Abstract
A 6-year-old, male southern chamois (Rupicapra pyrenaica) had an absence of flight response and was captured by hand in the Catalan Pyrenees in northeast Spain. On clinical examination, the animal was in good body condition, and only atrophy of the right eye was observed. Blood samples were collected and hematologic analysis performed, but no alterations were observed. The animal was sent to a Wildlife Rescue Centre, where it developed chronic wasting and died after 32 days in captivity. At necropsy, the animal was cachectic and had edematous, mottled lungs. Histopathologic examination revealed systemic toxoplasmosis and acute Gram-negative septicemia. The protozoan organisms were identified as Toxoplasma gondii based on immunohistochemistry. An indirect fluorescent antibody test was performed, and the animal was positive with an antibody titer of 1:50.
Keywords
Toxoplasma gondii (family Sarcocystidae, genus Toxo-plasma) is a protozoan parasite of worldwide distribution that can infect many species of warm-blooded animals, including humans. It has an indirect life cycle; wild and domestic felids are the definitive hosts, and a wide range of animals serve as intermediate hosts. Hosts can become infected by ingestion of food and water contaminated with sporulated T. gondii oocysts, by consumption of tissue cysts in infected animal tissues, or by congenital transmission of protozoal infection. 5
Toxoplasmosis is a major cause of abortion and stillbirth in domestic sheep and goats but also has been described in many exotic ungulates in zoos. Among free-ranging ungulates, toxoplasmosis has been reported in many host species, mainly feral pigs and cervids. Thus, wild game is a potential source of T. gondii infection for humans worldwide. 8 In North America, white-tailed deer (Odocoi-leus virginianus) and mule deer (Odocoileus hemionus) constitute reservoirs of T. gondii among wildlife. 3,12 In Scotland, viable T. gondii has been isolated from red deer (Cervus elaphus), the main game deer species in the region. 24 Studies of antibody production to T. gondii in wild ungulates in Spain, including chamois, have recently been reported. 10,11 A significantly higher prevalence of infection (44.2%) was observed in red deer from Catalonia (northeast Spain) compared with other areas. 11 In the same study, positive antibody titers were detected in 2 of 10 southern chamois (Rupicapra pyrenaica), but these specimens were from chamois in the Cantabrian Mountains (northern Spain). The study suggested that a high exposure to oocysts in the Catalonian environment was probably the main factor that contributed to seropositivity. In fact, cats, especially feral cats from urban Barcelona (Catalonia), have shown high prevalence of T. gondii antibodies (45%) and may excrete oocysts and contaminate the environ-ment. 9 Furthermore, the wild cat (Felis silvestris) population has been increasing in recent years and is widely distributed throughout the Pyrenees (http://mediambient.gencat.net/cat/el_medi/fauna/fauna_auctoctona/especies_protegides/gat_fer.jsp [In Spanish]). All together, the results seem to identify Catalonia as one of the most important areas of T. gondii infection in Spain. In the current study, a case of systemic toxoplasmosis and Gram-negative septice-mia in a free-ranging southern chamois, manifested by neurologic signs and lesions, is described.
In December 2002, a 6-year-old, male southern chamois was found near a village in the Vallfosca Valley, near the Aiguestortes i Estany de Sant Maurici National Park, in the central Pyrenees (Catalonia, northeastern Spain; 42°27′N, 2°12′E). The chamois was easily captured by hand because it had an absence of flight response and lacked apparent fear of humans. The animal had a normal body condition. On clinical examination, no alterations were found, except atrophy of the right eye, presumably a result of trauma or infectious keratoconjunctivitis, which is common in this species. The left eye was unremarkable and had normal vision based on the presence of a menace response and the ability of the chamois to avoid obstacles. The animal was sent to the Wildlife Rescue Centre of Vallcalent (Lleida, NE Spain), kept in an enclosure, and provided with water and alfalfa hay ad libitum.
At the time of the current case, an outbreak of a pestivirus-associated disease was decimating the chamois population in the area. Because this animal had similar neurologic signs, 17 serologic and virologic analyses for pestivirus were performed. A blocking enzyme-linked immunosorbent assay (ELISA) a and a reverse transcription polymerase chain reaction (RT-PCR), using previously described pan-pestivirus primers (Pesti 3 and Pesti D), 13,15 were performed. Both analyses yielded negative results.
The health and body condition of the chamois deteriorated progressively, and the animal died after 32 days in captivity. At necropsy, the animal weighed 15 kg (reference range: 25–40 kg) and lacked subcutaneous and abdominal fat. The lungs were edematous and mottled. No other macroscopic lesions were observed, except for the atrophy of the right eye. Tissue samples from major organs were collected, fixed in 10% neutral buffered formalin, and processed routinely for histopathologic examination. Paraffin-embedded tissues were sectioned at 4–5 μm and stained with hematoxylin and eosin. Retrospectively, selected paraffin-embedded sections were stained immunohistochemically with T. gondii and Neospora caninum rabbit polyclonal antibodies using methods described previously. 16 Both positive and negative control sections were included for each antibody. For Toxoplasma immunohisto-chemistry, positive controls were from the liver of a cat with feline immunodeficiency and disseminated toxoplasmosis. For Neospora immunohistochemistry, positive controls were from the spinal cord of a young dog with neosporosis. On the negative controls (all tissues examined), the primary antibody was omitted. Selected sections of brain, liver, lung, and heart were also stained with periodic acid–Schiff (PAS) and modified Gram staining technique. Serum obtained from the animal on arrival at the Wildlife Rescue Centre was assayed for antibodies to T. gondii by modified agglutination test (MAT), as described previously, 6 and tested at dilutions of 1:25, 1:50, and 1:500. Positive and negative controls were included for each test.
