Accepted Manuscript

Disseminated Mycobacterium genavense Infection in a chinchilla (Chinchilla lanigera)

M. Huynh, J.-L. Pingret, A. Nicolier

PII: S0021-9975(14)00049-8

DOI: 10.1016/j.jcpa.2014.03.003

Reference: YJCPA 1726

To appear in: Journal of Comparative Pathology

Received Date: 13 September 2013

Revised Date: 16 January 2014

Accepted Date: 4 March 2014

Please cite this article as: Huynh, M., Pingret, J.-L., Nicolier, A., Disseminated Mycobacteriumgenavense Infection in a chinchilla (Chinchilla lanigera), Journal of Comparative Pathology (2014), doi:10.1016/j.jcpa.2014.03.003.

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DISEASE IN WILDLIFE OR EXOTIC SPECIES

Short Title: Mycobacteriosis in a Chinchilla

Disseminated Mycobacterium genavense Infection in a chinchilla (Chinchilla lanigera)

M. Huynh *, J-L. Pingret† and A. Nicolier‡

*CHV Frégis, 43 Avenue Aristide Briand 94110 Arcueil, †Scanelis, 9 allée Charles Cros,

31771 Colomiers and ‡Vetdiagnostics, 14 Avenue Rockefeller, 69008 Lyon, France

Correspondence to: M. Huynh (e-mail: nacologie@gmail.com).

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Summary

A 1-year-old neutered male chinchilla was presented with emaciation and a 1-month history

of progressive weight loss. The animal was bright and responsive on clinical examination,

but had poor body condition. Serum biochemical analysis revealed elevated alanine amino

transferase and alkaline phosphatase. Ultrasound examination was unremarkable. Thoracic

radiography showed changes consistent with bullous emphysema and severe pneumonia.

Antibiotic therapy was initiated, but the chinchilla died 6 weeks later. Necropsy examination

revealed granulomatous lesions in the lungs and liver. Numerous acid-fast bacilli were

present in the cytoplasm of macrophages. Sequencing of genetic material isolated from fixed

tissue classified the pathogen as Mycobacterium genavense.

Keywords: chinchilla; Mycobacterium genavense

Mycobacterium genavense is a non-tuberculous bacterium first detected in a human patient

with acquired immunodeficiency syndrome (Bottger, 1990; Bottger et al., 1993). Clinical

and histopathological features of this infection were indistinguishable from those caused by

members of the Mycobacterium avium–intracellulare complex (Bottger et al., 1993). M.

genavense is a fastidious organism with special growth requirements, making the diagnosis of

infections challenging (Bottger, 1994). This bacterium is thought to be ubiquitous and

concerns have been expressed about the potential threat to public health (McClure, 2012).

Infection with this organism is considered to be an emerging disease, which can affect

immunocompromised human beings (Bessesen et al., 1993; Bottger, 1994; Kiehn et al.,

1996). M. genavense has also been isolated from a dog (Kiehn et al., 1996), a cat with feline

immunodeficiency virus infection (Hughes et al., 1999) and birds (Palmieri et al., 2013). To

our knowledge, this is the first report of mycobacteriosis in a chinchilla.

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A 1-year-old neutered male chinchilla was presented with a 1-month history of

progressive weight loss. The chinchilla weighed 500g and had lost 170g over that period,

although it had normal appetite. The chinchilla lived in a private colony of 11 unrelated

chinchillas. The medical record of this animal indicated a previous episode of paraphimosis

and castration 3 months before presentation. The chinchilla was smaller than the other

animals in the group and the owners reported stunted growth.

The animal was bright and responsive on clinical examination, but had poor body

condition with a body condition score of 2/5. Rectal temperature was normal (38.2°C) and

there was no evidence of dental disease.

A blood sample was taken and radiographs were obtained under general anaesthesia.

Serum biochemical analysis revealed elevated alanine amino transferase (234 U/l, reference

range 10–35 U/l; Mayer, 2013) and alkaline phosphatase (101U/l, reference range 6–72 U/l;

Mayer, 2013). Thoracic radiographs showed lesions consistent with bullous emphysema in

the left caudal lung lobe and severe bronchopneumonia (Fig.1). Abdominal ultrasound

examination was unremarkable.

Antibiotic therapy was initiated with enrofloxacin (Baytril 2.5%, Bayer, Loos, France)

at 5 mg/kg q12h and doxycycline (Ronaxan 20, Merial, Lyon, France) at 5 mg/kg q12h for 15

days. However, despite initial stabilization in body weight, the status of the animal

deteriorated. Repeat radiography showed no improvement in the pulmonary lesion.

Azithromycin (Zithromax; Pfizer, Paris, France) at 15 mg/kg q12h was prescribed. Despite

therapy, the chinchilla died 6 weeks after presentation.

At necropsy examination nodular lesions were observed in the lungs and the liver was

congested. Samples from the lungs, liver, heart, intestine, spleen, kidney and brain were

fixed in 10% neutral buffered formalin.

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Microscopical examination of the lungs revealed marked intra-alveolar accumulation

of foamy macrophages and giant cells associated with severe interstitial fibrosis and

lymphocytic infiltration (Figs. 2 and 3). Multifocal rupture of alveolar walls led to formation

of emphysematous bulla. The remaining alveolar spaces were filled with fibrin, heterophils

and oedema fluid. The liver parenchyma showed several necrotic and haemorrhagic foci. On

Ziehl Neelsen staining, numerous acid-fast bacilli were visible in the cytoplasm of

macrophages and giant cells in the lungs and the liver (Fig. 4). Other tissues were

histologically normal.

