J. Comp. Path. 2015, Vol. 153, 185e189

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jcpa

DISEASE IN WILDLIFE OR EXOTIC SPECIES

Sarcocystis spp. Infection in two Red Panda Cubs (Ailurus fulgens) W. M. Zoll*,†, D. B. Needle‡, S. J. French*, A. Lim*, S. Bolin*, I. Langohr*,x and D. Agnew* * Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI, † Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, ‡ Comparative Medicine and Integrated Biology, Michigan State University, Lansing, MI and x Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA

Summary Two neonatal male red panda (Ailurus fulgens) littermates were submitted for necropsy examination. One animal was found dead with no prior signs of illness; the other had a brief history of laboured breathing. Post-mortem examination revealed disseminated protozoal infection. To further characterize the causative agent, transmission electron microscopy (TEM), immunohistochemistry (IHC), polymerase chain reaction (PCR) and amplification and nucleic acid sequencing were performed. IHC was negative for Toxoplasma gondii and Neospora caninum, but was positive for a Sarcocystis spp. TEM of cardiac muscle and lung revealed numerous intracellular apicomplexan protozoa within parasitophorous vacuoles. PCR and nucleic acid sequencing of partial 18S rRNA and the internal transcribed spacer (ITS)-1 region confirmed a Sarcocystis spp. that shared 99% sequence homology to Sarcocystis neurona and Sarcocystis dasypi. This represents the first report of sarcocystosis in red pandas. The histopathological, immunohistochemical, molecular and ultrastructural findings are supportive of vertical transmission resulting in fatal disseminated disease. Ó 2015 Elsevier Ltd. All rights reserved. Keywords: disseminated infection; immunohistochemistry; red panda; Sarcocystis spp.

The red panda (Ailurus fulgens) is an endangered mammal and sole member of the family Ailuridae, native to central China and the eastern Himalayas. Females typically give birth to two altricial cubs that are completely dependent on their mother for immunity and protection. Some serological immunity is derived from the mother during pregnancy, as red pandas have vasochorial discoid placentation (Benirschke, 2011). The neonatal period is a time when red pandas are particularly susceptible to infectious diseases (Preece, 2011), but there are no reports of such infections in neonatal red pandas. The three main causes of death within the first 10 days of life are Correspondence to: W.M. Zoll (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2015.04.009

cannibalism (secondary to other illness), hypothermia and starvation, often attributed to poor mothering skills, trauma and pneumonia (Preece, 2011). There have been no descriptions of sarcocystosis in red panda adults or neonates (Preece, 2011). In this report we describe two red panda cub littermates that died in the first 2 weeks of life from disseminated Sarcocytis spp. infection. A 1-week-old, 0.13 kg male red panda cub (case 1), from a zoo in Michigan, was found dead in its exhibit with no premonitory signs. One week later, the 0.182 kg, male, sole littermate of the first red panda, died (case 2). It had a 3-day history of hypothermia and laboured breathing, followed by brief improvement and then peracute precipitous decline. It died 5 days after the onset of illness. Ó 2015 Elsevier Ltd. All rights reserved.

186

W.M. Zoll et al.

A complete routine gross necropsy examination was performed on each animal and findings were non-specific. Samples of brain, lung, heart, spleen, liver, kidney, adrenal gland, lymph node, pancreas, stomach, jejunum, duodenum, ileum, caecum, colon, skeletal muscle, tongue, brown fat and bone marrow from case 1 and brain, liver, lung, kidney, gallbladder, adrenal gland, heart, pancreas, oesophagus, jejunum, colon, trachea, peripheral nerve, mesenteric lymph node, tracheobronchial lymph node, urinary bladder, skin and skeletal muscle from case 2 were collected. Samples were fixed in 10% neutral buffered formalin for 24 h, trimmed, processed routinely, sectioned (5 mm), mounted on frosted glass slides and stained with haematoxylin and eosin (HE). For indirect immunohistochemistry (IHC), sections (5 mm) of the formalin-fixed, paraffin waxembedded heart and lung were mounted on negatively charged glass slides. Slides with tissue sections were heated in a 60 C oven for 1 h. IHC for Toxoplasma, Neospora and Sarcocystis spp. were performed as previously described (Soldati et al., 2004; Mullaney et al., 2005; Hoffman et al., 2012). A series of polymerase chain reaction (PCR) assays were performed on DNA extracted from fresh brain tissue (case 1) and fresh lung tissue (case 2), using the DNeasyÒ Blood & Tissue Kit (Qiagen Inc., Valencia, California, USA). The initial testing included a PCR assay for Toxoplasma gondii, which used PCR primers derived from the 35-fold repetitive B1 gene of that organism (Burg et al., 1989; Bretagne et al., 1993) and a PCR assay targeting the gene for 18S ribosomal RNA (18S rDNA) that can detect several members of the apicomplexan group of parasites (Harrison et al., 2007). Based on results from the latter assay, multiple additional PCR assays targeting the 18S rDNA or the internal transcribed spacer (ITS)1 region were designed from aligned nucleic acid sequences of Sarcocystis neurona, Sarcocystis canis, Sarcocystis felis and Sarcocystis dasypi. The PCR amplicons were visualized in ethidium bromide-stained 1.5% agarose gels after electrophoresis in sodium borate buffer (Brody and Kern, 2004). PCR amplicons of the appropriate size were excised from the gel, purified with QIAquick Gel Extraction Kit (Qiagen) and eluted in 30 ml of nuclease-free water. DNA of the PCR amplicons was then directly sequenced in each direction using the aforementioned PCR primers. Sequences were edited with Sequencher software (Gene Codes Corporation, Ann Arbor, Michigan, USA), trimmed to remove primer sites and analyzed with the Basic Local Alignment Search Tool (BLAST), available through the National Center for Biotechnology Information.

