control. Scientific Publication No. 256. Pan American Health Organization, Washington, D.C. 1973.
157
ABSTRACT A 57 cow dairy herd, believed to be in excellent condition and producing well, was found to contain 30 tuberculous cattle after cervical skin testing employing bovine PPD tuberculin with the single intradermal comparative test. Testing was initiated after two cows from this herd were found at meat inspection to have tuberculosis. The reliability of bovine PPD tuberculin for testing as well as significant epidemiological, laboratory and meat inspection factors are presented.
RESUMe L'epreuve cutanee cervicale, a l'aide d'une seule injection intra-dermique comparative de tuberculine bovine PPD, revela qu'un troupeau de 57 vaches laitieres qui semblait en excellente condition et dont la production etait satisfaisante, recelait neanmoins 30 sujets tuberculeux. On decida de soumettre ce troupeau a l'epreuve de la tuberculine apres que les inspecteurs d'un abattoir eurent diagnostique la tuberculose chez deux de ses sujets. Les auteurs commentent l'efficacite de la tuberculine bovine PPD et certains facteurs epidemiologiques de la tuberculose, ainsi que le role du laboratoire et de l'inspection des viandes dans la lutte contre cette maladie.
INTRODUCTION During meat inspection and follow up studies in 1977 gross and microscopic *Pan American Health Organization/World Health Organization, Bridgetown, Barbados (Wilson) and Tuberculosis Laboratory, Central Laboratories, Ontario Ministry of Health, P.O. Box 9000, Terminal A, Toronto, Ontario M5W 1R5 (Howes).
Submitted June 14, 1978.
Volume 43 -April, 1979
lesions consistent with tuberculosis were detected in lymph nodes and abdominal and thoracic organs from two dairy cows. These mature cows originated from a 57 cow dairy herd which was composed of 39 Holstein-Friesian, 14 Guernsey, one Zebu and three Ayrshire animals. The Ayrshire cattle were imported from Canada while the remainder of the herd was of local origin. Th herd had been maintained on sugarcane tops, pangola grass hay, concentrate, molasses and mineral salts and was permanently housed in a 60' x 40' corral which included a sunshade over approximately one third of the corral. Improper drainage under the sunshade caused that area to be extremely wet. The herd was considered by the owner and attending veterinarian to be in excellent condition and producing well. This report describes the pathological, epidemiological and testing data relevant to this epizootic. Microbiological information regarding the first isolation of Mycobacterium bovis from Barbados is included as well as pertinent public health considerations. Included also are comments regarding the reliability of the recently introduced bovine PPD tuberculin (purified protein derivative tuberculin prepared from Mycobacterium bovis) for cattle tuberculosis skin testing.
MATERIALS AND METHODS Pooled four-quarter milk samples were obtained from 18 randomly selected cows on the farm and prepared for microscopic examination for acid fast bacilli (40). During the interval between initial diagnosis of the disease and testing, six cows were removed from the herd including the two index animals and one cow with suspected tubercular mastitis. Three cows
151
died of unknown causes and were burned. Fifty mature cows and one bull were skin tested in the cervical area employing the single intradermal comparative test. Tuberculin used was PPD, mammalian (bovine), 1 mg/ml (B1/77) and avian, 0.5 mg/ml (A1/77) prepared by the Ministry of Agriculture, Central Veterinary Laboratory, Weybridge, England. The standard interpretation was applied to the first test while subsequent tests were severely interpreted (3). Animals that failed the test and those which had inconclusive results were field slaughtered and necropsied under veterinary supervision. Complete necropsies were performed with particular attention being given to lymph nodes. Selected specimens were placed in 10% buffered formialin, routinely prepared for histological study and stained with haematoxylin and eosin and Zeihl-Neelsen stains (19). Nonencapsulated or poorly encapsulated noncalcified pulmonary lesions that had actual or potential access to the external environment via the airways were classified as open lesions. Selected microbiological specimens obtained at necropsy were refrigerated or frozen and airshipped to Canada. Thirteen samples comprising four lungs, two livers and seven lymph nodes from six bovine carcasses were ground up and treated with 3% hydrochloric acid for one and one-half hours. Smears were made from this sediment and stained with fluorochrome and Zeihl-Neelsen and examined for the presence of acid fast bacilli (37, 40). Treated samples were then inoculated to four different egg media: LowensteinJensen (Jensen, 1932), American Trudeau Society mediump modified LowensteinJensen with pyruvic acid instead of glycerol, and Wooley's medium containing potato water, eggs and no glycerol (1, 13). Biochemical tests performed were niacin production (31), nitrate reduction (41) and amidase activity (12, 34). Also, Tween 80 hydrolysis, arylsulphatase and catalase activity were performed according to standard methods (40). Susceptibility was measured to thiophene-2-carboxylic acid hydrazide and thiosemicarbazone (39) and sensitivity tests to first-line antituberculosis drugs were conducted using the proportion method and Middlebrook 7H 10 agar medium (6, 40). 152
Five, two month old guinea pigs weighing between 250 to 350 g were injected intramuscularly in the right thigh with untreated separate lymph node material. Palpation of the animals at regular intervals for the presence of enlarged lymph nodes was carried out. The animals were euthanized after four weeks on test and macroscopic and microscopic examinations for evidence of tuberculosis conducted (32, 40).
