Identification of Ehrlichia chaffeensis by nested PCR in ticks from Southern China

Abstract

A total of 717 ticks collected from southern China were examined by nested PCR for the presence of Ehrlichia chaffeensis. Sixteen (55. 2%) of 29 adult Amblyomma testudinarium ticks and 28 (11.7%) of 240 adult and at least 4.2% of 215 nymphal (pooled specimens) Haemaphysalis yeni ticks tested positive. Four other species of ticks were negative. Selected positive amplicons were confirmed by DNA sequencing.

FIG. 1

Analytical sensitivity of nested PCR for detection of E. chaffeensis 16S rRNA genes in Chinese ticks. Lanes M, molecular standards. Sizes (in base pairs) are indicated on the left. (A) Products of the primary amplification using serial dilutions of plasmids containing the E. chaffeensis 16S rRNA gene as templates. Lanes 1 through 7, template copy numbers of 8 × 10⁴, 8 × 10³, 8 × 10², 80, 8, 4, and 2, respectively. Lane 8, negative (water) control. The expected size of the primary amplified product is 587 bp. (B) Products of the nested PCR using 1 μl of the corresponding primary product as the template. The expected product size is 389 bp.

FIG. 2

Nested PCR products amplified from representative tick samples. Lane M, DNA marker. Sizes (in base pairs) are indicated on the left. Lane 1, positive control (plasmid containing the E. chaffeensis 16S rRNA gene); lane 2, A. testudinarium adult from cattle; lane 3, H. yeni adult from M. reevesi; lane 4, H. yeni adult from cattle; lane 5, H. yeni adult from Caprologus sinensis; lane 6, H. yeni nymph from L. sinensis; lane 7, water (as a negative control). The expected product size is 389 bp.