J Comp Path 2016, Vol -, 1e4 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jcpa DISEASE IN WILDLIFE OR EXOTIC SPECIES Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens) N Fuke*, T Hirai*, N Makimura†, Y Goto†, W A Habibi*, S Ito*, N T Trang*,‡, K Koshinox, M Takedax and R Yamaguchi* * Department of Veterinary Pathology, † Department of Veterinary Microbiology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan, ‡ Department of Veterinary Public Health, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, Vietnam and x Miyazaki City Phoenix Zoo, Miyazaki, Japan Summary A 9-year-old male red panda (Ailurus fulgens) became emaciated and died Necropsy examination revealed systemic lymphadenomegaly The liver, lungs and left kidney contained multifocal yellow nodules Microscopical examination revealed granulomatous inflammation in the liver, lungs, kidney, spleen and lymph nodes, with numerous acid-fast bacilli Sequencing of genetic material isolated from the tissues classified the pathogen as Mycobacterium gastri Lymphoma was found in the liver, lungs, kidney and lymph nodes The neoplastic cells were strongly labelled for expression of CD3, Ki67 and proliferating cell nuclear antigen by immunohistochemistry This is the first report of M gastri infection with T-cell lymphoma in a red panda Ó 2016 Elsevier Ltd All rights reserved Keywords: Mycobacterium gastri; non-tuberculous mycobacteriosis; red panda; T-cell lymphoma Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and are potential pathogens for man and animals More than 140 mycobacterial species have been reported in people and NTM are common pathogens in immunodeficient patients (Tortoli et al., 2000) In man, the main pathogens causing non-tuberculous mycobacteriosis are Mycobacterium aviumeintracellulare complex (MAC) (Tsukamura et al., 1988; Biet et al., 2005), which induce respiratory disease Non-tuberculous mycobacteriosis is mainly caused by MAC in many animals including, deer, horses, pigs and birds In pigs, M avium usually causes granulomatous lesions in the mesenteric lymph nodes, liver, lungs and spleen (Biet et al., 2005) NTM can affect immunosuppressed human patients with lymphoma and acquired immunodeficiency syndrome (AIDS) (Jacobson et al., 1991; Chin et al., 1994) Mycobacterial infection has also Correspondence to: T Hirai (e-mail: t-hirai@cc.miyazaki-u.ac.jp) 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2016.06.001 been reported in an immunosuppressed ferret (Saunders and Thomosen, 2006) Chronic inflammation can also cause tumour formation; for example, macrophages stimulated by Helicobacter pylori release a proliferation-inducing ligand, which belongs to the tumour necrosis factor family B lymphocytes are stimulated to maturity by this cytokine and mucosa-associated lymphoid tissue (MALT) lymphoma develops (Westbrook et al., 2010; Sebastian and Hana, 2014) Mycobacterium gastri is a NTM first detected by human gastric lavage (Wayne, 1966) and is a known factor in the development of peritonitis and septic metacarpophalangeal arthritis, seminal vesiculitis and lymphadenitis in man (Linton et al., 1986; Indudhara et al., 1991; Perandones et al., 1991; Velayati et al., 2005) However, there are no reports of M gastri infection in animals We present a case of non-tuberculous mycobacteriosis due to M gastri with T-cell lymphoma formation in a red panda (Ailurus fulgens) To our knowledge, this is the first report Ó 2016 Elsevier Ltd All rights reserved Please cite this article in press as: Fuke N, et al., Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.001 N Fuke et al of M gastri infection with T-cell lymphoma in this species A 9-year-old neutered male red panda in a zoological garden in Japan was observed to have a cataract in the right eye and dyskeratosis throughout the body after transportation from another zoo in winter Two months later, lymph node biopsy was performed because of lymphadenomegaly and microscopical examination revealed lymphadenitis Neoplastic changes were not observed at that time Four months later, the animal showed emaciation and died A necropsy examination was performed at the Department of Veterinary Pathology, University of Miyazaki The other red pandas in the group appeared clinically normal At necropsy examination, the lymph nodes, especially the right axillary, inguinal, mandibular, popliteal and mesenteric lymph nodes were enlarged Multiple, diffuse, yellow foci, 2e5 cm in diameter, were observed in all lung and liver lobes The cut surface of these foci and of the