RESULTS Microscopic examination of milk sediment smears from 18 cows on the farm revealed short, plump, acid fast bacteria in one sample. This cow was removed from the herd prior to testing and at necropsy gross and microscopic lesions consistent with tuberculosis were found in the liver and retropharyngeal lymph nodes. Twenty-three cows failed the first test and seven animals yielded inconclusive results. Retesting employing the same testing procedures in 60 days detected seven additional failures totalling 30 test failures and seven inconclusive animals (Table I), all of which were removed from the herd and euthanized. Necropsies were conducted on 28 test failure cows and six test inconclusive cows and tabulated (Table I and II) (Fig. 1). Macroscopic lesions were observed in various lymph nodes, lungs and livers and open lesions in the lungs were detected in 18 cows (Table II). The macroscopic lesions were very variable in size and ranged from focal lesions 3-4 mm in diameter to tubercular mediastinal lymph nodes 20-25 cm TABLE I. Tuberculosis Skin Test and Necropsy Results of 51 Dairy Cattle, Barbados, West Indies
Skin Test + Number Skin Test + Necropsy + Animals Necropsied for Tuberculosis 28 25(89%) 30 Number Skin Test Necropsy + Skin Test Inconclusive Inconclusive Animals for Tuberculosis Necropsied 6 7 3(50%) 34 28(82%) Total 37
Can. J. comp. Med.
TABLE II. The Distribution of Gross Tubercular Lesions in 27 Mature Milk Cows and One Bull, Barbados, West Indies
z hi
'.
. .
z
z .
z . ,..
z ..
.
i..
.
'-.
'.
-
.-
..
..
0. hi
0
..
..
.0
hi
.
.
.
'.
..
e.
.i
hi
hi
.
G.
.
iihi
.
.
2
15
28
0
2
28
28
28
18
16
14
28
28
3
2
28
28
,.
0
28
0
2
27
28
0 28
18 28
28
Fig. 1. Guernsey cow displaying enlarged tubercular parotid and submandibular lymph nodes, Barbados, W.I. (Arrows).
Fig. 2. Very enlarged, calcified tubercular mediastinal lymph nodes from a mature dairy cow, Barbados, W.I. (Arrows).
long and 7-10 cm in diameter (Figs. 2, 3 and 4). The majority of the encapsulated lesions were yellow, firm and calcified while some were caseous and only slightly calcified. No macroscopic lesions were observed in any mammary glands or supramammary lymph nodes. Histologically, lesions in the lung, liver and lymph nodes were characterised by
All smears contained acid-fast bacilli but in very small numbers, usually less than ten bacilli per slide. Initial cultural isolation of 11 mycobacterial strains was achieved in two to four weeks on the egg media with best growth on media devoid of glycerol (Lowenstein-Jensen with pyruvic acid, and Woolley's). The strains were dysgonic on primary isolation, becoming luxuriant on subculture. They grew at 36°C, not at 22°C or 450C and colonies were nonpigmented and rough. All isolates were negative for niacin production, nitrate reduction and Tween 80 hydrolysis. They exhibited positive arylsulphatase activity in two weeks, only slight catalase activity and only a positive urease in the test for amidase activity. They were
necrotic centers, often calcified, surrounded by an admixture of reticuloendothelial cells, lymphocytes and Langhans' giant cells. Acid fast organisms were rarely found. Interalveolar septa in the mammary gland were thickened and infiltrated by reticuloendothelial cells, lymphocytes and the occasional Langhans' giant cell. Acid-fast bacilli were observed in some giant cells.
