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

Autores
Moré, Gastón Andrés; Schares, Susann; Maksimov, Aline; Conraths, Franz J.; Venturini, María Cecilia; Schares, Gereon
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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 real time 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. 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. Friedrich-Loeffler-Institute; Alemania
Fil: Maksimov, Aline. Friedrich-Loeffler-Institute; Alemania
Fil: Conraths, Franz J.. Friedrich-Loeffler-Institute; Alemania
Fil: Venturini, María Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina
Fil: Schares, Gereon. Friedrich-Loeffler-Institute; Alemania
Materia
ARGENTINA
BEEF
MULTIPLEX REAL TIME PCR
SARCOCYSTIS SPP.
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/3431
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/3431
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattleMoré, Gastón AndrésSchares, SusannMaksimov, AlineConraths, Franz J.Venturini, María CeciliaSchares, GereonARGENTINABEEFMULTIPLEX REAL TIME PCRSARCOCYSTIS SPP.https://purl.org/becyt/ford/4.3https://purl.org/becyt/ford/4Cattle 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 real time 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. 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; ArgentinaFil: Schares, Susann. Friedrich-Loeffler-Institute; AlemaniaFil: Maksimov, Aline. Friedrich-Loeffler-Institute; AlemaniaFil: Conraths, Franz J.. Friedrich-Loeffler-Institute; AlemaniaFil: Venturini, María Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Schares, Gereon. Friedrich-Loeffler-Institute; AlemaniaElsevier2013-10-18info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/3431Moré, Gastón Andrés; Schares, Susann; Maksimov, Aline; Conraths, Franz J.; Venturini, María Cecilia; et al.; Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle; Elsevier; Veterinary Parasitology; 197; 1-2; 18-10-2013; 85-940304-4017enginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0304401713002367info:eu-repo/semantics/altIdentifier/doi/10.1016/j.vetpar.2013.04.024info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:14:59Zoai:ri.conicet.gov.ar:11336/3431instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 12:15:00.165CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
title Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
spellingShingle Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
Moré, Gastón Andrés
ARGENTINA
BEEF
MULTIPLEX REAL TIME PCR
SARCOCYSTIS SPP.
title_short Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
title_full Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
title_fullStr Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
title_full_unstemmed Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
title_sort Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle
dc.creator.none.fl_str_mv Moré, Gastón Andrés
Schares, Susann
Maksimov, Aline
Conraths, Franz J.
Venturini, María Cecilia
Schares, Gereon
author Moré, Gastón Andrés
author_facet Moré, Gastón Andrés
Schares, Susann
Maksimov, Aline
Conraths, Franz J.
Venturini, María Cecilia
Schares, Gereon
author_role author
author2 Schares, Susann
Maksimov, Aline
Conraths, Franz J.
Venturini, María Cecilia
Schares, Gereon
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv ARGENTINA
BEEF
MULTIPLEX REAL TIME PCR
SARCOCYSTIS SPP.
topic ARGENTINA
BEEF
MULTIPLEX REAL TIME PCR
SARCOCYSTIS SPP.
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.3
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv 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 real time 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. 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. Friedrich-Loeffler-Institute; Alemania
Fil: Maksimov, Aline. Friedrich-Loeffler-Institute; Alemania
Fil: Conraths, Franz J.. Friedrich-Loeffler-Institute; Alemania
Fil: Venturini, María Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina
Fil: Schares, Gereon. Friedrich-Loeffler-Institute; Alemania
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 real time 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.
publishDate 2013
dc.date.none.fl_str_mv 2013-10-18
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/3431
Moré, Gastón Andrés; Schares, Susann; Maksimov, Aline; Conraths, Franz J.; Venturini, María Cecilia; et al.; Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle; Elsevier; Veterinary Parasitology; 197; 1-2; 18-10-2013; 85-94
0304-4017
url http://hdl.handle.net/11336/3431
identifier_str_mv Moré, Gastón Andrés; Schares, Susann; Maksimov, Aline; Conraths, Franz J.; Venturini, María Cecilia; et al.; Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle; Elsevier; Veterinary Parasitology; 197; 1-2; 18-10-2013; 85-94
0304-4017
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0304401713002367
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.vetpar.2013.04.024
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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