J Vet Diagn Invest 4:428-433 (1992)
Gross and microscopic lesions of naturally occurring tuberculosis in a captive herd of wapiti (Cervus elaphus nelsoni) in Colorado Jack C. Rhyan, Dennis A. Saari, Elizabeth S. Williams, Michael W. Miller, Arthur J. Davis, Arach J. Wilson Abstract. A Mycobacterium bovis-infected herd of captive wapiti (Cervus elaphus nelsoni) in Colorado was depopulated after lesions of bovine tuberculosis were confirmed in 8 of 10 tuberculin skin test reactors. Of the 43 animals > 1 year of age, 26 had gross lesions suggestive of tuberculosis, 24 had microscopic lesions of tuberculosis, and 23 had acid-fast bacilli associated with the lesions. Lungs and retropharyngeal lymph nodes were the most frequently affected sites. Most lesions grossly and microscopically resembled tuberculosis in cattle; however, some lesions resembled abscesses or ovine caseous lymphadenitis lesions. Special stains and immunohistochemical techniques labeled few to numerous mycobacteria associated with the lesions.
Until recently, bovine tuberculosis was considered an unusual and sporadic disease in cervids. Reported cases involved captive and wild cervids and were often attributed to contact with infected cattle or bison.14,16,24 With recent development and expansion of game farming, the prevalence, economic impact, and public health significance of tuberculosis in captive cervids have greatly increased. 6,9,32 In New Zealand, tuberculosis was diagnosed in wild red deer (Cervus elaphus elaphus) in 19702 and in farmed deer in 1978.17 It is now considered the most important bacterial disease in New Zealand’s farmed deer population.6,13,32 Bovine tuberculosis has also been diagnosed in several species of free-living and captive cervids from other countries1,8,11,15,19,20,25,29-31,33,35,36,43 In North America, reports of tuberculosis in cervids have been limited to sporadic cases in wild and captive white-tailed deer (Odocoileus virginianus) 3,10,12,24 a n d 2 2 to outbreaks in captive sika deer (Cervus nippon), 22,29,41 captive fallow deer (Dama dama), and captive wapiti (Rocky Mountain elk, Cervus elaphus nel37,39 soni). In addition, gross lesions consistent with tuberculosis were reported in wapiti, mule deer (Odocoileus hemionus), and moose (Alces alces) in the Canadian National Buffalo Park near Wainwright, Alberta, in 1939 and 1940.16 This finding was concurrent
with a high prevalence of M. bovis infection in the Park bison (Bison bison). There are no recent reports of bovine tuberculosis in free-ranging cervids in North America. In June 1991, an M. bovis-infected herd of captive wapiti in Colorado was depopulated after gross and histologic lesions of bovine tuberculosis were confirmed in 8 of 10 tuberculin skin test reactors.26 This report provides more detailed descriptions of all gross and microscopic lesions of bovine tuberculosis observed in this herd. Materials and methods All animals were killed by gunshot. Necropsies were done, gross lesions recorded and photographed, and tissue specimens collected.26 Samples of grossly observed lesions and/ or parotid, mandibular, retropharyngeal, mediastinal, bronchial, hepatic, and mesenteric lymph nodes that were routinely bisected were placed in 10% neutral buffered formalin and saturated sodium borate transport solution for mycobacterial culture. Specimens were transported within 4 days to the National Veterinary Services Laboratories, Ames, Iowa, for histopathologic examination and mycobacterial culture. Formalin-fixed tissues were routinely processed using paraffin embedding, and 5-µm sections were stained with hematoxylin and eosin (HE). Significantly mineralized tissues were decalcified and routinely processed. Additional sections from tissues with lesions suggestive of tuberculosis were stained by a new fuchsin-methylene blue procedure (NF)18 and acridine orange-auramine O (AOA0)27 and were examined for the presence of acid-fast bacilli and AOAO fluorescent bacilli. Some sections were also stained with Giemsa and Gram’s stains. Selected specimens were stained by a labeled streptavidinbiotin immunohistochemical technique to better demonstrate antigen location. Four-micrometer paraffin sections were deparaffinized, treated with 3% H2O2 to quench endog-
From the Pathobiology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, US Department of Agriculture, PO Box 844, Ames, IA 50010 (Rhyan, Saari, Davis, Wilson), the Wyoming State Veterinary Laboratory, Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82070 (Williams), and the Colorado Division of Wildlife, Wildlife Research Center, 317 W. Prospect Road, Fort Collins, CO 80526 (Miller). Received for publication February 27, 1992. 428
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Lesions of tuberculosis in wapiti Table 1.
429
Signalment of wapiti with gross and/or microscopic tuberculous lesions and location of grossly observed lesions.
enous peroxidase, trypsin digested (20 min at 37 C), blocked with goat serum, and incubated with commercially available polyclonal anti-M. bovis BCG antibodya (30 min at 37 C). Following 3 rinses in buffer,b sections were incubated with biotinylated goat origin anti-rabbit Iga (30 min at 37 C). After 3 rinses, peroxidase-labeled streptavidina was applied (10 min at 37 C) and rinsed, and the color was developed with 3-amino-9-ethylcarbazole (AEC).a Sections were counterstained with Gill II hematoxylin,c and coverslips were mounted with water-base medium.a Nonimmunized rabbit Ig fractiona was substituted for the primary antibody as a negative control. A commercially available capillary gap systemd was used for immunohistochemical staining.
