History

A 5-year-old 16.3-kg intact male mixed-breed dog was referred to the São Paulo State University Veterinary Hospital for a 3-month history of apathy, anorexia, lethargy, and weakness as well as a 3-day duration of severe acute respiratory distress. The owner was unable to provide information regarding previous diseases or vaccination history. The patient was from a rural area and had direct contact with 2 more dogs in the house, also chickens, and cattle that were not clinically affected.

Clinical and Gross Findings

During physical examination, the haired skin surrounding both eyes and eyelids were covered by light yellow to brown dry exudate and an infestation of brown ticks (Rhipicephalus sanguineus) in the hair coat. Marked peripheral lymphadenopathy and severe splenomegaly were noted. The CBC and biochemistry evaluation revealed severe normochromic normocytic anemia, thrombocytopenia, lymphopenia, hypoalbuminemia, hyperglobulinemia, and azotemia (Table 1). The abdominal ultrasound examination showed irregular contours, severely enlarged dimensions, and heterogeneous echotexture of the splenic parenchyma. The dog died from a cardiorespiratory arrest during hospital care. Gross postmortem examination revealed multiple enlarged lymph nodes (popliteal, axillary, prescapular, and mesenteric) ranging from 2.0 X 1.5 X 0.9 cm to 2.4 X 1.8 X 1.2 cm. The spleen was severely enlarged with diffuse irregular capsular thickening (Figure 1). On cut surface, the splenic parenchyma had meaty consistency with prominent white pulp. No other significant postmortem findings were noted.

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Photograph of the spleen of a 5-year-old dog with apathy, anorexia, and weakness for approximately a 3-month period. The spleen was severely enlarged with meaty consistency on cut surface. The splenic capsule was diffusely thickened and roughed (bar = 2 cm).

Citation: Journal of the American Veterinary Medical Association 262, 1; 10.2460/javma.23.09.0498

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Cytologic and Histopathologic Findings

Cytologic examination of postmortem touch impressions of the spleen and popliteal lymph node revealed numerous intra- and extra-cytoplasmic amastigotes with an eccentric, round to ovoid, basophilic nucleus, a < 1 µm, linear, basophilic kinetoplast (consistent with Leishmania spp), marked histiocytic and lymphoplasmacytic infiltrate, rare Mott cells, and increased numbers of lymphoglandular bodies (Figure 2). Erythrophagocytosis, hemosiderophages, and a few neutrophils were seen in the splenic samples.

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Photomicrographs of cytology of the spleen (A, B) and histopathology of the popliteal lymph node (C, D) from the dog of Figure 1. A—Lymphohistioplasmacytic infiltration with myriad intralesional intracytoplasmic amastigotes Leishmania spp within the macrophages. Note on the background lymphoglandular bodies (arrowhead A). Romanowski-type stain; bar = 10 μm. B—Higher magnification of the intracytoplasmic amastigotes. Romanowski-type; bar = 5 μm. C—Macrophages had intracytoplasmic amastigotes Leishmania spp (red arrows) and brown granular pigment (hemosiderosis; black arrows). H&E stain; bar = 10 μm. D—Intracytoplasmic amastigotes had strong immunolabeling with an anti-Leishmania spp antibody (arrows), bar = 10 µm.

Citation: Journal of the American Veterinary Medical Association 262, 1; 10.2460/javma.23.09.0498

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Multiple tissue samples from the gastrointestinal tract including popliteal, axillary, prescapular, and mesenteric lymph nodes, lungs, heart, liver, kidneys, and spleen were collected, fixed in neutral buffered 10% formalin, and routinely processed for histologic examination. Histologically, the splenic parenchyma architecture was diffusely replaced and effaced by numerous macrophages and plasma cells, fewer lymphocytes, and occasional neutrophils. Macrophages contained numerous 2 to 4 µm, ovoid, intracytoplasmic amastigotes (consistent with Leishmania spp; Figure 2). Moderate erythrophagocytosis and hemosiderin-laden macrophages were noted. The splenic capsule was expanded by fibrosis and infiltrated by multifocal inflammatory aggregates composed of viable and degenerate neutrophils, lymphocytes, and macrophages. Lymph nodes has expansion of cortical follicles and paracortex by lymphocytes and plasma cells (lymphoid hyperplasia). Immunohistochemical staining labeled the structure of the parasites within macrophages of the spleen and lymph node using a mouse polyclonal anti–Leishmania amazonensis antibody, dilution 1:1000 (Figure 2).¹

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: Histiocytic and plasmacytic splenitis and lymphadenitis, subacute, diffuse, severe, with numerous intralesional Kinetoplastida protozoa (consistent with protozoal amastigotes of Leishmania spp).

Case summary: canine visceral leishmaniasis (CanL).

Comments

The clinical and pathological findings confirmed canine visceral leishmaniasis. Leishmaniasis is a vector-borne infectious disease that affects humans, domestic and wild animals, and is caused by an obligate intracellular parasitic protozoa of the genus Leishmania.^2,3 Leishmaniasis is transmitted to humans and other mammals by the bite of sandflies of the genus Phlebotomus or Lutzomyia.^2,3 There are 3 clinical forms of leishmaniasis in humans: cutaneous, mucocutaneous, and visceral.^2,3 The visceral form is the most serious and can result in 100% mortality if left untreated.^2,3

