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An Unusual Presentation of Mycobacterium avium spp. paratuberculosis Infection in a Captive Tundra Reindeer (Rangifer tarandus tarandus)
J. Comp. Path. 2013, Vol. 149, 126e131
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DISEASE IN WILDLIFE OR EXOTIC SPECIES
An Unusual Presentation of Mycobacterium avium spp. paratuberculosis Infection in a Captive Tundra Reindeer (Rangifer tarandus tarandus) J. Del-Pozo*, S. Girling†, J. McLuckie‡, E. Abbondatix and K. Stevenson‡ * Veterinary Pathology Unit, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Roslin, Midlothian EH25 9RG, † Royal Zoological Society of Scotland, Edinburgh Zoo, 134 Corstorphine Road, EH12 6TS Edinburgh, ‡ Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ and x School of Veterinary Medicine, University of Glasgow, Bearsden, Glasgow G611QH, UK
Summary This report describes an unusual presentation of paratuberculosis in a captive, 4-year-old female tundra reindeer (Rangifer tarandus tarandus). The gross and histological presentation was consistent with clinical paratuberculosis as previously reported for other ruminants, with poor body condition, subcutaneous oedema, granulomatous ileitis (multibacillary), mesenteric lymphadenitis and hepatitis. However, this animal also presented with unusual lung lesions, with necrosis and mineralization similar to that reported for Mycobacterium bovis in other wild and domestic ruminants. The presence of DNA of Mycobacterium avium subsp. paratuberculosis was confirmed by polymerase chain reaction (PCR) in intestine and lung tissue (IS900, Hsp65) and PCR tests for the detection of Mycobacterium tuberculosis complex and other members of the M. avium complex were negative. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: Johne’s disease; Mycobacterium avium spp. paratuberculosis; pneumonia; reindeer
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne’s disease. Paratuberculosis affects a wide range of species, including domestic ruminants (Clarke, 1997), wild ruminants (Polikarpov, 1966; Williams et al., 1979; Chiodini and Van Kruiningen, 1983; Cook et al., 1997; Pavlik et al., 2000; Marco et al., 2002; Nettles et al., 2002; Tryland et al., 2004; Rafferty and Harley, 2009; Johnson et al., 2010; Kim et al., 2012) and non-ruminant wild species (Beard et al., 2001; Hutchings et al., 2010). Information on the histological presentation of paratuberculosis in reindeer is not readily available in the literature and in this report we discuss an unusual presentation of paratuberculosis in this species. A 4-year-old female tundra reindeer (Rangifer tarandus tarandus), kept in captivity, developed weight loss and diarrhoea. Routine serum biochemical and haematological examinations at this time revealed Correspondence to: J. Del-Pozo (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.10.004
low serum copper concentration (<1.9 mmol/l; normal range 6.7e18 mmol/l; Nieminen and Timisj€ arvi, 1983) and elevated g-globulin concentration (20.3 g/l; normal range 7.3e15.4 g/l; Nieminen and Timisj€ arvi, 1983). Albumin concentration was normal (27 g/l; normal range 24e45 g/l; Nieminen and Timisj€ arvi, 1983). Faecal sedimentation analysis revealed a strongyle count of 80 eggs/g. This faecal sample was negative for acid-fast bacteria and no significant bacterial isolate was recovered from it. Serological testing for MAP was negative (ID SCREENÒ, ID.vet, Montpellier, France). Treatment with ivermectin/clorsulon, copper and oxytetracycline resulted in temporary improvement with remission of diarrhoea and weight gain. However, diarrhoea with associated weight loss recurred 5 months later and treatment with ivermectin/clorsulon, copper and oxytetracycline was ineffective at that time. The reindeer became weakened and recumbent and was humanely destroyed on welfare grounds. Ó 2012 Elsevier Ltd. All rights reserved.
Mycobacteriosis in a Tundra Reindeer
At necropsy examination, the carcass was in poor bodily condition and there were dried faeces on the skin of the perineal area and caudal aspect of both hindlimbs. The thoracic cavity contained 300 ml of dark red, cloudy fluid (serosanguineous exudate) and there was a well demarcated, round, flattened, 3.5 cm diameter, subpleural nodule of pale yellow, caseous material in the apical lobe of the right lung (Fig. 1). The abdomen contained 60 ml of red, translucent fluid and the abomasal mucosa was diffusely thickened and corrugated. All of the remaining organs examined were grossly normal. On microscopical examination, the most significant changes were noted in the lungs, bronchial lymph node, ileum, mesenteric lymph nodes, liver and abomasum. The lesion within the right apical pulmonary lobe was comprised of multifocal to coalescing, well demarcated and variably encapsulated granulomas with central coagulative and liquefactive necrosis and mineralization (caseous necrosis). These granulomas consisted of large numbers of macrophages, fewer lymphocytes and plasma cells, numerous multinucleated giant cells (Langhans type) and
Fig. 1. Ventral view of the thoracic cavity. Note the focally extensive lesion with caseous necrosis of the right cranial lobe (arrow). This has resulted in adhesion of the medial aspect of this lobe to the mediastinum.
