Detecção de tripanosomatídeos em gatos domésticos de região endêmica para leishmaniose visceral por técnicas parasitológicas, sorológicas e moleculares
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Data
2017-06-26
Autores
Alves-Martin, Maria Fernanda [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A família Trypanosomatidae inclui protozoários flagelados com dois gêneros de importância médica e veterinária: Leishmania, causador das leishmanioses e Trypanosoma, responsáveis pelas tripanossomíases de relevância epidemiológica, ambos com o envolvimento de hospedeiros mamíferos e vetores em seu ciclo de vida. O gato doméstico passou a ser considerado um hospedeiro reservatório de Leishmanias, após comprovação por xenodiagnóstico, da transmissibilidade de formas promastigotas de Leishmania infantum para o vetor flebotomíneo. Devido a inespecificidade dos achados clínicos na Leishmaniose Felina (LF), à semelhança dos aspectos clínicos dessa enfermidade com outras comuns em gatos, e à provável resistência felina a infecção, é possível que, dentro de uma área endêmica para leishmaniose ocorra um percentual elevado de indivíduos infectados, mas sem apresentarem sinais clínicos, e a falta de diagnóstico precoce da LF nessas áreas pode implicar com que o animal continue a representar risco potencial de transmissão de leishmanias aos vetores. Assim sendo, pretendeu-se investigar possíveis associações entre sorologia positiva para L. infantum, com o parasitismo tecidual em sangue, órgãos e células conjuntivais de gatos de região endêmica para LV. Foram incluídos neste estudo 36 gatos adultos, cedidos pelo Centro de Controle de Zoonoses (CCZ) de Bauru - São Paulo, com 31 animais apresentando pelo menos um tipo de alteração clínica. A partir dos diagnósticos sorológicos, verificou-se a positividade em 26 animais, sendo que (66,7%) (24/36) foram reagentes à Reação de Imunofluorescência Indireta (RIFI), (61,1%) (22/36) animais reagentes ao ELISA e 10 animais não foram reagentes (K=0,64). A observação de formas sugestivas de amastigotas se deu em 12 animais (33,3%), quatro animais (11,1%) e 12 animais (33,3%), a partir das técnicas parasitológicas de esfregaço sanguíneo, imprint de órgãos e histoquímica, respectivamente. O diagnóstico molecular foi realizado com a utilização dos primers ITS-1, ITS-2 e HSP70, cuja detecção molecular pela PCR de sangue se deu em 15 animais (41,6%), pela PCR de tecidos em oito animais (22,2%) e pela PCR de suabes em quatro animais (11,1%). A quantificação da carga parasitária pela qPCR de sangue se deu em nove animais (25%) e pela qPCR de tecidos em dois animais (5,5%). Após o sequenciamento genético, foram identificadas três amostras com Trypanosoma theileri, cinco com Leishmania donovani e uma amostra com Leishmania braziliensis. Dessa forma, verificamos que os gatos avaliados, sorologicamente negativos e positivos apresentaram sinais clínicos, presença do parasito em células e DNA parasitário em sangue e/ou tecidos, atuando como reservatórios de Leishmanias na região estudada. A detecção de tripanosomatídeos em amostras biológicas dos gatos do estudo sugere sua participação na manutenção do parasito no ambiente urbano.
