Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis
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.
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Elsevier
2021-12-10T14:54:34Z
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Subjects: | Enfermedades de los Animales, Babesia bigemina, Babesia bovis, Diagnóstico, Infección, Animal Diseases, Diagnosis, PCR, Infection, |
Online Access: | 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 |
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Enfermedades de los Animales Babesia bigemina Babesia bovis Diagnóstico Infección Animal Diseases Diagnosis PCR Infection Enfermedades de los Animales Babesia bigemina Babesia bovis Diagnóstico Infección Animal Diseases Diagnosis PCR Infection |
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Enfermedades de los Animales Babesia bigemina Babesia bovis Diagnóstico Infección Animal Diseases Diagnosis PCR Infection Enfermedades de los Animales Babesia bigemina Babesia bovis Diagnóstico Infección Animal Diseases Diagnosis PCR Infection De La Fourniere, Sofía Ana María Paoletta, Martina Guillemi, Eliana Carolina Sarmiento, Nestor Fabian Donati, Pablo Alejandro Wilkowsky, Silvina Elizabeth Farber, Marisa Diana Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis |
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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. |
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Enfermedades de los Animales Babesia bigemina Babesia bovis Diagnóstico Infección Animal Diseases Diagnosis PCR Infection |
author |
De La Fourniere, Sofía Ana María Paoletta, Martina Guillemi, Eliana Carolina Sarmiento, Nestor Fabian Donati, Pablo Alejandro Wilkowsky, Silvina Elizabeth Farber, Marisa Diana |
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De La Fourniere, Sofía Ana María Paoletta, Martina Guillemi, Eliana Carolina Sarmiento, Nestor Fabian Donati, Pablo Alejandro Wilkowsky, Silvina Elizabeth Farber, Marisa Diana |
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De La Fourniere, Sofía Ana María |
title |
Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis |
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 |
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Elsevier |
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2021-12-10T14:54:34Z |
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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 |
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oai:localhost:20.500.12123-108842021-12-10T14:58:00Z Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis De La Fourniere, Sofía Ana María Paoletta, Martina Guillemi, Eliana Carolina Sarmiento, Nestor Fabian Donati, Pablo Alejandro Wilkowsky, Silvina Elizabeth Farber, Marisa Diana Enfermedades de los Animales Babesia bigemina Babesia bovis Diagnóstico Infección Animal Diseases Diagnosis PCR Infection 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 info:eu-repo/date/embargoEnd/2022-12-10 2021-12-10T14:54:34Z 2021-12-10T14:54:34Z 2021-08 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion 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 eng info:eu-repo/semantics/embargoedAccess application/pdf Elsevier Veterinary Parasitology 296 : 109493 (August 2021) |