Molecular and parasitological detection of Leishmania spp. in dogs caught in Palmas, TO, Brazil

Abstract

This study evaluated occurrences of Leishmania infantum in dogs in the municipality of Palmas, Tocantins, comparing diagnostic data obtained using the polymerase chain reaction (PCR) and parasitological diagnosis. Blood samples and lymph node aspirates were collected from 63 dogs of males and females and various ages and races, with or without owners, between August 2009 and June 2010. Slides containing smears of lymph node aspirates were stained with Giemsa stained. In PCR, the 145 bp target sequence of the LT1 fragment, located in the Leishmania donovani kDNA minicircle was detected using the RV1 and RV2 oligonucleotide primers. The chi-square test revealed that there was a significant relationship between the symptoms and dogs that were positive for visceral leishmaniasis (VL). The parasitological investigation showed concordance of 66.7% with PCR on blood and 84.1% with PCR on lymph node aspirate. In addition to these tests, evaluations of the diagnoses in parallel and in series were conducted, which showed concordances with the parasitological test of 76.2% and 74.6%, respectively. The results make it possible to suggest that PCR on lymph nodes should be used in evaluating large populations (surveys) and that the parasitological test should be used for initial clinical evaluations in veterinary consultation offices.Keywords: PCR, blood, lymph node aspirate, parasite, diagnosis. ResumoAvaliou-se a ocorrência de Leishmania infantum em cães do município de Palmas-TO, comparando dados diagnósticos obtidos pela Reação em Cadeia da Polimerase (PCR) e pelo diagnóstico parasitológico. Foram coletadas amostras de sangue e de aspirado de linfonodo de 63 cães machos e fêmeas, várias idades e raças, domiciliares ou não de agosto de 2009 a junho de 2010. As lâminas contendo esfregaço dos aspirados de linfonodos foram coradas pelo corante Giemsa. Na PCR, a sequência alvo de 145 pb do fragmento LT1, situado no minicírculo do kDNA do grupo Leishmania donovani, foi detectada através dos oligonucleotídeos iniciadores RV1 e RV2. O teste χ 2 (Qui-quadrado), demonstrou haver relação significativa entre a sintomatologia e a positividade dos cães para Leishmaniose Visceral (LV). O exame parasitológico mostrou uma concordância de 66,7% com a PCR em sangue e 84,1% com a PCR de aspirado de linfonodo. Além destas análises, houve a avaliação dos diagnósticos em paralelo e em série, onde as concordâncias com o exame parasitológico foram de 76,2% e 74,6%, respectivamente. Os resultados permitem sugerir a utilização da PCR de linfonodos na avaliação de grandes populações (inquéritos), e o exame parasitológico para a avaliação clínica inicial em consultórios veterinários.Palavras-chaves: PCR, sangue, aspirado de linfonodos, parasita, diagnóstico.

Seroprevalence and molecular characterization of Leishmania in dogs from an endemic area of zoonotic visceral leishmaniasis in Brazil

“…In the present study, LnPCR reached almost 100% of positivity when lymph node and spleen were used as DNA source, independently of the clinical state of the animal. This result is in accordance with other author reports [24] , [25] . Still, in practical terms, the best sample would be canine skin because it is more easily obtained.…”

Dogs, cats, parasites, and humans in Brazil: opening the black box

“…For instance, L. infantum is widespread in all Brazilian regions [81,113,149,150], but less frequent in southern parts of the country [151,152]. However, L. longipalpis , the principal vector for L. infantum , is present in northern Argentina [153], Paraguay [154], and has recently been detected in Uruguay [155].…”

“…The use of molecular tools for diagnosing protozoan parasites (e.g., B. vogeli , H. canis , and L. infantum ) is becoming more and more popular, but it is still mostly restricted to research [121,199-204]. Indeed, current PCR protocols have shown a good level of concordance with parasitological methods [150]. Unfortunately, the costs of molecular tools are still prohibitive for most pet owners living in endemic areas and this severely impairs the diagnosis of diseases such as visceral and cutaneous leishmaniosis in dogs.…”

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

“…In fact, the conventional PCR showed a low sensitivity for the detection of Leishmania spp. in samples of asymptomatic dogs 34 , and a strong association between the frequency of animals with symptoms and the frequency of positive animals by PCR has been shown 37 . Thus, although improving strategies for CVL diagnosis is important, samples should be carefully selected to avoid inappropriate interpretation of results.…”