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few neutrophils (Figs. 2, 3). In the necrotic areas and in the areas of granulomatous inflammation, there were multifocal clear, oval, 150e200 mm diameter spaces surrounded by neutrophilic infiltration, which contained very rare, 3e5 mm elongate, thin acid-fast rods (Fig. 4). There was no evidence of any other specific organisms in any of the tissue sections, when stained with Gram or periodic acideSchiff stains. In the immediately adjacent pulmonary parenchyma, there was an occasional increase in the number of alveolar macrophages and compression caused by the granulomas. The parenchyma of the right caudal lobe and left lung was diffusely congested and alveoli contained scattered aggregates of plant material, ciliated protozoal organisms or bacteria. These were not associated with inflammation, suggesting that their presence was the result of perimortem inspiration of digestive contents. The bronchial lymph node had sinus histiocytosis and oedema, but there were no acid-fast bacteria. There was mild to severe stunting of villi in the distal jejunum and ileum. The lamina propria and submucosa were infiltrated multifocally by small to moderate numbers of vacuolated macrophages, with fewer eosinophils and occasional multinucleated giant cells (granulomatous enteritis). The macrophages often contained moderate to large numbers of slender acid-fast rods. Submucosal lymphatic vessels were dilated by intraluminal and intramural clusters of lymphocytes and macrophages with intracytoplasmic acid-fast rods. The mesenteric lymph node was partially effaced by infiltration of macrophages with interspersed giant cells, also containing acid-fast rods
Fig. 2. Low-power microscopical view of an area of the right cranial lung lobe. In the lower two-thirds of the image, there are coalescing nodules of granulomatous inflammation delimited by haphazard trabeculae of fibrous tissue. Note the clear spaces at the centre of microabscesses (arrowheads). Haematoxylin and eosin. Bar, 500 mm.
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Fig. 3. High-power view of a clear space (*) within the lung rimmed by neutrophils and adjacent to an area of necrosis (+). To the left of the image is an area of dense infiltration by macrophages with giant cells (arrowheads). Haematoxylin and eosin. Bar, 100 mm.
(granulomatous lymphadenitis). In the liver, there were multifocal, randomly distributed similar granulomas, with very rare intracellular acid-fast rods. To further identify the acid-fast bacteria in the lungs and ileum, various polymerase chain reactions (PCRs) were performed on DNA extracted from the formalin-fixed, paraffin-wax embedded tissues (Tripathi and Stevenson, 2012). Four PCRs were conducted: MAP-specific IS900 (Sanderson et al., 1992), IS901 specific to M. avium subsp. silvaticum (MAS; Moss et al., 1992), the 30 variable region of Hsp65 in order to differentiate between subspecies of the M. avium complex (Turenne et al., 2006) and IS6110, which is specific for Mycobacterium tuberculosis complex organisms (Miller et al., 1997). Negative controls were sections of healthy equine tongue tissue and sterile distilled water. Positive controls were sections of ovine
Fig. 4. High-power view of a clear space with small numbers of acid-fast organisms at the periphery (arrowheads). Ziehl Neelsen stain. Bar, 20 mm.
