Distribución de garrapatas duras (acari: ixodoidea) y detección molecular de bacterias del género Borrelia, Ehrlichia y Rickettsia provenientes de diferentes ecorregiones de Chile
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Date
2023
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Universidad de Concepción
Abstract
Las enfermedades transmitidas por vectores (CVBD), y especialmente por garrapatas, han aumentado a un ritmo desconocido en los últimos años, generando repercusión en la salud pública y animal. En vista de la escasa información epidemiológica sobre la presencia de microorganismos en garrapatas duras en Chile, el objetivo general de esta propuesta fue evaluar la presencia de bacterias Ehrlichia, Borrelia y Rickettsia asociadas a garrapatas duras (Acari: Ixodoidea) provenientes de diferentes ecorregiones de Chile, para responder a la hipótesis que la presencia de bacterias de los géneros Ehrlichia, Borrelia y/o Rickettsia se asocia a zonas con mayor riqueza de garrapatas, esperando encontrar una mayor presencia de estas bacterias en las ecorregiones con climas templados y fríos en Chile. Para esto, se recolectaron 624 garrapatas provenientes de siete ecorregiones de Chile, las cuales se identificaron taxonómica y molecularmente usando un fragmento parcial (≈460-pb) del gen ARNr 16S mitocondrial. Luego, en cada garrapata, se buscó mediante técnicas moleculares la presencia de bacterias del género Borrelia (Spirochaetaceae), Rickettsia (Rickettsiaceae) y Ehrlichia Anaplasmataceae), empleando la reacción en cadena de la polimerasa convencional (PCR) con los genes flaB, gltA y ARNr 16S como screening, respectivamente, para luego amplificar los genes ospC, p66 e IGS para Borrelia, dsb y gltA para Ehrlichia, y ompA, ompB y htrA para Rickettsia. Del total de garrapatas, en 108 (18,68%) se obtuvo ADN de alguna de estas bacterias, siendo la presencia para los géneros Ehrlichia del 0,85%, Rickettsia del 17,5% y para Borrelia del 0,34%. Para Borrelia se efectuó confirmación de su identidad en dos garrapatas positivas (Ixodes abrocomae) utilizando los genes p66, IGS y ospC, los cuales, según BLAST, fueron 93% (611/657 pb), 84% (486/577 pb) y 78,4% (259/329 pb) idénticas a la secuencia de la genoespecie Borrelia chilensis VA1. En el caso de Rickettsia, del total de 103 muestras positivas al gen gltA empleado como screening, solo 10 muestras fueron secuenciadas (9,7%) obtenidas de Amblyomma tigrinum (80%) y de Rhipicephalus sanguineus (20%). Los haplotipos recuperados de A. tigrinum y R. sanguineus formaron un clado con secuencias de Candidatus Rickettsia andaenae obtenidas de Ixodes boliviensis (Perú), Amblyomma parvum (Brasil) y Amblyomma triste (Chile). En el caso de los genes htrA, ompA y ompB, según BLAST, fueron 100% idénticas a secuencias de Ca. R. andaenae obtenida de A. parvum en Brasil (KY402193; MK522488.1; KF030933.1). Para Ehrlichia, 12 ejemplares de tres especies de garrapatas (Ixodes uriae, Ixodes auritulus e Ixodes taglei) fueron positivas al gen ARNr 16S empleado como screening, de estas, 2 (16,6%) obtenidas desde I. uriae (1 ninfa, 1 hembra) fueron secuenciadas, donde la secuencia obtenida de la ninfa fue 100% (306 pb/306 pb) idéntica a Ehrlichia spp. aislada en estudios previos de I. uriae en Chile (MK049840.1). Mientras que, la otra secuencia fue 99% (331 pb/335 pb) idéntica a Candidatus Midichloria