Trypanosomatidae family includes flagellate protozoa of two genera of medical and veterinary importance: Leishmania, the causative agent of leishmaniasis and Trypanosoma, responsible for trypanosomiasis of epidemiological relevance, with the involvement of mammalian hosts and vectors in their life cycle. Nowadays, the domestic cat is considered a host reservoir of leishmanias, after proving by xenodiagnosis, the transmissibility of promastigotes of Leishmania infantum to the phlebotomine vector. Due to the lack of specificity of the clinical findings in Feline Leishmaniasis (FL), similar to the clinical aspects of this disease with other common ones in cats, and to the probable feline resistance to infection, it is possible that, within an endemic area for leishmaniasis, a high percentage of individuals with no clinical signs, and lack of early diagnosis of FL in these areas may imply that the animal continues to pose a potential risk of transmission of leishmaniasis to the vectors. Thus, it was intended to investigate possible associations between positive serology for L.infantum, with tissue parasitism in blood, organs and conjunctival cells of cats from endemic region to visceral leishmaniasis. Thirty-six adult cats, assigned by the Zoonoses Control Center (ZCC) of Bauru-SP, with 31 animals presenting at least one type of clinical alteration, were included in this study. From the serological diagnoses, the positivity was verified in 26 animals, and (66.7%) (24/36) were reactive to the Immunofluorescence Antibody Test (IFAT), (61.1%) (22/36) animals reactives to ELISA test and 10 animals were non-reactive (K = 0.64). The observation of suggestive forms of amastigotes occurred in 12 animals (33.3%), four animals (11.1%) and 12 animals (33.3%), by the parasitological techniques of blood smear, imprint of organs and histochemistry, respectively. Molecular diagnosis was performed using the ITS-1, ITS-2 and HSP70 primers, whose molecular detection by blood PCR was done in 15 animals (41.6%), by tissue PCR in eight animals (22.2 %) and by PCR of conjunctival swabs in four animals (11.1%). Quantification of the parasitic load by blood qPCR occurred in nine animals (25%) and in the qPCR of tissues in two animals (5.5%). After the genetic sequencing, three samples were identified as Trypanosoma theileri, five as Leishmania donovani and one as Leishmania braziliensis. In this way, we verified that the evaluated cats, serologically negative and positive, presented clinical signs, presence of the parasite in cells and parasitic DNA in blood and / or tissues, possibly acting as reservoirs of leishmaniasis in the studied region. The detection of trypanosomatids in biological samples of the studied cats suggests their participation in the maintenance of the parasite in the urban environment.
Trypanosomatidae family includes flagellate protozoa of two genera of medical and veterinary importance: Leishmania, the causative agent of leishmaniasis and Trypanosoma, responsible for trypanosomiasis of epidemiological relevance, with the involvement of mammalian hosts and vectors in their life cycle. Nowadays, the domestic cat is considered a host reservoir of leishmanias, after proving by xenodiagnosis, the transmissibility of promastigotes of Leishmania infantum to the phlebotomine vector. Due to the lack of specificity of the clinical findings in Feline Leishmaniasis (FL), similar to the clinical aspects of this disease with other common ones in cats, and to the probable feline resistance to infection, it is possible that, within an endemic area for leishmaniasis, a high percentage of individuals with no clinical signs, and lack of early diagnosis of FL in these areas may imply that the animal continues to pose a potential risk of transmission of leishmaniasis to the vectors. Thus, it was intended to investigate possible associations between positive serology for L.infantum, with tissue parasitism in blood, organs and conjunctival cells of cats from endemic region to visceral leishmaniasis. Thirty-six adult cats, assigned by the Zoonoses Control Center (ZCC) of Bauru-SP, with 31 animals presenting at least one type of clinical alteration, were included in this study. From the serological diagnoses, the positivity was verified in 26 animals, and (66.7%) (24/36) were reactive to the Immunofluorescence Antibody Test (IFAT), (61.1%) (22/36) animals reactives to ELISA test and 10 animals were non-reactive (K = 0.64). The observation of suggestive forms of amastigotes occurred in 12 animals (33.3%), four animals (11.1%) and 12 animals (33.3%), by the parasitological techniques of blood smear, imprint of organs and histochemistry, respectively. Molecular diagnosis was performed using the ITS-1, ITS-2 and HSP70 primers, whose molecular detection by blood PCR was done in 15 animals (41.6%), by tissue PCR in eight animals (22.2 %) and by PCR of conjunctival swabs in four animals (11.1%). Quantification of the parasitic load by blood qPCR occurred in nine animals (25%) and in the qPCR of tissues in two animals (5.5%). After the genetic sequencing, three samples were identified as Trypanosoma theileri, five as Leishmania donovani and one as Leishmania braziliensis. In this way, we verified that the evaluated cats, serologically negative and positive, presented clinical signs, presence of the parasite in cells and parasitic DNA in blood and / or tissues, possibly acting as reservoirs of leishmaniasis in the studied region. The detection of trypanosomatids in biological samples of the studied cats suggests their participation in the maintenance of the parasite in the urban environment.
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Palavras-chave
Leishmaniose felina, Tripanosomatídeos, Gato doméstico, Diagnóstico, Feline leishmaniasis, Trypanosomatids, Domestic cat, Diagnosis