Naturally Acquired Visceral Leishmaniosis in a Captive Bennett’s Wallaby ( Macropus rufogriseus rufogriseus )

Leishmaniosis is a zoonotic parasitic disease caused by an intracellular protozoan of the genus Leishmania (family Trypanosomatidae). Members of the genus are parasites of humans, dogs, and other mammals. This intracellular parasite of the mononuclear-phagocytic system is transmitted by blood-sucking sandflies (Phlebotomus spp). The parasite reproduces itself within the host’s macrophages, causing an intense inflammatory reaction consisting of mononuclear immune cells.^6,7 Leishmania infantum is a zoonotic agent that is endemic in Mediterranean countries. L. infantum is the species usually implicated in both animal and human visceral leishmaniosis, a severe systemic form of disease characterized by progressive wasting due to involvement of multiple organs, such as spleen, liver, lymph nodes, bone marrow, kidney, and skin.^6,8,9 The clinical presentation of visceral leishmaniosis varies widely, depending on both the organ(s) affected and the extent of functional impairment caused by the infection. Diagnosis of leishmaniosis may be challenging, particularly if the disease appears subclinical. ²

Reports of leishmaniosis in Australian macropods describe only skin lesions.^3,7 This short communication describes macroscopic, histopathologic, and immunohistochemical features associated with visceral infection by Leishmania in a Bennett’s wallaby (Macropus rufogriseus rufogriseus) that died suddenly in a zoo in Madrid, Spain.

Pathologic Findings

A captive 2-year-old female Bennett’s wallaby was found dead in its cage. No previous clinical signs were observed. The animal had been housed with others of its species, none of which showed clinical signs suggestive of disease. At necropsy, external examination yielded normal results. The spleen was dark brown to black, 2 times or more normal size, with numerous granulomas, occasionally coalesced, which measured 0.5 to 2 cm in diameter. The liver was uniformly enlarged and dark brown containing white spots and small granulomas (< 0.5 cm in diameter). Pulmonary edema and generalized hyperemia were also observed.

Samples of liver and spleen from the wallaby were submitted in 10% neutral buffered formalin for histopathologic analysis. The referred material was routinely processed and embedded in paraffin wax. Sections 3 μm thick were stained with hematoxylin and eosin (HE), with Giemsa, and by the periodic acid–Schiff method for examination under light microscopy. Histologically, the spleen contained multifocal, variable-sized granulomas with abundant necrosis, cellular debris, and mineralization. Marked lymphoid hypoplasia affecting mainly the periarteriolar lymphatic sheaths was observed. Macrophages located in the splenic marginal zone, within the lymphoid follicles, and surrounding the arterioles contained a high number of cytoplasmic round to oval, 1- to 2-μm-wide, and 2- to 4-μm-long organisms, which had a round basophilic nucleus and distinct bar-shaped paranuclear kinetoplast (Fig. 1). These organisms appeared to reside within cytoplasmic parasitophorous vacuoles. Organisms were accentuated with Giemsa staining but not with periodic acid–Schiff staining. Morphologic features were compatible with Leishmania amastigotes. Occasionally, the lymphocytes were virtually missing, being replaced by plasma cells and parasite-containing macrophages. The red pulp was enlarged with sinus areas diffusely occupied by large macrophages heavily laden with intracytoplasmic organisms, numerous plasma cells, and megakaryocytes. Proliferation of the sinus endothelial cells was also seen. In the liver, a heavy infiltrate composed mainly of macrophages was evident in portal areas and in small foci between hepatocytes (Fig. 2). Macrophages contained numerous amastigotes. Variable numbers of lymphocytes and plasma cells were also observed in the hepatic infiltrates. Megakaryocytes were occasionally observed in the hepatic parenchyma. Capillaries and medium-sized blood vessels in the spleen and liver contained homogeneous acidophilic microthrombi, consistent with disseminated intravascular coagulation.

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Figure 1. Spleen; Bennet’s wallaby. High-power magnification showing granulomatous inflammation formed by large foamy macrophages containing numerous Leishmania amastigotes. HE. Inset: Intense immunoreaction to L. infantum antibody directed toward amastigotes in macrophages. ABC method, Mayer’s hematoxylin counterstain. Figure 2. Liver; Bennet’s wallaby. Section of liver showing intense inflammation formed by macrophages, lymphocytes, and plasma cells. HE.

The etiologic diagnosis and identification of parasitized macrophages was greatly facilitated by the immunohistochemical detection of Leishmania antigens. The avidin–biotin–peroxidase complex method (Vector Laboratories, Burlingame, CA) was used. Sections 3 μm thick were labeled with a rabbit polyclonal antibody specific for L. infantum (Departamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Murcia, Spain ⁴ ). Labeling was detected by incubation with the liquid DAB+Substrate Chromogen System (Dako, Carpinteria, CA). The slides were counterstained in Mayer’s hematoxylin. A sample of skin from a dog with leishmaniosis was used as a positive control. Intracytoplasmic organisms showed a strong positive labeling for the L. infantum antibody in both the spleen (Fig. 1) and the liver.