mitochondrii obtenida de Ixodes ricinus en Francia (KU559921.1). Para el gen dsb la secuencia fue 94% (209 pb/307 pb) idéntica a una secuencia de Ehrlichia spp. obtenida de bazo de pingüino magallánico (Spheniscus magellanicus) de la Isla Magdalena (MK049838.1), y para el gen gltA fue del 86% (526 pb/611 pb) idénticas a una secuencia de Ehrlichia muris obtenida de bazo de un roedor silvestre de Japón (CP006917). Respecto a la ocurrencia de ADN de bacterias a través de las ecorregiones analizadas, la mayor ocurrencia para bacterias se registró en Puna (85,7%), seguida de Coquimbo (33,5%), Maule (24%), Bosque Valdiviano (23%), Atacama (16,2%), Patagonia central (1,54%) y Santiago (0%), además se evidenció asociación entre la presencia de Rickettsia y Ehrlichia con las ecorregiones. En relación con las variables bioclimáticas, las variables seleccionadas mediante el análisis de componentes principales (PCA) fueron: la temperatura promedio anual (Bio 1), temperatura máxima del periodo más caliente (Bio 5), temperatura mínima del periodo más frio (Bio 6), precipitación anual, rango medio diurno (Bio 2), Precipitación anual (mm) (Bio 12) y Precipitación en el trimestre más caluroso (mm) (Bio 18). Mientras que, al análisis de regresión logística binaria para género de bacteria y de garrapata recolectada, solo se evidenció significancia estadística para el género Ixodes, para las variables Bio1, Bio2, Bio6 y Bio15 (Estacionalidad de la precipitación (Coeficiente de variación). Finalmente, al evaluar la correlación entre la riqueza de garrapatas y el gradiente latitudinal, se obtuvo una correlación baja y no significativa entre estas variables. En el presente estudio, se logró detectar ADN de bacterias del género Borrelia, Ehrlichia y Rickettsia en garrapatas Ixodidae, existiendo asociación estadística entre la mayor ocurrencia de bacterias en las ecorregiones de la zona norte que en la sur.
Vector-borne diseases (CVBD), and especially tick-borne diseases, have increased at an unknown rate in recent years, generating repercussions on public and animal health. In view of the scarce epidemiological information on the presence of microorganisms in hard ticks in Chile, the general objective of this proposal was to evaluate the presence of Ehrlichia, Borrelia and Rickettsia bacteria associated with hard ticks (Acari: Ixodoidea) from different ecoregions of Chile, to respond to the hypothesis that the presence of bacteria of the Ehrlichia, Borrelia and/or Rickettsia genera is associated with areas with greater tick richness, hoping to find a greater presence of these bacteria in ecoregions with temperate and cold climates in Chile. For this, 624 ticks were collected from seven ecoregions of Chile, which were taxonomically and molecularly identified using a partial fragment (≈460-bp) of the mitochondrial 16S rRNA gene. Then, in each tick, the presence of bacteria of the genus Borrelia (Spirochaetaceae), Rickettsia (Rickettsiaceae) and Ehrlichia (Anaplasmataceae) was searched using molecular techniques, using the conventional polymerase chain reaction (PCR) with the genes flaB, gltA and 16S rRNA as screening, respectively, to then amplify the genes ospC, p66 and IGS for Borrelia, dsb and gltA for Ehrlichia, and ompA, ompB and htrA for Rickettsia. Of the total number of ticks, DNA from some of these bacteria was obtained in 108 (18.68%), with the presence