Determining the analytical sensitivity of polymerase chain reaction targeting Ehrlichia spp. disulfide oxidoreductase gene: Molecular diagnosis of ehrlichiosis in a dog clinically suspected with leishmaniasis

Abstract

Ehrlichia spp. is tick-borne zoonotic pathogen that can infect humans and animals. Nowadays, among the tests used in the diagnosis of ehrlichiosis, the importance of molecular methods is increasing steadily due to their high sensitivity and specificity. The aim of this study was to determine the analytical sensitivity of a conventional polymerase chain reaction (PCR) targeting Ehrlichia spp. disulfide oxidoreductase (DSB) gene. Ehrlichia spp. DSB gene was cloned into the TOPO vector. After TOPO plasmid containing DSB gene were serially diluted, PCR targeting the Ehrlichia spp. DSB gene was performed. While working on this research, blood and skin scraping samples of a stray dog clinically suspected with leishmaniasis as well as treated for leishmaniasis arrived to our laboratory. Thereafter, PCRs targeting Ehrlichia spp. DSB and 16S rRNA and Leishmania kinetoplast DNA (kDNA) genes were performed to identify the pathogen in blood and skin scraping samples of the stray dog. The analytical sensitivity of the PCR assay targeting Ehrlichia spp. DSB gene was 1 ≥ copy plasmid/reaction using serially diluted TOPO plasmid containing DSB gene. PCR targeting the Ehrlichia spp. DSB gene was positive and PCR targeting Leishmania spp. kDNA was negative in blood and skin samples of the stray dog clinically suspected with leishmaniasis. Using nested PCR targeting Ehrlichia spp. 16S rRNA, E. canis was identified in blood and skin scraping samples of the stray dog. In this study, PCR targeting Ehrlichia spp. DSB gene has been shown to have high sensitivity. Also it was shown molecular methods can help clinicians in differential diagnosis of ehrlichiosis and leishmaniasis to prevent inappropriate treatment.