Results Gross observations. Of the 43 captive wapiti examined that were 21 year of age, 26 had gross lesions considered suggestive of tuberculosis.26 Tuberculous lesions occurred most often in retropharyngeal lymph nodes and lung (Table 1). Lung lesions were concentrated in the dorsocaudal half of caudal lobes (Fig. I). Several animals had disseminated lesions in the thoracic cavity, and 3 animals had lesions in thoracic and abdominal cavities. In addition, No. 22 had extensive lesions in the mammary gland and supramammary lymph nodes. Pleural lesions involving the thoracic
wall were noted in 6 cases and diaphragmatic granulomas in 4 cases. Extensive subcutaneous thoracic abscesses were noted in 2 animals, and 2 animals had lesions in the mesenteric lymph nodes. Gross lesions varied in size from small miliary foci 1 mm in diameter to large tubercles or abscesses >30 cm in diameter. Lesions ranged from calcified caseous tubercles to thin-walled abscesses. Some lesions contained white to grey-white viscous exudate with scattered tiny flakes of mineralized material. Several round encapsulated lesions contained caseous necrotic material arranged in concentric lamellar layers. In some of these, mineralized material was located in a lamellar arrangement near the outer fibrous capsule. Abscesses and calcified caseous tubercles were present in close proximity in the same organ in several cases. Mastitis in animal No. 22 was characterized by numerous small to large (2-8 cm diameter) caseous abscesses virtually replacing all normal mammary gland tissue. The caseous material varied from white or greywhite to tan, and some of the abscesses had cavitated centers containing white liquid. Occasional mammary gland abscesses contained caseous material arranged in distinct concentric laminations (Fig. 2). There was direct extension of mammary lesions to supramam-
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Figure 1. Caudal and middle right lung lobes of a captive wapiti. Note incised granulomas with caseous centers in dorsocaudal portion of caudal lobe. Figure 2. Abscess in wapiti mammary gland with concentric laminae of caseous and mineralized exudate surrounding central zone of liquefactive necrosis. Figure 3. Portion of granuloma with caseous center from a wapiti. Note central area of mineralization (arrows), caseous necrosis, neutrophils and peripheral mineralization (arrowheads) extending into inflammatory cell mantle. HE stain. Figure 4. Tuberculous lung lesion from a wapiti. Note bronchiole containing necrotic exudate and adjacent caseous abscess with focal mineralization. HE stain.
mary lymph nodes. These nodes were enlarged (3 x 5 cm), and normal parenchyma was replaced with caseous necrotic material. In the same animal, caseous material in some pleural tubercles was also arranged in concentric lamellar layers. Microscopic features. Microscopic examination confirmed lesions from 24 animals as suggestive or typical of tuberculosis, and acid-fast- or AOAO-positive bacilli were observed in lesions from 23 of the affected animals. Lymph node lesions typically consisted of round, oval, or irregular central zones of caseous necrosis and mineralization surrounded by a nar-
row to wide mantle of mixed inflammatory cells and a layer of dense collagenous connective tissue. The finely granular caseous necrotic material was stained faintly to moderately eosinophilic with focal basophilic stippling or areas of diffuse basophilia due to chromatin debris. In many lesions, few to numerous neutrophils and degenerating leukocytes were scattered throughout the necrotic material. Mineralization, usually present, was centrally and peripherally located with small to large mineralized foci often extending into the surrounding inflammatory cell infiltrate (Fig. 3). The mantle of inflammatory cells surrounding the
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Figure 5. Large caseous granuloma in lymph node of a wapiti. Immunohistochemical stain labels strata of antigen near periphery of lesion. Inset: high magnification of same area. Note numerous mycobacteria clustered between collagen fibers. Acid-fast stain. Figure 6. Caseous granuloma in lymph node of a wapiti. Note immunoperoxidase-labeled mycobacteria scattered (arrows) near periphery of lesion and concentrated (arrowheads) in caseous center. Also note peripheral mineralization.
