Isolamento, propagação, caracterização genética e mecanismo de evasão de Ehrlichia canis in vitro

Abstract

Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis (CME), one of the most important infectious diseases of dogs in Brazil. The amount of information about the genetic variability and the mechanisms used by this pathogen to ensure their immune evasion remains insufficient. Therefore, the present study reports the first isolation and culture of Ehrlichia canis in Uberlândia, from a naturally infected dog using the DH82 cell line. Partial sequences of dsb and p28 genes of the new E. canis isolate were obtained and aligned with the corresponding sequences of other Ehrlichia strains accessible in GenBank. Furthermore, the possible fusion of lysosomes with endosomes containing E. canis was investigated in infected DH82 cells. Lysosomes in cells with 50% infectivity were labeled cytochemically with acid phosphatase to document endosome-lysosome (E-L) fusion. The new strain, designated as Uberlândia isolate, showed 99─100% of similarity with the dsb gene sequence of E. canis from different geographic areas including: Brazil, Africa and North America. On the other hand, the p28 partial sequence for E. canis Uberlândia isolate differed in several nucleotides from the corresponding sequences of the E. canis strains from Brazil, Venezuela and North America. Through these variations the antigenic index predicted potential antigenic determinants of E. canis strains and similarities in antigenicity were observed, however, some differences in deduced epitopes suggest that this protein is antigenically different among strains, regardless of origin. These findings suggest that new studies are necessary to evaluate the utility of this antigen for serodiagnosis of CME. In relation to cytochemical analysis, E-L fusion was generally not observed in active E. canis containing vacuoles, indicating that E. canis is able to inhibit this mechanism of host defense.