Infection with Toxoplasma gondii in a red kangaroo ( Macropus rufus ) and a Patagonian mara ( Dolichotis patagonum ) in captivity

Toxoplasmosis is an infectious, zoonotic and parasitic disease, caused by Toxoplasma gondii. In this manucript, two cases of infection with T. gondii in captive animals from a zoological park in the central region of Chile are described. One case was a red kangaroo (Macropus rufus), which is highly susceptible to the infection, and the other was a Patagonian mara (Dolichotis patagonum), a rodent in which there is no previous report of the infection. Both animals had myocarditis, with the presence of intralesional tachizoites and cysts suggestive of infection with T. gondii. This infection was confirmed by immunohistochemistry in both animals. The origin of the infection is unknown, but it is likely that free ranging domestic felines were associated with the dissemination of the parasites. This highlights the importance of controlling the domestic animal populations in zoological parks. To the best of our knowledge, this is the first time that T. gondii infection is described in a Patagonian mara, adding a new host for this infectious agent.


Introduction
Toxoplasmosis is a globally distributed parasitic disease caused by the protozoan Toxoplasma gondii.The definitive hosts of the parasite are members of the Felidae family.A wide variety of warm-blooded animals can act as intermediate hosts, including human beings (DUBEY & BEATTIE, 1988).This disease has been reported in numerous wild animals in zoological parks, and it is believed that the domestic cat plays a role in the dissemination of the parasite (HARRISON et al., 2007;DUBEY, 2010).Toxoplasmosis is mainly transmitted through the fecal-oral route, but it can also be transmitted by carnivorism and through the placenta (FREDES, 2010).Diagnosis can be achieved through serology and confirmed with molecular techniques, histopathology, immunohistochemistry and isolation (DUBEY, 2010) Despite the vast amount of literature on the impact of T. gondii in wild animals, to the best of the authors' knowledge there are no reports of this disease in animals from zoological parks in the scientific literature in Chile, nor of the infection or disease in Patagonian maras (Dolichotis patagonum).Two cases of disseminated infection with T. gondii in animals from a zoological park of the Metropolitan area are described.

Materials and Methods
The carcasses of a red kangaroo (Macropus rufus) and a Patagonian mara (Dolichotis patagonum) were submitted to the Department of Pathology, School of Veterinary Medicine, University of Chile for post mortem examination.A full necropsy was performed and in both animals, samples from the brain, trachea, lung, heart, liver, kidneys, spleen, stomach, small intestine, large intestine, adrenal gland and thyroid gland were collected.These samples were fixed in 10% buffered formalin, routinely processed for histopathology and stained with hematoxylin and eosin (H&E).Liver sections of the mara were also stained with Brown and Brenn Gram stain (CHURUKIAN & SCHENK, 1982).
Immunohistochemistry for T. gondii detection, using samples of heart of both animals, was performed at the California Animal Health and Food Safety Laboratory, San Bernardino branch.Briefly, after antigen retrieval with pepsin (Sigma P7000; 0.4% HCl), rabbit anti-T.gondii polyclonal antibody (UCDavisRbt#58; 1:1500) was used, followed by anti-rabbit polymer (Dako K4003, Carpinteria, CA).The reaction was visualized with Novared for peroxidase following the manufacturer's instructions (VectorLab SK4800, Burlingame, CA).Slides were counterstained with Mayer hematoxylin, air dried and cover slipped.As a positive control, heart from a cat with a confirmed infection with T. gondii was used.

