CONICET | Buscador de Institutos y Recursos Humanos

tCattle are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis hominiswhich use canids, felids or primates as definitive hosts (DH), respectively, and in additionof Sarcocystis sinensis from which the DH is unknown. The aims of the present study were todevelop and optimize a multiplex real time PCR for a sensitive and specific differentiation ofSarcocystis spp. affecting cattle and to estimate the prevalence of Sarcocystis spp. in Argen-tinean cattle. The 18S rRNA genes from individual sarcocysts were amplified and clonedto serve as controls. For the amplification of bovine Sarcocystis spp. a total of 3 primerswere used in combination with specific individual probes. Each assay was evaluated andoptimized individually and subsequently combined in a multiplex assay (BovSarcoMulti-plex real time PCR). The analytical specificity of the multiplex assay was assessed using5 ng of DNA of heterologous Sarcocystis spp. and other apicomplexan parasites, and no pos-itive reactions were observed other than for the species the PCR targeted. The analyticalsensitivity ranged between 0.0125 and 0.125 fg of plasmid DNA (equivalent to the DNA of2?20 plasmid DNA copies) or resembling DNA of 0.1?0.3 bradyzoites. A total of 380 DNAloin samples from Argentina were tested and 313, 29, 14 and 2 were positive for S. cruzi,S. sinensis, S. hirsuta and S. hominis, respectively. S. sinensis was the most prevalent speciesamong thick walled Sarcocystis spp. in Argentinean cattle. Mixed infections were detectedin 8.9% of all samples. Diagnostic sensitivity and specificity for the BovSarcoMultiplex realtime PCR relative to previous microscopic examination for thin and thick-walled cyst were91.5% and 41.7%, 36.3% and 95.9% respectively. Improved DNA extraction methods mayallow to further increase the specific and sensitive detection of Sarcocystis spp. in meatsamples.

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