The purpose of the present study was to evaluate the immunohistochemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for Canine Visceral Leishmaniasis (CVL) diagnosis and compare the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitological test (microscopic direct examination of the parasite stained with haematoxylin and eosin--HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5%) but also healthy skins (31.8%) were positives by IMHC and confirmed by PCR in 97.8% of skin samples. In asymptomatic group, 87.5% dogs were negatives by serological tests, but positives by IMHC in 50% and by PCR in 100%. In oligosymptomatic group, 100%, 85.7% and 28.6% of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7% of polisymptomatic dogs were serum positive and had intact parasites in the skin. In general, PCR showed higher positivity (100%). The efficiency of each test varied with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR in inconclusive cases after HE and IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this canine disease.

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The aim of this work was a correlation study and histopathological description of alterations associated with the presence of Leishmania infantum amastigote in the intestinal wall of dogs infected with canine visceral leishmaniasis (CVL). Three groups were used: G1 (n = 8), comprising naturally infected dogs with CVL with amastigotes of L. infantum in the small and large intestines; G2 (n = 9), infected dogs with CVL, without intestinal amastigotes; and G3 (n = 3), uninfected dogs. Histochemistry and immunohistochemistry methods were used for histopathology and amastigotes identification. 47.1% (8/17) of dogs from G1 group had amastigotes in the mucosa, submucosa and muscle layers of the small and large intestines and it was observed a prominent inflammatory reaction characterized by chronic infiltration of mononuclear cells: macrophages, lymphocytes and plasma cells. Comparison between the groups showed only a significant difference in relation to mucosal microscopic structural alterations in dogs from G1 in relation to G2 and G3. Parasite burden showed significant correlations with the microscopic alterations and clinical status of dogs in G1. By the conclusion, the inflammatory reactions caused by the parasites in the intestines might have contributed towards alterations in digestive processes, worsening the dogs' clinical status of CVL.Keywords: Dog, histology, immunohistochemistry, intestine, leishmaniasis, amastigotes. ResumoO objetivo foi realizar um estudo de correlação e descrição histopatológica das lesões associadas à presença de amastigotas de Leishmania infantum na parede intestinal de cães infectados com leishmaniose visceral canina (LVC). Os cães foram subdivididos em três grupos: G1 (n = 8) cães naturalmente infectados com LVC e com amastigotas de L. infantum no intestino; G2 (n = 9) com LVC, mas sem o parasitismo intestinal; e G3 (n = 3) cães não infectados. Métodos histoquímicos e imunoistoquímicos foram utilizados para a histopatologia e a identificação das amastigotas, respectivamente. 47,1% (8/17) dos cães infectados (grupo G1) apresentavam formas amastigotas na mucosa, submucosa e camada muscular do intestino delgado e grosso, destacando-se uma reação inflamatória caracterizada por infiltrado crônico de células mononucleares; macrófagos, linfócitos e plasmócitos. Observou-se uma diferença significativa somente com relação às alterações estruturais microscópicas intestinais nos cães do G1 quando comparadas com G2 e G3. A intensidade parasitária intestinal teve correlação significativa com as alterações microscópicas e os sinais clínicos dos cães do G1. Concluiu-se que as amastigotas de L. infantum por causarem reações inflamatórias na parede intestinal dos cães podem ter contribuído para as alterações dos processos digestórios, agravando ainda mais o quadro clínico dos animais.Palavras-chave: Cão, histologia, imunoistoquímica, intestino, leishmaniose, amastigotas.

The aim of the present study was to evaluate the serological methods using ELISA with recombinant-rK39 (ELISA-rK-39) and soluble extract-SE (ELISA-SE) antigens, the indirect fluorescence antibody test (IFAT) in comparison to an immunochromatography rapid diagnostic test (RDT-rK39) and with a direct parasitological exam (PA) for Canine Visceral Leishmaniasis (CVL) diagnosis. The results showed that 89% (60/67) of the dogs were positive for at least one serological diagnostic test. ELISA-SE was the test that detected anti-Leishmania antibodies in the serum of the highest number of dogs (71.6%) followed by ELISA-rK39 (65.7%), IFAT (65.7%) and RDT-rK39 (55.2%). PA detected the lowest numbers (40.3%) of positive dogs. In relation to the total of examined dogs, the Kappa indexes (p ≤ 0.05) showed a good agreement between ELISA-SE and IFAT (88.1%; k = 0.7237), and it was also observed in the comparison of RDT-rK39 with ELISA-SE (83.6%, k= 0.6561), IFAT (83.5%, k= 0.6605) and PA (85.0%, k= 0.7074). A bad agreement was detected in any association of ELISA-rk39 with the other tests in either symptomatic or asymptomatic animals. ELISA as well as RDT using recombinant antigenic protein (rK39) were the methods that detected the lowest prevalence rates (33.3%) of CVL in asymptomatic dogs. In conclusion, only one test does not adequately identify dogs with CVL and it is necessary the association of two or more diagnostic tests. Because of the good agreement indexes of RDT-rK39 when evaluated with ELISA-SE, IFAT and PA it was suggested as a complementary method to be used in association with either ELISA-SE or IFAT, particularly in the symptomatic dogs. Furthermore, new studies are recommended in order to improve the sensitivity of tests mainly for asymptomatic dogs.Keywords: CVL, diagnosis, dogs, Leishmania infantum, rK39, RDT. ResumoO objetivo do presente estudo foi avaliar os métodos sorológicos usando ELISA (Ensaio Imunoenzimático Indireto) com o antígeno recombinante rK39 (ELISA-rK39) e o antígeno extrato solúvel bruto (ELISA-ES) e a RIFI (Reação de Imunofluorescência Indireta) em comparação com o método imunocromatográfico rápido (RDT-rK39) e o parasitológico direto (PA), para o diagnóstico da Leishmaniose Visceral Canina (LVC) em cães de Ilha Solteira, São Paulo, Brasil. Os resultados mostraram que 89% (60/67) dos cães foram positivos por pelo menos um teste diagnóstico sorológico (RIFI, ELISA-ES, ELISA-rk39 ou RDT-rK39) e somente 40,3% (27/67) foram positivos pelo PA. O ELISA-ES foi o teste que detectou anticorpos anti-Leishmania em maior número de cães (71,6%) seguido por ELISArK39, RIFI (65,7%) e por 2%). No total de cães analisados (assintomáticos e sintomáticos), o índice Kappa de concordância (p ≤ 0,05) foi considerado de boa concordância entre ELISA-ES e IFAT (88,1%; k= 0,7237) e entre RDT-rK39 com ELISA-ES (83,6%, k= 0,6561), RIFI (83,5%, k= 0,6605) e PA (85,0%, k= 0,7074). O índice de concordância ruim foi observado em qualquer associação de ELISA-rk39 com todos os outros testes nos animais sintomáticos ...