lymph nodes was white and roughened There was serous atrophy of the perirenal fat Many white spots (3e4 mm diameter) were seen in the cortex of the left kidney The abdominal cavity contained yellow fluid Samples of the lungs, liver, heart, intestine, spleen, kidneys and lymph nodes were collected, fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin wax Sections (2 mm) were stained with haematoxylin and eosin (HE) and ZiehleNeelsen stain Immunohistochemistry (IHC) was performed with primary reagents specific for CD3, CD20, proliferating cell nuclear antigen (PCNA) and Ki67 (Dako, Glostrup, Denmark) Parts of the excised liver lesion were submitted for bacterial culture on 2% Ogawa egg yolk medium at the Department of Veterinary Microbiology, University of Miyazaki Polymerase chain reaction (PCR), using primers for the Mycobacterium16S rRNA gene, was performed on colonies from the culture medium and sequencing was conducted (Shin et al., 2009) A DNAeDNA hybridization method for 22 Mycobacterium spp was conducted using DDHemycobacteria Kyokuto (Kyokuto, Tokyo, Japan), according to methods described previously (Kusunoki et al., 1991) Conventional biochemical tests, photochromogenicity and nitrate reduction, were conducted to distinguish between M gastri and Mycobacterium kansasii (Tsukamura, 1967; Tsukamura, 1973) Microscopical examination of the liver (Fig 1), lungs, left kidney and the lymph nodes revealed a mixture of granulomatous inflammation and neoplastic lymphocytes The liver parenchyma showed diffuse granulomatous inflammation with central necrosis surrounded by macrophages, multi- Fig A mixture of granulomatous inflammation (G) and foci of neoplastic lymphocytes (N) in the liver HE Bar, 200 mm nucleated giant cells, neutrophils and lymphocytes (Fig 2) There was abundant fibrosis and mineralization around these areas of granulomatous inflammation Similar findings were observed in the lungs, left kidney, spleen and lymph nodes ZiehleNeelsen staining revealed numerous acid-fast bacilli in the cytoplasm of macrophages and extracellular areas in the liver, lungs, left kidney, spleen and lymph nodes (Fig 3) Normal lymph node structure was replaced by granulomatous inflammation and necrosis Foci of neoplastic cells were found around the portal vein and invading lymphatic vessels in the liver (Fig 4A) Neoplastic cells were round and pleomorphic with large, irregular nuclei that contained a single prominent nucleolus The mitotic rate was (average number of mitoses over ten Â400 fields) Fig Granulomatous inflammation consisting of necrosis, macrophages, multinucleated giant cells and neutrophils in the liver HE Bar, 100 mm Inset: multinucleated giant cells HE Bar, 20 mm Please cite this article in press as: Fuke N, et al., Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.001 Mycobacteriosis and Lymphoma in a Red Panda Fig Section of axillary lymph node Numerous acid-fast bacilli (arrows) are found intracellularly and outside of macrophages ZN stain Bar, 20 mm Fig (A) Neoplastic cells invade lymphatic vessels in the liver HE Bar, 50 mm (B) Neoplastic lymphocytes around the portal vein The cells are round and pleomorphic with large oval or irregular nuclei, anisokaryosis and a single prominent nucleolus Mitotic figures are observed frequently HE Bar, 20 mm (C) Neoplastic cells express CD3 IHC Bar, 20 mm (Fig 4B) Foci of neoplastic cells were also found in the lungs and kidney The lymph node cells had been replaced by neoplastic cells expressing CD3 (Fig 4C), Ki67 and PCNA, but not CD20 Therefore, the neoplasm was diagnosed as a T-cell lymphoma Sequence data from the 16S rRNA gene showed 100% and 99% nucleotide identity with M gastri and M kansasii, respectively Isolates reacted with both M gastri and M kansasii probes, especially M gastri, by the DNAeDNA hybridization method The red panda was diagnosed with an M gastri infection after photochromogenic yellow pigment and nitrate reduction tests were negative These two species can be differentiated by biochemical characteristics such as photochromogenicity and nitrate reduction (Tsukamura, 1973) M gastri cannot produce yellow pigment even if exposed to light and not react with nitrate reduction In this case, M gastri infection was confirmed by both of these tests being negative Non-tuberculous mycobacteriosis is induced by NTM, which are frequently pathogenic in immunodeficient human patients and cause infection via the oral or respiratory routes (Tortoli et al., 2000; Biet et al., 2005) In pigs, non-tuberculous mycobacteriosis is