ileum with multibacillary paratuberculosis and purified MAP DNA (for IS900 and Hsp65), purified DNA from MAS (for IS901 and Hsp65) and sections from a confirmed case of Mycobacterium bovis and M. bovis DNA (for IS6110). To assess reproducibility, two separate rounds of DNA extraction and PCR were performed for all the tests. The reindeer ileum and lung were both negative for IS901 and IS6110. Both ileum and lung were positive for IS900 and Hsp65, consistent with MAP infection. For further confirmation, all positive IS900 PCR products were sequenced and the sequence was found to be identical to that reported by Green et al. for MAP (1989). Several features of the presentation of this reindeer are consistent with clinical paratuberculosis, as previously reported in other wild ruminants. Grossly, these included the presence of faecal soiling, chronic weight loss and subcutaneous oedema (Clarke, 1997; Marco et al., 2002; de Lisle et al., 2003; Balseiro et al., 2008). The histological lesions of the ileum, mesenteric lymph node and liver of this reindeer were also consistent with paratuberculosis and the results of PCR testing confirmed the presence of IS900 specific to MAP in the sections of ileum. In fallow deer, the histological presentation of intestinal paratuberculosis has been classified on the basis of the severity of the lesions (Balseiro et al., 2008), using a system derived from previous studies in cattle and sheep (Perez et al., 1996; Gonzalez et al., 2005). Another study in red deer proposed a scoring system to assess the severity of the lesions, ranging from 0 to 13 (Clark et al., 2010). According to these classification systems, the presentation of the present case was consistent with diffuse multibacillary disease (Balseiro et al., 2008) with a severity score of 10 (Clark et al., 2010), suggesting an advanced stage of the disease. Lymphangiectasia and granulomatous lymphangitis were prominent in this case, a feature rarely noted in fallow deer (Balseiro et al., 2008). The most striking feature of this case compared with previous reports of paratuberculosis in wild ruminants was the pulmonary lesions. These lesions had areas of caseous necrosis and granulomatous inflammation, with frequent giant cell formation. Ascites was not present and hypoproteinaemia was not reported in the clinical history. It is therefore likely that the pleural effusion noted was a result of exudation from these lesions. The interesting aspect of this pulmonary presentation lies with its similarity to that previously reported in deer infected with M. bovis (Kim et al., 2002; Palmer et al., 2002; Mackintosh et al., 2004; Martin-Hernando et al., 2010). This may lead to misinterpretation, which is a serious issue as M. bovis is a zoonosis (Thoen et al., 2006) that is a notifiable disease in the UK (section 88 of the
129
Mycobacteriosis in a Tundra Reindeer
Animal Health Act 1981). Co-infection of M. bovis and MAP has been reported in cattle (Aranaz et al., 2006). This possibility was examined by PCR testing for several mycobacterial species, which revealed that only DNA from MAP was present in the samples examined. These results are suggestive of mono infection by MAP in this animal, although coinfection with other mycobacteria cannot be ruled out completely due to poor sensitivity of PCR on fixed tissue containing few bacteria. Extraintestinal lesions are not uncommon in MAP infection and these have been reported previously in domestic and wild ruminants. In domestic ruminants, systemic paratuberculosis has been reported in cattle (Hines et al., 1987) and lung and liver lesions have been noted in 3% and 61% of multibacillary cases in sheep, respectively (Clarke and Little, 1996). In the latter study, granulomatous lesions with sparse acidfast rods and no calcification were noted in the lungs in 3% of multibacillary cases (Clarke and Little, 1996), but in these cases the presentation was peribronchiolar only, in contrast with the present case. In wild ruminants granulomatous foci have been noted in extraintestinal lymph nodes and the liver of bighorn sheep and Rocky Mountain goats, the spleen and lung of an experimentally infected mule deer (Williams et al., 1983) and the lung, liver and head lymph nodes in farmed deer (de Lisle et al., 1993, 2003). Calcified (caseous) lesions have been previously reported in the lymph nodes of MAP-infected sheep (Perez et al., 1996), goats and deer (Williams et al., 1983; de Lisle et al., 1993; Paolicchi et al., 2001; Clark et al., 2010). Several authors have specifically highlighted the similarity of lesions of different mycobacterial species in deer (de Lisle et al., 1993; Mackintosh et al., 2004) and lesions in Mycobacterium kansasii-infected reindeer closely resemble those seen in M. bovis-inoculated reindeer (Palmer et al., 2006). However, these similarities refer mainly to the presentation in the lymph nodes and intestine, and the report of the histological presentation of pulmonary lesions of deer with paratuberculosis does not mention calcification and necrosis specifically (Williams et al., 1983). Another unusual feature of the presentation in this case was the presence of small, clear spaces associated with a rim of neutrophilic infiltration within the lung lesions. To our knowledge, this pattern has not been reported previously in animals. A similar pattern has been noted in immunosuppressed people with mycobacterial dermatitis caused by rapidly growing, non-tuberculous mycobacteria such as Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum (Gable et al., 2008). In this report, these structures (‘microabscesses with pseudocyst formation’) contained large numbers of acid-fast rods. Interest-
ingly, in the lung lesions of the present reindeer it was only within these spaces that acid-fast rods were noted, but in very small numbers. In conclusion, this case report describes an unusual presentation associated with infection of a reindeer with MAP. This consisted of ‘typical’ intestinal, mesenteric and hepatic lesions, but with additional ‘atypical’ pulmonary lesions similar to those noted in M. bovis infection in other deer species. Because of the lack of published information on the presentation of paratuberculosis in reindeer, it is not possible to determine whether this is likely to be an isolated occurrence. A description of the presentation of paratuberculosis in a larger number of reindeer, including samples of lung, will be required to determine the frequency of this presentation of MAP infection.
