Detecção molecular de Ehrlichia canis em cães e em órgãos de seus respectivos carrapatos
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Data
2016-05-02
Autores
Oliveira, Bruno César Miranda [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A erliquiose canina apresenta elevada ocorrência na rotina da clínica médica de pequenos animais. Esta bactéria multiplica-se nas células epiteliais do intestino, nos hemócitos e nas células das glândulas salivares do carrapato Riphicephalus sanguineus, onde ocorre a transmissão transestadial e provavelmente não ocorre transmissão transovariana. O objetivo do presente estudo foi detectar molecularmente Ehrlichia canis em amostras de linfonodos e medulas ósseas de cães e nos intestinos, ovários e glândulas salivares de seus respectivos carrapatos R. sanguineus. Assim, 720 artrópodes fêmeas (dez de cada animal) foram removidos dos 72 cães examinados. Em seguida, foram efetuadas punções aspirativas de linfonodos e medulas ósseas. Após a dissecação dos carrapatos, seus órgãos (intestinos, ovários e glândulas salivares), bem como as amostras puncionadas de linfonodos e medulas ósseas dos cães foram testadas por meio da Reação em Cadeia da Polimerase (nested-PCR) para amplificação de um fragmento do gene 16S do ácido ribonucleico ribossomal (rRNA) de E. canis e testados também por meio da PCR em Tempo Real Quantitativa (qPCR) para E. canis, baseada em um fragmento do gene dsb. A detecção por meio da nested-PCR foi de 80,5% nos linfonodos e 44,4% nas medulas ósseas, havendo diferença significativa (p<0,05). Já em relação às nested-PCRs dos órgãos dos carrapatos, observamos positividade de 22% nos intestinos, 11% nos ovários e 7% nas glândulas salivares. Na qPCR a detecção foi de 70,8% e 44,4% nos linfonodos e medulas ósseas, respectivamente (p<0,05) e de 31,9% nos intestinos, 10,0% nos ovários e 15, 2% nas glândulas salivares dos carrapatos estudados. As cargas parasitárias médias nos linfonodos foram de 1473,79 Cópias de um fragmento do gene dsb de E. canis/µL, nas medulas ósseas de 2080,09 Cópias/µL e nos intestinos, ovários e glândulas salivares as quantificações foram de 1211,53 Cópias/µL, 2602,01 Cópias/µL e de 49,23 Cópias/µL respectivamente. Nós concluímos que houve maior detecção molecular em linfonodos tanto na nested-PCR quanto na qPCR e que a bactéria estava presente e a níveis quantificáveis pela primeira vez em ovários de R. sanguineus sensu lato.
The canine erlichiosis presents a high level of occurrence in the routine of the medical clinic of small animals. This bacteria multiplies in the epithelial cells of the intestine, in the hemocytes and in the salivary glands of the tick Riphicephalus sanguineus, where there is a transestadial transmission and probably does not occur a transovarial transmission. The objective of our study was to molecularly detect Ehrlichia canis in samples of lymph nodes and bone marrow of dogs and in the intestines, ovaries and salivary glands of their respective ticks R. sanguineus. Therefore, 720 female arthropods (ten of each animal) were removed from the 72 evaluated dogs. Next, aspirative punches were made of lymph nodes e bone marrow. After the dissection of the ticks, their organs (intestines, ovaries and salivary glands), like the punches samples of lymph nodes and bone marrow of the dogs were tested by Polymerase Chain Reaction (nested-PCR) for amplification of the gene 16S rRNA of E. canis and also tested by the Quantitative Real Time PCR (qPCR) to E. canis, based on a fragment of the gene dsb. The detection of the refer pathogen by the nested-PCR was 80,5% in lymph nodes and 44,4% in bone marrows, showing a significant difference (p<0,05). ). In relation to the nested-PCRs of the tick’s organs, we observed positivity of 22% on the intestines, 11% on the ovaries and 7% on the salivary glands. In the qPCR the detection was of 70,8% and 44,4% on the lymph nodes e bone marrows, respectively (p<0,05) and of 31,9% on the intestines, 10,0% on the ovaries and 15, 2% on the salivary glands of the studied ticks. The absolute quantification on the lymph nodes and bone marrow of the dogs was of 1473,79 Copies of a fragment of the gene dsb of E. canis/µL, in bone marrows of 2080,09 copies/µL and in the intestines, ovaries and salivary glands the quantification were of 1211,53 copies/µL, 2602,01 copies/µL and of 49,23 copies/µL respectively. We concluded that there was a major molecular detection on lymph nodes on the nested-PCR as much as the qPCR and that the bacteria was present and in quantifiable levels for the first time in ovaries of R. sanguineus sensu lato.
The canine erlichiosis presents a high level of occurrence in the routine of the medical clinic of small animals. This bacteria multiplies in the epithelial cells of the intestine, in the hemocytes and in the salivary glands of the tick Riphicephalus sanguineus, where there is a transestadial transmission and probably does not occur a transovarial transmission. The objective of our study was to molecularly detect Ehrlichia canis in samples of lymph nodes and bone marrow of dogs and in the intestines, ovaries and salivary glands of their respective ticks R. sanguineus. Therefore, 720 female arthropods (ten of each animal) were removed from the 72 evaluated dogs. Next, aspirative punches were made of lymph nodes e bone marrow. After the dissection of the ticks, their organs (intestines, ovaries and salivary glands), like the punches samples of lymph nodes and bone marrow of the dogs were tested by Polymerase Chain Reaction (nested-PCR) for amplification of the gene 16S rRNA of E. canis and also tested by the Quantitative Real Time PCR (qPCR) to E. canis, based on a fragment of the gene dsb. The detection of the refer pathogen by the nested-PCR was 80,5% in lymph nodes and 44,4% in bone marrows, showing a significant difference (p<0,05). ). In relation to the nested-PCRs of the tick’s organs, we observed positivity of 22% on the intestines, 11% on the ovaries and 7% on the salivary glands. In the qPCR the detection was of 70,8% and 44,4% on the lymph nodes e bone marrows, respectively (p<0,05) and of 31,9% on the intestines, 10,0% on the ovaries and 15, 2% on the salivary glands of the studied ticks. The absolute quantification on the lymph nodes and bone marrow of the dogs was of 1473,79 Copies of a fragment of the gene dsb of E. canis/µL, in bone marrows of 2080,09 copies/µL and in the intestines, ovaries and salivary glands the quantification were of 1211,53 copies/µL, 2602,01 copies/µL and of 49,23 copies/µL respectively. We concluded that there was a major molecular detection on lymph nodes on the nested-PCR as much as the qPCR and that the bacteria was present and in quantifiable levels for the first time in ovaries of R. sanguineus sensu lato.
Descrição
Palavras-chave
Erliquiose monocítica canina – diagnóstico, Parasitologia, Rhipicephalus sanguineus, Canine monocytic ehrlichiosis – diagnosis, Parasitology