mainly caused by MAC and usually induces granulomatous lesions in abdominal tissues Therefore, the route of infection for MAC is suspected to be oral, via food and water (Biet et al., 2005) The route of infection for M gastri is suspected to be oral via water, due to detection of the bacteria in gastric washings (Wayne, 1966; Velayati et al., 2005) In the present case, severe granulomatous lesions were found in the abdominal organs, especially liver, iliac lymph nodes and mesenteric lymph nodes, and these findings may suggest an oral route of infection In man, there are only a few reports of M gastri infection (Linton et al., 1986; Indudhara et al., 1991; Perandones et al., 1991; Velayati et al., 2005) and clinical symptoms include peritonitis, seminal vesiculitis and arthritis After treatment, these symptoms improve, except for two patients who developed disseminated lymphadenitis However, in the present case, infection had disseminated to the liver, lungs, kidney, spleen and lymph nodes, because this animal only received antibiotics and not specific drugs for treatment of tuberculosis Differences in the lesion distribution may have been influenced by the treatment In summary, immunodeficient human patients with lymphoma and AIDS are susceptible to NTM (Jacobson et al., 1991; Chin et al., 1994), although chronic inflammation can also induce malignant tumours For example, lymphoma can develop in the pleural cavity after a long-standing history of pyothorax (Nakatsuka et al., 2002) and H pylori infection is associated with MALT lymphoma (Sebastian and Hana, 2014) Nontuberculous and tuberculous infections may cause B-cell lymphoma (Inadome et al., 2001; Gaur et al., 2004), but there are only a few reports of Tcell lymphoma induced by chronic inflammation (Nakatsuka et al., 2002; Santini et al., 2008) The red panda reported here was transferred from another zoo during winter and the combination of transport and low temperature may have led to stress and subsequent immunosuppression, allowing infection by M gastri Other red pandas in the Please cite this article in press as: Fuke N, et al., Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.001 N Fuke et al same group that had not travelled were not affected The affected red panda had both granulomatous lymphadenitis and T-cell lymphoma and the background of chronic inflammation may have predisposed to the development of lymphoma Acknowledgments The authors thank the Miyazaki City Phoenix Zoo staff including the director, Mr T Deguchi, and the animal keeper, Mrs N Seki Conflict of Interest Statement The authors declare no potential conflicts of interest with respect to the research, authorship or publication of this article References Biet F, Boschiroli ML, Thorel MF, Guilloteau LA (2005) Zoonotic aspects of Mycobacterium bovis and Mycobacterium aviumeintracellulare complex (MAC) Veterinary Research, 36, 411e436 Chin DP, Hopewell PC, Yajiko DM, Vittinghoff E, Horsburgh CRJ et al (1994) Mycobacterium avium complex in the respiratory or gastrointestinal tract and the risk of M avium complex bacteremia in patients with human immunodeficiency virus infection Journal of Infectious Diseases, 169, 289e295 Gaur S, Trayner E, Aish L, Weinstein R (2004) Bronchusassociated lymphoid tissue lymphoma arising in a patient with bronchiectasis and 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39, 284e287 Wayne LG (1966) Classification and identification of mycobacteria American Review of Respiratory Disease, 93, 919e928 Westbrook AM, Szakmary A, Schiestl RH (2010) Mechanisms of intestinal inflammation and development of associated cancers: lessons learned from mouse models Mutant Research, 705, 40e59 January 22nd, 2016 ½ Received, Accepted, June 3rd, 2016 Please cite this article in press as: Fuke N, et al., Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens), Journal of Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.06.001 ... Fuke et al of M gastri infection with T- cell lymphoma in this species A 9-year-old neutered male red panda in a zoological garden in Japan was observed to have a cataract in the right eye and dyskeratosis... lymphoma can develop in the pleural cavity after a long-standing history of pyothorax (Nakatsuka et al., 2002) and H pylori infection is associated with MALT lymphoma (Sebastian and Hana, 2014) Nontuberculous... lymphadenitis Neoplastic changes were not observed at that time Four months later, the animal showed emaciation and died A necropsy examination was performed at the Department of Veterinary Pathology,