Aportaciones al diagnóstico serológico y molecular de la piroplasmosis equina

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Aportaciones al diagnóstico serológico y molecular de la piroplasmosis equina

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Title: Aportaciones al diagnóstico serológico y molecular de la piroplasmosis equina
Author: Montes Cortés, María Guadalupe
Abstract: La piroplasmosis equina es una enfermedad protozoaria transmitida por garrapatas extendida mundialmente, que genera grandes pérdidas económicas. Hemos realizado un estudio seroepidemiológico en una amplia zona de España (n=3100), empleando la Inmunofluorescencia Indirecta (IFI) como método diagnóstico. El estudio reveló una seroprevalencia superior a 50%. Adicionalmente, desarrollamos una técnica combinando la PCR múltiple y la PCR anidada (mn-PCR), basada en los genes “18SrRNA”, “β-tubulina”, “EMA-1”, “RAP-1” y citocromo B (“CytB”). Esta metodología demostró ser eficaz, mostrando una prevalencia aún más elevada (72,77%, n=235). Mediante ambas técnicas, la presencia de “Theileria equi” fue superior a la de “Babesia caballi”. Además, se hicieron dos estudios comparativos entre técnicas usando el índice κ de Cohen. Las dos comparaciones, IFI vs. kit comercial cELISA y mn-PCR vs. kit comercial cELISA, mostraron que la concordancia fue superior para “T. equi” que para “B. caballi”. Al secuenciar un fragmento del gen “RAP-1” de “B. caballi” de cepas españolas, y tras el pertinente análisis filogenético usando dicho gen, se constató que las cepas españolas se situaban en uno de los dos superclados. En el otro, las americanas, cuyos antígenos se emplean para el diseño del kit cELISA. Así, se ratificaría que la diversidad genética podría impedir un diagnóstico eficaz de las cepas españolas de “B. caballi”. También se aportó una secuencia del gen de la “β-tubulina” de “B. caballi” de origen español. Se confirmó que la procedencia geográfica, la raza y la edad (fundamentalmente para “T. equi”) eran factores de riesgo para la incidencia de esta enfermedad.Equine piroplasmosis is a worldwide spread protozoan disease transmitted by ticks, which produces great economic losses. A seroepidemiological study was conducted in a large area of Spain (n=3100), using the Immunofluorescence Antibody Test (IFAT). This study showed a higher seroprevalence than 50%. Additionally, we developed a technique, a multinested PCR (mn-PCR), which combined the multiple PCR with the nested PCR, using the “18S rRNA”, “β-tubulin”, “EMA-1”, “RAP-1” and cytochrome b (“cytB”) genes. This technique was effective and it showed a higher prevalence (72.77%, n=235) than the IFAT. Also, two comparative studies between different tests were made utilizing the Cohen´s κ index. Both comparisons, IFAT vs. cELISA commercial kit and mn-PCR vs. cELISA commercial kit, showed that the concordance was higher for “Theileria equi” than for “Babesia caballi”. The prevalence of T. equi was higher than that of “B. caballi” using the three techniques. A fragment of the “B. caballi RAP-1” gene from three Spanish strains were sequenced and a phylogenetic analysis was performed using that gene. The Spanish strains were found in one of the two superclades. The American strains were in the other superclade, whose antigens are used for the design of the cELISA kit. It would ratify that genetic variability could avoid the effective diagnosis of the “B. caballi” Spanish strains. A sequence of the “β-tubulin” gene of “B. caballi” from Spain was also provided. The geographical location, the breed and the age (mainly for “T. equi”) were found as risk factors for the presence of this disease.
Description: Tesis por compendio de publicaciones
URI: http://hdl.handle.net/10662/9733
Date: 2019-09-20


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