Acknowledgments The authors extend their sincere thanks to the histology laboratory staff at Easter Bush Veterinary Centre (EBVC), Edinburgh, UK, for the histological processing of the samples. This work was carried out as part of a diagnostic investigation commissioned by the Royal Zoological Society of Scotland, based at Edinburgh Zoo, Edinburgh, UK, and was funded in part by the Scottish Government Rural and Environment Research and Analysis Directorate.
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and other tissues. Journal of Comparative Pathology, 114, 419e437. Cook WE, Cornish TE, Shideler S, Lasley B, Collins MT (1997) Radiometric culture of Mycobacterium avium paratuberculosis from the faeces of tule elk. Journal of Wildlife Diseases, 33, 635e637. de Lisle GW, Yates GF, Collins DM (1993) Paratuberculosis in farmed deer: case reports and DNA characterization of isolates of Mycobacterium paratuberculosis. Journal of Veterinary Diagnostic Investigation, 5, 567e571. de Lisle GW, Yates GF, Montgomery H (2003) The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand e a review of 619 cases. New Zealand Veterinary Journal, 51, 58e62. Gable AD, Marsee DK, Milner DA, Granter SR (2008) Suppurative inflammation with microabscess and pseudocyst formation is a characteristic histologic manifestation of cutaneous infections with rapid-growing Mycobacterium species. American Journal of Clinical Pathology, 130, 514e517. Gonzalez J, Geijo MV, Garcia-Pariente C, Verna A, Corpa JM et al. (2005) Histopathological classification of lesions associated with natural paratuberculosis infection in cattle. Journal of Comparative Pathology, 133, 184e196. Green EP, Tizard ML, Moss MT, Thompson J, Winterbourne DJ et al. (1989) Sequence and characteristics of IS900, an insertion element identified in a human Crohn’s disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Research, 17, 9063e9073. Hines SA, Buergelt CD, Wilson JH, Bliss EL (1987) Disseminated Mycobacterium paratuberculosis infection in a cow. Journal of the American Veterinary Medical Association, 190, 681e683. Hutchings MR, Stevenson K, Greig A, Davidson R, Marion G et al. (2010) Infection of non-ruminant wildlife by Mycobacterium avium subsp. paratuberculosis. In: Paratuberculosis: Organism, Disease, Control, MA Behr, DM Colins, Eds., CAB International, London, pp. 188e200. Johnson D, Harms NJ, Larter NC, Elkin BT, Tabel H et al. (2010) Serum biochemistry, serology, and parasitology of boreal caribou (Rangifer tarandus caribou) in the Northwest Territories, Canada. Journal of Wildlife Diseases, 46, 1096e1107. Kim JH, Sohn HJ, Kang KI, Kim WI, An JS et al. (2002) Mycobacterium bovis infection in a farmed elk in Korea. Journal of Veterinary Science, 3, 163e166. Kim JM, Ku BK, Lee HN, Hwang IY, Jang YB et al. (2012) Seroprevalence of Mycobacterium avium subspecies paratuberculosis in elks in Korea. Veterinary Record, 170, 102. Mackintosh CG, de Lisle GW, Collins DM, Griffin JFT (2004) Mycobacterial diseases of deer. New Zealand Veterinary Journal, 52, 163e174. Marco I, Ruiz M, Juste R, Garrido JM, Lavin S (2002) Paratuberculosis in free-ranging fallow deer in Spain. Journal of Wildlife Diseases, 38, 629e632. Martin-Hernando MP, Torres MJ, Aznar J, Negro JJ, Gandia A et al. (2010) Distribution of lesions in red and fallow deer naturally infected with Mycobacterium bovis. Journal of Comparative Pathology, 142, 43e50.