Pulmonary and cardiac tissues were dewaxed, fixed in a mixture of 2.5% glutaraldehyde and 2.5 % paraformaldehyde in 0.1 M cacodylate buffer at 4 C for 24 h, post-fixed in 1% osmium tetroxide and dehydrated in a graded acetone series. Samples were infiltrated and embedded in Poly/Bed 812 resin (Polysciences, Warrington, Pennsylvania, USA). Thin sections (70 nm) obtained with a PTXL ultramicrotome (RMC, Boeckeler Instruments, Tucson, Arizona, USA) were placed on 200 mesh copper grids (w90 mm), stained with uranyl acetate and lead citrate and imaged with a JEOL 100CX transmission electron microscope (JEOL, Tokyo, Japan) at a 100 kV accelerating voltage. Both animals were in poor nutritional condition and the carcasses were mildly autolyzed. The only significant gross lesions were seen in the thorax of case 2. The visceral pleura overlying the lateral aspect of the right cranial lung lobe and the medial aspect of the right caudal lung lobe had two 1e3 mm diameter, white, friable foci. Additionally, there was a 4 mm, round, dark red, firm, slightly raised area on the left caudal lung lobe. In sections of lung (Figs. 1 and 2), heart (Fig. 3), brain, tongue and brown fat from case 1, and heart, skeletal muscle, adrenal gland, brain and lung from case 2, there were multiple protozoal organisms (2.0  2.0e5.0 mm) with a distinct basophilic nucleus and amphophilic cytoplasm. The protozoa were encysted in aggregates of 6e25 organisms within neurons, cardiac myocytes (Fig. 3), pneumocytes (Figs. 1 and 2), macrophages or endothelial cells, or were free as individual merozoites within the interstitium.

Fig. 1. Section of lung from case 1 showing mild to moderate expansion of the interstitium by lymphocytes, plasma cells and macrophages, and multiple intracellular parasites (black arrows). HE.

Sarcocystosis in Red Panda Cubs

Fig. 2. Higher magnification of Fig. 1 showing endopolygeny (black arrow), consisting of a peripheral ring of zoites with central clearing. HE.

Fig. 3. Section of heart from case 1 showing a capillary with endothelial cells that have a markedly expanded cytoplasm, containing a myriad of protozoal zoites. HE.

Intracellular organisms were observed mostly in haphazard aggregates, suggestive of schizogony, although rare instances of endopolyogeny were observed. Within the lung there were small foci of haemorrhage and coagulative necrosis. In addition to the apicomplexan protozoa, the affected portions of brain from both cases contained multifocal

