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Detec??o de Anaplasma platys em c?es e em carrapatos: padroniza??ode qPCR e an?lise epidemiol?gica no Estado do Rio de Janeiro, Brasil e na regi?o ocidental de Cuba / Detection of Anaplasma platys in dogs and ticks: standardization of qPCR and epidemiological analysis in the State of Rio de Janeiro, Brazil and in western Cuba

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Previous issue date: 2016-03-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / and investigate the circulation of this agent in dogs in the Itaguai microregion, Rio de
Janeiro, Brazil, and dogs and ticks in two provinces of the island of Cuba, analyzing
epidemiological aspects associated with infections caused by this bacterium in dogs. A new
real-time polymerase chain reaction method (qPCR) was patterned to target the citrate
synthase gene (gltA) for the identification of A. platys in naturally infected dogs. The primers
and probe were designed to amplify a fragment of 84 base pairs based on gltA gene sequences
of A. platys available in GenBank. 186 blood samples of dogs from Itaguai microregion, Rio
de Janeiro, Brazil, were tested by qPCR. The same samples were tested by cytology and
nested polymerase chain reaction (nPCR, 16S rDNA) to determine the performance of qPCR
front of these techniques. 17.20% of the samples tested positive by qPCR were significantly
more than that detected by nPCR (13.98%). The qPCR technique was more specific than
cytology, due to false-positive results obtained by optical microscopy. The prevalence of A.
platys in dogs from Itaguai microregion was 14.4%. Dogs less than six months, infested by
ticks, that spend the most of the time restrict to domestic environment and without shelter are
factors associated with infection by this hemoparasite in dogs in the study area. During
research, A. platys held in Cuba, 100 blood samples were collected from residents dogs in
four cities located in the provinces of Havana and Mayabeque. When inspecting the animals,
found ticks were collected, identified and carefully grouped, forming a total of 49 pools. DNA
extracted from blood samples from dogs and ticks were subjected nPCR (16S rDNA). Positive
samples in nPCR were also subjected to conventional PCR (gltA gene), and the products were
sequenced. Only the species Rhipicephalus sanguineus sensu lato was found in Cuban dogs
and 10.2% (n=5/49) of these ticks added to 16.0% (n=16/100) dogs were considered positive
for A. platys. All sequences analyzed of the gltA and 16S rDNA genes, respectively, showed a
99-100% identity with sequences from A. platys reported in other countries. Phylogenetic
analysis showed two clusters defined for the 16S rDNA gene and three clusters defined for the
gltA gene. Based on the gltA gene, the deduced amino acid sequence showed two points of
non-synonymous mutations at positions 88 and 168 compared to the reference sequence
DQ525687. A preliminary study on the epidemiological aspects associated with infection with
A. platys showed no statistical association with the variables studied (p> 0.05). This study
also to report the first evidence of A. platys in both dogs and ticks in Cuba also presents for
the first time the development of a new qPCR method that contributes to the advancement of
research involving A. platys. The epidemiological study in Brazil allowed us to identify
significant factors in the occurrence of canine anaplasmosis, while in Cuba, it can be
concluded that more research is needed to assess what the deciding factors in the transmission
and spread of A. platys in that country. / platys, e investigar a circula??o deste agente em c?es na microrregi?o de Itagua?, Rio de Janeiro,
Brasil, e c?es e carrapatos em duas prov?ncias da ilha de Cuba, analisando aspectos
epidemiol?gicos associados ? infec??o causada por esta bact?ria em c?es. Um novo m?todo de
rea??o em cadeia da polimerase em tempo real (qPCR) foi padronizado com alvo no gene citrato
sintase (gltA) para a identifica??o de A. platys em c?es naturalmente infectados. Os
oligoiniciadores e a sonda foram desenhados para amplificar um fragmento de 84 pares de base
baseado em sequ?ncias do gene gltA de A. platys dispon?veis no GenBank. 186 amostras de
sangue de c?es da microrregi?o de Itagua?, Rio de Janeiro, Brasil, foram testados pela qPCR. As
mesmas amostras foram testadas pela citologia e rea??o em cadeia da polimerase nested (nPCR,
16S rDNA) para determinar o desempenho da qPCR frente ? essas t?cnicas. 17,20% das amostras
testadas pela qPCR foram positivas, significativamente mais do que detectado pela nPCR
(13,98%). A t?cnica de qPCR foi mais espec?fica que a citologia, em virtude dos resultados falsopositivos
obtidos pela microscopia ?ptica. A preval?ncia de A. platys em c?es da microrregi?o de
Itagua? foi de 14,4%. C?es com menos de seis meses, infestados por carrapatos, que possam maior
tempo restrito ao ambiente dom?stico e sem abrigo s?o fatores associados a infec??o por este
hemoparasito em c?es na regi?o do estudo. Durante investiga??o de A. platys realizada em Cuba,
100 amostras de sangue foram coletadas de c?es residentes em quatro cidades localizadas nas
prov?ncias de Habana e Mayabeque. Ao inspecionar os animais, carrapatos encontrados foram
coletados, identificados e criteriosamente agrupados, formando um total de 49 pools. Amostras de
DNA extra?das do sangue dos c?es e de carrapatos foram submetidas a nPCR (16S rDNA).
Amostras positivas na nPCR foram tamb?m submetidas a PCR convencional (gene gltA), e os
produtos foram sequenciados. Somente a esp?cie Rhipicephalus sanguineus sensu lato foi
encontrada em c?es cubanos, e 10,2% (n=5/49) desses carrapatos somado aos 16,0% (n=16/100)
de c?es foram considerados positivos para A. platys. Todas as sequ?ncias analisadas dos genes
gltA e 16S rDNA, respectivamente, mostraram uma identidade de 99-100% com sequ?ncias de A.
platys reportadas em outros pa?ses. A an?lise filogen?tica mostrou dois clusters definidos para o
gene 16S rDNA e tr?s clusters definidos para o gene gltA. Com base no gene gltA, a sequ?ncia de
amino?cidos deduzidos demonstrou dois pontos de muta??es n?o-sin?nimas nas posi??es 88 e 168
comparados com sequ?ncia de refer?ncia DQ525687. Um estudo preliminar sobre os aspectos
epidemiol?gicos associados com a infec??o por A. platys demonstrou nenhuma associa??o
estat?stica com as vari?veis avaliadas (p > 0,05). O presente estudo al?m de relatar a primeira
evid?ncia de A. platys em ambos c?es e carrapatos em Cuba, tamb?m apresenta pela primeira vez
o desenvolvimento de um novo m?todo de qPCR que contribui para o avan?o da pesquisa
envolvendo A. platys. O estudo epidemiol?gico realizado no Brasil permitiu identificar fatores
importantes na ocorr?ncia da anaplasmose canina, enquanto em Cuba, pode-se concluir que mais
investiga??es s?o necess?rias para avaliar quais os fatores decisivos na transmiss?o e dispers?o de
A. platys nesse pa?s.

Identiferoai:union.ndltd.org:IBICT/oai:localhost:jspui/2107
Date11 March 2016
CreatorsSilva, Claudia Bezerra da
ContributorsMassard, Carlos Luiz, Santos, Huarrisson Azevedo, Coelho, Irene da Silva, Guedes, Daniel da Silva, Barreira, Jairo Dias, Macieira, Daniel Barros
PublisherUniversidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria
Source SetsIBICT Brazilian ETDs
LanguagePortuguese
Detected LanguageEnglish
Typeinfo:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis
Formatapplication/pdf
Sourcereponame:Biblioteca Digital de Teses e DissertaƧƵes da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ
Rightsinfo:eu-repo/semantics/openAccess
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