Volume 43 - April, 1979
153
TABLE III. Biochemical, Enzymatic and Drug Susceptibility Patterns of 13 Mycobacterium Strains Isolated from Barbados Cattle, West Indies
Fig. 3. Very eniarged calcied-caseous tubercular mediastinal lymph nodes from a mature dairy cow, Barbados, W.I. (Arrows).
Biochemical and Enzyme Results Niacin Negative Nitrate reduction Negative Arylsulphatase Negative three day; positive two weeks Tween 80 hydrolysis Negative 12 days Catalase activity Very slight Amidase activity Positive urease Drug Susceptibility Growth inhibited by 10 pLg/ml Thiosemicarbazone 10 iug/ml Thiophenecarboxylic acid hydrazide 2 ag/ml Streptomycin 2 wg/ml PAS 0.2 ,ug/ml Isoniazid 5 ILg/ml Ethambutol 1 ;Lg/ml Rifampin
tenuated variant of M. bovis, by the more luxuriant growth of BCG and absence of virulence of BCG for rabbits and guinea pigs (7, 11, 38).
DISCUSSION Fic. geal
4. Ver
enlarged caseous tubercular Barbados, W.I. lymph node,
retropharyn-
susceptible to 10 /g/ml thiophene-2-carboxylic acid hydrazide (TCH), 10 /Lg/ml thiosemicarbazone, and also fully susceptible to 2 glg/ml streptomycin, 2 ug/ml PAS, 0.2 pug/ml isoniazid, 5 ,ug/ml ethambutol and 1 ylg/ml rifampicin (Table III). Postmortem examination of all five guinea pigs showed typical tuberculous infection, with the presence of macroscopic, caseating lesions in the inguinal and lumbar lymph glands. The spleen was also grossly affected in two cases. Microscopically, the lesions revealed the presence of numerous, short, acid-fast bacilli. Based on the above results all strains were identified as M. bovis. This organism, recognised as a distinct species (17), is distinguished from Mycobacterium tuberculosis on the basis of its slower growth rate, inhibition by glycerol, inhibition by TCH, negative nitrate reduction, negative niacin production and differences in the amidase patterns. Mycobacterium bovis is differentiated from M. bovis BCG, the at154
In 1966 Barbados initiated a cattle tuberculosis testing program which revealed a very low reactor rate to mammalian tuberculin of 0.172% (14). Some further testing was conducted. However, due to staff and financial constraints island wide coverage was never achieved. Because of the very low reactor rate and the lack of confirmed tuberculosis cases at the slaughterhouse the island was declared free of bovine tuberculosis. This epizootic clearly demonstrates that bovine tuberculosis control/ eradication schemes must rely on continuous testing programs and thorough slaughterhouse inspection programs (2,4,8,16,30,33). Additionally, as depicted in this outbreak and others reported (5,8,16,25), bovine tuberculosis can be a chronic disease, often present for many months or years in a productive herd and only being detected by skin test or at slaughter after the majority of the herd has becomie infected. Significant improvements in materials and methods relevant to bovine tuberculosis testing have taken place. Recently there has been considerable research conducted
Can. J. comp. Med.