Visceral leishmaniasis (VL) is caused by geographic variants of the Leishmania donovani complex (L donovani, L infantum, L chagasi).³ Dogs are considered the main reservoir in endemic urban areas, but several mammals are considered to be potential reservoirs, such as black rats, domestic cats, hares, crab-eating foxes and opossums.² In the US, CanL is mostly reported as being a travel-associated disease, but sporadic cases have been documented in dogs with no history of travel to enzootic areas.² A study of more than 12,000 foxhounds in the US and Canada revealed that canine leishmaniasis is enzootic in 18 states in the US and 2 Canadian provinces.³ This may be the result of environmental changes that provide ideal conditions for vectors to survive and, consequently, increase the incidence of the disease.³

Canine VL presents with a wide range of nonspecific clinical signs: anorexia or increased appetite, lethargy, wasting, cachexia, peripheral lymphadenopathy, exercise intolerance, temporal muscle atrophy, splenomegaly, polyuria/polydipsia, epistaxis, eye lesions, lameness, vomiting and diarrhea.² The systemic changes observed in the CBC and within the serum biochemistry profile of infected dogs vary with their immune capacity in response to disease. Symptomatic dogs usually show a humoral response, plasmacytosis, and hyperglobulinemia.² The proliferation of B lymphocytes, plasma cells, histiocytes, and macrophages leads to lymphadenomegaly and splenomegaly.² Polyclonal hyperglobulinemia is considered the clinical hallmark of the disease and involves the deposition of immune complexes causing a type III hypersensitivity reaction that leads ultimately to chronic kidney disease.² The anemia, which is usually normochromic, normocytic, and non-regenerative may be due to immune-mediated mechanisms generating hemolysis, splenic sequestration, blockage in the production of the erythrocyte lineage in the bone marrow secondary to the presence and immune response against Leishmania spp and chronic kidney disease. Hypoalbuminemia is associated with renal failure and reduced hepatic function in response to acute-phase protein production, leading to dysproteinemia.²

In this case, findings such as lymphadenomegaly, splenomegaly associated with normocytic and normochromic anemia with hyperproteinemia due to hyperglobulinemia and hypoalbuminemia were highly suggestive of canine VL, especially in endemic areas.²

The diagnosis of canine VL was confirmed by cytology and histopathology based on the presence of intra- and extra- cytoplasmic forms of amastigotes consistent with Leishmania spp. Although cytology and histopathology are confirmatory for the diagnosis of canine VL, these techniques are not very sensitive. This is especially true in asymptomatic dogs who may have a low parasitic load, generating false negatives. The polymerase chain reaction can offer greater accuracy in the diagnosis of CanL in this sense, presenting high specificity and sensitivity, with the disadvantage of having a higher cost.²

Co-infection between Leishmania spp, and other etiological agents such as Anaplasma, Bartonella, Dirofilaria, Hepatozoon, Rickettsia, Trypanosoma, Ehrlichia spp, and Babesia spp has been reported. Ehrlichiosis and babesiosis were possible coinfections and differential diagnoses; however, the dog in this report was negative for ehrlichiosis and babesiosis by ELISA serology.²

The treatment of CanL is based on the clinical stage in which the animal is at the time of diagnosis; however, the parasitological cure is extremely difficult to achieve.¹ Dogs can survive longer when monitored during and after therapy.⁴

Treatment options are limited to the use of allopurinol, which is nontoxic and effective against the organisms at a dose of 7 to 20 mg/kg, PO, every 8 to 12 hours for 3 to 12 months, or in some cases the rest of dog’s life.⁵ When allopurinol is used alone, clinical remission can be achieved.⁵ Relapses are common, but the 4-year survival rate of treated dogs without renal failure is 80%.⁵ Combined therapies include the use of pentavalent antimony like meglumine antimoniate (100 mg/kg, IV/SC, q 48 h, 1 month) and sodium stibogluconate (30-50 mg/kg, IV/SC, q 24 h, 1 month).⁵ Pentavalent antimonials are not licensed in the US and can only be obtained through the CDC.⁵ Amphotericin B is effective against the parasite and can be used at a dose of 0.25-0.5 mg/kg, reconstituted in 5% dextrose, IV/SC 48h or 3 times a week for an undetermined period dependent on the animals’ renal function. However, it is more toxic and less effective than allopurinol.⁵ The prognosis of infected animals is worse when emaciation and renal insufficiency are present. Regardless of therapeutical options, due to the zoonotic nature of the disease, the common relapses, and the persistence of the organisms, euthanasia is promoted in the US.^2,5 Hematology, serum biochemistry, serum protein electrophoresis, and complete urinalysis including urine protein: creatinine (UPC) should be monitored at the end of the first month of treatment, followed by every 3-4 months until recovery and then every 6-12 months.⁴ Antibody titers are meaningless before 6 months, but it is useful afterward since more than a 2-fold increase is suggestive of relapse.⁴ The dog in this report was native to Araçatuba city in the Northwest of São Paulo State, which is an endemic region for CanL. Two other dogs were present in this household and should be considered likely potential reservoirs and/or asymptomatic dogs for leishmaniasis. The owner had little information about canine leishmaniasis and its zoonotic potential, despite residing in an endemic area for the disease. Therefore, the owner did not use prophylactic methods. In addition to testing other dogs for CanL, recommendations for prophylactic measures include targeting the use of repellents, vaccinations, and sanitary measures to reduce vector density in the environment.² Vaccination decreases de incidence of clinical disease but may not prevent the infection.² There is no licensed vaccines for use in the US.² The diagnosis of CanL is of great importance for public health and raises the need for prophylactic measures to prevent and contain this disease, which is an important zoonosis.

Disclosures

The author, Tatiane Terumi Negrão Watanabe, is employed by Antech Diagnostics. The remaining authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors have nothing to disclose.