Toxoplasma gondii infection in two captive Patagonian maras

Abstract

Toxoplasma gondii infection was diagnosed in 2 captive Patagonian maras (Dolichotis patagonum). One animal developed fatal systemic toxoplasmosis and had concurrent localized bacterial and fungal infections; its daughter remained clinically healthy. Microscopic findings included acute, coagulative necrosis, lymphohistiocytic inflammatory infiltrates, and extra- and intracellular parasites in the liver, myocardium, urinary bladder, and adrenal glands of the diseased animal. PCR and subsequent genotyping of parasites from fresh tissue from both cases revealed infection with T. gondii genotype II. Direct agglutination testing of blood from the healthy individual revealed high levels of T. gondii IgG antibodies. T. gondii is a potential cause of disease and lethality in captive and wild Patagonian maras, and toxoplasmosis should be considered when managing and providing veterinary care for this species.

Toxoplasma gondii is a ubiquitous, heteroxenous, apicomplexan, protozoan parasite with felids as its definitive host.⁵ A large range of mammals and birds can serve as intermediate hosts, and the wide distribution of T. gondii oocysts in the environment has led to infection and disease in a wide variety of animals.⁵ Although T. gondii is common all over the world, molecular studies have identified various prevalence and distribution patterns among specific genotypes within this species. In Europe and North America, types I and III are the dominant clonal lineages. Of these, type II is responsible for most infections in humans and livestock.¹⁶ In South America, there are 15 T. gondii lineages, forming 6 major genetic groups.¹⁷ Experimental and surveillance data have shown that different T. gondii lineages and strains have differing pathogenic potential.^1,2,11

The Patagonian mara (syn. Patagonian cavy; Dolichotis patagonum) is a large species of herbivorous rodent of the family Caviidae originating in Argentina and is closely related to capybaras and guinea pigs. The species is classed as “near threatened” by the International Union for the Conservation of Nature.¹⁴ Although they are relatively common in zoologic collections, little is known about diseases affecting the Patagonian mara.

We describe herein a case of fatal systemic toxoplasmosis and localized fungal infection in a Patagonian mara, as well as subclinical T. gondii infection in that animal’s offspring. PCR and subsequent genotyping of parasites from fresh tissue from both cases revealed infection with genotype II, suggestive of infection originally occurring in Europe.

Both animals were housed at Tangen Zoo, Norway, and were the only Patagonian maras in the collection. They were housed in an indoor winter enclosure (~25 m²) at the time of presentation. From spring to autumn, they were housed in a grassy outdoor enclosure (~200 m²). Their diet was a mixture of fresh grass, hay, guinea pig pellets, as well as fresh vegetables.

Case 1, a 5-y-old intact female was presented to the Faculty of Veterinary Medicine, Norwegian University of Life Sciences, weak, recumbent, and severely depressed. Depression developed and gradually worsened over a span of 48 h; the animal became progressively ataxic over the last 24 h. At presentation, the animal was hypothermic (body temperature < 32°C), nearly emaciated (marked loss of body fat and muscle), hypotensive, and showed little response to external stimuli. A bony swelling expanded the right rostral maxilla, and halitosis was present. Despite emergency treatment, the animal died 2 h after presentation.

Case 2 was a 2-y-old intact female mara that was the sole cage mate and daughter of case 1. Euthanasia was performed 4 mo after the death of case 1 because a life in solitude was considered incompatible with good animal welfare, and the zoo had no plans of obtaining more maras. At the time of euthanasia, this mara was considered clinically healthy. Additionally, it had not displayed any signs of disease before, during, or after clinical signs were observed in case 1.

Both animals were autopsied within 24 h of death. Gross lesions in case 1 included splenomegaly, gray-white mottling of the hepatic cut surface, and petechial hemorrhages in the brain. Additionally, a bony swelling surrounded the right maxillary incisor, the urinary bladder contained white fibrin strands, and the bladder mucosa was hyperemic. Abundant thick, yellow pus filled the vagina. No gross abnormalities were found in case 2.