animals lesions were present only in the lymph nodes of the head while the major pulmonary lymph nodes were lesion free. Additionally, 11 of 28 (39%) cows had gross lesions in the major pulmonary lymph nodes while designated lymph nodes of the head had no lesions. Meat inspection staff must make complete and detailed gross examinations of the major lymph nodes of the head and lungs. This should be particularly stressed in developing countries where oftentimes the mneat inspection service is in its very formative stages. The proper laboratory follow-up of granulomatous lesions is a most important activity in bovine tuberculosis programs and should be strongly encouraged (2,4). One important aspect of this laboratory study of suspect granulomatous lesions is a thorough histological examination of the lesions. However, over reliance on histopathology as a definitive diagnostic tool must be avoided. Acid fast bacteria were rarely observed histologically in a variety of tissues from 28 cattle on this farm and this observation was supported by the rarity of such organisms under fluorescent and acid fast examination of smears from various diseased tissue. One study on bovine tuberculosis (35) completely disregarded acid fast histological staining as it did not aid significantly in the diagnosis of bovine tuberculosis when microbiological culturing was done. Histopathological find(2). Depopulation of the entire herd in ings where possible should be correlated large outbreaks of bovine tuberculosis is with the isolation and identification of the generally considered to be a most appro- bacteria. priate procedure (2,4,8,25,42). Immediate Microbiological identification of the economic consideration, unfortunately, pre- causative agent in bovine tuberculosis is cluded entire herd depopulation on this essential (2,4,29) as it is well recognised farm and eight animals from the original that atypical mycobacterium may cause herd remain. tuberculosis-like lesions in cattle and proAn attempt was made to correlate the duce sensitization to the skin test (28). Adresults of the skin test (dimensions and ditionally, microbiological identification is characteristics of reaction) to the size and most important in those geographical areas number of gross lesions found at necropsy. where little information exists about boNo simple relationship appeared to exist in vine tuberculosis. this situation. Mycobacterium bovis isolates from this Meat inspection is considered to be one herd clearly indicate it to be the etiological of the most important surveillance activi- agent and more importantly, add valuable ties in bovine tuberculosis control pro- information relevant to the reliability of grams and is today responsible for the bovine PPD tuberculin under field testing identification of many tuberculous cattle conditions. Little information (10, 14, 15) herds (2,4,8,16,25,26,29,35,42). Special sig- exists on the epidemiology of bovine tubernificance relevant to the distribution of the culosis in Barbados or the English speakgross lesions and meat inspection is evident ing Caribbean and this report should prowhen one notes that in five of 28 (17%) vide a clearer understanding of this imon the use of bovine PPD tuberculin in cattle tuberculosis testing in place of the widely used human PPD tuberculin and several studies have indicated a high degree of sensitivity and specificity for PPD tuberculin of bovine origin (9,18,2024,27,36). Our results tabulated from a natural field epizootic revealed a high degree of reliability in the use of bovine PPD tuberculin. This herd was gradually built up over a ten-year period by purchasing cattle in small numbers from around the island. The tuberculosis testing history of the major herd and the animals added to the herd was not clear. Investigations concerning cattle movement in and out of this herd and trace back tuberculosis testing have failed to reveal the source of infection. Three cows found to be infected were imported from Canada. However, because of Canadian testing programs this is an unlikely source of infection (42). Lack of proper records of many of the small farmers considerably complicated the epidemiological follow-up. It is quite clear, however, that after introduction the disease may have spread very rapidly and diffusely. The close confinement of the cattle under very wet conditions would have allowed opportunity for repeated exposure to the infectious agent which may have caused the development of the very large lesions noted, especially in the pulmonary lymph nodes
Volume 43 - April, 1979
155
portant zoonosis. Tuberculosis of the bovine udder may take several forms including disseminated miliary, lobular infiltrating and caseous mastitis (2,33). Lobular infiltrating tubercular mastitis is reported to be the most common form and is unique in that the macroscopic changes in the udder and regional lymph nodes are very minimal. Examination of milk samples from a portion of this herd revealed one cow that was shedding acid fast bacilli resembling M. bovis in the milk. Necropsy of this cow revealed gross tubercular lesions in several sites, excluding the udder. However, histological lesions consistent with lobular infiltrating tuberculosis of the udder were observed. Although no macroscopic changes were detected in the udder or suprammamary lymph nodes of 27 cows, histological lesions consistent with lobular infiltrating tuberculosis were detected in five cattle. Microbiological tests were not conducted on mammary tissue. Bovine tuberculosis, in particular the shedding of M. bovis in the milk, has very important livestock and public health implications (2). In 1977 fresh milk production in Barbados amounted to approximately ten million pounds of which six million pounds was pasteurised prior to public sale. Four million pounds or 40% of the total milk supply was available to the public as a raw product. Because of the well recognised threat of M. bovis to human health an epidemiological study was initiated by health officials to trace any persons in close contact with the farm animals or farm milk products. Additionally health officials have advised all those consuming raw milk to boil it before use. In preparation of an island wide cattle tuberculosis testing program health officials have com,pleted a cattle census detailing all owners who sell raw milk to the public. Bovine tuberculosis occurs throughout the world and can have a serious impact on cattle development in both developed and developing countries. It is clear that through proper implementation of both testing and slaughterhouse surveillance this important zoonosis can be efficiently controlled. It is particularly important for those countries embarking on cattle development schemes to plan and implement modern tuberculosis control programs and avoid unnecessary livestock losses.
