Detección coprológica y molecular de Anoplocephala perfoliata, en equinos del departamento del Valle del Cauca

Abstract

El objetivo del presente trabajo fue la detección coprológica y molecular del céstodo Anoplocephala perfoliata, parásito de equinos, a partir de heces extraídas directamente del recto de 112 caballos del Departamento del Valle del Cauca. Para aislar huevos del céstodo en las muestras de heces se usó el método reportado por Proudman y Edwards (1992 Se utilizó un kit para extraer ADN de un ejemplar de la A. perfoliata, como control positivo. Para la extracción del ADN fecal se utilizó el método fenol-cloroformo descrito por Menghi et al., 2006. Debido a su alta tasa de sustitución de nucleótidos, los espaciadores internos transcritos (ITS-1 e ITS-2) del ADN ribosomal (ADNr) se han convertido en regiones muy populares en la investigación de la variación inter e intraespecífica de los diferentes grupos de céstodos. Se amplificaron las regiones ITS (Internal Transcribed Spacer) del ADN de Anoplocephala perfoliata para todas las muestras extraídas basados en el protocolo de la PCR anidada (Drogemuller et al., 2004).

//Abstract: The aim of this study was the detection of tapeworm stool and molecular Anoplocephala perfoliata, a parasite of horses, from faeces taken directly from the rectum of 112 hp from the Department of Valle del Cauca. To isolate tapeworm eggs in stool samples was used the method reported by Proudman and Edwards 1992. Kit was used to extract DNA from a specimen of A. perfoliata as a positive control. For fecal DNA extraction method was used described by phenol-chloroform Menghi et al., 2006. Due to its high rate of nucleotide substitution, internal transcribed spacers (ITS- 1 and ITS-2) of ribosomal DNA (rDNA) regions have become very popular in the investigation of inter-and intraspecific variation of the different groups cestodes. Regions were amplified ITS (Internal Transcribed Spacer) Anoplocephala perfoliata DNA for all samples taken based on the nested PCR protocol (Drogemuller et al., 2004). In the first PCR primers were used to amplify partial regions as: 18S, ITS-1, 5.8S, ITS-2, and 28S rDNA of A. perfoliata. (Jousson et al. 1999). 1. S18 (5'-TAACAGGTCTGTGATGCC-3 ') 2. L3T (5'-CAACTTTCCCTCACGGTACTTG-3 ') (kept in the tapeworm).Based on the sequences obtained from A. perfoliata were used for nested PCR two primers that amplify the ITS-2 region, these primers amplify a 254pb fragment: 1. Set AP-ITS-2-3F (5´-AATTGTGGGGGCTTCTCTTA-3´) 2. AP-ITS-2-2R (5´-ACCTCGTGCCTTTCTTTAT-3´) (Drogemuller et al., 2004). With the technique stool was detected 16.96% of positive animals to the parasite and the molecular 20.53% of the 23 positive, only 10 were detected with the method stool, which represents an efficiency of 43.75%, found 9 false positive addition to the method stool due to the difficulty of clearly differentiating Anoplocephala perfoliata eggs with eggs of other genres such as Moniezia cestodes or Anoplocephala magna. The sensitivity and specificity of the technique were 63.98% and stool 91.96% respectively. With molecular techniques achieved a sensitivity and specificity of 100%. he results were analyzed by chi-square test to assess the independence of the parasite with the variables sex, age, ability, found no relationship of dependency between these variables and infestation