The relevance of the dog as a source of visceral leishmaniasis infection is known, but the role of cats as reservoir hosts for leishmaniasis is not yet fully clear. This study assessed the efficacy of conjunctival swab PCR (CS-PCR) in the detection of cats infected by Leishmania spp. The results were seven (13.5%) cats positive for Leishmania spp. in the PCR, in 52 cats tested from Pirassunuga-SP and Ilha Solteira-SP. From the city of Pirassununga -SP 28.6% (2/7) were positive and from the city of Ilha Solteira -SP 11.1% (5/45) were positive. The results showed that CS-PCR was capable of detecting cats infected by this protozoan. Conjunctival swab samples proved easier to perform in cats, which might facilitate studies on the frequency and distribution of feline leishmaniasis.Keywords: Conjunctival swab, PCR, cats. ResumoA importância do cão como fonte de infecção da leishmaniose visceral já é conhecida, mas o papel dos gatos como reservatórios das leishmanioses ainda não está totalmente esclarecido. O presente estudo avaliou a eficácia da PCR de suabe conjuntival (PCR-SC) na detecção de gatos infectados por Leishmania spp. Foram encontrados sete (13,5%) gatos positivos para Leishmania spp. na PCR de suabe conjuntival, dentre 52 animais de Pirassununga -SP e Ilha Solteira -SP testados. Sendo positivos 28,6% (02/07) dos gatos do município de Pirassununga e 11,1% (5/45) dos gatos do município de Ilha Solteira. Os resultados demonstraram que o suabe de conjuntiva ocular foi capaz de detectar gatos infectados por esse protozoário. A coleta de amostras da conjuntiva mostrou ser um método simples, menos invasivo e pouco estressante para os gatos e seus proprietários, o que pode facilitar estudos sobre a frequência e distribuição da leishmaniose felina.Palavras-chave: Suabe conjuntival, PCR, gatos.

Introduction:Conjunctival swab PCR was evaluated as a tool to diagnose visceral leishmaniasis in dogs. Methods: Conjunctival swab PCR was compared to indirect immunofl uorescence antibody test and blood PCR. Results: Indirect immunofl uorescence was signifi cantly correlated with conjunctival swab PCR (p < 0.05), but not with blood PCR (p > 0.05). In addition, conjunctival swab PCR was signifi cantly associated with presence of clinical symptoms (p < 0.05), whereas blood PCR was associated with absence of clinical symptoms (p < 0.05). Conclusions: Results indicate that conjunctival swab PCR is useful in epidemiological surveys of canine visceral leishmaniasis.

Leishmaniasis is a zoonotic disease that affects 12 million people worldwide. Several mammalian species can serve as a reservoir for this disease. Dogs are the main reservoir for visceral leishmaniasis in urban areas, which has become a serious public health concern in Brazil. The aim of this study was to evaluate the presence of Leishmania spp. in captive wild animals from Ilha Solteira, São Paulo, Brazil. Blood and various tissues samples were collected from animals of five different species: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, and Procyon cancrivorus. Antibodies against Leishmania spp. were detected in three wild canids by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). PCR analyses of blood and bone marrow from all animals were negative, but Leishmania DNA was found in the tissues and skin of seropositive animals. Positive PCR samples were also positive for Leishmania donovani complex. Analysis of sequenced PCR products showed similarities with different regions of Leishmania (Leishmania) infantum and Leishmania (Leishmania) chagasi kinetoplastids. Measures to control visceral leishmaniasis in wild animals kept in Brazilian zoos should be established, as no disease control programs are currently available.

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