?Identificaci?n molecular de los estadios de plerocercoide y adulto de Diphyllobothrium sp. obtenidos de humanos, lobos marinos y peces.?

Abstract

La difilobotriosis es una zoonosis causada por la ingesti?n accidental de la larva plerocercoide de Diphyllobothrium, familia Diphyllobothridae, que se encuentra enquistada o libre en la superficie visceral de los peces tele?steos marinos, considerados hospederos parat?nicos o intermediarios. El presente trabajo tuvo como objetivo identificar molecularmente los estadios de plerocercoide y adulto de Diphyllobothrium sp. obtenidos de humanos, lobos marinos y peces en el Per?. Se muestrearon 3668 ejemplares de Engraulis ringens ?anchoveta?, 22 de Sciaena deliciosa ?lorna?, 6 de Sciaena callaensis ?lorna grande?, 6 de Trachurus symmetricus murphyi ?jurel? frescas y congeladas en diferentes periodos estacionales, adquiridos de diferentes Puertos del Norte (Chiclayo), Centro (Lima) y Sur (Ica) de la costa peruana. Los espec?menes adultos de Otaria byronia se obtuvieron de Barranca y Huacho (Lima) y tres aislados de humanos. Para la extracci?n del ADN gen?mico de los estadios larvario y adulto, se utiliz? el Kit de extracci?n de ADN de tejidos siguiendo el protocolo del fabricante. La calidad de las muestras de ADN se evalu? por electroforesis; la concentraci?n y pureza por espectrofotometr?a utilizando un Nanodrop 2000. Se amplific? el ADNmt parasitario mediante PCR utilizando el gen cox1 de las larvas plerocercoides y adultos. Las pruebas moleculares revelaron la presencia de Adenocephalus pacificus (=Diphyllobothrium pacificum) en las muestras de adultos (3 de humanos y 2 de O. byronia) y cinco plerocercoides. Se reporta por primera vez la presencia de la larva plerocercoide de A. pacificus en Engraulis ringens ?anchoveta?, siendo un probable 2? hospedero intermediario. Se demuestra que los progl?tidos gr?vidos del estadio adulto y el esc?lex de la larva plerocercoide presentan una alta plasticidad morfol?gica. Por vez primera se identifica mediante el marcador molecular cox1 a dos aislados de O. byronia como A. pacificus. De acuerdo a las inferencias filogen?ticas basadas en el gen cox1, se determin? que los adultos y cinco larvas plerocercoides corresponden a A. pacificus. Dos larvas plerocercoides se ubicaron en un clado hermano de Diphyllobothrium. Diphyllobothriasis is a zoonosis caused by the accidental ingestion of Diphyllobothrium plerocercoid larvae, Diphyllobothridae family, which is found to be cystic or free in the visceral surface of marine teleost fish, considered as paratenic hosts or intermediates. The aim of the present work was to identify molecularly the stages of plerocercoide larvae and adult Diphyllobothrium sp. obtained from humans, sea lions and fish in Peru. Twenty-two specimens of Sciaena deliciosa "lorna", 6 of Sciaena callaensis "big lorna ", 6 of Trachurus symmetricus murphyi "mackerel" and 3668 of Engraulis ringens "anchoveta" were sampled fresh and frozen in different seasonal periods acquired from different Northern Ports (Chiclayo), Centro (Lima) and South (Ica) of the Peruvian coast. The adult specimens of Otaria byronia were obtained from Barranca and Huacho (Lima) and three human isolates. For the extraction of genomic DNA from the larvae and adult stages, the tissue DNA extraction kit was used following the manufacturer's protocol. The quality of the DNA samples was evaluated by electrophoresis; Concentration and purity by spectrophotometry using a Nanodrop 2000. Parasitic mtDNA was amplified by PCR using the cox1 gene of the stages of larvae and adults. Molecular tests revealed the presence of Adenocephalus pacificus (= Diphyllobothrium pacificum) in adult samples (3 human and 2 O. byronia) and five plerocercoids. The presence of A. pacificus plerocercoid larvae in Engraulis ringens "anchoveta" is reported for the first time, being a probable 2nd intermediate host. It is demonstrated that the pregnant proglottids of the adult stage and the scolex of the plerocercoid larva have a high morphological plasticity. For the first time, the molecular marker cox1 is identified as two isolates of O. byronia as A. pacificus. According to phylogenetic inferences based on the cox1 gene, it was determined that adults and five plerocercoid larvae correspond to A. pacificus. Two plerocercoid larvae were located in a sister clade of Diphyllobothrium.