DISEASE IN WILDLIFE OR EXOTIC SPECIES

Short Title: Yersiniosis in a Ring-tailed Lemur (Lemur catta)

Systemic Yersinia pseudotuberculosis as a Cause of Osteomyelitis in a Captive Ring-

tailed Lemur (Lemur catta)

D. Walker*, J. Gibbons†, J. D. Harris*, C. S. Taylor*, C. Scott‡, G. K. Paterson*

and L. R. Morrison*

* Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh,

Easter Bush, Edinburgh, UK, †School of Veterinary Medicine, University College Dublin,

Belfield, Dublin, Ireland and ‡Struthers and Scott Veterinary Practice, Doune, Perthshire,

UK.

Correspondence to: D. Walker (e-mail: dave.walker@ed.ac.uk).

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Summary

Yersinia pseudotuberculosis and Y. enterocolitica are ubiquitous pathogens with wildlife and

domestic animal reservoirs. Outbreaks of ‘non-plague’ yersiniosis in man and non-human

primates are reported frequently (including zoological specimens and research breeding

colonies), and are usually characterized by enteritis, mesenteric lymphadenitis and

occasionally organ abscessation. In people, non-septic reactive arthritis (ReA) is a common

sequela to yersiniosis. However, there have been rare reports in people of septic arthritis and

osteomyelitis because of active systemic infection with Y. pseudotuberculosis. Osteomyelitis

has also been reported rarely in historical yersiniosis outbreaks in farmed turkeys in England

and the USA. This paper reports the first case of osteomyelitis caused by systemic infection

with Y. pseudotuberculosis O:1 in a non-human primate, a captive ring-tailed lemur (Lemur

catta). The lemur had a short clinical history of hyporexia and weight loss with reduction in

mobility, especially of the left hindlimb. On post-mortem examination there was evidence of

multi-organ abscessation. In addition, severe necrosis, inflammation and large bacterial

colonies were present in the musculature, periosteum and bone marrow in the hip, ribs and a

vertebra at the cervicothoracic junction. Osteomyelitis should be considered as a rare clinical

presentation in non-human primates with systemic Y. pseudotuberculosis infection.

Keywords: yersiniosis; Yersinia pseudotuberculosis; lemur; osteomyelitis

Yersinia pseudotuberculosis is a gram-negative, facultative anaerobic coccobacillus of the

family Enterobacteriaceae. Together with Y. enterocolitica, it is the causative agent of

yersiniosis (non-plague), which typically presents as ileitis and mesenteric lymphadenitis

(Smego et al., 1999; Loftus et al., 2002). Y. enterocolitica usually only causes clinical

disease in human beings, although outbreaks have been reported infrequently in non-human

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primates (Nakamura et al., 2010). Y. pseudotuberculosis is reported in a broad range of host

species including free-ranging species in Europe and Japan (Mair 1973; Smego et al., 1999).

Yersiniosis is recognized as an important food-borne zoonosis worldwide (Giannitti et al.,

2014; Bonardi et al., 2016).

Typical reservoirs are found in production animals (e.g. cattle, sheep, pigs and goats;

Slee and Button, 1990; Milnes et al., 2008), companion animals (e.g. dogs and cats; Stamm et

al., 2013) and wildlife (e.g. rodents, hares and wild boar; Mair 1973; Wacheck et al., 2010),

which are often apparently healthy. Y. pseudotuberculosis is transmitted by the faecal–oral

route; pigs are determined to be a significant source of infection for pathogenic zoonotic

strains (Smego et al., 1999). Yersinia spp. cross the intestinal barrier through translocation

via M cells (Clark et al., 1998; Schulte et al., 2000). The bacteria are thought to replicate in

Peyer’s patches and local lymph nodes before seeding other tissues, although evidence

suggests that Y. pseudotuberculosis can replicate in the intestinal lumen before dissemination

to distant sites (Barnes et al., 2006). Clinical signs of yersiniosis in people include abdominal

pain (sometimes presenting as a ‘pseudoappendicitis’), diarrhoea and fever. In more severe

infections, intestinal necrosis and haemorrhage occur, in addition to septicaemia and multi-

organ abscessation (Smego et al., 1999). Reactive polyarthritis (i.e. synovial fluid

demonstrating a polymorphonuclear pleocytosis) is a sequela in people (occurring in

approximately 10 to 30% of patients) and can persist for weeks to months (Smego et al.,

1999; Vasala et al., 2014).