caseous necrosis usually consisted of an inner layer of few to numerous neutrophils surrounded by a narrow to wide zone of epithelioid macrophages admixed with small or moderate numbers of lymphocytes and macrophages and rare or occasional Langhan’s giant cells. Lesions were partially or completely encapsulated with focal capsular invasion and extension into the adjacent lymph node parenchyma. Small unencapsulated focal accumulations of epithelioid macrophages and giant cells were located in some lymph node cortices. The single suggestive lesion noted in the mandibular lymph node of animal No. 21 consisted of a small (1 mm diameter), round, thinly encapsulated focal accumulation of neutrophils, epithelioid macrophages, and Langhans’s giant cells. Lung, pleural, and mammary gland lesions were histologically similar to lymph node lesions. Pleural lesions were usually surrounded by a wider zone of collagenous connective tissue. Lung lesions occasionally lacked mineralization and frequently contained large central accumulations of neutrophils. In some sections, bronchiectatic caseous abscesses were confluent with tuberculous lesions in the adjacent pulmonary parenchyma (Fig. 4). Microscopic examination of lesions with gross lamellar architecture revealed concentrically arranged layers of finely granular eosinophilic material and collagen fibers, occasionally interspersed with layers of mineralized material, neutrophils, and chromatin debris. The necrotic centers were usually bordered by small numbers of mixed inflammatory cells and a collagenous connective tissue capsule. New fuchsin- and AOAO-stained sections contained
rare to numerous mycobacteria associated with lesions. Organisms were not observed in the single small lesion from animal No. 21. In most cases, rare or occasional organisms (1 organism/25 250 x fields) were scattered in the caseous necrotic material or surrounding mantle of macrophages and giant cells. Lesions from 5 animals, however, contained numerous mycobacteria (> 25 organisms/25 250 x fields). Organisms were concentrated in the caseous centers, focally or diffusely scattered in the cellular mantle, or randomly scattered throughout the lesions. In some of the concentric lamellar lesions, mycobacteria were concentrated in lamellar zones in the caseous material near the periphery of the lesions or clustered between collagen layers in the capsule (Fig. 5). Other organisms were not observed in Gram-stained sections of these lesions. In immunohistochemically stained sections, antigen was present in distribution patterns identical to those seen with NF and AOAO stains. Immunoperoxidaselabeled mycobacteria stained strongly positive and were usually surrounded by a small smudge or halo of lighter staining material. The labeled antigen was easily detected on low magnification (Figs. 5, 6). The quantity and distribution of mycobacteria in lesions were more easily determined in immunoperoxidase- and AOAOstained sections than in NF-stained sections. Incidental lesions noted were pyogranulomas containing Splendore-Hoeppli material and gram-negative rod-shaped organisms morphologically typical of Actinobacillus lignieresi in a mandibular lymph node and retropharyngeal lymph node, 1 sublingual abscess with associated gram-positive coccobacilli, scattered mineralized foci in 1 lymph node, and a focal lymphocytic
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middle lung lobes were also most frequently affected in an M. bovis-infected herd of fallow deer.41 Granulomas with caseous centers and abscesses have been previously described in wapiti and red deer. 11,16,36 Discussion Concentrically laminated lesions, however, have not There are few published descriptions of bovine tu- been reported with cervid tuberculosis. These lesions berculosis in wapiti. Previous anecdotal reports men- grossly and microscopically resembled lesions of ovine tioned the isolation of M. bovis from 1 wapiti40 and caseous lymphadenitis.21 The microscopic features of caseous and suppurative the presence of tuberculous lesions in 73 wapiti at slaughter. 16 A report of an outbreak of tuberculosis in lesions in these wapiti are similar to those described confined wapiti herds described granuloma-type pul- in tuberculous red deer.4,35,36 These lesions usually posmonary lesions, and lymph node and subcutaneous sessed the hallmark features of bovine tuberculosis, abscesses.37 Lymph node and lung lesions were recently i.e., caseous necrosis, mineralization, epithelioid macnoted in a captive M. bovis-infected wapiti in Mon- rophages, giant cells, and fibrosis. However, several tana.39 Lesions of M. bovis infection in the conspecific differences from lesions in cattle were noted: 1) lesions European red deer4,7,35,36 and other cervidsl,l0,15,22,29-31,33,41 contained moderate or large numbers of neutrophils, 2) mineralization was not as extensive as in bovine have been described and recently reviewed.5,13 Mycobacterium bovis can produce high disease prev- lesions and was often located around the periphery of alence in a captive herd of wapiti. Twenty-three of 43 the necrotic centers even extending into the layer of (5 3.5%) wapiti had granulomatous or pyogranuloma- macrophages, 3) the caseous necrotic material did not tous lesions with associated acid-fast bacilli, and M. stain as intensely eosinophilic and frequently conbovis was isolated from 16 animals with lesions and 1 tained abundant chromatin debris and/or neutrophils, animal without observed lesions.26 In a previously re- 4) lesions contained fewer giant cells than do tuberported outbreak of tuberculosis in captive wapiti,37 8 culous lesions in cattle, and 5) several lesions contained of 10 animals that died from a herd of 62 had sugges- large numbers of acid-fast bacilli, an unusual finding tive lesions, and M. bovis was isolated from 2 animals. in cattle. Varied patterns of mycobacterial distribution in leAlso, 1 of 5 slaughtered wapiti from a different herd had gross lesions suggestive of tuberculosis from which sions were demonstrated with acid-fast, AOAO, and M. bovis was subsequently isolated. The prevalence of immunohistochemical staining. The organisms’ lamdisease in this group of captive wapiti is similar to that inar distribution in the concentric lamellar lesions has reported in farmed red deer. In 1 group of 51 red deer not, to our knowledge, been previously described. The imported to the United Kingdom from Eastern Eu- staining reaction of anti-M. bovis BCG antibody to M. rope, M. bovis was isolated from 20 (39.2%) animals, bovis antigen was similar to results in studies using the and 13 (25.5%) had visible lesions.35,36 A 90% infection antibody to demonstrate other mycobacteria.28,42 The rate in adult animals was described in a herd of New immunoperoxidase stain allowed detection of mycoZealand farmed red deer.13 Other than the report of bacteria at a much lower magnification than the NF gross lesions suggestive of tuberculosis in wapiti from stain. The AOAO stain also allowed demonstration of National Buffalo Park near Wainwright, Alberta, Can- mycobacteria at a low magnification but required the ada 16,34,38 tuberculosis has not been encountered in free- use of a fluorescent scope and did not allow concurrent detailed assessment of the surrounding lesion. ranging wapiti. This report is the first detailed description of gross Wide variations in lesion distribution and gross and microscopic features can occur with M. bovis infection and microscopic lesions of M. bovis infection in wapiti. in wapiti. The high prevalence of lung and retropha- The location and morphology of M. bovis lesions in ryngeal lymph node lesions is consistent with other this herd of wapiti were widely varied. The gross and reports in wapiti and red deer.2,6,7,11,l3,32,36,37 Pleural and microscopic appearance of lesions resembled bovine diaphragmatic lesions, subcutaneous abscesses, and tuberculosis in cattle, abscesses, and ovine caseous mesenteric lymph node involvement have also been lymphadenitis lesions. Subcutaneous abscesses and tudescribed. 11,36,37 Tuberculous lesions in the mammary berculous mastitis were also present. These findings gland of cervids are considered rare.5 Occurrence of indicate that M. bovis should be considered as a pospulmonary lesions, predominantly in the subpleural sible etiologic agent whenever abscesslike lesions are parenchyma of the dorsocaudal portions of the caudal encountered in cervids. 11 lobes, is similar to tuberculosis in adult cattle21 and in Acknowledgements sika and fallow deer. 23 In experimentally infected red deer, however, intratracheal inoculation of M. bovis We gratefully acknowledge the technical assistance of Krisproduced anterior lung lobe lesions.7 Anterior and tin Wilson, Julie Campbell, and Brenda Burleson. We also
hepatitis of unknown etiology. Also present was a focal eosinophilic lymphadenitis in 1 lymph node considered suggestive of a parasitic etiology.
