Detection and characterization of four novel genotypes of Ehrlichia canis from dogs

The genetic diversity of Ehrlichia canis strains worldwide is currently poorly defined. The present study aimed to characterize E. canis strains in naturally infected dogs in Taiwan, using a combination of PCR and sequence analysis of the 16S rDNA and two antigen-encoding genes, gp19 and gp36. Genomic DNA was extracted from 34 parasitemic dogs and the genes of the pathogen were separately amplified, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rDNA sequences (1623 bp) amplified from the Taiwanese isolates were identical and had very high similarity (99.4-100%) with previously reported E. canis sequences. Nevertheless, most of the gp19 gene sequences (414 bp) from the Taiwanese isolates had three specific nucleotide substitutions at positions 9, 323 and 371 that resulted in three amino acid changes. The gp36 gene of the Taiwanese isolates consists of three regions: a 5' end pre-repeat region (426 bp), a tandem repeat region with variable numbers of the 27-bp repeat unit depending on the isolate, and a 3' end region (87 bp). The nucleotide sequences of the 5' end region of gp36 from Taiwanese isolates were identical to each other, but unexpectedly, quite distinct from the sequences of eleven other E. canis strains previously published, with 86.7-87.2% identities only. A phylogenetic tree of E. canis strains based on the gp36 amino acid sequences showed that the Taiwanese isolates fell into a separate clade, indicating the presence of a novel strain that had not yet been characterized.