Miller J, Jenny A, Rhyan J, Saari D, Suarez D (1997) Detection of Mycobacterium bovis in formalin-fixed, paraffinembedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms. Journal of Veterinary Diagnostic Investigation, 9, 244e249. Moss MT, Sanderson JD, Tizard ML, HermonTaylor J, el-Zaatari FA et al. (1992) Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn’s disease and control tissues. Gut, 33, 1209e1213. Nettles VF, Quist CF, Lopez RR, Wilmers TJ, Frank P et al. (2002) Morbidity and mortality factors in key deer (Odocoileus virginianus clavium). Journal of Wildlife Diseases, 38, 685e692. Nieminen M, Timisj€arvi J (1983) Blood composition of the reindeer: II. Blood chemistry. Rangifer, 3, 16e32. Palmer MV, Waters WR, Thacker TC, Stoffregen WC, Thomsen BV (2006) Experimentally induced infection of reindeer (Rangifer tarandus) with Mycobacterium bovis. Journal of Veterinary Diagnostic Investigation, 18, 52e60. Palmer MV, Waters WR, Whipple DL (2002) Lesion development in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis. Veterinary Pathology, 39, 334e340. Paolicchi FA, Vagnozzi A, Morsella CG, Verna AE, Massone AR et al. (2001) Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study. Journal of Veterinary Medicine B. Infectious Diseases and Veterinary Public Health, 48, 313e320. Pavlik I, Bartl J, Dvorska L, Svastova P, du Maine R et al. (2000) Epidemiology of paratuberculosis in wild ruminants studied by restriction fragment length polymorphism in the Czech Republic during the period 1995e1998. Veterinary Microbiology, 77, 231e251. Perez V, Garcia Marin JF, Badiola JJ (1996) Description and classification of different types of lesion associated with natural paratuberculosis infection in sheep. Journal of Comparative Pathology, 114, 107e122. Polikarpov VA (1966) [Diagnosis of paratuberculosis in reindeer]. Veterinariia, 43, 29e32. Rafferty A, Harley J (2009) Reindeer health and welfare. Veterinary Record, 164, 346. Sanderson JD, Moss MT, Tizard ML, Hermon-Taylor J (1992) Mycobacterium paratuberculosis DNA in Crohn’s disease tissue. Gut, 33, 890e896. Thoen C, LoBue P, de Kantor I (2006) The importance of Mycobacterium bovis as a zoonosis. Veterinary Microbiology, 112, 339e345. Tripathi BN, Stevenson K (2012) Detection of Mycobacterium avium subsp. paratuberculosis in formalin-fixed, paraffin embedded tissues of goats by IS900 polymerase chain reaction. Small Ruminant Research, 102, 84e88. Tryland M, Olsen I, Vikoren T, Handeland K, Arnemo JM et al. (2004) Serologic survey for antibodies against Mycobacterium avium subsp. paratuberculosis in freeranging cervids from Norway. Journal of Wildlife Diseases, 40, 32e41.
Mycobacteriosis in a Tundra Reindeer
Turenne CY, Semret M, Cousins DV, Collins DM, Behr MA (2006) Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex. Journal of Clinical Microbiology, 44, 433e440. Williams ES, Snyder SP, Martin KL (1983) Pathology of spontaneous and experimental infection of North American wild ruminants with Mycobacterium paratuberculosis. Veterinary Pathology, 20, 274e290.