Brain, kidney, myocardium, liver, lung, adrenal gland, and spleen from both animals were formalin-fixed for histologic examination. In addition, urinary bladder, vagina, and the right rostral maxilla were collected from case 1. Bone tissue was demineralized in 10% EDTA. All samples were processed routinely, and sections stained with hematoxylin and eosin for histologic examination. Additional staining included Fontana Masson staining of the nasal mucosa and Grocott–Gomori methenamine silver staining of the lung tissue from case 1.

Case 1 had randomly distributed foci of acute, coagulative necrosis and mild-to-moderate, periportal, lymphohistiocytic inflammatory infiltrates in the liver (Fig. 1). Similar necrotizing lesions and mild-to-moderate lymphohistiocytic inflammation were found in the myocardium, spleen, adrenal gland, and urinary bladder smooth muscle. Moderate-to-marked histiocytic and lymphoplasmacytic interstitial pneumonia with alveolar histiocytosis, fibrin exudation, and multinucleate cells was present. Absent or mild inflammatory infiltrates, including rare neutrophils and extracellular tachyzoites of 2–6 µm diameter, were found in necrotic foci in the myocardium and liver. Intracytoplasmic parasitophorous vacuoles containing tachyzoites were found in cardiomyocytes, hepatocytes, Kupffer cells, and in macrophages in the urinary bladder. Glial nodules and mild hemorrhage were found throughout the gray and white matter of the cerebrum, cerebellum, and medulla oblongata. Rare intracytoplasmic parasites were found in cerebral endothelium and neurons.

Figures 1, 2.

Toxoplasmosis in a Patagonian mara (case 1). Figure 1. Multifocal, random, acute necrosis (*), and periportal hepatitis. H&E. Bar = 200 μm. Figure 2. Degenerate leukocytes and necrotic debris surround pigmented fungal hyphae (arrows) in the nasal mucosa. H&E. Bar = 50 μm.

Additional lesions in case 1 included focally extensive, necrotizing rhinitis with intralesional gram-positive bacteria, and filamentous, pigmented fungal hyphae (Fig. 2), purulent vaginitis, and degeneration and loss of the right incisor tooth root. Fungal hyphae were found in necrotic debris and exudate lining the ulcerated nasal mucosa, but not in the underlying tissue. In case 2, mild lymphohistiocytic infiltrates were found in the myocardium and liver. The remaining examined tissues were considered normal.

Immunohistochemistry was performed with a polyclonal T. gondii antiserum (VRMD, Pullman, WA; Supplementary Data 1). Abundant T. gondii antigen–positive intracellular and extracellular parasites were found in the liver, myocardium, and lungs of case 1 (Fig. 3); no tissue cysts or tachyzoites were observed in liver, myocardium, or brain from case 2. Bacterial culture of the spleen and liver of case 1 was negative, and mixed, nonspecific bacterial flora suggestive of sample contamination was cultured from the brain. Abundant mixed bacterial flora including Streptococcus sp. was cultured from a vaginal swab. For detection of antibodies to T. gondii, a commercial direct agglutination test (Toxo-Screen; bioMérieux, Lyon, France) was used according to the manufacturer’s instructions. Blood collected from the heart of case 2 was analyzed in duplicate at the dilutions of 1:40 and 1:4,000 and revealed high levels of toxoplasma IgG antibodies with a titer of 1:54,000 equal to 5,400 IU/mL.

Figure 3.

Toxoplasmosis in a Patagonian mara (case 1). Toxoplasma gondii antigen–positive extracellular and intracellular tachyzoites in a focus of hepatic necrosis. IHC. Bar = 50 μm.

For detection of T. gondii genome fragments, fresh frozen liver, spleen, and brain from both cases were examined using PCR. DNA was isolated from 200 mg of tissue samples (QIAcube; QIAamp DNA mini tissue kit; Qiagen, Sollentuna, Sweden) and eluted to 50 µL using PCR-grade water in 1.5-mL microcentrifuge tubes. Tubes containing isolated DNA were stored at −20°C before further processing. PCR and subsequent sequencing of the SAG-1, SAG-2, GRA6, and CS3 genes were used to confirm the presence of T. gondii and to determine the strain or genotype^6,10,11,18 (Supplementary Data 2, Supplementary Table 1). PCR was successful from all primers, with DNA isolated from at least one tissue sample (liver, spleen, or brain) from both animals. For case 1, all PCR products were sequenced successfully; only SAG2 and GRA6 were successful for case 2. All products were identical (100% homology) to the genotype II ME49 strain (Table 1).