187

rarefaction of the neuropil with gliosis and mild lymphoplasmacytic and histiocytic encephalomyelitis. Both animals also had multifocal, moderate, lymphoplasmacytic, histiocytic and neutrophilic interstitial pneumonia (Figs. 1 and 2), multifocal alveolar histiocytosis and multifocal pleuritis. Non-specific lesions included lymphocytolysis in the mesenteric and tracheobronchial lymph nodes. IHC for T. gondii and Neospora caninum were negative in both cubs. There was strong intracellular immunohistochemical labelling for Sarcocystis spp. in both cases. IHC also labelled individual extracellular organisms (merozoites) in the cardiac and pulmonary tissues of both cubs. The PCR assay for T. gondii was negative for both cases. The PCR assay for apicomplexan generated an amplicon of expected size (w500 base pairs [bp]). Nucleic acid sequence analysis showed the amplification products from both cubs were identical and were 100% similar with published sequences of multiple Sarcocystis spp. Amplification products for multiple segments of the 18S rDNA gene and the ITS-1 were sequenced and the sequences were assembled for analysis. A 1,679 bp sequence from the 18S rDNA showed 99% similarity with sequences of multiple Sarcocystis spp. including S. neurona, S. canis, S. felis and several avian Sarcocystis spp. A sequence from 547 bp of the ITS-1 region showed 99% similarity with corresponding sequences of S. neurona and S. dasypi. Numerous elliptical, unicellular, nucleated, apicomplexan protozoa were observed within numerous cardiac myocytes (Fig. 3), cardiac endomyseal fibrocytes and pneumocytes (Figs. 1 and 2). The protozoa were often present in aggregates of 2e11, within a parasitophorous vacuole characterized by a thin bilayered membrane that had a thicker, more electron dense outer lamina and a thinner less electron dense inner lamina (Fig. 4). A conoid was visible in some organisms (Fig. 4), with fragmented, electron dense, tapered, linear structures in close proximity to the conoid probably representing partially necrotic rhoptries. Some mitochondria were visible within few parasites (Fig. 4). Sarcocystis spp. are heterogeneous organisms with indirect life cycles that involve a carnivorous direct host and an omnivore or herbivore as an intermediate host (Lindsay et al., 1995; Bowman, 2009). Sarcocystis spp. undergo multiple development stages within different host cells and can be an incidental finding or cause pathological changes (Lindsay et al., 1995; Bowman, 2009). Systemic sarcocystosis is characterized by invasion of vascular endothelium, skeletal and cardiac myocytes, parenchymal cells and leucocytes by obligate intracellular apicomplexan

188

W.M. Zoll et al.

Fig. 4. Transmission electron micrograph of tissue from the heart showing protozoa within a parasitophorous vacuole. The conoid of one parasite is in the plane of section (circle), as are some mitochondria (m). The host cell nucleus is visible in the bottom right corner of the image (N). The protozoa bilaminar cell wall is clearly visible. Bar, 1,000 nm.

protozoa of the genus Sarcocystis (Lindsay et al., 1995; Bowman, 2009). Sarcocystosis has been described in numerous domestic and wildlife animals as an agent of neurological disease or abortions. The pathogenicity of Sarcocystis spp. depends on the host, species of the parasite, location of infection in the host and intensity of the infection (Lindsay et al., 1995). Clinical signs can be broad and non-specific, ranging from weakness and hair loss to encephalomyelitis and fetal or neonatal death (Lindsay et al., 1995). Sarcocystis spp. have been reported to infect endothelial cells, and this may contribute to disease progression and lesions by inciting local vasculitis and secondary tissue infarction (Lindsay et al., 1995). Sarcocystis neurona is known as the causative agent of equine protozoal myeloencephalitis (EPM), eosinophilic myositis in cattle and other ruminants, and as a potential cause of ruminant abortion due to vertical transmission from the dam to fetus (Dubey and Bergeron, 1982; Dubey et al., 2001; Duarte et al., 2004; Zachary, 2012). In the present cases, common protozoal causes of neonatal death (i.e. T. gondii and N. caninum), were ruled out by negative IHC for both agents and negative PCR for T. gondii. IHC for Sarcocystis in both cases was positive in multiple tissues. PCR and nucleic acid sequencing was strongly suggestive of a Sarcocyctis spp. that genetically closely resembled S. neurona and S. dasypi. Sarcocystis spp. cause abortion, early neonatal death and non-specific clinical signs in numerous animal species including ruminants, horses, wildlife and companion animals (Lindsay et al., 1995; Dubey and

Lindsay, 2006). Disease occurs because of aberrant systemic infection in dead-end hosts as appears to be the case in the two red panda cubs presented in this report (Dubey et al., 2001). Although histological identification of Sarcocystis spp. organisms in tissue samples may indicate a clinically irrelevant incidental finding, the findings of haemorrhage, necrosis and inflammation in the affected tissues in both cubs suggest infection rather than an incidental finding (Dubey and Lindsay, 2006). In these two red panda cubs, protozoa were identified with associated necrosis and inflammation in the heart, skeletal muscle, brain, lungs, adrenal glands, tongue and brown fat. Although the cubs had been in contact with the mother and her environment throughout their brief lives, we speculate that the route of protozoal transmission in these two panda cubs was either transplacental or transmammary. The vasochorial placentation of the red panda gives the potential for transuterine infection (Benirschke, 2011). Further support for the theory of vertical transmission is that in studies involving oral inoculation of neonatal animals with high doses of Sarcocystis spp., the interval from inoculation to the onset of clinical signs was 25 days in neonatal bison and 26 days in calves (Dubey et al., 1982; Dubey, 1982a). In another study, death or humane destruction due to severe clinical signs occurred 31e55 days after inoculation of 7e10-dayold calves (Dubey, 1982b). In comparison, clinical disease in these panda cubs became apparent 7e9 days post partum. While the immunological competence and inherent resistance to Sarcocystis spp. of neonatal domesticated ruminants and red pandas is likely different, the rapidly progressing fatal disease occurring in these cubs at least suggests transplacental infection. Sarcocystis infection should be recognized as a potential cause of early neonatal death in red panda cubs. Additionally, new management protocols should be put in place to ensure that the environment of the red panda is free from potential sources of protozoal organisms (e.g. opossum faeces) and other potential intermediate hosts that could have been ingested.