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Williams ES, Spraker TR, Schoonveld GG (1979) Paratuberculosis (Johne’s disease) in bighorn sheep and a Rocky Mountain goat in Colorado. Journal of Wildlife Diseases, 15, 221e227. October 10th, 2012 ½ Received, Accepted, October 30th, 2012
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
DISEASE IN WILDLIFE OR EXOTIC SPECIES
An Unusual Presentation of Mycobacterium avium spp. paratuberculosis Infection in a Captive Tundra Reindeer (Rangifer tarandus tarandus) J. Del-Pozo*, S. Girling†, J. McLuckie‡, E. Abbondatix and K. Stevenson‡ * Veterinary Pathology Unit, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Roslin, Midlothian EH25 9RG, † Royal Zoological Society of Scotland, Edinburgh Zoo, 134 Corstorphine Road, EH12 6TS Edinburgh, ‡ Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ and x School of Veterinary Medicine, University of Glasgow, Bearsden, Glasgow G611QH, UK
Summary This report describes an unusual presentation of paratuberculosis in a captive, 4-year-old female tundra reindeer (Rangifer tarandus tarandus). The gross and histological presentation was consistent with clinical paratuberculosis as previously reported for other ruminants, with poor body condition, subcutaneous oedema, granulomatous ileitis (multibacillary), mesenteric lymphadenitis and hepatitis. However, this animal also presented with unusual lung lesions, with necrosis and mineralization similar to that reported for Mycobacterium bovis in other wild and domestic ruminants. The presence of DNA of Mycobacterium avium subsp. paratuberculosis was confirmed by polymerase chain reaction (PCR) in intestine and lung tissue (IS900, Hsp65) and PCR tests for the detection of Mycobacterium tuberculosis complex and other members of the M. avium complex were negative. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: Johne’s disease; Mycobacterium avium spp. paratuberculosis; pneumonia; reindeer
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne’s disease. Paratuberculosis affects a wide range of species, including domestic ruminants (Clarke, 1997), wild ruminants (Polikarpov, 1966; Williams et al., 1979; Chiodini and Van Kruiningen, 1983; Cook et al., 1997; Pavlik et al., 2000; Marco et al., 2002; Nettles et al., 2002; Tryland et al., 2004; Rafferty and Harley, 2009; Johnson et al., 2010; Kim et al., 2012) and non-ruminant wild species (Beard et al., 2001; Hutchings et al., 2010). Information on the histological presentation of paratuberculosis in reindeer is not readily available in the literature and in this report we discuss an unusual presentation of paratuberculosis in this species. A 4-year-old female tundra reindeer (Rangifer tarandus tarandus), kept in captivity, developed weight loss and diarrhoea. Routine serum biochemical and haematological examinations at this time revealed Correspondence to: J. Del-Pozo (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.10.004
low serum copper concentration (<1.9 mmol/l; normal range 6.7e18 mmol/l; Nieminen and Timisj€ arvi, 1983) and elevated g-globulin concentration (20.3 g/l; normal range 7.3e15.4 g/l; Nieminen and Timisj€ arvi, 1983). Albumin concentration was normal (27 g/l; normal range 24e45 g/l; Nieminen and Timisj€ arvi, 1983). Faecal sedimentation analysis revealed a strongyle count of 80 eggs/g. This faecal sample was negative for acid-fast bacteria and no significant bacterial isolate was recovered from it. Serological testing for MAP was negative (ID SCREENÒ, ID.vet, Montpellier, France). Treatment with ivermectin/clorsulon, copper and oxytetracycline resulted in temporary improvement with remission of diarrhoea and weight gain. However, diarrhoea with associated weight loss recurred 5 months later and treatment with ivermectin/clorsulon, copper and oxytetracycline was ineffective at that time. The reindeer became weakened and recumbent and was humanely destroyed on welfare grounds. Ó 2012 Elsevier Ltd. All rights reserved.
Mycobacteriosis in a Tundra Reindeer
At necropsy examination, the carcass was in poor bodily condition and there were dried faeces on the skin of the perineal area and caudal aspect of both hindlimbs. The thoracic cavity contained 300 ml of dark red, cloudy fluid (serosanguineous exudate) and there was a well demarcated, round, flattened, 3.5 cm diameter, subpleural nodule of pale yellow, caseous material in the apical lobe of the right lung (Fig. 1). The abdomen contained 60 ml of red, translucent fluid and the abomasal mucosa was diffusely thickened and corrugated. All of the remaining organs examined were grossly normal. On microscopical examination, the most significant changes were noted in the lungs, bronchial lymph node, ileum, mesenteric lymph nodes, liver and abomasum. The lesion within the right apical pulmonary lobe was comprised of multifocal to coalescing, well demarcated and variably encapsulated granulomas with central coagulative and liquefactive necrosis and mineralization (caseous necrosis). These granulomas consisted of large numbers of macrophages, fewer lymphocytes and plasma cells, numerous multinucleated giant cells (Langhans type) and
Fig. 1. Ventral view of the thoracic cavity. Note the focally extensive lesion with caseous necrosis of the right cranial lobe (arrow). This has resulted in adhesion of the medial aspect of this lobe to the mediastinum.
