Caracteriza??o molecular de Ehrlichia canis (Donatien e Lestoquard, 1935) em c?es do estado do Rio de Janeiro

• Ano de defesa: 2018
• Autor(a) principal: Costa, Renata Lins da
• Orientador(a): Massard, Carlos Luiz
• Banca de defesa: Souza, Aline Moreira de, Almosny, N?dia Regina Pereira, Silva, Cl?udia Bezerra da, Souza, Miliane Moreira Soares de
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal Rural do Rio de Janeiro
• Programa de Pós-Graduação: Programa de P?s-Gradua??o em Ci?ncias Veterin?rias
• Departamento: Instituto de Veterin?ria
• País: Brasil
• Palavras-chave em Português: Erliquiose canina; Marcador molecular; Filogenia; Aspectos epidemiol?gicos
• Palavras-chave em Inglês: Canine ehrlichiosis; Molecular marker; Phylogeny; Epidemiologic aspects
• Área do conhecimento CNPq: Medicina Veterin?ria; Parasitologia; Microbiologia
• Link de acesso: https://tede.ufrrj.br/jspui/handle/jspui/4985

The aim of the present study was to characterize molecularly Ehrlichia canis using the membrane glycoprotein genes involved in the host parasite interaction (gp19, gp36, and p28), and also, the thermo unstable elongation factor (tuf) gene for characterization together with the 16S rDNA gene from dog blood samples, as well as to evaluate the distribution of E. canis in dogs from the mesoregions of Rio de Janeiro state. 267 blood samples from dogs from the state of Rio de Janeiro were obtained and tested by the Real-Time PCR (qPCR) targeting a fragment of 93 base pairs (bp) of 16S rDNA gene as a screening method for detection of the positive samples. Before evaluating the genetic diversity of E. canis, the samples were submitted to the molecular detection by 16S rDNA-qPCR. A total of 267 blood samples of dogs from the state of Rio de Janeiro were collected. Among the samples analyzed, 42.3% (n=113/267) were qPCR positive for 16S rDNA gene of E. canis. The average value of Cq observed in positive samples was 34,1 ? 5,1, ranging between 18 to 40 cycles. The detection limit of the qPCR was 10 copies of the plasmid per ?L containing a 16S rDNA gene from E. canis. The determination coefficient of the seven dilutions tested in the standard curve was 99.9% and efficiency de 95.7%. When performing gp19-PCR and gp36-PCR, 100% (n=113/113) and 5.3% (n=6/113) of the samples were positive, respectively. The six PCR positive samples for the 16S rDNA gene also amplified the p28 gene. Only one PCR positive sample for the three genes (gp19, gp36 and p28) in each of the six mesoregions were selected and subjected to analysis of amino acid and nucleotide sequences. The frequency in each mesoregion for the 113 animals positive for E. canis, by the 16S rDNA gene, was 59,29% (n=67/113) belonged to Metropolitan mesoregion, 13.27% (n=15/113) to South Fluminense, 15.04% (n=17/113) to Northern Fluminense, 5.3% (n=6/113) Center Fluminense, 4.42% (n=5/113) to Northwest Fluminense and 2.65% (n=3/113) to Coastal Baixada. The characterization based on the gp19 gene demonstrated that this gene is highly conserved and the samples showed 100% similarity with the Brazilian and worldwide isolates available in GenBank. Using the gp36 gene, it was possible to observe that there are polymorphic points between the sequences. Cluster analysis shows the formation of 3 clades, the sequences of the present study were allocated to the genogroup of the United States, suggesting a genetic similarity between the Brazilian and North American strains. In the protein analysis, all the samples presented repeated sequences (Tandem Repeat Sequences), with 11 replicates. Seven high entropy amino acid sites were observed, with variations of Hx: 0.5 to 0.67 generating mean entropy of 0.6 detected along the TRP36, suggesting a high degree of polymorphism. The ratio of non-silent to silent mutations using both genes showed significant genetic differences (p <0.05) among the isolates of the present study demonstrating that there was positive selection pressure in both genes analyzed. The molecular detection based on the tuf gene revealed that both samples from the Metropolitan and the Baixada Litor?nea mesoregions that presented adequate amplification. In the optimization of the reaction we observed a detection limit of 100 copies for the tuf gene. In the molecular characterization, E. canis-BaixLit and E. canis-Met presented 100% and 99% identity for the tuf and 16S rDNA genes when compared to the reference sequence Jake (CP000107) and Oklahoma (M73221), respectively. In the analysis of the tuf and 16S rDNA genes, the Baixada Coastal sample presented 99% identity. Non-synonymy mutation reflected in the substitution of a Glutamic Acid (E) for a Lysine (K). In the 16S rDNA and tuf genes analysis, we observed the formation of two well defined clades, where the A containing samples of Ehrlichia genus, and the B with other organisms of the genus Anaplasma suggesting genetic distance (0,34 tuf gene and 0,09 to 16S rDNA). To evaluate the distribution of E. canis in the mesoregions of Rio de Janeiro state, the cytological and molecular diagnosis was performed targeting the gp19 gene. From 267 samples, 54.68% (n=146/267) presented basophilic inclusions in monocytes suggestive of parasitism in the cytological evaluation. In 43.80% of the analyzed samples (n=117/267) it was possible to observe the amplification for gp19 gene, which demonstrated a disagreement between cytological and molecular technique results (p=0.0004). Among the 117 animals in which E. canis DNA was detected, 60.68% (n=71/117) belonged to Metropolitan mesoregion, 12.82% (n=15/117) to Fluminense South, 14.52% (n=17/117) to Northern Fluminense, 5.12% (n=6/117) Fluminense Center, 4.27% (n=5/117) to Fluminense Northwest and 2.56% (n=3/117) to Coastal Baixada. It was observed that race and gender had no association (p>0.05) with E. canis positivity. The investigation of gene differences in the isolates of E. canis becomes useful for understanding the parasite-host relationship; in addition, determining the distribution of this bacterium in the evaluated municipalities is advantageous for the updating of the epidemiological chain of canine ehrlichiosis.