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Lesions of tuberculosis in wapiti
acknowledge the great contributions to this project made by personnel from the following agencies: Colorado Division of Wildlife; Colorado Department of Agriculture; US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services; and the US Forest Service.
Sources and manufacturers a. b. c. d.
DAK0 Corp., Carpenteria, CA. 10X Automation Buffer, Biomeda Corp., Foster City, CA. Surgipath Medical Industries, Grayslake, IL. Microprobe, Fisher Scientific, Pittsburgh, PA.
References 1. Basak DK, Chatterjee A, Neogi MK, et al.: 1976, Tuberculosis in captive deer. Indian J Anim Health 15:135-137. 2. Beatson NS: 1985, Tuberculosis in red deer in New Zealand. In: Biology of deer production. R Soc NZ Bull 22: 147-150. 3. Bell LB: 1962, Bovine tuberculosis in a white-tailed deer (Odecoileus virginianus). Can Vet J 3:356-358. 4. Buchan GS, Griffin JFT: 1990, Tuberculosis in domesticated deer (Cervus elaphus): a large animal model for human tuberculosis. J Comp Pathol 103:11-22. 5. Clifton-Hadley RS, Wilesmith JW: 1991, Tuberculosis in deer: a review. Vet Rec 129:5-12. 6. de Lisle GW, Havill PF: 1985, Mycobacteria isolated from deer in New Zealand from 1970-1983. NZ Vet J 33:138-140. 7. de Lisle GW, Welch PJ, Havill PF, et al.: 1983, Experimental tuberculosis in red deer (Cervus elaphus). NZ Vet J 31:2 13-216. 8. Dodd K: 1984, Tuberculosis in free-living deer. Vet Rec 115: 592-593. 9. Fanning A, Edwards S: 1991, Mycobacterium bovis infection in human beings in contact with elk (Cervus elaphus) in Alberta, Canada. Lancet 338:1253-1255. 10. Ferris DH, Beamer PD, Alberts JO, et al.: Tuberculosis in transported deer. J Am Vet Med Assoc 138:326-328. 11. Fleetwood AJ, Stuart FA, Bode R, et al.: 1988, Tuberculosis in deer. Vet Rec 123:279-280. 12. Friend M, Kroll ET, Gruft H: 1963, Tuberculosis in a wild white-tailed deer. NY Fish Game J 10: 118-123. 13. Griffin JFT: 1988, The aetiology of tuberculosis and mycobacterial diseases in farmed deer. Ir Vet J 42:23-26. 14. Griffith AS: 1928, Tuberculosis in captive wild animals. J Hyg 28:198-218. 15. Gunning RF: 1985, Bovine tuberculosis in roe deer. Vet Rec 116:300-301. 16. Hadwen S: 1942, Tuberculosis in the buffalo. J Am Vet Med Assoc 100: 19-22. 17. Helstrom J: 1979, Tuberculosis in deer. NZ Vet J 27: 151. 18. Henry EJ, Cassidy DR: 1966, Rapid decalcification followed by a new fuchsin-methylene blue stain for acid-fast bacilli in frozen sections. Stain Technol 41:345-346. 19. Jones DM, Manton VJA: 1976, Tuberculosis in a herd of axis deer (Axis axis) at Whipsnade Park. Vet Rec 98:525-526. 20. Jorgensen JB, Husum P, Sorensen CI: 1988, Bovine tuberculosis in a deer farm. Dan Veterinaertidsskr 71:806-808. 21. Jubb KVF, Kennedy PC, Palmer N: 1985, Pathology of domestic animals, 3rd ed., vol. 2, pp. 498-500. Academic Press, Orlando, FL. 22. Kollias GV: 1978, Clinical and pathologic features of mycobacterial infections in sika and fallow deer. Proc Symp Natl Zool Park-Mycobacter Infect Zoo Anim, pp. 173-177.