Case 1: Red kangaroo (Macropus rufus)
An adult female red kangaroo was found dead without premonitory signs.At necropsy, the carcass displayed good body condition, bilateral serosanguinous nasal secretion, pulmonary edema, rib imprints in the visceral pleura and small amounts of a red tinged fluid in the thoracic cavity, pericardium and abdomen.Microscopically, discrete infiltrates of lymphocytes, plasma cells and macrophages were identified in the myocardium, associated with segmental degeneration and necrosis of myofibers and the presence of small numbers of tissue cysts in the sarcoplasm.These cysts contained round to oval, basophilic bradizoites, which measured 2-3 µm in diameter (Figure 1a).In the lung, there was interstitial pneumonia, with abundant fibrin in alveolar spaces, thickening of alveolar septa with mononuclear cells, type II pneumonocyte hyperplasia and occasional parasitic structures.In the brain, there were multiple perivascular infiltrates of mononuclear cells, with occasional areas of gliosis, presence of neutrophils and macrophages and intralesional tissue cysts.Toxoplasma gondii in a red kangaroo and a mara Case 2: Patagonian mara (Dolichotis patagonum) An adult male mara with a history of anorexia, progressive weight loss and lethargy was presented for post mortem examination.The necropsy revealed poor body condition.The lungs were edematous and displayed ecchymotic hemorrhages in all lobes, and serous atrophy of fat was detected in the heart.In the abdominal cavity, there was abundant serosanguinous fluid and fibrin, and there were numerous abscesses in the liver and small intestine.Microscopically, there was multifocal degeneration and necrosis of the myocardium, with intrasarcoplasmic tissue cysts, associated with mild lympho-histiocytic infiltrates (Figure 1b).The abscess of the liver and intestine had numerous bacterial colonies composed of Gram negative bacilli were identified with Brown and Brenn stain.
In both cases, T. gondii was identified in the heart with immunohistochemistry (Figure 1c, d).

Discussion
To the best of the author's knowledge, this is the first descriptive study of the infection with T. gondii in captive wild animals in Chile, and new host record for T. gondii in a Patagonian mara.Previously, Gorman (1993) reported seropositivity in different wild animals in a zoological park, with a prevalence of 27.5%.
The origin of T. gondii in zoological parks in unknown, although the presence of domestic cats prowling in the parks has been linked to contamination of water and food with oocysts (HARRISON et al., 2007;PATRONEK, 1998).On the other hand, it is less likely that oocysts were excreted from other animals in the collection due to no connection of the habitat of different species.
Australian marsupials are highly susceptible to infection with T. gondii, which frequently occurs with death without premonitory signs (DUBEY & CRUTCHLEY, 2008).Clinical signs have been described, characterized by diarrhea, lethargy, poor appetite, respiratory distress and neurological signs (CANFIELD et al., 1990).Gross lesions associated with this infection are usually minimal or absent, and consist mostly of congestion, edema and hemorrhages in the heart, lung and gastrointestinal tract.(BASSO et al., 2007;CANFIELD et al., 1990;OCHOA-AMAYA et al., 2012;PORTAS, 2010).Histopathology is a good diagnostic tool to diagnose the infection with T. gondii, because free or encysted bradizoites and tachizoites can be identified in the spleen, heart, lung, brain and liver, and are frequently associated with lympohoplasmacytic to granulomatous inflammation (STERN, 2010;OCHOA-AMAYA et al., 2012).In the brain, free or encysted parasites has been described, together with multifocal necrosis, mineralization and glial nodules.In the lung, it is common to find interstitial pneumonia associated with aggregates of macrophages in alveolar spaces and a fibrinonecrotic exudate (BOORMAN et al., 1977;CANFIELD et al., 1990;BASSO et al., 2007;BERMÚDEZ et al., 2009;PORTAS, 2010;OCHOA-AMAYA et al., 2012).
There are descriptions of sudden death and neurological signs in rodents with Toxoplasmosis.Grossly, it is common to find splenomegaly together with white mottled areas in the myocardium and liver (FORZÁN & FRASCA, 2004;JOKELAINEN & NYLUND, 2012).Histologic lesions in rodents consist of lymphoplasmacytic to granulomatous myocarditis and hepatitis, and the presence of parasitic cysts (PERCY & BARTHOLD, 2007).In rodents, the ingestion of water or vegetables contaminated with oocysts from members of the Felidae family has been suggested as the source of infection (PATRONEK, 1998;FORZÁN & FRASCA, 2004;PERCY & BARTHOLD, 2007;JOKELAINEN & NYLUND, 2012).
Although there are no reports of infection with T. gondii in Patagonian maras, findings in the heart are consistent with lesions observed in other hosts.Moreover, abscesses with intralesional Gram negative bacteria were identified in the liver and intestine.The cause of death of this animal is not fully clear and the infection with T. gondii may be an incidental finding.It is speculated that the presence of a bacterial infection with multiple abscesses is the most probable cause of death.

Conclusion
The inclusion of T. gondii as a differential diagnosis in captive wild animals in Chile is recommended.In addition, programs of domestic feline prevention and control should be implemented in zoological parks, in order to limit the impact of these animals on the health of animals kept in captivity.We highlight the presence of T. gondii in the heart of the Patagonian mara, adding a new host to the parasite and a new disease for the species.