156
ACKNOWLEDGMENTS Field work and testing was assisted in part by Doctors D. Armstrong, W. Huey, M. Proverbs, T. King and S. St. John. We thank them for their assistance. The authors are particularly indebted to Dr. W. J. McCabe, Regional Veterinary Director, Health of Animals Branch, Toronto and staff for their assistance in shipment and entry of tissue specimens. Special thanks to Dr. 0. W. Kelton, Contagious Diseases Division, for his assistance. Funds were provided by the United Nations Development Program (UNDP). REFERENCES 1. ANONYMOUS. Trudeau Mycobacterial Culture Collection. Trudeau Institute Incorporated. Saranac, N.Y. 1972. 2. ANONYMOUS. First International Seminar on Bovine Tuberculosis for the Americas. Scientific Publication no. 258, Pan American Health Organization, Washington, D.C. 1972. 3. ANONYMOUS. The Single Intradermal Comparative Tuberculin Test. Ministry of Agriculture, Fisheries and Food, Government Buildings, Garrison Lane,
Chessington, Surrey, England. 1975. 4. ANONYMOUS. Proceedings of the National Bovine Tuberculosis Conference. pp. 1-41. Animal and Plant Health Insp. Service, U.S.D.A. 1976. 5. BALDWIN, J.H. Pulmonary bovine tuberculosis in an owner and in his dairy herd. Cornell Vet. 58: 81-87. 1968. 6. CANETTI, G., S. FROMAN, J. GROSSET, P. HAUDUROY, M. LANGEROVA, H.T. MAHLER, G. MEISSNER. D.A. MITCHISON and L. SULA. Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bull. Wld Hlth Org. 29: 565-578. 1963.
7. DAMSKER, B., E.J. BOTTONE and S. SCHNEIRSON. Human infections with Mycybacterium bovis. Am. Rev. resp. Dis. 110: 446-449. 1974. 8. DIEHL, K.E. An epizootic of bovine tuberculosis traced from slaughter. J. Am. vet. med. Ass. 159: 1534-1537. 1971. 9. FRANCIS, J., C. L. CHOI and A.J. FROST. The diagnosis of tuberculosis in cattle with special reference to bovine PPD tuberculin. Aust. vet. J. 49: 246-251. 1973. 10. GUILBRIDGE, P.D.L. Veterinary public health. The importance of animal disease to public health in the Caribbean with special reference to Jamaica. W.I. med. J. 105: 1-21. 1952. 11. HARRINGTON, R. and A. G. KARLSON. Differentiation between M. tuberculosis and M. bovis by in vitro procedures. Am. J. vet. Res. 27: 1193-1196. 1966. 12. HELLBECQUE, D.M., V. HANDZEL and L. EIDUS. Simple amidase test for identification of mycobacteria. J. clin. Microbiol. 1: 50-53. 1975. 13. HUGHES, M.H. Pyruvic acid egg medium for tubercle bacilli. J. Clin. Path. 19: 73-75. 1966. 14. HUTSON, L.R. A review of tuberculin testing of cattle in Barbados. Can. vet. J. 8: 228-230. 1967. 15. HUTSON, L.R. Clinical tuberculosis in a goat. Can. J. comp. Med. 5: 259-262. 1941. 16. JOHNSON. D.C., A.N. ROGERS, J.F. ANDREWS. J.A. DOWNARD and C.O. THOEN. An epizootic of bovine tuberculosis in Georgia. J. Am. vet. med. Ass. 167: 833-837. 1975. 17. KARLSON, A.G. and E.F. LESSEL. Mycobacterium bovis nom - nov. Int. J. syst. Batc. 20: 273-282. 1970. 18. KONYHA, L.D. Replacement of mammalian tuberculin with an M. bovis PPD tuberculin for field testing for tuberculosis. pp). 345-350. 19th Annual Proc. Am. Ass. Vet. Lab. Diag. 1976. 19. LUNA, L.G. Manual of Histologic Staining Methodq of the Armed Forces Institute of Pathology, 3rd Edition. New York: McGraw-Hill. 1968.
Can. J. comp. Med.