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23. Kollias GV: 1980, Immunopathologic studies of mycobacterial infections in captive cervids. Proc Symp Natl Zool Park-Comp Pathol Zoo Anim, pp. 295-303. 24. Levine PP: 1934, A report on tuberculosis in wild deer (Odocoileus virginianus). Cornell Vet 241264-266. 25. McDiarmid A: 1975, Some disorders of wild deer in the United Kingdom. Vet Rec 97:6-9. 26. Miller MW, Williams JM, Schiefer TJ, Seidel JW: 1991, Bovine tuberculosis in a captive elk herd in Colorado: epizootiology, diagnosis, and management. Proc Annu Meet US Anim Health Assoc 95:533-542. 27. Mote RF, Muhm RL, Gigstad DC: 1975, A staining method using acridine orange and auramine O for fungi and mycobacteria in bovine tissue. Stain Technol 50:5-9. 28. Mshana RN, Humber DP, Harboe M, et al.: 1983, Demonstration of mycobacterial antigens in nerve biopsies from leprosy patients using peroxidase-antiperoxidase immunoenzyme technique. Clin Immunol Immunopathol 29:359-368. 29. Quinn JF, Tower D: 1963, Tuberculosis problems at a deer park in Michigan. Proc 100th Annu Meet Am Vet Med Assoc 144:262-265. 30. Robinson RC, Phillips PH, Stevens G, et al.: 1989, An outbreak of Mycobacterium bovis infection in fallow deer (Dama dama). Aust Vet J 66:195-197. 31. Sawa TR, Thoen CO: 1974, Mycobacterium bovis infection in wild axis deer in Hawaii. J Am Vet Med Assoc 165:998-999. 32. Selwyn P, Hathaway S: 1990, A study of the prevalence and economic significance of diseases and defects of slaughtered farmed deer. NZ Vet J 38:94-97. 33. Singh CDN, Prasad LN, Thakur HN: 1986, Some observations on tuberculosis in deers. Indian Vet J 63:867-868. 34. Smits JEG: 1991, A brief review of infectious and parasitic diseases of wapiti, with emphasis on western Canada and the northwestern United States. Can Vet J 32:471-479. 35. Stuart FA: 1988, Tuberculosis in farmed red deer (Cervus elaphus). In: The management and health of farmed deer, ed. Reid HW, pp. 101-111. Kluwer Academic Publishers, Dordrecht, The Netherlands. 36. Stuart FA, Manser PA, McIntosh FG: 1988, Tuberculosis in imported red deer (Cervus elaphus). Vet Rec 122:508-511. 37. Stumpff CD: 1982, Epidemiological study of an outbreak of bovine TB in confined elk herds. Proc Annu Meet US Anim Health Assoc 86:524-527. 38. Tesaro SV: 1986, The existing and potential importance of brucellosis and tuberculosis in Canadian wildlife: a review. Can Vet J 27:119-124. 39. Thoen CO, Quinn WJ, Miller LD, et al.: 1992, Mycobacterium bovis infection in North American elk (Cervus elaphus). J Vet Diagn Invest 4:423-427. 40. Thoen CO, Richards WD, Jarnagin JL: 1977, Mycobacteria isolated from exotic animals. J Am Vet Med Assoc 170:987990. 41. Towar DR, Scott RM, Goyings LS: 1965, Tuberculosis in a captive deer herd. Am J Vet Res 26:339-346. 42. Wiley EL, Mulhollan TJ, Beck B, et al.: 1990, Polyclonal antibodies against Bacillus calmette-guerin, Mycobacterium duvalli, and Mycobacterium paratuberculosis used to detect mycobacteria in tissue with the use of immunohistochemical techniques. Am J Clin Pathol 94:307-312. 43. Wilson P, Harrington R: 1976, A case of bovine tuberculosis in fallow deer. Vet Rec 98:74.
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Gross and microscopic lesions of naturally occurring tuberculosis in a captive herd of wapiti (Cervus elaphus nelsoni) in Colorado Jack C. Rhyan, Dennis A. Saari, Elizabeth S. Williams, Michael W. Miller, Arthur J. Davis, Arach J. Wilson Abstract. A Mycobacterium bovis-infected herd of captive wapiti (Cervus elaphus nelsoni) in Colorado was depopulated after lesions of bovine tuberculosis were confirmed in 8 of 10 tuberculin skin test reactors. Of the 43 animals > 1 year of age, 26 had gross lesions suggestive of tuberculosis, 24 had microscopic lesions of tuberculosis, and 23 had acid-fast bacilli associated with the lesions. Lungs and retropharyngeal lymph nodes were the most frequently affected sites. Most lesions grossly and microscopically resembled tuberculosis in cattle; however, some lesions resembled abscesses or ovine caseous lymphadenitis lesions. Special stains and immunohistochemical techniques labeled few to numerous mycobacteria associated with the lesions.
