Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle
A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPC...
| Autores principales: | , , , , , , |
|---|---|
| Formato: | Artículo |
| Lenguaje: | Inglés |
| Publicado: |
Elsevier
2022
|
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12123/11835 https://www.sciencedirect.com/science/article/abs/pii/S1877959X22000577 https://doi.org/10.1016/j.ttbdis.2022.101952 |
| _version_ | 1855484857779486720 |
|---|---|
| author | Primo, María Evangelina Bellezze, Julio Morel, Nicolas Mazzucco Panizza, Matilde Nahimé Valentini, Beatriz Susana Torioni, Susana Marta Thompson, Carolina Soledad |
| author_browse | Bellezze, Julio Mazzucco Panizza, Matilde Nahimé Morel, Nicolas Primo, María Evangelina Thompson, Carolina Soledad Torioni, Susana Marta Valentini, Beatriz Susana |
| author_facet | Primo, María Evangelina Bellezze, Julio Morel, Nicolas Mazzucco Panizza, Matilde Nahimé Valentini, Beatriz Susana Torioni, Susana Marta Thompson, Carolina Soledad |
| author_sort | Primo, María Evangelina |
| collection | INTA Digital |
| description | A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPCR specific for Anaplasma marginale (nPCR-Am) based on the msp1β gene and an nPCR specific for A. centrale (nPCR-Ac) based on the msp2 operon (msp2-o) gene. Amplicons dilutions of msp1β and msp5 of A. marginale and msp2-o and msp5 of A. centrale and dilutions of parasited erythrocytes (PE) with A. marginale and A. centrale were used to determine the detection limits. The results were 20 DNA copies/reaction and 30 PE for A. marginale and A. centrale by nPCR-RLFP and nPCR-Am/Ac. A mix of msp5-Am and msp5-Ac was used to evaluate the interference of msp5 from one species for the detection of the other. Co-amplification of the DNA from both species was observed up to a 1:7 ratio of one species to the other. Field samples positive for Anaplasma spp. antibodies (n = 260) from 32 herds were evaluated. Strength of agreement between results by nPCR-RFLP and nPCR-Am or nPCR-Ac was 78% (κ = 0.44) and 94% (κ = 0.85), respectively. Thirty-four samples were positive for A. marginale by nPCR-RFLP but negative by nPCR-Am. msp1β amplicons of 10 samples from 5 herds with discrepancies between nPCR-Am and nPCR-RFLP results were cloned and sequenced. The analysis of the msp1β sequence showed several mutations in the target region of the internal forward primer that would explain the failure in the amplification. Only 10 of the 20 coinfections identified by nPCR-Ac/nPCR-Am were detected by nPCR-RFLP. nPCR-RFLP is a sensitive, low-cost and accessible molecular method for low-complexity laboratories. More studies are needed to establish in which circumstances coinfections can be underestimated. |
| format | Artículo |
| id | INTA11835 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| record_format | dspace |
| spelling | INTA118352022-05-09T12:05:17Z Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle Primo, María Evangelina Bellezze, Julio Morel, Nicolas Mazzucco Panizza, Matilde Nahimé Valentini, Beatriz Susana Torioni, Susana Marta Thompson, Carolina Soledad Ganado Bovino RFLP Anaplasma marginale Anaplasma centrale Experimentación en Campo Cattle PCR Restriction Fragment Length Polymorphism Field Experimentation A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPCR specific for Anaplasma marginale (nPCR-Am) based on the msp1β gene and an nPCR specific for A. centrale (nPCR-Ac) based on the msp2 operon (msp2-o) gene. Amplicons dilutions of msp1β and msp5 of A. marginale and msp2-o and msp5 of A. centrale and dilutions of parasited erythrocytes (PE) with A. marginale and A. centrale were used to determine the detection limits. The results were 20 DNA copies/reaction and 30 PE for A. marginale and A. centrale by nPCR-RLFP and nPCR-Am/Ac. A mix of msp5-Am and msp5-Ac was used to evaluate the interference of msp5 from one species for the detection of the other. Co-amplification of the DNA from both species was observed up to a 1:7 ratio of one species to the other. Field samples positive for Anaplasma spp. antibodies (n = 260) from 32 herds were evaluated. Strength of agreement between results by nPCR-RFLP and nPCR-Am or nPCR-Ac was 78% (κ = 0.44) and 94% (κ = 0.85), respectively. Thirty-four samples were positive for A. marginale by nPCR-RFLP but negative by nPCR-Am. msp1β amplicons of 10 samples from 5 herds with discrepancies between nPCR-Am and nPCR-RFLP results were cloned and sequenced. The analysis of the msp1β sequence showed several mutations in the target region of the internal forward primer that would explain the failure in the amplification. Only 10 of the 20 coinfections identified by nPCR-Ac/nPCR-Am were detected by nPCR-RFLP. nPCR-RFLP is a sensitive, low-cost and accessible molecular method for low-complexity laboratories. More studies are needed to establish in which circumstances coinfections can be underestimated. EEA Rafaela Fil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Primo, María Evangelina. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentina Fil: Bellezze, Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Bellezze, Julio. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentina Fil: Morel, Nicolas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Morel, Nicolas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Mazzucco Panizza, Matilde Nahimé. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Mazzucco Panizza, Matilde Nahimé. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentina Fil: Valentini, Beatriz Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Valentini, Beatriz Susana. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentina Fil: Torioni, Susana Marta. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Torioni, Susana Marta. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentina Fil: Thompson, Carolina Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Thompson, Carolina Soledad. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentina 2022-05-09T12:02:38Z 2022-05-09T12:02:38Z 2022-07 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion http://hdl.handle.net/20.500.12123/11835 https://www.sciencedirect.com/science/article/abs/pii/S1877959X22000577 1877-959X https://doi.org/10.1016/j.ttbdis.2022.101952 eng info:eu-repo/semantics/embargoedAccess application/pdf Elsevier Ticks and Tick-borne Diseases 13 (4) : 101952 (July 2022) |
| spellingShingle | Ganado Bovino RFLP Anaplasma marginale Anaplasma centrale Experimentación en Campo Cattle PCR Restriction Fragment Length Polymorphism Field Experimentation Primo, María Evangelina Bellezze, Julio Morel, Nicolas Mazzucco Panizza, Matilde Nahimé Valentini, Beatriz Susana Torioni, Susana Marta Thompson, Carolina Soledad Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle |
| title | Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle |
| title_full | Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle |
| title_fullStr | Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle |
| title_full_unstemmed | Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle |
| title_short | Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle |
| title_sort | development and field evaluation of a nested polymerase chain reaction restriction fragment length polymorphism npcr rflp analysis to identify a marginale infected and a centrale vaccinated cattle |
| topic | Ganado Bovino RFLP Anaplasma marginale Anaplasma centrale Experimentación en Campo Cattle PCR Restriction Fragment Length Polymorphism Field Experimentation |
| url | http://hdl.handle.net/20.500.12123/11835 https://www.sciencedirect.com/science/article/abs/pii/S1877959X22000577 https://doi.org/10.1016/j.ttbdis.2022.101952 |
| work_keys_str_mv | AT primomariaevangelina developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle AT bellezzejulio developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle AT morelnicolas developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle AT mazzuccopanizzamatildenahime developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle AT valentinibeatrizsusana developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle AT torionisusanamarta developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle AT thompsoncarolinasoledad developmentandfieldevaluationofanestedpolymerasechainreactionrestrictionfragmentlengthpolymorphismnpcrrflpanalysistoidentifyamarginaleinfectedandacentralevaccinatedcattle |