127
few neutrophils (Figs. 2, 3). In the necrotic areas and in the areas of granulomatous inflammation, there were multifocal clear, oval, 150e200 mm diameter spaces surrounded by neutrophilic infiltration, which contained very rare, 3e5 mm elongate, thin acid-fast rods (Fig. 4). There was no evidence of any other specific organisms in any of the tissue sections, when stained with Gram or periodic acideSchiff stains. In the immediately adjacent pulmonary parenchyma, there was an occasional increase in the number of alveolar macrophages and compression caused by the granulomas. The parenchyma of the right caudal lobe and left lung was diffusely congested and alveoli contained scattered aggregates of plant material, ciliated protozoal organisms or bacteria. These were not associated with inflammation, suggesting that their presence was the result of perimortem inspiration of digestive contents. The bronchial lymph node had sinus histiocytosis and oedema, but there were no acid-fast bacteria. There was mild to severe stunting of villi in the distal jejunum and ileum. The lamina propria and submucosa were infiltrated multifocally by small to moderate numbers of vacuolated macrophages, with fewer eosinophils and occasional multinucleated giant cells (granulomatous enteritis). The macrophages often contained moderate to large numbers of slender acid-fast rods. Submucosal lymphatic vessels were dilated by intraluminal and intramural clusters of lymphocytes and macrophages with intracytoplasmic acid-fast rods. The mesenteric lymph node was partially effaced by infiltration of macrophages with interspersed giant cells, also containing acid-fast rods
Fig. 2. Low-power microscopical view of an area of the right cranial lung lobe. In the lower two-thirds of the image, there are coalescing nodules of granulomatous inflammation delimited by haphazard trabeculae of fibrous tissue. Note the clear spaces at the centre of microabscesses (arrowheads). Haematoxylin and eosin. Bar, 500 mm.
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Fig. 3. High-power view of a clear space (*) within the lung rimmed by neutrophils and adjacent to an area of necrosis (+). To the left of the image is an area of dense infiltration by macrophages with giant cells (arrowheads). Haematoxylin and eosin. Bar, 100 mm.
(granulomatous lymphadenitis). In the liver, there were multifocal, randomly distributed similar granulomas, with very rare intracellular acid-fast rods. To further identify the acid-fast bacteria in the lungs and ileum, various polymerase chain reactions (PCRs) were performed on DNA extracted from the formalin-fixed, paraffin-wax embedded tissues (Tripathi and Stevenson, 2012). Four PCRs were conducted: MAP-specific IS900 (Sanderson et al., 1992), IS901 specific to M. avium subsp. silvaticum (MAS; Moss et al., 1992), the 30 variable region of Hsp65 in order to differentiate between subspecies of the M. avium complex (Turenne et al., 2006) and IS6110, which is specific for Mycobacterium tuberculosis complex organisms (Miller et al., 1997). Negative controls were sections of healthy equine tongue tissue and sterile distilled water. Positive controls were sections of ovine
Fig. 4. High-power view of a clear space with small numbers of acid-fast organisms at the periphery (arrowheads). Ziehl Neelsen stain. Bar, 20 mm.
ileum with multibacillary paratuberculosis and purified MAP DNA (for IS900 and Hsp65), purified DNA from MAS (for IS901 and Hsp65) and sections from a confirmed case of Mycobacterium bovis and M. bovis DNA (for IS6110). To assess reproducibility, two separate rounds of DNA extraction and PCR were performed for all the tests. The reindeer ileum and lung were both negative for IS901 and IS6110. Both ileum and lung were positive for IS900 and Hsp65, consistent with MAP infection. For further confirmation, all positive IS900 PCR products were sequenced and the sequence was found to be identical to that reported by Green et al. for MAP (1989). Several features of the presentation of this reindeer are consistent with clinical paratuberculosis, as previously reported in other wild ruminants. Grossly, these included the presence of faecal soiling, chronic weight loss and subcutaneous oedema (Clarke, 1997; Marco et al., 2002; de Lisle et al., 2003; Balseiro et al., 2008). The histological lesions of the ileum, mesenteric lymph node and liver of this reindeer were also consistent with paratuberculosis and the results of PCR testing confirmed the presence of IS900 specific to MAP in the sections of ileum. In fallow deer, the histological presentation of intestinal paratuberculosis has been classified on the basis of the severity of the lesions (Balseiro et al., 2008), using a system derived from previous studies in cattle and sheep (Perez et al., 1996; Gonzalez et al., 2005). Another study in red deer proposed a scoring system to assess the severity of the lesions, ranging from 0 to 13 (Clark et al., 2010). According to these classification systems, the presentation of the present case was consistent with diffuse multibacillary disease (Balseiro et al., 2008) with a severity score of 10 (Clark et al., 2010), suggesting an advanced stage of the disease. Lymphangiectasia and granulomatous lymphangitis were prominent in this case, a feature rarely noted in fallow