Until recently, bovine tuberculosis was considered an unusual and sporadic disease in cervids. Reported cases involved captive and wild cervids and were often attributed to contact with infected cattle or bison.14,16,24 With recent development and expansion of game farming, the prevalence, economic impact, and public health significance of tuberculosis in captive cervids have greatly increased. 6,9,32 In New Zealand, tuberculosis was diagnosed in wild red deer (Cervus elaphus elaphus) in 19702 and in farmed deer in 1978.17 It is now considered the most important bacterial disease in New Zealand’s farmed deer population.6,13,32 Bovine tuberculosis has also been diagnosed in several species of free-living and captive cervids from other countries1,8,11,15,19,20,25,29-31,33,35,36,43 In North America, reports of tuberculosis in cervids have been limited to sporadic cases in wild and captive white-tailed deer (Odocoileus virginianus) 3,10,12,24 a n d 2 2 to outbreaks in captive sika deer (Cervus nippon), 22,29,41 captive fallow deer (Dama dama), and captive wapiti (Rocky Mountain elk, Cervus elaphus nel37,39 soni). In addition, gross lesions consistent with tuberculosis were reported in wapiti, mule deer (Odocoileus hemionus), and moose (Alces alces) in the Canadian National Buffalo Park near Wainwright, Alberta, in 1939 and 1940.16 This finding was concurrent
with a high prevalence of M. bovis infection in the Park bison (Bison bison). There are no recent reports of bovine tuberculosis in free-ranging cervids in North America. In June 1991, an M. bovis-infected herd of captive wapiti in Colorado was depopulated after gross and histologic lesions of bovine tuberculosis were confirmed in 8 of 10 tuberculin skin test reactors.26 This report provides more detailed descriptions of all gross and microscopic lesions of bovine tuberculosis observed in this herd. Materials and methods All animals were killed by gunshot. Necropsies were done, gross lesions recorded and photographed, and tissue specimens collected.26 Samples of grossly observed lesions and/ or parotid, mandibular, retropharyngeal, mediastinal, bronchial, hepatic, and mesenteric lymph nodes that were routinely bisected were placed in 10% neutral buffered formalin and saturated sodium borate transport solution for mycobacterial culture. Specimens were transported within 4 days to the National Veterinary Services Laboratories, Ames, Iowa, for histopathologic examination and mycobacterial culture. Formalin-fixed tissues were routinely processed using paraffin embedding, and 5-µm sections were stained with hematoxylin and eosin (HE). Significantly mineralized tissues were decalcified and routinely processed. Additional sections from tissues with lesions suggestive of tuberculosis were stained by a new fuchsin-methylene blue procedure (NF)18 and acridine orange-auramine O (AOA0)27 and were examined for the presence of acid-fast bacilli and AOAO fluorescent bacilli. Some sections were also stained with Giemsa and Gram’s stains. Selected specimens were stained by a labeled streptavidinbiotin immunohistochemical technique to better demonstrate antigen location. Four-micrometer paraffin sections were deparaffinized, treated with 3% H2O2 to quench endog-
From the Pathobiology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, US Department of Agriculture, PO Box 844, Ames, IA 50010 (Rhyan, Saari, Davis, Wilson), the Wyoming State Veterinary Laboratory, Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82070 (Williams), and the Colorado Division of Wildlife, Wildlife Research Center, 317 W. Prospect Road, Fort Collins, CO 80526 (Miller). Received for publication February 27, 1992. 428
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Signalment of wapiti with gross and/or microscopic tuberculous lesions and location of grossly observed lesions.
enous peroxidase, trypsin digested (20 min at 37 C), blocked with goat serum, and incubated with commercially available polyclonal anti-M. bovis BCG antibodya (30 min at 37 C). Following 3 rinses in buffer,b sections were incubated with biotinylated goat origin anti-rabbit Iga (30 min at 37 C). After 3 rinses, peroxidase-labeled streptavidina was applied (10 min at 37 C) and rinsed, and the color was developed with 3-amino-9-ethylcarbazole (AEC).a Sections were counterstained with Gill II hematoxylin,c and coverslips were mounted with water-base medium.a Nonimmunized rabbit Ig fractiona was substituted for the primary antibody as a negative control. A commercially available capillary gap systemd was used for immunohistochemical staining.
Results Gross observations. Of the 43 captive wapiti examined that were 21 year of age, 26 had gross lesions considered suggestive of tuberculosis.26 Tuberculous lesions occurred most often in retropharyngeal lymph nodes and lung (Table 1). Lung lesions were concentrated in the dorsocaudal half of caudal lobes (Fig. I). Several animals had disseminated lesions in the thoracic cavity, and 3 animals had lesions in thoracic and abdominal cavities. In addition, No. 22 had extensive lesions in the mammary gland and supramammary lymph nodes. Pleural lesions involving the thoracic
wall were noted in 6 cases and diaphragmatic granulomas in 4 cases. Extensive subcutaneous thoracic abscesses were noted in 2 animals, and 2 animals had lesions in the mesenteric lymph nodes. Gross lesions varied in size from small miliary foci 1 mm in diameter to large tubercles or abscesses >30 cm in diameter. Lesions ranged from calcified caseous tubercles to thin-walled abscesses. Some lesions contained white to grey-white viscous exudate with scattered tiny flakes of mineralized material. Several round encapsulated lesions contained caseous necrotic material arranged in concentric lamellar layers. In some of these, mineralized material was located in a lamellar arrangement near the outer fibrous capsule. Abscesses and calcified caseous tubercles were present in close proximity in the same organ in several cases. Mastitis in animal No. 22 was characterized by numerous small to large (2-8 cm diameter) caseous abscesses virtually replacing all normal mammary gland tissue. The caseous material varied from white or greywhite to tan, and some of the abscesses had cavitated centers containing white liquid. Occasional mammary gland abscesses contained caseous material arranged in distinct concentric laminations (Fig. 2). There was direct extension of mammary lesions to supramam-
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Figure 1. Caudal and middle right lung lobes of a captive wapiti. Note incised granulomas with caseous centers in dorsocaudal portion of caudal lobe. Figure 2. Abscess in wapiti mammary gland with concentric laminae of caseous and mineralized exudate surrounding central zone of liquefactive necrosis. Figure 3. Portion of granuloma with caseous center from a wapiti. Note central area of mineralization (arrows), caseous necrosis, neutrophils and peripheral mineralization (arrowheads) extending into inflammatory cell mantle. HE stain. Figure 4. Tuberculous lung lesion from a wapiti. Note bronchiole containing necrotic exudate and adjacent caseous abscess with focal mineralization. HE stain.