D.A. NEWTON-TABRETT, 20. LEPPER, A.W.D., L.A. CORNER, M.T. CARPENTER, W.A. SCANLAN, O.J. WILLIAMS and D.M. HELWIG. The use of bovine PPD tuberculin in the single caudal fold test to detect tuberculosis in beef cattle. Aust. vet. J. 53: 208-213. 1977. 21. LESSLIE, I.W., C.N. HERBERT, K.J. BURN and B.N. MacCLANCY. Comparison of the specificity of human and bovine tuberculin PPD for testing cattle. 1 - Republic of Ireland. Vet. Rec. 96: 332-334. 1976. 22. LESSLIE, I.W., C.N. HERBERT and D.N. BARNETT. Comparison of the specificity of human and bovine tuberculin PPD for testing cattle. 2 Southeastern England. Vet. Rec. 96: 335-338. 1975. 23. LESSLIE, I.W. and C.N. HERBERT. Comparison of the specificity and sensitivity of human and bovine tuberculin PPD for testing cattle. 3 - National trial in Great Britain. Vet. Rec. 96: 338-341. 1975. 24. LESSLIE, I.W., C.N. HERBERT and G.N. FRERICHS. Practical application of bovine tuberculin PPD in testing cattle in Great Britain. Vet. Rec. 98: 170-172. 1976. 25. McLAUGHLIN, A.R. and A.I. MOYLE. An epizootic of bovine tuberculosis. J. Am. vet. med. Ass. 164: 396-397. 1974. 26. MEDLAR, E.M. Pulmonary tuberculosis in cattle. Am. Rev. Tuber. 41: 282-306. 1940. 27. O'REILLY, L.M. and B.N. MacCLANCY. A comparison of the accuracy of a human and a bovine tuberculin PPD for testing cattle with a comparativecervical test. Irish vet. J. 29: 63-70. 1975. 28. PRITCHARD, D.G., D.A. FRANCIS, R. GRIPP. R.B. HARDING, E.P. JONES, C. MINTERN and P.T. McGOVERN. An abattoir survey of bovine tuberculosis in the Karamoja Region of Uganda. Br. vet. J. 131: 120-127. 1975. 29. ROSWURM, J.D. and A.F. RANNEY. Laboratory responsibility in diagnosis of mycobacterial disease. Hlth Lab. Sci. 9: 139-144. 1972. 30. ROSWURM, J.D. and A.F. RANNEY. Sharpening the attack on bovine tuberculosis. Am. J. publ. Hlth 63: 884-886. 1973.
Volume 43- April, 1979
31. RUNYON, E.H., M.J. SELIN and H.W. HARRIS. Distinguishing mycobacteria by the niacin test. Am. Rev. Tuber. 79: 663-665. 1959. 32. SOLTYS, M.A. Tubercle Bacillus and Laboratory Methods in Tuberculosis. Edinburgh: E. and S. Livinstone Ltd. 1952. 33. STEELE, J.H. and A.F. RANNEY. Animal tuberculosis. Am. Rev. resp. Dis 77: 908-922. 1958. 34. TACQUET, A., F. TISON, P. ROOS et B. DEVULDER. Activite amidasique des mycobacteries. Annls Inst. Pasteur, Paris 3: 876-383. 1967. 35. TAMMEMAGI, L., G.C. SIMMONS, R. KELMAN and W.T.K. HALL. A study of tuberculosis-like lesions in cattle slaughtered in Queensland meatworks. Aust. vet. J. 49: 507-511. 1973. 36. THOEN, C.O., R.D. ANGUS and M. SWANSON. Method for evaluation of Mycobacterium bovis purified protein derivative tuberculin in experimentally infected cattle. Am. J. vet. Res. 38: 1019-1022. 1977. 37. TRUANT, J.P., W.A. BRETT and W. THOMAS, JR. Fluorescence. Microscopy of Tubercle bacilli stained with auramine and rhodamine. Henry Ford Hosp. Bull. 10: 287-296. 1962. 38. TSUKAMURA, M. Niacin-negative Mycobacterium tuberculosis. Am. Rev. resp. Dis. 110: 101-103. 1974. 39. VESTAL, A.L. and G.P. KUBICA. Differential identification of mycobacteria, 111 use of thiacetozone, Thiophene-2-carboxylic acid hydrazide and triphenyltetrazolium chloride. Scand. J. resp. Dis. 48: 142-148. 1967. 40. VESTAL, A.L. Prcedures for the isolation and identification of mycobacteria. National Communicable Disease Center, Atlanta. Hew Publication No. 76-8203. 1976. 41. VIRTANEN, S. A Study of nitrate reduction by mycobacteria. Acta tuber. scand. Supp. 48: 1-119. 1960. 42. WELLS, K.F. The administration of bovine tuberculosis eradication in Canada. V InterAmerican Meeting on Foot and Mouth Disease and Zoonoses Control. Scientific Publication No. 256. Pan American Health Organization, Washington, D.C. 1973.
157
