Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis

Autores
De La Fourniere, Sofía Ana María; Paoletta, Martina; Guillemi, Eliana Carolina; Sarmiento, Nestor Fabian; Donati, Pablo Alejandro; Wilkowsky, Silvina Elizabeth; Farber, Marisa Diana
Año de publicación
2021
Idioma
inglés
Tipo de recurso
artículo
Estado
versión aceptada
Descripción
Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10−12 % parasitemia for B. bigemina and of 1 × 10−6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.
Instituto de Biotecnología
Fil: De La Fourniere, Sofía Ana María. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: De La Fourniere, Sofía Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Paoletta, Martina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Paoletta, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Guillemi, Eliana Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Guillemi, Eliana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sarmiento, Nestor Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; Argentina
Fil: Donati, Pablo Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Anestesiología y Manejo del Dolor; Argentina
Fil: Wilkowsky, Silvina Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Farber, Marisa Diana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Farber, Marisa Diana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fuente
Veterinary Parasitology 296 : 109493 (August 2021)
Materia
Enfermedades de los Animales
Babesia bigemina
Babesia bovis
Diagnóstico
Infección
Animal Diseases
Diagnosis
PCR
Infection
Nivel de accesibilidad
acceso restringido
Condiciones de uso
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/10884
Acceder
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oai_identifier_str oai:localhost:20.500.12123/10884
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network_name_str INTA Digital (INTA)
spelling Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovisDe La Fourniere, Sofía Ana MaríaPaoletta, MartinaGuillemi, Eliana CarolinaSarmiento, Nestor FabianDonati, Pablo AlejandroWilkowsky, Silvina ElizabethFarber, Marisa DianaEnfermedades de los AnimalesBabesia bigeminaBabesia bovisDiagnósticoInfecciónAnimal DiseasesDiagnosisPCRInfectionBovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10−12 % parasitemia for B. bigemina and of 1 × 10−6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.Instituto de BiotecnologíaFil: De La Fourniere, Sofía Ana María. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: De La Fourniere, Sofía Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paoletta, Martina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Paoletta, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guillemi, Eliana Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Guillemi, Eliana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sarmiento, Nestor Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Donati, Pablo Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Anestesiología y Manejo del Dolor; ArgentinaFil: Wilkowsky, Silvina Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farber, Marisa Diana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Farber, Marisa Diana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaElsevierinfo:eu-repo/date/embargoEnd/2022-12-102021-12-10T14:54:34Z2021-12-10T14:54:34Z2021-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/10884https://www.sciencedirect.com/science/article/abs/pii/S03044017210015270304-4017https://doi.org/10.1016/j.vetpar.2021.109493Veterinary Parasitology 296 : 109493 (August 2021)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccess2026-03-26T11:23:32Zoai:localhost:20.500.12123/10884instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2026-03-26 11:23:32.697INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
title Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
spellingShingle Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
De La Fourniere, Sofía Ana María
Enfermedades de los Animales
Babesia bigemina
Babesia bovis
Diagnóstico
Infección
Animal Diseases
Diagnosis
PCR
Infection
title_short Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
title_full Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
title_fullStr Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
title_full_unstemmed Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
title_sort Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
dc.creator.none.fl_str_mv De La Fourniere, Sofía Ana María
Paoletta, Martina
Guillemi, Eliana Carolina
Sarmiento, Nestor Fabian
Donati, Pablo Alejandro
Wilkowsky, Silvina Elizabeth
Farber, Marisa Diana
author De La Fourniere, Sofía Ana María
author_facet De La Fourniere, Sofía Ana María
Paoletta, Martina
Guillemi, Eliana Carolina
Sarmiento, Nestor Fabian
Donati, Pablo Alejandro
Wilkowsky, Silvina Elizabeth
Farber, Marisa Diana
author_role author
author2 Paoletta, Martina
Guillemi, Eliana Carolina
Sarmiento, Nestor Fabian
Donati, Pablo Alejandro
Wilkowsky, Silvina Elizabeth
Farber, Marisa Diana
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Enfermedades de los Animales
Babesia bigemina
Babesia bovis
Diagnóstico
Infección
Animal Diseases
Diagnosis
PCR
Infection
topic Enfermedades de los Animales
Babesia bigemina
Babesia bovis
Diagnóstico
Infección
Animal Diseases
Diagnosis
PCR
Infection
dc.description.none.fl_txt_mv Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10−12 % parasitemia for B. bigemina and of 1 × 10−6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.
Instituto de Biotecnología
Fil: De La Fourniere, Sofía Ana María. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: De La Fourniere, Sofía Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Paoletta, Martina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Paoletta, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Guillemi, Eliana Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Guillemi, Eliana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Sarmiento, Nestor Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; Argentina
Fil: Donati, Pablo Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Anestesiología y Manejo del Dolor; Argentina
Fil: Wilkowsky, Silvina Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Farber, Marisa Diana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina
Fil: Farber, Marisa Diana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10−12 % parasitemia for B. bigemina and of 1 × 10−6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.
publishDate 2021
dc.date.none.fl_str_mv 2021-12-10T14:54:34Z
2021-12-10T14:54:34Z
2021-08
info:eu-repo/date/embargoEnd/2022-12-10
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/acceptedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/10884
https://www.sciencedirect.com/science/article/abs/pii/S0304401721001527
0304-4017
https://doi.org/10.1016/j.vetpar.2021.109493
url http://hdl.handle.net/20.500.12123/10884
https://www.sciencedirect.com/science/article/abs/pii/S0304401721001527
https://doi.org/10.1016/j.vetpar.2021.109493
identifier_str_mv 0304-4017
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
eu_rights_str_mv restrictedAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv Veterinary Parasitology 296 : 109493 (August 2021)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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