Table 1.

PCR target genes and accessions for top hits for Toxoplasma gondii using BLAST.

PCR target gene	ME49 accession	Identity	Genotype
CS3*	XM_002367739.2	100%	II
SAG1*	XM_002368164.2	100%	II
SAG2	XM_018781602.1	100%	II
GRA6	XM_002371898.2	100%	II

^*Sequencing of CS3 and SAG1 PCR products were only successful for Patagonian mara case 1.

Although T. gondii has been reported in the Patagonian mara previously,³ our cases underscore that toxoplasmosis can be a cause of disease in this species. Understanding the health and disease of the Patagonian mara is important for their captive management as well as the conservation of wild populations. In addition, given that the genotypes of T. gondii and their pathogenicity differ in Europe and South America, and high levels of congenital transmission of T. gondii have been documented in other species,⁴ we considered it important to clarify if our cases were infected with South American or local European T. gondii lineages. This is particularly relevant for biosecurity and health-and-safety protocols in zoos, which routinely transport animals, and possibly pathogens, between continents.

The source of T. gondii infection in our cases is unknown, but the finding of T. gondii genotype II suggests that initial infection occurred in Europe and not via intrauterine infection from ancestors in South America. Given that identical isolates of T. gondii genotype II were found in both cases, and high levels of T. gondii antibodies were found in case 2, a common point-of-exposure occurring relatively recently were considered most likely.

The origin of case 1 was Tromsø Mini Zoo, a small zoo in northern Norway, and it was reported to be captive bred there, not imported directly from South America. However, as the origin of the ancestry of case 1 was uncertain, it is possible that case 1 (dam of case 2) was born of a wild-caught mara. In utero infection of case 1 and subsequently case 2 might have occurred. However, although T. gondii genotype II is found in South America, it is not the dominant genotype isolated from that continent.^15,17 Domestic cats entering zoologic parks have been associated with contamination of feed and water with T. gondii oocysts.⁷ Further, even with strict biosecurity measures to prevent cats entering the zoo, feedstuffs may be contaminated earlier along the supply chain.¹²

T. gondii genotype II is endemic in Norway and northern Europe,¹³ and is generally considered to have lower pathogenicity than South American genotypes, which can be more virulent in humans and mice.^1,2,11 However, infection with genotype II has caused fatal toxoplasmosis in adult European brown hares (Lepus europaeus), mountain hares (Lepus timidus), and Eurasian red squirrels (Sciurus vulgaris), indicating that several species of adult lagomorphs and rodents are susceptible to disease caused by this genotype.^8,9

It remains unknown how susceptible Patagonian maras are to toxoplasmosis; however, systemic fatal infections in healthy adult animals generally occur in species that evolved in the absence of cats (e.g., Australian marsupials, Madagascan lemurs, and neotropical monkeys).⁴ Several studies have documented a high seroprevalence of T. gondii antibodies, but not toxoplasmosis, in wild and captive capybaras.¹⁹ Given that capybaras are closely related to Patagonian maras, and descriptions of toxoplasmosis in maras also are limited, it is possible that maras are not particularly susceptible to toxoplasmosis. This is consistent with only one of our maras displaying clinical signs of disease. The poor body condition and the concurrent localized infections in case 1 support an underlying chronic disease process potentially leading to immunosuppression and increased susceptibility to infection or reactivation of chronic infection, although this could not be confirmed by autopsy or clinical examination. Additionally, in a captive mara with toxoplasmosis described previously, lesions ascribed to T. gondii infection were limited to the myocardium, and a bacterial infection, characterized by fibrinous peritonitis with intestinal and hepatic abscesses, was believed to have been the most likely cause of death.³

Unfortunately, samples were not collected from the nasal tissue for fungal or bacterial culture, making exact identification of the fungi and bacteria present in the nasal cavity impossible. The presence of melanin in the fungal cell wall is consistent with phaeohyphomycosis (infection with dematiaceous pigmented fungi). These fungi are found ubiquitously in the environment, with infections most frequently acquired by inhalation or trauma. These fungi may cause disease in immunocompetent individuals. Inhalation of fungal spores from soil or plant or other organic debris is likely in our case, given their herbivorous nature.