Spontaneous yersiniosis outbreaks are reported in captive mammal breeding and

research colonies in Europe, North America and Asia, including non-human primates

(MacArthur and Wood, 1983; Taffs and Dunn, 1983; Nakamura et al., 2010). The source of

disease is often speculated to be contamination of the environment by wild rodents or birds

(Taffs and Dunn, 1983). Yersiniosis in non-human primates often presents with non-specific

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clinical signs such as lethargy, depression, dehydration, diarrhoea and weight loss. Other

affected animals may die acutely with few preceding clinical signs (Buhles Jr et al., 1981;

Taffs and Dunn, 1983; Nakamura et al., 2010). On post-mortem examination, intestinal

erosion and ulceration is often present, together with multi-organ abscessation, usually

involving the intestinal tract, liver, spleen and abdominal lymph nodes (e.g. in red-bellied

tamarins; Saguinus labiatus; Taffs and Dunn, 1983).

Yersiniosis has not been reported as a cause of septic arthritis or osteomyelitis in non-

human primates and is rare in human beings (Katamura et al., 2001; van Zonneveld et al.,

2002). In this report a novel case of multifocal osteomyelitis in a non-human primate is

described; this presented as part of a severe systemic infection with Y. pseudotuberculosis in a

ring-tailed lemur (Lemur catta). The lemur was an approximately 3-year-old, neutered male

and was housed at a safari park in Scotland, UK. It was presented to the referring veterinarian

with a 10-day history of lethargy, hyporexia and weight loss. Diagnostic work performed

ante mortem revealed the presence of haematuria and mild proteinuria using a commercial

urine dipstick. Serum biochemistry indicated elevated amylase levels (1239 U/l, reference

108–954 U/l; Greendale Veterinary Diagnostics Limited, Surrey, UK), amongst otherwise

unremarkable biochemical and haematological results. The animal was treated

symptomatically with amoxicillin/clavulanic acid (approximately 25 mg/kg q12h), but

morbidity progressed to decreased function of the left hindlimb and continued loss of body

condition, resulting in the decision to humanely destroy the lemur.

A post-mortem examination was performed at Easter Bush Pathology, Royal (Dick)

School of Veterinary Studies, University of Edinburgh, UK. The lemur weighed 1.65 kg and

possessed minimal subcutaneous and intra-abdominal fat reserves. The following abnormal

findings were grossly evident. On the ribs, there were scattered, pale-yellow, raised, 2 to 5

mm diameter nodules, containing pus, which elevated the overlying pleura. There was a focal

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accumulation of pus at the cervicothoracic junction (Fig. 1) with thickening of the left ventral

spinal nerve root. There was a focal abscess in the musculature cranial to the left hip joint,

which extended into the surrounding tissues of the left hemipelvis and associated

musculature. The left hemipelvis was easily separated from the right side with a poorly

demarcated, approximately 5 cm diameter, region of pus and soft granular material replacing

the musculature and bone. These findings were consistent with a regionally extensive,

necrotising and suppurative cellulitis, myositis and osteomyelitis with fibrosis in the left

hemipelvis and hip joint. There was no evidence of external wounds in this area.

The lungs were mottled dark red and the left kidney contained a focal, 3 mm diameter

abscess. The gastric mucosa had multifocal to coalescing areas of mucosal erosion and

ulceration (possibly compatible with chronic stress). The liver was grossly normal, although

the gall bladder was distended and contained inspissated bile.

A swab of the pelvic lesion was taken during post-mortem examination. Gram-

negative bacilli grew as small, grey colonies on Columbia horse blood agar and as non-lactose

fermenters on MacConkey agar. The bacteria were identified as Y. pseudotuberculosis by the

VITEK 2 system (bioMérieux, Basingstoke, UK) using the VITEK 2 GN ID (gram-negative

identification) card and following the manufacturer’s instructions. Antimicrobial

susceptibility was performed using the VITEK 2 AST-GN65 card, with interpretation

according to Clinical and Laboratory Standards Institute Veterinary Criteria VET01-S2. The

isolate was susceptible to all antimicrobials tested. The O serogroup was determined with the

Mast Assure Yersinia pseudotuberculosis O Grouping Antisera (Mast Group, Liverpool, UK)

following the manufacturer’s guidelines and was found to be O:1.

A panel of tissues, including targeted lesions and routinely collected samples, were

sampled during the post-mortem examination and fixed in 10% neutral buffered formalin.

Tissues were processed routinely and embedded in paraffin wax. Sections were stained with

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haematoxylin and eosin (HE). Slides were digitized using the NanoZoomer-XR system

(Hamamatsu, Welwyn Garden City, Hertfordshire, UK).

The main histopathological findings were as follows. In the left hemipelvis and

surrounding musculature there was evidence of regionally extensive myositis and multifocal

osteomyelitis. This was characterized by a marked, extensive, predominantly neutrophilic,

inflammatory infiltrate in the external pelvic musculature. Inflammation was centred on

multiple, large bacterial colonies and was associated with marked myofibre degeneration and

atrophy. The inflammation and bacterial colonies extended to the periosteum and into the

bone marrow. Similar findings were observed in samples from the macroscopically evident

rib nodules and at the cervicothoracic junction (Fig. 2.). Osteoclasts and evidence of bone

remodelling and periosteal woven bone proliferation was frequently observed in affected bone

(Fig. 3). The inflammation extended around the nerve roots of the spinal cord.

