Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle

Authors
Moré, Gastón Andrés; Schares, Susann; Maksimov, Aline; Conraths, Franz J.; Venturini, Maria Cecilia; Schares, Gereon
Publication Year
2013
Language
English
Format
article
Status
Published version
Description
Cattle are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis hominis which use canids, felids or primates as definitive hosts (DH), respectively, and in addition of Sarcocystis sinensis from which the DH is unknown. The aims of the present study were to develop and optimize a multiplex realtime PCR for a sensitive and specific differentiation of Sarcocystis spp. affecting cattle and to estimate the prevalence of Sarcocystis spp. in Argentinean cattle. The 18S rRNA genes from individual sarcocysts were amplified and cloned to serve as controls. For the amplification of bovine Sarcocystis spp. a total of 3 primers were used in combination with specific individual probes. Each assay was evaluated and optimized individually and subsequently combined in a multiplex assay (BovSarcoMultiplex real time PCR). The analytical specificity of the multiplex assay was assessed using 5 ng of DNA of heterologous Sarcocystis spp. and other apicomplexan parasites, and no positive reactions were observed other than for the species the PCR targeted. The analytical sensitivity ranged between 0.0125 and 0.125 fg of plasmid DNA (equivalent to the DNA of 2–20 plasmid DNA copies) or resembling DNA of 0.1–0.3 bradyzoites. A total of 380 DNA loin 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 species among thick walled Sarcocystis spp. in Argentinean cattle. Mixed infections were detected in 8.9% of all samples. Diagnostic sensitivity and specificity for the BovSarcoMultiplex real time PCR relative to previous microscopic examination for thin and thick-walled cyst were 91.5% and 41.7%, 36.3% and 95.9% respectively. Improved DNA extraction methods may allow to further increase the specific and sensitive detection of Sarcocystis spp. in meat samples.
Fil: Moré, Gastón Andrés. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemania. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnológico La Plata; Argentina
Fil: Schares, Susann. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemania
Fil: Maksimov, Aline. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemania
Fil: Conraths, Franz J.. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemania
Fil: Venturini, Maria Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina
Fil: Schares, Gereon. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemania
Subject
Sarcocystis spp,
Cattle
Multiplex real time PCR
Argentina
Ciencias Veterinarias
Ciencias Veterinarias
CIENCIAS AGRÍCOLAS
Access level
Restricted access
License
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/3431