Molecular Survey and Genetic Analysis of Ehrlichia canis in Rhipicephalus sanguineus Ticks Infesting Dogs in Northern Taiwan

Abstract

Ehrlichia canis is a tick-transmitted zoonotic pathogen in dogs. We conducted a molecular survey for screening of E. canis infection in Rhipicephalus sanguineus ticks infesting dogs and identified its genetic identity in Taiwan. A total of 1195 R. sanguineus ticks were collected and examined for Ehrlichia infection by nested-PCR assay targeting the 16S ribosomal RNA (rRNA) gene. In general, Ehrlichia infection was detected in 1.5% of examined ticks, and was detected in nymph, male and female stages with infection rates of 0.6%, 1.31% and 2.76%, respectively. The highest monthly prevalence was observed in August with an infection rate of 5.91%. Genetic identity was analyzed by comparing the 16S rRNA gene sequences obtained from 11 Taiwan strains and 15 other strains representing five genospecies of Ehrlichia spp., including two outgroups (Anaplasma phagocytophilum and Rickettsia rickettsii). Results revealed that all Taiwan strains were genetically affiliated to the same clade within various E. canis strains documented in GenBank with a high sequence similarity (99.7-100%) and that they can be clearly distinguished from other genospecies of Ehrlichia. This study provides the first evidence of E. canis identified in R. sanguineus ticks and highlights the potential threat for human infections in Taiwan.

Keywords: Ehrlichia canis; Rhipicephalus sanguineus; Taiwan; tick; zoonosis.

Figure 1

Molecular detection of E. canis infection in Rhipicephalus sanguineus ticks infesting dogs of Taiwan by a nested PCR assay targeting the 16S rRNA gene. M, 100 bp DNA marker; 1–14, sample numbers; 15, negative control. The expected PCR product is 390 bp for the 16S rRNA gene.

Figure 2

Monthly prevalence of E. canis infection and the number of R. sanguineus ticks collected from dogs in northern Taiwan.

Figure 3

Phylogenetic tree based on the 16S rRNA gene. The aligned sequences of eleven Taiwan strains (indicated as ●) detected in R. sanguineus ticks of Taiwan were compared with available sequences from GenBank, including thirteen strains of Ehrlichia spp. and two outgroup strains identified from different biological and geographical origins. The constructed tree was analyzed by the Maximum Likelihood (ML) method using 1000 bootstrap replicates. Branch length is drawn proportional to the estimated sequence divergence. Numbers at the nodes indicate the percentage reliability of the tree.

Figure 4

Phylogenetic tree based on the 16S rRNA gene. The aligned sequences of eleven Taiwan strains (indicated as ●) detected in R. sanguineus ticks of Taiwan were compared with available sequences from GenBank, including thirteen strains of Ehrlichia spp. and two outgroup strains identified from different biological and geographical origins. The constructed tree was analyzed by the neighbor-joining (NJ) method using 1000 bootstrap replicates. Branch length is drawn proportional to the estimated sequence divergence. Numbers at the nodes indicate the percentage reliability of the tree.