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Resumen
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. [ver mas...]
dc.contributor.authorPrimo, María Evangelina
dc.contributor.authorBellezze, Julio
dc.contributor.authorMorel, Nicolas
dc.contributor.authorMazzucco Panizza, Matilde Nahimé
dc.contributor.authorValentini, Beatriz Susana
dc.contributor.authorTorioni, Susana Marta
dc.contributor.authorThompson, Carolina Soledad
dc.date.accessioned2022-05-09T12:02:38Z
dc.date.available2022-05-09T12:02:38Z
dc.date.issued2022-07
dc.identifier.issn1877-959X
dc.identifier.otherhttps://doi.org/10.1016/j.ttbdis.2022.101952
dc.identifier.urihttp://hdl.handle.net/20.500.12123/11835
dc.identifier.urihttps://www.sciencedirect.com/science/article/abs/pii/S1877959X22000577
dc.description.abstractA 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.eng
dc.formatapplication/pdfes_AR
dc.language.isoenges_AR
dc.publisherElsevieres_AR
dc.rightsinfo:eu-repo/semantics/embargoedAccesses_AR
dc.sourceTicks and Tick-borne Diseases 13 (4) : 101952 (July 2022)es_AR
dc.subjectGanado Bovinoes_AR
dc.subjectCattleeng
dc.subjectPCReng
dc.subjectRFLPes_AR
dc.subjectRestriction Fragment Length Polymorphismeng
dc.subjectAnaplasma marginalees_AR
dc.subjectAnaplasma centralees_AR
dc.subjectExperimentación en Campoes_AR
dc.subjectField Experimentationeng
dc.titleDevelopment 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 cattlees_AR
dc.typeinfo:ar-repo/semantics/artículoes_AR
dc.typeinfo:eu-repo/semantics/articlees_AR
dc.typeinfo:eu-repo/semantics/acceptedVersiones_AR
dc.description.origenEEA Rafaelaes_AR
dc.description.filFil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Primo, María Evangelina. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentinaes_AR
dc.description.filFil: Bellezze, Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Bellezze, Julio. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentinaes_AR
dc.description.filFil: Morel, Nicolas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Morel, Nicolas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Mazzucco Panizza, Matilde Nahimé. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Mazzucco Panizza, Matilde Nahimé. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentinaes_AR
dc.description.filFil: Valentini, Beatriz Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Valentini, Beatriz Susana. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentinaes_AR
dc.description.filFil: Torioni, Susana Marta. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Torioni, Susana Marta. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentinaes_AR
dc.description.filFil: Thompson, Carolina Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentinaes_AR
dc.description.filFil: Thompson, Carolina Soledad. Instituto de Investigación de la Cadena Láctea (IDICAL, INTA-CONICET); Argentinaes_AR
dc.subtypecientifico


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