mary lymph nodes. These nodes were enlarged (3 x 5 cm), and normal parenchyma was replaced with caseous necrotic material. In the same animal, caseous material in some pleural tubercles was also arranged in concentric lamellar layers. Microscopic features. Microscopic examination confirmed lesions from 24 animals as suggestive or typical of tuberculosis, and acid-fast- or AOAO-positive bacilli were observed in lesions from 23 of the affected animals. Lymph node lesions typically consisted of round, oval, or irregular central zones of caseous necrosis and mineralization surrounded by a nar-
row to wide mantle of mixed inflammatory cells and a layer of dense collagenous connective tissue. The finely granular caseous necrotic material was stained faintly to moderately eosinophilic with focal basophilic stippling or areas of diffuse basophilia due to chromatin debris. In many lesions, few to numerous neutrophils and degenerating leukocytes were scattered throughout the necrotic material. Mineralization, usually present, was centrally and peripherally located with small to large mineralized foci often extending into the surrounding inflammatory cell infiltrate (Fig. 3). The mantle of inflammatory cells surrounding the
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Lesions of tuberculosts in wapiti
Figure 5. Large caseous granuloma in lymph node of a wapiti. Immunohistochemical stain labels strata of antigen near periphery of lesion. Inset: high magnification of same area. Note numerous mycobacteria clustered between collagen fibers. Acid-fast stain. Figure 6. Caseous granuloma in lymph node of a wapiti. Note immunoperoxidase-labeled mycobacteria scattered (arrows) near periphery of lesion and concentrated (arrowheads) in caseous center. Also note peripheral mineralization.
caseous necrosis usually consisted of an inner layer of few to numerous neutrophils surrounded by a narrow to wide zone of epithelioid macrophages admixed with small or moderate numbers of lymphocytes and macrophages and rare or occasional Langhan’s giant cells. Lesions were partially or completely encapsulated with focal capsular invasion and extension into the adjacent lymph node parenchyma. Small unencapsulated focal accumulations of epithelioid macrophages and giant cells were located in some lymph node cortices. The single suggestive lesion noted in the mandibular lymph node of animal No. 21 consisted of a small (1 mm diameter), round, thinly encapsulated focal accumulation of neutrophils, epithelioid macrophages, and Langhans’s giant cells. Lung, pleural, and mammary gland lesions were histologically similar to lymph node lesions. Pleural lesions were usually surrounded by a wider zone of collagenous connective tissue. Lung lesions occasionally lacked mineralization and frequently contained large central accumulations of neutrophils. In some sections, bronchiectatic caseous abscesses were confluent with tuberculous lesions in the adjacent pulmonary parenchyma (Fig. 4). Microscopic examination of lesions with gross lamellar architecture revealed concentrically arranged layers of finely granular eosinophilic material and collagen fibers, occasionally interspersed with layers of mineralized material, neutrophils, and chromatin debris. The necrotic centers were usually bordered by small numbers of mixed inflammatory cells and a collagenous connective tissue capsule. New fuchsin- and AOAO-stained sections contained
rare to numerous mycobacteria associated with lesions. Organisms were not observed in the single small lesion from animal No. 21. In most cases, rare or occasional organisms (1 organism/25 250 x fields) were scattered in the caseous necrotic material or surrounding mantle of macrophages and giant cells. Lesions from 5 animals, however, contained numerous mycobacteria (> 25 organisms/25 250 x fields). Organisms were concentrated in the caseous centers, focally or diffusely scattered in the cellular mantle, or randomly scattered throughout the lesions. In some of the concentric lamellar lesions, mycobacteria were concentrated in lamellar zones in the caseous material near the periphery of the lesions or clustered between collagen layers in the capsule (Fig. 5). Other organisms were not observed in Gram-stained sections of these lesions. In immunohistochemically stained sections, antigen was present in distribution patterns identical to those seen with NF and AOAO stains. Immunoperoxidaselabeled mycobacteria stained strongly positive and were usually surrounded by a small smudge or halo of lighter staining material. The labeled antigen was easily detected on low magnification (Figs. 5, 6). The quantity and distribution of mycobacteria in lesions were more easily determined in immunoperoxidase- and AOAOstained sections than in NF-stained sections. Incidental lesions noted were pyogranulomas containing Splendore-Hoeppli material and gram-negative rod-shaped organisms morphologically typical of Actinobacillus lignieresi in a mandibular lymph node and retropharyngeal lymph node, 1 sublingual abscess with associated gram-positive coccobacilli, scattered mineralized foci in 1 lymph node, and a focal lymphocytic
