Detección y caracterización molecular de Anaplasma Phagocytophilum en garrapatas de perros y sangre de perros y equinos de diversas regiones de Costa Rica
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2015
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Anaplasma phagocytophilum es el agente causal de la anaplasmosis granulocítica que afecta tanto a los humanos como a diversas especies de animales domésticos y silvestres por lo que tiene impacto en la salud veterinaria y pública. En Costa Rica, hasta el momento, no se han realizado estudios moleculares para determinar su presenciaenposibles vectores y animales domésticos hospederos; por lo que el principal objetivo de este trabajo fue determinar la presencia A. phagocytophilum en muestras de garrapatas de perros, así como sangre de perros y caballos de diferentes regiones de Costa Rica, mediante reacción en cadena de la polimerasa (PCR). Además se buscó identificar molecularmente las cepas presentes mediante análisis de secuenciación. Para ello se analizaron fragmentos del gen ARNr 16S de A. phagocytophilum de 161 muestras de garrapatas de perros, 420 muestras de sangre de perros y 339 muestras de sangre de equinos. Las muestras que resultaron positivas para este gen fueron analizadas con pruebas de PCR de diferentes regiones genómicas de especies de Anaplasma específicas: msp2 de A. phagocytophilum y groEL de A. phagocytophilum, Anap!asma p!atys, Anap!asma p/atys-likey Anaplasma phagocytophilum-variante Japón. Se determinó que dos (1.24%) muestras de garrapatas (Rhipicephalus sanguineus s.I.), tres (0.71 %) muestras sanguíneas de perros y ninguna muestra sanguínea de equino fueron positivas en el PCR del ARNr 16S. El análisis de las secuencias obtenidas para este gen confirmaron la presencia de A. phagocytophilum en las dos muestras de garrapatas pero no fueron concluyentes para establecer la especie de Anaplasma presente en las tres muestras de sangre de perros, ya que presentaron porcentajes de homología similares con A. phagocytophilum, A. platys y Candidatus Anap/asma camelii. La comparación general de las cinco secuencias obtenidas, muestra que no son idénticas entre sí...
Inglés: Anaplasmaphagocytophilum is the causal agent of the zoonotic granulocytic anaplasmosis, which can affect human and different species of domestic and wild animals, and therefore has significant impact in veterinary and public health. Molecular studies to establish the presence of this bacteria! species in hosts and vectors are currently lackingin Costa Rica; thus the main goal of this work was to investigate the presence of A. phagocytophilumin dog ticks and in blood of dogs and equines from different regions of Costa Rica by polymerase chain reaction (PCR). Also, this investigation aimed to characterize molecularly the strains by sequencing. Far this purpose, the presence of the A. phagocytophilum 16S rRNA gene was investigated in 161 dog ticks, 420 blood samples collected from dogs, and 339 blood samples from equines. DNA samples positive to 16S rRNA were analyzed with additional PCR tests targeting different genomic regions of selected Anaplasma species: the msp2of A. phagocytophi/um, and the groELgene of A. phagocytophilum, Anaplasma platys, Anaplasma p/atys-like and Anap/asmaphagocytophi/um- Japan variant. Two (1.24%) tick samples (Rhipicepha/ussanguineuss.1.), three (0.71 %) samples from dogs, and none of the equine samples were positive by 16S rRNA PCR. Sequencing confirmed the presence of A. phagocytophilum in the two tick samples, but failed to identify the species of Anap/asma present in the 3 dog samples since sequences had similar homology with A. phagocytophilum, A. p/atys and CandidatusAnaplasmacamelii. Overall, com parison of the five 16S sequences showed that they were not identical among them, suggesting the presence of more than one variant and allowed to classify them in two main groups with substantial differences that show the possible presence of a new Anap/asma species. None of the five samples were positive far the msp2PCR, whereas the analysis of the groEL gene confirmed the...
Inglés: Anaplasmaphagocytophilum is the causal agent of the zoonotic granulocytic anaplasmosis, which can affect human and different species of domestic and wild animals, and therefore has significant impact in veterinary and public health. Molecular studies to establish the presence of this bacteria! species in hosts and vectors are currently lackingin Costa Rica; thus the main goal of this work was to investigate the presence of A. phagocytophilumin dog ticks and in blood of dogs and equines from different regions of Costa Rica by polymerase chain reaction (PCR). Also, this investigation aimed to characterize molecularly the strains by sequencing. Far this purpose, the presence of the A. phagocytophilum 16S rRNA gene was investigated in 161 dog ticks, 420 blood samples collected from dogs, and 339 blood samples from equines. DNA samples positive to 16S rRNA were analyzed with additional PCR tests targeting different genomic regions of selected Anaplasma species: the msp2of A. phagocytophi/um, and the groELgene of A. phagocytophilum, Anaplasma platys, Anaplasma p/atys-like and Anap/asmaphagocytophi/um- Japan variant. Two (1.24%) tick samples (Rhipicepha/ussanguineuss.1.), three (0.71 %) samples from dogs, and none of the equine samples were positive by 16S rRNA PCR. Sequencing confirmed the presence of A. phagocytophilum in the two tick samples, but failed to identify the species of Anap/asma present in the 3 dog samples since sequences had similar homology with A. phagocytophilum, A. p/atys and CandidatusAnaplasmacamelii. Overall, com parison of the five 16S sequences showed that they were not identical among them, suggesting the presence of more than one variant and allowed to classify them in two main groups with substantial differences that show the possible presence of a new Anap/asma species. None of the five samples were positive far the msp2PCR, whereas the analysis of the groEL gene confirmed the...
Descripción
Tesis (maestría académica en microbiología)--Universidad de Costa Rica. Sistema de Estudios de Posgrado. Programa de Estudios de Posgrado en Microbiología, Parasitología, Análisis Clínicos e Inmunología, 2015
Palabras clave
ANAPLASMA PHAGOCYTOPHILUM, ANAPLASMA PHAGOCYTOPHILUM - COSTA RICA, ANAPLASMA PHAGOCYTOPHILUM - MICROBIOLOGIA, BACTERIOLOGIA VETERINARIA, DIAGNOSTICO MICROBIOLOGICO, GENOTIPOS, MICROBIOLOGIA MOLECULAR - PRUEBAS, REACCION EN CADENA DE LA POLIMERASA