for the Ehrlichia genera being 0.85%, Rickettsia 17.5% and for Borrelia 0.34%. For Borrelia, confirmation of its identity was carried out in two positive ticks (Ixodes abrocomae) using the p66, IGS and ospC genes, which, according to BLAST, were 93% (611/657 bp), 84% (486/577 bp) and 78.4% (259/329 bp) identical to the sequence of the genospecies Borrelia chilensis VA1. In the case of Rickettsia, of the total of 103 samples positive for the gltA gene used as screening, only 10 samples were sequenced (9.7%) obtained from Amblyomma tigrinum (80%) and Rhipicephalus sanguineus (20%). The haplotypes recovered from A. tigrinum and R. sanguineus formed a clade with Candidatus Rickettsia andaenae sequences obtained from Ixodes boliviensis (Peru), Amblyomma parvum (Brazil) and Amblyomma triste (Chile). In the case of the htrA, ompA and ompB genes, according to BLAST, they were 100% identical to sequences of Ca. R. andaenae obtained from A. parvum in Brazil (KY402193; MK522488.1; KF030933.1). For Ehrlichia, 12 specimens of three species of ticks (Ixodes uriae, Ixodes auritulus and Ixodes taglei) were positive for the 16S rRNA gene used as screening, of these, 2 (16.6%) obtained from I. uriae (1 nymph, 1 female) were sequenced, where the sequence obtained from the nymph was 100% (306 bp/306 bp) identical to Ehrlichia spp. isolated in previous studies of I. uriae in Chile (MK049840.1). While the other sequence was 99% (331 bp/335 bp) identical to Candidatus Midichloria mitochondrii obtained from Ixodes ricinus in France (KU559921.1). For the dsb gene the sequence was 94% (209 bp/307 bp) identical to a sequence from Ehrlichia spp. obtained from the spleen of a Magellanic penguin (Spheniscus magellanicus) from Magdalena Island (MK049838.1), and for the gltA gene it was 86% (526 bp/611 bp) identical to a sequence of Ehrlichia muris obtained from the spleen of a wild rodent from Japan (CP006917). Regarding the occurrence of bacterial DNA across the ecoregions analyzed, the highest occurrence for bacteria was recorded in Puna (85.7%), followed by Coquimbo (33.5%), Maule (24%), Bosque Valdiviano (23%), Atacama (16.2%), central Patagonia (1.54%) and Santiago (0%), in addition, an association was evident between the presence of Rickettsia and Ehrlichia with the ecoregions. In relation to the bioclimatic variables, the variables selected through principal component analysis (PCA) were the average annual temperature (Bio 1), maximum temperature of the hottest period (Bio 5), minimum temperature of the coldest period (Bio 6), annual precipitation, mean diurnal range (Bio 2), Annual precipitation (mm) (Bio 12) and Precipitation in the hottest quarter (mm) (Bio 18). While, in the binary logistic regression analysis for the genus of bacteria and the collected tick, statistical significance was only evident for the genus Ixodes, for the variables Bio1, Bio2, Bio6 and Bio15 (Seasonality of precipitation (Coefficient of variation). Finally, when evaluating the correlation between tick richness and the latitudinal gradient, a low and non-significant correlation was obtained between these variables. In the present study, it was possible to detect DNA from bacteria of the genus Borrelia, Ehrlichia and Rickettsia in Ixodidae ticks, existing statistical association between the greater occurrence of bacteria in the ecoregions of the northern zone than in the south.