Acknowledgment Funds for preparation of samples for electron microscopical examination were from the department of Pathobiology and Diagnostic Investigation, Michigan State University.

Conflict of Interest Statement The author(s) declare no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Sarcocystosis in Red Panda Cubs

References Benirschke K (2011) Placentation of the red panda. In: Red Panda: Biology and Conservation of the First Panda, AR Glaston, Ed., Academic Press, San Diego, pp. 147e156. Bowman DD (2009) Sarcocystis. In: Georgis’ Parasitology for Veterinarians, 9th Edit., DD Bowman, Ed., Saunders Elsevier, St. Louis, pp. 104e105. Bretagne S, Costa JM, Vidaud M, Tran J, Nhieu V et al. (1993) Detection of Toxoplasma gondii by competitive DNA amplification of bronchoalveolar lavage samples. Journal of Infectious Diseases, 168, 1585e1588. Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. BioTechniques, 36, 214e216. Burg JL, Grover CM, Pouletty P, Boothroyd JC (1989) Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. Journal of Clinical Microbiology, 27, 1787e1792. Duarte PC, Conrad PA, Barr BC, Wilson WC, Ferraro GL et al. (2004) Risk of transplacental transmission of Sarcocysits neurona and Neospora hughesi in California horses. Journal of Parasitology, 90, 1345e1351. Dubey JP (1982a) Sarcocystosis in neonatal bison fed Sarcocystis cruzi sporocysts derived from cattle. Journal of the American Veterinary Medical Association, 181, 1272e1274. Dubey JP (1982b) Quantitative parasitemia in calves fed Sarcocystis cruzi sporocysts from coyotes. American Journal of Veterinary Research, 43, 1085e1086. Dubey JP, Bergeron JA (1982) Sarcocystis as a cause of placentitis and abortion in cattle. Veterinary Pathology, 19, 315e318. Dubey JP, Lindsay D (2006) Neosporosis, toxoplasmosis and sarcocystosis in ruminants. Veterinary Clinics of North America: Food Animal Practice, 22, 645e671. Dubey JP, Lindsay D, Saville W, Reed S, Granstrom D et al. (2001) A review of Sarcocystis neurona and equine

189

protozoal myeloenephalitis (EPM). Veterinary Parasitology, 95, 89e131. Dubey JP, Speer CA, Epling GP (1982) Sarcocystosis in newborn calves fed Sarcocystis cruzi sporocysts from coyotes. American Journal of Veterinary Research, 43, 2147e2164. Harrison TM, Moorman JB, Bolin SR, Grosjean NL, Lim A et al. (2007) Toxoplasma gondii in an African crested porcupine (Hystrix cristata). Journal of Veterinary Diagnostic Investigation, 19, 191e194. Hoffman AR, Cadieu J, Kiupel M, Lim A, Bolin SR et al. (2012) Cutaneous toxoplasmosis in two dogs. Journal of Veterinary Diagnostic Investigation, 24, 636e640. Lindsay D, Blagburn B, Braund K (1995) Sarcocystis spp. and sarcocystosis. European Journal of Translational Myology, 5, 249e254. Mullaney T, Murphy AJ, Kiupel M, Bell JA, Rossano MG et al. (2005) Evidence to support horses as natural intermediate hosts for Sarcocystis neurona. Veterinary Parasitology, 133, 27e36. Preece B (2011) Red panda pathology. In: Red Panda: Biology and Conservation of the First Panda, AR Glaston, Ed., Academic Press, San Diego, pp. 287e302. Soldati S, Kiupel M, Wise A, Maes R, Botteron C et al. (2004) Meningoencephalitis caused by Neospora caninum in a juvenile fallow deer (Dama dama). Journal of Veterinary Medicine Series A; Physiology, Pathology, Clinical Medicine, 51, 280e283. Zachary J (2012) Nervous system. In: Pathologic Basis of Veterinary Disease, 5th Edit., J Zachary, M McGavin, Eds., Mosby Elsevier, St. Louis, pp. 771e870.

April 8th, 2015 ½ Received, Accepted, April 28th, 2015 

Sarcocystis spp. Infection in two Red Panda Cubs (Ailurus fulgens).

Two neonatal male red panda (Ailurus fulgens) littermates were submitted for necropsy examination. One animal was found dead with no prior signs of il...
1MB Sizes 2 Downloads 8 Views