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Fasciolose em Portugal e no Brasil: diagnóstico e genotipagem nos hospedeiros definitivo e intermediário
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A fasciolose é uma helmintose cosmopolita causada por duas espécies de tremátodes, Fasciola hepatica e Fasciola gigantica, cujos hospedeiros intermediários são moluscos limneídeos e o reservatório são ruminantes. Esta parasitose é considerada reemergente pela OMS e é um dos fatores limitantes na produção em pasto de ruminantes, incluindo Brasil e Portugal. O controlo desta parasitose requer métodos de diagnósticos rápidos e precisos, e o conhecimento sobre as populações de moluscos hospedeiros se faz necessário. As técnicas de diagnóstico molecular são mais sensíveis que as parasitológicas, mas são necessários métodos para implementação em laboratórios com poucos recursos, como a técnica de Amplificação Isotérmica Mediada por Loop (LAMP) de DNA, que pode apresentar alta sensibilidade e especificidade.
O presente trabalho se propôs desenvolver e testar a técnica de LAMP na deteção e genotipagem de F. hepatica em populações de hospedeiros bovinos e moluscos intermediários e verificar sua aplicação em campo, em amostras provenientes de Portugal e Brasil. Desenhou-se e implementou-se a técnica de LAMP com um novo conjunto selecionado de primers LAMP na região mitocondrial do gene NAD5, denominado Fh_1, para deteção específica de F. hepatica. O método LAMP, e a reação de PCR usando os primers externos, apresentou alta especificidade em amostras de DNA total de parasita adulto, fezes de ruminantes e em DNA extraído da região cefalopodal de moluscos, em relação a F. gigantica e outros helmintas. A sensibilidade em PCR foi de 10-4 ng e em LAMP de 10-2 ng de DNA, com um tempo para se obter o resultado de 3:00 h por PCR e 1:12 h por LAMP. De 222 moluscos Lymnaeidae, dos 1049 colhidos no distrito de Setúbal, confirmou-se infeção por F. hepatica em nove (4 %) através de PCR, e seis (1,8 %) por LAMP. Dos nove, seis foram identificados por métodos moleculares como Galba truncatula, principal hospedeiro intermediário de F. hepatica na Europa, um como a espécie gémea Galba schirazensis, e dois como Ampullaceana balthica. Avaliou-se o método de LAMP e PCR de NAD5 na deteção de F. hepatica em fezes bovinas em amostras de matadouro e numa manada tratada com albendazol no Brasil. Das 28 amostras de fezes recolhidas em matadouro, em Portugal e Brasil, com presença de parasitas adulto no fígado, quatro apresentaram ovos em exame parasitológico direto, mas 13 foram positivas através de PCR e três por LAMP. Nas amostras de manada antes e após tratamento, todas as 55 amostras de 11 bovinos foram positivas por PCR, mas, por LAMP, a infeção foi confirmada somente em 20% das amostras. Com os novos primers para amplificação por LAMP e PCR de NAD5 detetou-se DNA de F. hepatica com alta especificidade, e, apesar da sua menor sensibilidade, considerando a maior rapidez até ao resultado por LAMP que por PCR, LAMP poderá ser usado para detetar infeção por F. hepatica em populações de moluscos e bovinos no campo.
