Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-03T15:45:32Z No. of bitstreams: 1 2010 - Raquel Silva Lisboa.pdf: 4230911 bytes, checksum: e7087ccd29d417f592ca09ec5f49c657 (MD5) / Made available in DSpace on 2016-08-03T15:45:32Z (GMT). No. of bitstreams: 1 2010 - Raquel Silva Lisboa.pdf: 4230911 bytes, checksum: e7087ccd29d417f592ca09ec5f49c657 (MD5) 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.