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Uso de m??todo de biologia molecular quantitativo (PCR real-time) na avalia????o de reservat??rios para leishmaniose visceral

By Fred da Silva Juli??o

Abstract

A leishmaniose visceral (LV) ?? uma zooantroponose sist??mica de import??ncia reconhecida em sa??de p??blica, 90% dos casos no novo mundo s??o oriundos do Brasil. Os c??es dom??sticos e as raposas s??o considerados como os principais reservat??rios. A persist??ncia da Leishmania em ??reas end??micas e o insucesso das medidas de preven????o, dirigidas exclusivamente ao reservat??rio canino, sugerem que outros animais podem ter import??ncia na manuten????o do ciclo de transmiss??o da LV. OBJETIVO: Avaliar potenciais reservat??rios para LV numa ??rea end??mica, utilizando m??todo de biologia molecular quantitativo (PCR real-time). M??TODOS: Foram estudados animais dom??sticos (bovinos, equ??deos, caprinos e ovinos) e animais silvestres (marsupial), no munic??pio de Salinas da Margarida, Bahia, de 2007 a 2009. Todos os animais dom??sticos de produ????o mantidos e/ou pernoitando nas ??reas urbanas do munic??pio foram inclu??dos. Os marsupiais foram capturados com armadilha animal modelo Tomahawk colocadas no peridomic??lio das resid??ncias onde ocorreram casos de LV humana e/ou canina na localidade de Encarna????o. Nos animais dom??sticos de produ????o foi coletado apenas amostra de sangue perif??rico e nos marsupiais, al??m de sangue, foi obtida uma amostra de pele atrav??s de bi??psia da orelha. Em todas as amostras foi realizado PCR real-time para investigar a presen??a de DNA do parasito e estimar a carga parasit??ria. Os primers e sondas utilizados foram selecionados em gene SSU rRNA, que aparece 160 vezes no genoma de Leishmania spp. e ?? altamente conservado entre as esp??cies de Leishmania. RESULTADOS: No total, foram avaliados 80 animais dom??sticos (20 bovinos, 33 equ??deos, 20 caprinos e 7 ovinos) e 103 marsupiais, todos da esp??cie Didelphis albiventris. Cinco bovinos foram positivos no teste de PCR real-time com carga parasit??ria variando de 12,7 a 183,5 parasitos/mL. Apenas um marsupial apresentou amostra de sangue positiva (6,0 parasitos/mL). Todos os demais animais testaram negativo. CONCLUS??O: A t??cnica de PCR real-time pode ser uma ferramenta ??til para avaliar o papel de potenciais reservat??rios dom??sticos e silvestres para LV. A execu????o do PCR real-time ?? menos trabalhosa e mais pr??tica do que a realiza????o do teste de xenodiagn??stico, al??m disso, ela poder ser automatizada, permitindo a an??lise de grande n??mero de amostras em estudos epidemiol??gicos. A detec????o de carga parasit??ria de Leishmania em sangue de bovinos, em quantidade compar??vel ?? encontrada em c??es, sugere que eles podem ser reservat??rios para LV. A relativa abund??ncia de bovinos nas ??reas end??micas para LV, assim como as evid??ncias da prefer??ncia alimentar do vetor por estes animais, ressaltam a import??ncia do papel que os bovinos podem ter na transmiss??o da LV. Mais trabalhos s??o necess??rios para elucidar estas quest??es.Visceral leishmaniasis (VL) is a systemic zooantroponose of public health relevance, 90% of cases in the new world are from Brazil. Domestic dogs and foxes are considered the main reservoirs. The persistence of Leishmania in endemic areas and the failure of preventive measures, directed exclusively to the canine reservoir, suggest that other animals may be important in maintaining the transmission cycle of Leishmania. OBJECTIVE: To investigate potential reservoirs for VL in an endemic area, using molecular biology quantitative method (real-time PCR). METHODS: We studied domestic animals (cattle, horses, goats and sheeps) and wildlife (marsupial), in the city of Salinas da Margarida, Bahia, from 2007 to 2009. All livestock animals maintained and/or staying overnight in the urban areas of the municipality were included. The marsupials were captured with Tomahawk model animal traps placed outside the home of homes where there were human and/or dog VL cases in the locality of the Encarna????o. In livestock animals, we collected only a sample of peripheral blood and in marsupials, in addition to blood we also collected a sample of ear skin biopsy. In all samples we carried out real-time PCR to detect the presence of parasite DNA and to estimate the parasite load. The primers and probes used were selected on SSU rRNA gene, which appears 160 times in the genome of Leishmania spp. and is highly conserved among species of Leishmania. RESULTS: In total, 80 livestock animals were evaluated (20 cattle, 33 horses, 20 goats and 7 sheep), and 103 marsupials, all Didelphis albiventris. Five cattle were positive by real-time PCR with parasite load ranging from 12.7 to 183.5 parasites/mL. Only one marsupial had a positive blood sample (6.0 parasites/mL). All other animals tested negative. CONCLUSION: The real-time PCR technique can be a useful tool for assessing the potential role of domestic and wild reservoir for LV. The implementation of real-time PCR is less laborious and more practical than the test of xenodiagnosis, in addition, it can be automated, allowing for the analysis of large number of samples in epidemiological studies. Detection of Leishmania parasite load in the blood of cattle, in an amount comparable to that found in dogs, suggests that they may be reservoirs for VL. The relative abundance of cattle in endemic areas for VL, as well as evidence of vector feeding preference for these animals, highlight the important role that cattle may exert in the transmission of VL. More research is needed to clarify these issues

Topics: Leishmaniose visceral, Rea????o em Cadeia da Polimerase, Ruminantes, Didelphis, Leishmaniasis, Polymerase chain reaction, Ruminants, Didelphis
Year: 2011
OAI identifier: oai:agregador.ibict.br.RI_FIOCRUZ:oai:localhost:icict/4206
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