Abstract
Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.
Highlights
Ehrlichia canis, an Anaplasmataceae agent with a ubiquitous distribution, is the etiological agent of canine monocytic ehrlichiosis (CME), a globally dispersed canine infectious disease (Ristic & Holland, 1993)
The genomic DNA extracted from E. canis cultivated in DH82 cells was used as the template for polymerase chain reaction (PCR) targeting a portion of the ehrlichial trp36 gene, which encodes the TRP36 protein
This protein was analyzed by 10% SDS-PAGE under denaturing and nonreducing conditions, revealing a protein band with a molecular mass of 44 kDa, which was expected, since TRP36 surface protein in E. canis species have identical tandem repeats sequences, but they differ in the number of repeats (Figure 3)
Summary
An Anaplasmataceae agent with a ubiquitous distribution, is the etiological agent of canine monocytic ehrlichiosis (CME), a globally dispersed canine infectious disease (Ristic & Holland, 1993). The disease is transmitted by the tick vector Rhipicephalus sanguineus sensu lato (Latreille) (Donatien & Lestoquard, 1935; Groves et al, 1975), which is more active in warm seasons (Harrus & Waner, 2011). This is a determinant factor for the occurrence of high-incidence areas of the disease in tropical and subtropical regions (Ristic & Holland, 1993). Ehrlichia canis expresses immunoreactive glycoproteins with serial repeat regions (tandem repeat protein; TRP) in its genome (Doyle et al, 2006; McBride et al, 2011). Another immunogenic glycoprotein is the 28-kDa outer surface protein (P28), which is encoded by a multigenic locus with at least 22 alleles of the p28 gene (p28-1 to p28-22) in the genome of E. canis (McBride et al, 1999)
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