Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks

[EN] Background: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spai...

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Autores: Fernández Escobar, Mercedes, Calero Bernal, Rafael, Benavides Silván, Julio, Regidor Cerrillo, Javier, Guerrero Molina, María Cristina, Gutiérrez Expósito, Daniel, Collantes Fernández, Esther, Ortega Mora, Luis Miguel
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2020
País:España
Institución:Universidad de León
Repositorio:BULERIA. Repositorio Institucional de la Universidad de León
OAI Identifier:oai:buleria.unileon.es:10612/19117
Acceso en línea:https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-020-04275-z
https://hdl.handle.net/10612/19117
Access Level:acceso abierto
Palabra clave:Sanidad animal
Toxoplasma gondii
Sheep
Abortion
Isolates
Genotyping
Sequencing
Population structure
Spain
3109 Ciencias Veterinarias
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oai_identifier_str oai:buleria.unileon.es:10612/19117
network_acronym_str ES
network_name_str España
repository_id_str
spelling Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocksFernández Escobar, MercedesCalero Bernal, RafaelBenavides Silván, JulioRegidor Cerrillo, JavierGuerrero Molina, María CristinaGutiérrez Expósito, DanielCollantes Fernández, EstherOrtega Mora, Luis MiguelSanidad animalToxoplasma gondiiSheepAbortionIsolatesGenotypingSequencingPopulation structureSpain3109 Ciencias Veterinarias[EN] Background: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain. Methods: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. Results: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. Conclusions: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversitySIThis research was supported by projects funded by the Spanish Ministry of Science and Innovation (AGL2016-75935-C2-R) and the Community of Madrid (PLATESA2-CM-P2018/BAA-4370). MF and RC were funded by UCMSantander/ 2017 pre-doctoral grants, and PLATESA2 post-doctoral grants, respectively. CG was funded by DGAPA, National Autonomous University of Mexico (UNAM). RC, EC and LO are part of the TOXOSOURCES consortium, supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No. 773830: One Health European Joint ProgrammeBMCSanidad AnimalFacultad de Veterinaria2020info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-020-04275-zhttps://hdl.handle.net/10612/19117reponame:BULERIA. Repositorio Institucional de la Universidad de Leóninstname:Universidad de LeónIngléshttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:buleria.unileon.es:10612/191172026-06-24T12:43:27Z
dc.title.none.fl_str_mv Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
title Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
spellingShingle Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
Fernández Escobar, Mercedes
Sanidad animal
Toxoplasma gondii
Sheep
Abortion
Isolates
Genotyping
Sequencing
Population structure
Spain
3109 Ciencias Veterinarias
title_short Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
title_full Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
title_fullStr Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
title_full_unstemmed Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
title_sort Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks
dc.creator.none.fl_str_mv Fernández Escobar, Mercedes
Calero Bernal, Rafael
Benavides Silván, Julio
Regidor Cerrillo, Javier
Guerrero Molina, María Cristina
Gutiérrez Expósito, Daniel
Collantes Fernández, Esther
Ortega Mora, Luis Miguel
author Fernández Escobar, Mercedes
author_facet Fernández Escobar, Mercedes
Calero Bernal, Rafael
Benavides Silván, Julio
Regidor Cerrillo, Javier
Guerrero Molina, María Cristina
Gutiérrez Expósito, Daniel
Collantes Fernández, Esther
Ortega Mora, Luis Miguel
author_role author
author2 Calero Bernal, Rafael
Benavides Silván, Julio
Regidor Cerrillo, Javier
Guerrero Molina, María Cristina
Gutiérrez Expósito, Daniel
Collantes Fernández, Esther
Ortega Mora, Luis Miguel
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Sanidad Animal
Facultad de Veterinaria
dc.subject.none.fl_str_mv Sanidad animal
Toxoplasma gondii
Sheep
Abortion
Isolates
Genotyping
Sequencing
Population structure
Spain
3109 Ciencias Veterinarias
topic Sanidad animal
Toxoplasma gondii
Sheep
Abortion
Isolates
Genotyping
Sequencing
Population structure
Spain
3109 Ciencias Veterinarias
description [EN] Background: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain. Methods: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. Results: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. Conclusions: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity
publishDate 2020
dc.date.none.fl_str_mv 2020
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-020-04275-z
https://hdl.handle.net/10612/19117
url https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-020-04275-z
https://hdl.handle.net/10612/19117
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv BMC
publisher.none.fl_str_mv BMC
dc.source.none.fl_str_mv reponame:BULERIA. Repositorio Institucional de la Universidad de León
instname:Universidad de León
instname_str Universidad de León
reponame_str BULERIA. Repositorio Institucional de la Universidad de León
collection BULERIA. Repositorio Institucional de la Universidad de León
repository.name.fl_str_mv
repository.mail.fl_str_mv
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