On histologic examination, intracellular groups of protozoa or cyst-like structures up to 35 μm in diameter were observed in the brain, lung, and liver (Fig. 1A–C). In the brain, there was a mild, multifocal, nonsuppurative inflammation affecting the cerebrum, brain stem, and cerebellum; multifocal glial nodules, spongiosis and necrosis with intralesional and extralesional protozoan cysts, and edema (evidenced by perivascular spongiosis and the presence of clear, eosinophilic, amorphous material phagocytized by glial cells within Virchow-Robin spaces) were observed. Protozoan cysts, glial nodules, and foci of necrosis were more numerous in the cerebrum and easily observed. A concomitant, acute, Gram-negative bacterial septicemia was also diagnosed because multifocal purulonecrotic foci with microcolonies of bacteria were seen in the lungs, kidneys, lymph nodes, liver, and spleen. The lungs also had extensive foci of acute edema and multifocal alveolar hemorrhages. Thromboses with intravascular bacterial microcolonies were present in lungs, liver, and kidneys. The source of the bacteria was not determined. Although samples from the lung, kidney, spleen, and liver were cultured by routine microbiologic methods, no bacteria were isolated. These negative results were most likely due to the long freezing storage before the samples were analyzed. Autolysis was too pronounced for proper evaluation of the intestines. Groups of protozoa or cysts reacted positively with anti–T. gondii antibodies (Fig. 1E) but not with anti-N. caninum antibodies. Protozoa within the intracellular cyst-like structures had many PAS-positive granules (Fig. 1D). The MAT antibody titer to T. gondii in the serum of this animal at 1 month before death was 1:50.
Toxoplasma gondii usually parasitizes the host without producing clinical signs and rarely causes severe clinical manifestations in nonpregnant animals. Fatal toxoplasmosis in wild ungulates described in the veterinary literature generally involves captive animals from zoos. 8,20 A free-ranging, 4-month-old Rocky Mountain bighorn sheep (Ovis canadensis) was reported with toxoplasmic encephalitis but was found dead, and pneumonic pasteurellosis was the final cause of death. 1 In other free-ranging species, such as Arctic foxes and mink, fatal cases of toxoplasmosis have been described recently. 14,21
In severe cases of toxoplasmosis, infected hosts usually die because of necrosis of the intestines and mesenteric lymph nodes before other organs are severely damaged. However, clinical signs are determined by the extent and severity of organ injury. 8 The chamois reported in the current study was a free-ranging animal with a severe neurologic disorder and progressive deterioration of the animal's condition during observation. It displayed abnormal behavior, manifested by the absence of a flight response, fearlessness of humans, and relative tameness (it was easily captured). It is possible that this behavior could be due to T. gondii infection. At the time of necropsy, the only lesions present in the brain were those derived from this parasite. Toxoplasma gondii infection may be responsible for changes in the host's behavior. 22 For example, T. gondii–infected rats are more likely to be caught by traps in the wild. Protozoal infection also reduces the rat's perception of cat predation risk, thus turning their innate aversion to this predator into an imprudent attraction. 2,23
When the chamois in the present study died, protozoa within most of the intracellular cysts were identified as bradyzoites. They contained many PAS-positive granules (amylopectin granules). In tachyzoites, this material is either in discrete particles or is absent. 7 Although cysts with bradyzoites may form as early as 3 days after infection, 7 the size of the cysts present in this chamois suggests that this was an older infection. Necrotic lesions in the lung and liver, despite the presence of protozoan cysts, were mainly attributed to the bacterial septicemia, which most likely was the proximate cause of death.
The MAT antibody titer to T. gondii in the serum of this animal was 1:50. Animals usually become seropositive for T. gondii by MAT approximately 3 weeks after ingesting infective oocysts. 5 This animal could have acquired the infection by ingestion of food or water contaminated with oocysts from cat feces. Because seroprevalence shows that cats and wildlife are frequently exposed to this parasite, 9,11 toxoplasmosis may be common, but unnoticed, in wild ungulates because diseased animals are difficult to visually detect in the wild.
Lesions and Toxoplasma gondii in different tissues of the chamois.
The possibility of zoonotic disease transmission from wild animals to humans has important public health implications. The population of chamois in Spain has grown over recent decades, primarily because of increased hunting management and human field abandonment. Chamois is one of the main wild ungulate species that is hunted in the Pyrenees and other mountain areas of Europe and represents a potential source of zoonosis. Cases of acute and ocular toxoplasmosis in deer hunters in the United States have been reported, and it has been pointed out thatevisceration, handling, and ingestion of inadequately cooked meat from infected wild ungulates represent risks for human infection. 4,18,19 Thus, chamois used as a source of food could represent a public health risk for persons that handle or consume undercooked meat.
Acknowledgements. The authors are grateful to the rangers of Pallars Jussa and staff of the Wildlife Rescue Centre of Vallcalent for the capture and care of the chamois. The authors also thank Sonia Almeria for helpful review of this manuscript, and to J. P. Dubey for providing the N. caninum-specific antiserum and T. gondii MAT. This research was supported by the Direccio del Medi Natural del Department de Medi Ambient de la Generalitat de Catalunya.
Footnotes
a.
Synbiotics, Lyon, France.