Total genomic DNA was extracted from paraffin wax embedded tissue using a silica-

based extraction kit (Nucleospin FFPE DNA; Macherey-Nagel, Hoerdt, France). Sequences

from the 5’end of the 16S ribosomal gene of Mycobacterium (Genbank) were aligned (data

not shown) in order to design a set of primers (Mycoseq1F: 5’-

ACACATGCAAGTCGAACG-3’; Mycoseq1R 5’-TGAGATTTCACGAACAACG-3’). A

polymerase chain reaction (PCR) was performed using Platinum Taq DNA polymerase

(Invitrogen, Paisley, UK). Cycling conditions were as follows: 95°C 5 min/35 cycles, 94°C

15 sec/55°C 20 sec/72°C 1 min/72°C 5 min. PCR products were separated by electrophoresis

in a 1.5% agarose gel. The primers amplified a 510 kb fragment. Similar bands were

obtained for extract from the lung and the positive control (DNA from confirmed

Mycobacterium avium culture), while there was no signal for the negative control (water).

The PCR product was sequenced for both strands (MilleGen, Labège, France). The sequence

analysis showed 100% nucleotide identity (461/461) with M. genavense (Genbank accession

number AF547928). The identity score fell to 99.1% (457/461) by comparison with

Mycobacterium triplex and to 95.8% (442/461) with Mycobacterium avium (accession

numbers GQ153279 and AF547898, respectively). The molecular identification confirmed

M. genavense infection.

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In exotic small mammals, M. genavense has been described in a dwarf rabbit (Ludwig

et al., 2009), a grizzled giant squirrel (Moreno et al., 2007; Theuss et al., 2010) and two

ferrets (Lucas et al., 2000). Wild rodents are known to be affected by mycobacterial

infection especially with Mycobacterium microti (Cavanagh et al., 2002). However

spontaneous mycobacteriosis is reported rarely in pet rodents (McClure, 2012). A case of

Mycobacterium avium subsp. avium infection has also been described in a pet ground squirrel

(Juan-Salles et al., 2009) and a pet Korean squirrel (Moreno et al., 2007). However, to our

knowledge, mycobacteriosis has never been reported in more common pet rodents such as

guinea pigs or chinchillas.

Affected organs are usually the liver, spleen, lymph node, skin, lungs and conjunctiva

(Kiehn et al., 1996; Lucas et al., 2000; Moreno et al., 2007; Ludwig et al., 2009). In the

present case, only the lungs and the liver were affected. The source of infection is usually

unknown and drinking water or airborne dissemination has been suggested (Portaels et al.,

1996; Ludwig et al., 2009). The source of infection was not identified in the present case.

Following diagnosis, all of the chinchillas from the colony were screened radiographically,

but no evidence of pulmonary lesions was found. Two chinchillas from the same colony died

within the following 2 years, but necropsy examination did not show any evidence of

mycobacterial lesions. The immune status of the infected individuals may be a crucial factor

allowing infection (Bessessen et al., 1993; Hughes et al., 1999).

Unlike in man or cats, there are no reports of viral disease causing immunodeficiency

in chinchillas (Mans and Donnelly, 2012). No pre-existing condition interfering with

immunity was demonstrated ante mortem or post mortem in the present case, although

environmental stress cannot be completely ruled out. Post-operative complications after

castration may have influenced the course of the disease; however, recovery from surgery and

anaesthesia was recorded as uneventful.

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Known predisposing factors for respiratory disease in chinchillas are overcrowding,

inadequate ventilation and poor hygiene (Mans and Donnelly, 2012). Respiratory disease is

uncommon in chinchillas and differential diagnosis usually includes infectious disease (Mans

and Donnelly, 2012). Primary lung disease can be caused by Bordetella bronchiseptica,

Streptococcus pneumoniae, Klebsiella pneumoniae, Proteus mirabilis or Enterobacter

aerogenes, under stressful conditions (Bartoszcze et al., 1990; Harkness and Wagner, 1995;

Girling, 2003; Bautista et al., 2007). Other differentials for granulomatous pulmonary lesions

would be fungal infection caused by Histoplasma capsulatum (Owens et al., 1975). As seen

in the present case, the prognosis is guarded to poor if body condition is affected (Mans and

Donnelly, 2012).

Regarding the potential zoonotic threat, treating mycobacterial infection in pet

animals may not be appropriate. Treatment with rifampicin has been successfully reported in

two ferrets (Lucas et al., 2000). However the diagnosis is usually made on post-mortem

examination in pet animals, which limits any therapeutic options (Martinez et al., 1993;

Moreno et al., 2007; Ludwig et al., 2009).

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Received, September 13th, 2013

Accepted, March 4th, 2014

List of Figures

Fig. 1. Lateral thoracic radiograph showing a large emphysematous bulla in the caudal

thoracic area.

Fig. 2. Section of lung. Alveoli are filled with macrophages admixed with fewer giant cells

and degenerate polymorphonuclear cells. Septa are slightly fibrotic and infiltrated by

lymphocytes. HE. ×40.

Fig. 3. Section of lung. Alveolar macrophages have abundant foamy cytoplasm. HE. ×200.

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Fig. 4. Section of lung. Numerous large aggregates of acid-fast bacilli are seen within the

cytoplasm of macrophages in the granulomatous lesions. Ziehl Neelsen stain. ×40.

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