Fasciolosis is a cosmopolitan helminth disease caused by two species of trematodes, Fasciola hepatica and Fasciola gigantica, whose intermediate hosts are limneid molluscs and the reservoir are ruminant. This parasitosis is considered re-emergent by the WHO and is one of the limiting factors in ruminant pasture production, including Brazil and Portugal. The control of this parasitosis requires rapid and accurate diagnostic methods, and knowledge about the host mollusk populations is necessary. Molecular diagnostic techniques are more sensitive than parasitological diagnosis, but methods are needed for implementation in laboratories with few resources, such as Loop-Mediated Isothermal Amplification (LAMP) of DNA, which can have high sensitivity and specificity. The present work aimed to develop and test the LAMP technique in the detection and genotyping of F. hepatica in populations of bovines and molluscs intermediate hosts and to verify its application in the field, in samples from Portugal and Brazil. The LAMP technique was designed and implemented with a new set of selected LAMP primers in the mitochondrial NAD5 gene, named Fh_1, for specific detection of F. hepatica. The LAMP method, and the PCR reaction using the external primers, showed high specificity in samples of total DNA from adult parasites, ruminant feces and in DNA extracted from the cephalopodal region of molluscs, in relation to F. gigantica and other helminths. The PCR sensitivity was 10-4 ng and in LAMP 10-2 ng of DNA, with a time to the result of 3:00 h by PCR and 1:12 h by LAMP. Of 222 Lymnaeidae molluscs, of the 1049 collected in the district of Setúbal, infection by F. hepatica was confirmed in nine (4%) by PCR, and six (1.8%) by LAMP. Of the nine, six were identified by molecular methods as Galba truncatula, the main intermediate host of F. hepatica in Europe, one as the twin species Galba schirazensis, and two as Ampullaceana balthica. The LAMP and NAD5 PCR method were evaluated in the detection of F. hepatica in bovine feces in slaughterhouse samples and in an albendazole-treated herd in Brazil. Of the 28 stool samples collected in slaughterhouses, in Portugal and Brazil, with adult parasites in the liver, four had eggs by direct parasitological examination, but 13 were positive by PCR and three by LAMP. In herd samples before and after treatment, all 55 samples from 11 bovine were positive by PCR, but by LAMP infection was confirmed in only 20% of samples. With the new primers for amplification by LAMP and PCR of NAD5, F. hepatica DNA was detected with high specificity, and, despite its lower sensitivity, considering the greater speed to the result by LAMP than by PCR, LAMP can be used to detect F. hepatica infection in mollusc and cattle populations in the field.
Fasciolosis is a cosmopolitan helminth disease caused by two species of trematodes, Fasciola hepatica and Fasciola gigantica, whose intermediate hosts are limneid molluscs and the reservoir are ruminant. This parasitosis is considered re-emergent by the WHO and is one of the limiting factors in ruminant pasture production, including Brazil and Portugal. The control of this parasitosis requires rapid and accurate diagnostic methods, and knowledge about the host mollusk populations is necessary. Molecular diagnostic techniques are more sensitive than parasitological diagnosis, but methods are needed for implementation in laboratories with few resources, such as Loop-Mediated Isothermal Amplification (LAMP) of DNA, which can have high sensitivity and specificity. The present work aimed to develop and test the LAMP technique in the detection and genotyping of F. hepatica in populations of bovines and molluscs intermediate hosts and to verify its application in the field, in samples from Portugal and Brazil. The LAMP technique was designed and implemented with a new set of selected LAMP primers in the mitochondrial NAD5 gene, named Fh_1, for specific detection of F. hepatica. The LAMP method, and the PCR reaction using the external primers, showed high specificity in samples of total DNA from adult parasites, ruminant feces and in DNA extracted from the cephalopodal region of molluscs, in relation to F. gigantica and other helminths. The PCR sensitivity was 10-4 ng and in LAMP 10-2 ng of DNA, with a time to the result of 3:00 h by PCR and 1:12 h by LAMP. Of 222 Lymnaeidae molluscs, of the 1049 collected in the district of Setúbal, infection by F. hepatica was confirmed in nine (4%) by PCR, and six (1.8%) by LAMP. Of the nine, six were identified by molecular methods as Galba truncatula, the main intermediate host of F. hepatica in Europe, one as the twin species Galba schirazensis, and two as Ampullaceana balthica. The LAMP and NAD5 PCR method were evaluated in the detection of F. hepatica in bovine feces in slaughterhouse samples and in an albendazole-treated herd in Brazil. Of the 28 stool samples collected in slaughterhouses, in Portugal and Brazil, with adult parasites in the liver, four had eggs by direct parasitological examination, but 13 were positive by PCR and three by LAMP. In herd samples before and after treatment, all 55 samples from 11 bovine were positive by PCR, but by LAMP infection was confirmed in only 20% of samples. With the new primers for amplification by LAMP and PCR of NAD5, F. hepatica DNA was detected with high specificity, and, despite its lower sensitivity, considering the greater speed to the result by LAMP than by PCR, LAMP can be used to detect F. hepatica infection in mollusc and cattle populations in the field.
Descrição
Palavras-chave
Parasitologia Fasciolose hepática Galba truncatula Identificação molecular Deteção LAMP (Loop-mediated isothermal amplification)