In the lung, there were multifocal areas of pyogranulomatous inflammation with large

accumulations of neutrophils and florid bacterial colonies (microabscesses). In the kidney,

effacing and replacing the cortical parenchyma, was a large pyogranuloma surrounding large

bacterial colonies.

There was no evidence of abscessation in the liver; however, there was mild,

multifocal, random hepatocellular necrosis and evidence of extramedullary haematopoiesis.

Examined sections of intestinal tract, brain, heart, oesophagus and adrenal gland were

unremarkable. The spleen showed evidence of reactive hyperplasia and extramedullary

haematopoiesis, but was otherwise unremarkable. A mild, chronic, pancreatitis was also

detected.

Therefore, in summary, there was evidence of severe, systemic infection with Y.

pseudotuberculosis, which included osteomyelitis in several bones. Unusually, there was no

observed evidence of enteric yersiniosis clinically or at the time of post-mortem examination.

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Although outbreaks and single case reports of Y. pseudotuberculosis are occasionally

reported, to the authors’ knowledge, this is the first report of osteomyelitis caused by Y.

pseudotuberculosis in a non-human primate and is therefore a rare clinical presentation.

Reactive arthritis (ReA; immune-mediated) is reported as a sequela to infection by

Yersinia spp. in people (Granfors et al., 1989; Hill Gaston et al., 1999). However,

osteomyelitis and septic arthritis have been reported uncommonly as a result of Y.

enterocolitica infections in human beings (Sebes et al., 1976; Spira and Kabins, 1976; Crowe

et al. 1996). Furthermore, Y. pseudotuberculosis as a cause of osteomyelitis is rare; it has

been confirmed in one case of chronic recurrent multifocal osteomyelitis (CRMO) in a human

being (Katamura et al., 2001) and was suspected to be the cause of bone lysis in an

immunosuppressed kidney transplant patient (van Zonneveld et al., 2002). More recently,

Ishihara et al. (2016) reported the first case of pyogenic vertebral osteomyelitis in a human

being caused by Y. pseudotuberculosis. Y. pseudotuberculosis has been reported rarely as a

cause of osteomyelitis in commercial turkey flocks (Wise and Uppal, 1971; Wallner-

Pendleton and Cooper, 1982).

Outbreaks of infection with Y. pseudotuberculosis are reported in captive non-human

primate colonies across the world, where it usually causes acute enteritis, mesenteric

lymphadenitis and often visceral abscessation (MacArthur and Wood, 1983; Taffs and Dunn,

1983; Iwata and Hayashidani, 2011). Y. enterocolitica and Y. pseudotuberculosis are

considered ubiquitous and can persist for long periods in the environment and are carried by a

variety of free-ranging species (Galindo et al., 2011); therefore, the source of infection in this

lemur was not determined. It is also noted that there was no overt evidence of underlying

immunosuppression in this relatively young ring-tailed lemur, which might have predisposed

to development of a severe multi-organ infection with bone involvement.

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In conclusion, this case importantly highlights that, together with Y. enterocolitica and

rarely Y. pseudotuberculosis in human beings, osteomyelitis may be a rare clinical

presentation in non-human primates systemically infected with Y. pseudotuberculosis.

Acknowledgments

With thanks to the technical staff in the Histopathology Laboratory, Easter Bush

Pathology, Royal (Dick) School of Veterinary Studies, for preparing the slides used in this

study. This research did not receive any specific grant from funding agencies in the public,

commercial, or not-for-profit sectors.

Conflict of Interest Statement

The authors declare no conflicts of interest with respect to the publication of this manuscript.

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Received, June 7th, 2018

Accepted,

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Figure Legends

Fig.1. Focal accumulation of pus in the vertebral canal (arrowed), observed post mortem

following dorsal laminectomy. Bar, 1 cm.

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Fig.2. Section of the left hemipelvis. Scalloped bone surfaces due to remodelling with

scattered osteoclasts is evident (inset). Predominately neutrophilic inflammation with

bacterial colonies (arrowheads) is evident in the bone marrow. Bar, 100 µm; inset 20 µm.

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Fig.3. Section of the transverse process of a vertebra at the cervicothoracic junction.

Periosteal woven bone proliferation is evident. Pyogranulomatous inflammation and bacterial

colonies are present in the surrounding soft tissue. Karyorrhectic debris is evident in the bone

marrow (arrowhead). Bar, 250 µm.

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