deer (Balseiro et al., 2008). The most striking feature of this case compared with previous reports of paratuberculosis in wild ruminants was the pulmonary lesions. These lesions had areas of caseous necrosis and granulomatous inflammation, with frequent giant cell formation. Ascites was not present and hypoproteinaemia was not reported in the clinical history. It is therefore likely that the pleural effusion noted was a result of exudation from these lesions. The interesting aspect of this pulmonary presentation lies with its similarity to that previously reported in deer infected with M. bovis (Kim et al., 2002; Palmer et al., 2002; Mackintosh et al., 2004; Martin-Hernando et al., 2010). This may lead to misinterpretation, which is a serious issue as M. bovis is a zoonosis (Thoen et al., 2006) that is a notifiable disease in the UK (section 88 of the
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Animal Health Act 1981). Co-infection of M. bovis and MAP has been reported in cattle (Aranaz et al., 2006). This possibility was examined by PCR testing for several mycobacterial species, which revealed that only DNA from MAP was present in the samples examined. These results are suggestive of mono infection by MAP in this animal, although coinfection with other mycobacteria cannot be ruled out completely due to poor sensitivity of PCR on fixed tissue containing few bacteria. Extraintestinal lesions are not uncommon in MAP infection and these have been reported previously in domestic and wild ruminants. In domestic ruminants, systemic paratuberculosis has been reported in cattle (Hines et al., 1987) and lung and liver lesions have been noted in 3% and 61% of multibacillary cases in sheep, respectively (Clarke and Little, 1996). In the latter study, granulomatous lesions with sparse acidfast rods and no calcification were noted in the lungs in 3% of multibacillary cases (Clarke and Little, 1996), but in these cases the presentation was peribronchiolar only, in contrast with the present case. In wild ruminants granulomatous foci have been noted in extraintestinal lymph nodes and the liver of bighorn sheep and Rocky Mountain goats, the spleen and lung of an experimentally infected mule deer (Williams et al., 1983) and the lung, liver and head lymph nodes in farmed deer (de Lisle et al., 1993, 2003). Calcified (caseous) lesions have been previously reported in the lymph nodes of MAP-infected sheep (Perez et al., 1996), goats and deer (Williams et al., 1983; de Lisle et al., 1993; Paolicchi et al., 2001; Clark et al., 2010). Several authors have specifically highlighted the similarity of lesions of different mycobacterial species in deer (de Lisle et al., 1993; Mackintosh et al., 2004) and lesions in Mycobacterium kansasii-infected reindeer closely resemble those seen in M. bovis-inoculated reindeer (Palmer et al., 2006). However, these similarities refer mainly to the presentation in the lymph nodes and intestine, and the report of the histological presentation of pulmonary lesions of deer with paratuberculosis does not mention calcification and necrosis specifically (Williams et al., 1983). Another unusual feature of the presentation in this case was the presence of small, clear spaces associated with a rim of neutrophilic infiltration within the lung lesions. To our knowledge, this pattern has not been reported previously in animals. A similar pattern has been noted in immunosuppressed people with mycobacterial dermatitis caused by rapidly growing, non-tuberculous mycobacteria such as Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum (Gable et al., 2008). In this report, these structures (‘microabscesses with pseudocyst formation’) contained large numbers of acid-fast rods. Interest-
ingly, in the lung lesions of the present reindeer it was only within these spaces that acid-fast rods were noted, but in very small numbers. In conclusion, this case report describes an unusual presentation associated with infection of a reindeer with MAP. This consisted of ‘typical’ intestinal, mesenteric and hepatic lesions, but with additional ‘atypical’ pulmonary lesions similar to those noted in M. bovis infection in other deer species. Because of the lack of published information on the presentation of paratuberculosis in reindeer, it is not possible to determine whether this is likely to be an isolated occurrence. A description of the presentation of paratuberculosis in a larger number of reindeer, including samples of lung, will be required to determine the frequency of this presentation of MAP infection.
Acknowledgments The authors extend their sincere thanks to the histology laboratory staff at Easter Bush Veterinary Centre (EBVC), Edinburgh, UK, for the histological processing of the samples. This work was carried out as part of a diagnostic investigation commissioned by the Royal Zoological Society of Scotland, based at Edinburgh Zoo, Edinburgh, UK, and was funded in part by the Scottish Government Rural and Environment Research and Analysis Directorate.
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Williams ES, Spraker TR, Schoonveld GG (1979) Paratuberculosis (Johne’s disease) in bighorn sheep and a Rocky Mountain goat in Colorado. Journal of Wildlife Diseases, 15, 221e227. October 10th, 2012 ½ Received, Accepted, October 30th, 2012