Non tuberculous Mycobacteriosis with T cell Lymphoma in a Red Panda (Ailurus fulgens)

Microscop-ical examination revealed granulomatous inflammation in the liver, lungs, kidney, spleen and lymph nodes, with numerous acid-fast bacilli.. Lymphoma was found in the liver, lun

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 andxMiyazaki 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 Microscop-ical 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 immunohistochem-istry 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

ubiqui-tous 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

Myco-bacterium aviumeintracellulare complex (MAC)

(Tsukamura et al., 1988; Biet et al., 2005), which

induce respiratory disease Non-tuberculous

myco-bacteriosis 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

pa-tients with lymphoma and acquired

immunodefi-ciency syndrome (AIDS) (Jacobson et al., 1991;

Chin et al., 1994) Mycobacterial infection has also

been reported in an immunosuppressed ferret (Saunders and Thomosen, 2006) Chronic inflamma-tion can also cause tumour formainflamma-tion; 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) lym-phoma develops (Westbrook et al., 2010; Sebastian and Hana, 2014)

Mycobacterium gastriis a NTM first detected by hu-man gastric lavage (Wayne, 1966) and is a known fac-tor 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 (Ai-lurus fulgens) To our knowledge, this is the first report

J Comp Path 2016, Vol -, 1e4 Available online atwww.sciencedirect.com

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Correspondence to: T Hirai (e-mail: t-hirai@cc.miyazaki-u.ac.jp ).

Ó 2016 Elsevier Ltd All rights reserved.

of M gastri infection with T-cell lymphoma in this

species

A 9-year-old neutered male red panda in a

zoolog-ical 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

ex-amination revealed lymphadenitis Neoplastic

changes were not observed at that time Four months

later, the animal showed emaciation and died A

nec-ropsy 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,

espe-cially the right axillary, inguinal, mandibular,

popli-teal and mesenteric lymph nodes were enlarged

Multiple, diffuse, yellow foci, 2e5 cm in diameter,

were observed in all lung and liver lobes The cut

sur-face of these foci and of the lymph nodes was white

and roughened There was serous atrophy of the

peri-renal 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,

Univer-sity 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

Mycobacte-riumspp was conducted using DDHemycobacteria

Kyokuto (Kyokuto, Tokyo, Japan), according to

methods described previously (Kusunoki et al.,

1991) Conventional biochemical tests,

photochro-mogenicity 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-nucleated giant cells, neutrophils and lymphocytes (Fig 2) There was abundant fibrosis and mineraliza-tion around these areas of granulomatous inflamma-tion 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 por-tal vein and invading lymphatic vessels in the liver (Fig 4A) Neoplastic cells were round and pleomor-phic with large, irregular nuclei that contained a sin-gle prominent nucleolus The mitotic rate was 2 (average number of mitoses over ten 400 fields)

Fig 1 A mixture of granulomatous inflammation (G) and foci of neoplastic lymphocytes (N) in the liver HE Bar, 200 mm.

Fig 2 Granulomatous inflammation consisting of necrosis, mac-rophages, multinucleated giant cells and neutrophils in the liver HE Bar, 100 mm Inset: multinucleated giant cells HE 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

infec-tion after photochromogenic yellow pigment and

ni-trate reduction tests were negative

These two species can be differentiated by biochemical characteristics such as photochromoge-nicity and nitrate reduction (Tsukamura, 1973) M gastricannot produce yellow pigment even if exposed

to light and do 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 immuno-deficient 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 granu-lomatous 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 infec-tion 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) Non-tuberculous and Non-tuberculous infections may cause B-cell lymphoma (Inadome et al., 2001; Gaur

et al., 2004), but there are only a few reports of T-cell 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

Fig 3 Section of axillary lymph node Numerous acid-fast bacilli

(arrows) are found intracellularly and outside of

macro-phages ZN stain Bar, 20 mm.

Fig 4 (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.

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

publica-tion of this article

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½ Received, January 22nd, 2016 Accepted, June 3rd, 2016