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middle lung lobes were also most frequently affected in an M. bovis-infected herd of fallow deer.41 Granulomas with caseous centers and abscesses have been previously described in wapiti and red deer. 11,16,36 Discussion Concentrically laminated lesions, however, have not There are few published descriptions of bovine tu- been reported with cervid tuberculosis. These lesions berculosis in wapiti. Previous anecdotal reports men- grossly and microscopically resembled lesions of ovine tioned the isolation of M. bovis from 1 wapiti40 and caseous lymphadenitis.21 The microscopic features of caseous and suppurative the presence of tuberculous lesions in 73 wapiti at slaughter. 16 A report of an outbreak of tuberculosis in lesions in these wapiti are similar to those described confined wapiti herds described granuloma-type pul- in tuberculous red deer.4,35,36 These lesions usually posmonary lesions, and lymph node and subcutaneous sessed the hallmark features of bovine tuberculosis, abscesses.37 Lymph node and lung lesions were recently i.e., caseous necrosis, mineralization, epithelioid macnoted in a captive M. bovis-infected wapiti in Mon- rophages, giant cells, and fibrosis. However, several tana.39 Lesions of M. bovis infection in the conspecific differences from lesions in cattle were noted: 1) lesions European red deer4,7,35,36 and other cervidsl,l0,15,22,29-31,33,41 contained moderate or large numbers of neutrophils, 2) mineralization was not as extensive as in bovine have been described and recently reviewed.5,13 Mycobacterium bovis can produce high disease prev- lesions and was often located around the periphery of alence in a captive herd of wapiti. Twenty-three of 43 the necrotic centers even extending into the layer of (5 3.5%) wapiti had granulomatous or pyogranuloma- macrophages, 3) the caseous necrotic material did not tous lesions with associated acid-fast bacilli, and M. stain as intensely eosinophilic and frequently conbovis was isolated from 16 animals with lesions and 1 tained abundant chromatin debris and/or neutrophils, animal without observed lesions.26 In a previously re- 4) lesions contained fewer giant cells than do tuberported outbreak of tuberculosis in captive wapiti,37 8 culous lesions in cattle, and 5) several lesions contained of 10 animals that died from a herd of 62 had sugges- large numbers of acid-fast bacilli, an unusual finding tive lesions, and M. bovis was isolated from 2 animals. in cattle. Varied patterns of mycobacterial distribution in leAlso, 1 of 5 slaughtered wapiti from a different herd had gross lesions suggestive of tuberculosis from which sions were demonstrated with acid-fast, AOAO, and M. bovis was subsequently isolated. The prevalence of immunohistochemical staining. The organisms’ lamdisease in this group of captive wapiti is similar to that inar distribution in the concentric lamellar lesions has reported in farmed red deer. In 1 group of 51 red deer not, to our knowledge, been previously described. The imported to the United Kingdom from Eastern Eu- staining reaction of anti-M. bovis BCG antibody to M. rope, M. bovis was isolated from 20 (39.2%) animals, bovis antigen was similar to results in studies using the and 13 (25.5%) had visible lesions.35,36 A 90% infection antibody to demonstrate other mycobacteria.28,42 The rate in adult animals was described in a herd of New immunoperoxidase stain allowed detection of mycoZealand farmed red deer.13 Other than the report of bacteria at a much lower magnification than the NF gross lesions suggestive of tuberculosis in wapiti from stain. The AOAO stain also allowed demonstration of National Buffalo Park near Wainwright, Alberta, Can- mycobacteria at a low magnification but required the ada 16,34,38 tuberculosis has not been encountered in free- use of a fluorescent scope and did not allow concurrent detailed assessment of the surrounding lesion. ranging wapiti. This report is the first detailed description of gross Wide variations in lesion distribution and gross and microscopic features can occur with M. bovis infection and microscopic lesions of M. bovis infection in wapiti. in wapiti. The high prevalence of lung and retropha- The location and morphology of M. bovis lesions in ryngeal lymph node lesions is consistent with other this herd of wapiti were widely varied. The gross and reports in wapiti and red deer.2,6,7,11,l3,32,36,37 Pleural and microscopic appearance of lesions resembled bovine diaphragmatic lesions, subcutaneous abscesses, and tuberculosis in cattle, abscesses, and ovine caseous mesenteric lymph node involvement have also been lymphadenitis lesions. Subcutaneous abscesses and tudescribed. 11,36,37 Tuberculous lesions in the mammary berculous mastitis were also present. These findings gland of cervids are considered rare.5 Occurrence of indicate that M. bovis should be considered as a pospulmonary lesions, predominantly in the subpleural sible etiologic agent whenever abscesslike lesions are parenchyma of the dorsocaudal portions of the caudal encountered in cervids. 11 lobes, is similar to tuberculosis in adult cattle21 and in Acknowledgements sika and fallow deer. 23 In experimentally infected red deer, however, intratracheal inoculation of M. bovis We gratefully acknowledge the technical assistance of Krisproduced anterior lung lobe lesions.7 Anterior and tin Wilson, Julie Campbell, and Brenda Burleson. We also
hepatitis of unknown etiology. Also present was a focal eosinophilic lymphadenitis in 1 lymph node considered suggestive of a parasitic etiology.
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Lesions of tuberculosis in wapiti
acknowledge the great contributions to this project made by personnel from the following agencies: Colorado Division of Wildlife; Colorado Department of Agriculture; US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services; and the US Forest Service.
Sources and manufacturers a. b. c. d.
DAK0 Corp., Carpenteria, CA. 10X Automation Buffer, Biomeda Corp., Foster City, CA. Surgipath Medical Industries, Grayslake, IL. Microprobe, Fisher Scientific, Pittsburgh, PA.
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