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Previous issue date: 2010-04-14 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq. / Dogs can be infected with various hemoparasites, and the occurrence of co-infections between
Ehrlichia canis, Babesia canis, Anaplasma platys, and Hepatozoon canis species is very
common, since they have the same tick vector. The objectives of this study were to delineate a
multiplex PCR technique for the simultaneous diagnostic of microorganisms of Babesia and
Ehrlichia genera in canine blood samples, and to realize the partial characterization of
fragments of the 16S rRNA gene of the family Anaplasmataceae agents and, of 18S rRNA
gene of Babesia detected in some samples PCR-positive, comparing the sequences obtained
with sequences of other strains previously deposited in GenBank. Total DNA of 119 blood
samples was extracted, of these, 40 were selected by showing cytoplasmatic inclusions in
leukocytes and/or platelets suggesting infection by agents of Anaplasmataceae family (1E to
40E), 37 by showing piroplasms (1B to 37B), and two by presenting structures of both agents
(M1 and M2), and finally, 40 samples with negative parasitological diagnostic and
hematological exam without alterations. All these samples were tested by PCR to confirm the
absence or presence of these hemoparasites, and them utilized in the multiplex PCR
delineation. In multiplex PCR reactions the primers A17/EC3 were used to amplify an
approximately 600bp region of the 16S rRNA gene of Ehrlichia species and the primers
PIRO-A1/PIRO-B were used to amplify an approximately 450bp region of the 18S rRNA
gene of Babesia species. Validation of multiplex PCR was performed by real time multiplex
PCR. The multiplex PCR was able to simultaneously detect both agents in a DNA sample of a
dog naturally co-infected and all the single infections by Babesia, but does not detected all the
Ehrlichia infections. The real-time multiplex PCR was more sensitive in detect both single
and also co-infections, as well as positive DNA mixtures for the two agents. The sequencing
results confirmed the isolates identity, and that the primers PIRO-A1/PIRO-B also amplified
the DNA of Hepatozoon canis. Phylogenetic analysis indicated that E. canis, A. platys, B.
canis and H. canis species found in this study showed close similarities with sequences
previously deposited in GenBank, forming monophyletic groups. / Os c?es podem se infectar com diversos hemoparasitos, sendo muito comum a ocorr?ncia de
coinfec??es entre as esp?cies Ehrlichia canis, Babesia canis, Anaplasma platys e Hepatozoon
canis, visto que possuem o mesmo carrapato vetor. Este estudo teve como objetivos delinear
uma t?cnica de PCR multiplex para diagnosticar simultaneamente microrganismos dos
g?neros Ehrlichia e Babesia em amostras de sangue de c?es e realizar a caracteriza??o parcial
de fragmentos do gene 16S rRNA de agentes da fam?lia Anaplasmataceae e do gene 18S
rRNA de Babesia detectados em algumas amostras positivas pela PCR, comparando as
sequ?ncias obtidas com as sequ?ncias de outras cepas depositadas previamente no GenBank.
O DNA total de 119 amostras de sangue foi extra?do. Destas, 40 foram selecionadas por
apresentar inclus?es citoplasm?ticas em leuc?citos e/ou plaquetas sugestivas de infec??o por
agentes da fam?lia Anaplasmataceae (1E a 40E), 37 por apresentar formas parasit?rias de
piroplasm?deos (1B a 37B), duas por apresentar estruturas de ambos os agentes (M1 e M2) e,
finalmente, 40 amostras com diagn?stico parasitol?gico negativo e exame hematol?gico sem
altera??es. Todas estas amostras foram testadas por PCR, para a confirma??o da aus?ncia ou
presen?a destes hemoparasitos, e depois utilizadas no delineamento da PCR multiplex. Nas
rea??es de PCR multiplex utilizou-se os oligonucleot?deos iniciadores A17/EC3 que
amplificam um produto de aproximadamente 600pb de uma por??o do gene 16S rRNA de
esp?cies de Ehrlichia e os oligonucleot?deos iniciadores PIRO-A1/PIRO-B que amplificam
um produto de aproximadamente 450pb de uma por??o do gene 18Sr RNA de esp?cies de
Babesia. A valida??o da PCR multiplex foi realizada por PCR multiplex em tempo-real. A
PCR multiplex foi capaz de detectar simultaneamente os dois agentes em uma amostra de
DNA de um c?o naturalmente coinfectado e todas as infec??es individuais por Babesia, mas
n?o detectou todas as infec??es por Ehrlichia. A PCR multiplex em tempo real foi mais
sens?vel em detectar tanto infec??es ?nicas quanto coinfec??es, al?m de misturas de DNA
positivo para os dois agentes. Os resultados dos sequenciamentos confirmaram a identidade
dos isolados, e que os oligonucleot?deos PIRO-A1/PIRO-B amplificaram tamb?m, o DNA de
Hepatozoon canis. As an?lises filogen?ticas indicaram que as esp?cies de E. canis, A. platys,
B. canis e H. canis encontradas neste estudo possuem similaridades pr?ximas com sequ?ncias
previamente depositadas no GenBank, formando grupos monofil?ticos.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1151 |
| Date | 14 April 2010 |
| Creators | Lisb?a, Raquel Silva |
| Contributors | Massard, Carlos Luiz, Oliveira, Carina Elisei de, Fonseca , Adivaldo Henrique da |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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