Vector-borne diseases (CVBD), and especially tick-borne diseases, have increased at an unknown rate in recent years, generating repercussions on public and animal health. In view of the scarce epidemiological information on the presence of microorganisms in hard ticks in Chile, the general objective of this proposal was to evaluate the presence of Ehrlichia, Borrelia and Rickettsia bacteria associated with hard ticks (Acari: Ixodoidea) from different ecoregions of Chile, to respond to the hypothesis that the presence of bacteria of the Ehrlichia, Borrelia and/or Rickettsia genera is associated with areas with greater tick richness, hoping to find a greater presence of these bacteria in ecoregions with temperate and cold climates in Chile. For this, 624 ticks were collected from seven ecoregions of Chile, which were taxonomically and molecularly identified using a partial fragment (≈460-bp) of the mitochondrial 16S rRNA gene. Then, in each tick, the presence of bacteria of the genus Borrelia (Spirochaetaceae), Rickettsia (Rickettsiaceae) and Ehrlichia (Anaplasmataceae) was searched using molecular techniques, using the conventional polymerase chain reaction (PCR) with the genes flaB, gltA and 16S rRNA as screening, respectively, to then amplify the genes ospC, p66 and IGS for Borrelia, dsb and gltA for Ehrlichia, and ompA, ompB and htrA for Rickettsia. Of the total number of ticks, DNA from some of these bacteria was obtained in 108 (18.68%), with the presence for the Ehrlichia genera being 0.85%, Rickettsia 17.5% and for Borrelia 0.34%. For Borrelia, confirmation of its identity was carried out in two positive ticks (Ixodes abrocomae) using the p66, IGS and ospC genes, which, according to BLAST, were 93% (611/657 bp), 84% (486/577 bp) and 78.4% (259/329 bp) identical to the sequence of the genospecies Borrelia chilensis VA1. In the case of Rickettsia, of the total of 103 samples positive for the gltA gene used as screening, only 10 samples were sequenced (9.7%) obtained from Amblyomma tigrinum (80%) and Rhipicephalus sanguineus (20%). The haplotypes recovered from A. tigrinum and R. sanguineus formed a clade with Candidatus Rickettsia andaenae sequences obtained from Ixodes boliviensis (Peru), Amblyomma parvum (Brazil) and Amblyomma triste (Chile). In the case of the htrA, ompA and ompB genes, according to BLAST, they were 100% identical to sequences of Ca. R. andaenae obtained from A. parvum in Brazil (KY402193; MK522488.1; KF030933.1). For Ehrlichia, 12 specimens of three species of ticks (Ixodes uriae, Ixodes auritulus and Ixodes taglei) were positive for the 16S rRNA gene used as screening, of these, 2 (16.6%) obtained from I. uriae (1 nymph, 1 female) were sequenced, where the sequence obtained from the nymph was 100% (306 bp/306 bp) identical to Ehrlichia spp. isolated in previous studies of I. uriae in Chile (MK049840.1). While the other sequence was 99% (331 bp/335 bp) identical to Candidatus Midichloria mitochondrii obtained from Ixodes ricinus in France (KU559921.1). For the dsb gene the sequence was 94% (209 bp/307 bp) identical to a sequence from Ehrlichia spp. obtained from the spleen of a Magellanic penguin (Spheniscus magellanicus) from Magdalena Island (MK049838.1), and for the gltA gene it was 86% (526 bp/611 bp) identical to a sequence of Ehrlichia muris obtained from the spleen of a wild rodent from Japan (CP006917). Regarding the occurrence of bacterial DNA across the ecoregions analyzed, the highest occurrence for bacteria was recorded in Puna (85.7%), followed by Coquimbo (33.5%), Maule (24%), Bosque Valdiviano (23%), Atacama (16.2%), central Patagonia (1.54%) and Santiago (0%), in addition, an association was evident between the presence of Rickettsia and Ehrlichia with the ecoregions. In relation to the bioclimatic variables, the variables selected through principal component analysis (PCA) were the average annual temperature (Bio 1), maximum temperature of the hottest period (Bio 5), minimum temperature of the coldest period (Bio 6), annual precipitation, mean diurnal range (Bio 2), Annual precipitation (mm) (Bio 12) and Precipitation in the hottest quarter (mm) (Bio 18). While, in the binary logistic regression analysis for the genus of bacteria and the collected tick, statistical significance was only evident for the genus Ixodes, for the variables Bio1, Bio2, Bio6 and Bio15 (Seasonality of precipitation (Coefficient of variation). Finally, when evaluating the correlation between tick richness and the latitudinal gradient, a low and non-significant correlation was obtained between these variables. In the present study, it was possible to detect DNA from bacteria of the genus Borrelia, Ehrlichia and Rickettsia in Ixodidae ticks, existing statistical association between the greater occurrence of bacteria in the ecoregions of the northern zone than in the south.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Veterinarias
Keywords
Garrapatas, Patología veterinaria