J Vet Sci.  2010 Dec;11(4):333-340. 10.4142/jvs.2010.11.4.333.

Sequence and phylogenetic analysis of the gp200 protein of Ehrlichia canis from dogs in Taiwan

• Huang CC
• Hsieh YC
• Tsang CL
• Chung YT

• KMID: 1072183
• DOI: http://doi.org/10.4142/jvs.2010.11.4.333

Abstract

Ehrlichia (E.) canis is a Gram-negative obligate intracellular bacterium responsible for canine monocytic ehrlichiosis. Currently, the genetic diversity of E. canis strains worldwide is poorly defined. In the present study, sequence analysis of the nearly full-length 16S rDNA (1,620 bp) and the complete coding region (4,269 bp) of the gp200 gene, which encodes the largest major immunoreactive protein in E. canis, from 17 Taiwanese samples was conducted. The resultant 16S rDNA sequences were found to be identical to each other and have very high homology (99.4~100%) with previously reported E. canis sequences. Additionally, phylogenetic analysis of gp200 demonstrated that the E. canis Taiwanese genotype was genetically distinct from other reported isolates obtained from the United States, Brazil, and Israel, and that it formed a separate clade. Remarkable variations unique to the Taiwanese genotype were found throughout the deduced amino acid sequence of gp200, including 15 substitutions occurring in two of five known species-specific epitopes. The gp200 amino acid sequences of the Taiwanese genotype bore 94.4~94.6 identities with those of the isolates from the United States and Brazil, and 93.7% homology with that of the Israeli isolate. Taken together, these results suggest that the Taiwanese genotype represents a novel strain of E. canis that has not yet been characterized.

Keyword

canine ehrlichiosis; Ehrlichia canis; gp200 gene; phylogenetic analysis; sequence analysis

MeSH Terms

Amino Acid Sequence
Animals
Bacterial Proteins/chemistry/*genetics
Dogs
Ehrlichia canis/*classification/*genetics
Genotype
Molecular Sequence Data
*Phylogeny
RNA, Ribosomal, 16S/genetics
Sequence Alignment
Sequence Analysis, Protein
Taiwan

Figure

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  Fig. 1 Photomicrographs of the Giemsa-stained thin blood smear showing (A) Ehrlichia (E.) canis morula and (B) E. canis inclusion bodies.

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  Fig. 2 Phylogenetic tree based on the partial 16S rDNA sequences of E. canis isolates. To root the tree, the corresponding sequence of Neorickettsia (N.) sennetsu was used as an outgroup. Accession numbers for E. canis isolates and the outgroup species N. sennetsu are given in parentheses. The scale bar indicates the number of substitutions per nucleotide position. The numbers at the nodes represent the percentage of 1,000 bootstrap resamplings.

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  Fig. 3 Alignment of the deduced amino acid sequences of E. canis gp200. Amino acids highlighted in grey represent residues divergent from the USA Jake1 isolate (US1) sequence, while dashes represent gaps. The underlined regions indicate known dominant species-specific antibody epitopes. The GenBank accession number for each sequence is given at the end of the sequence. Abbreviations of specific E. canis strains - US2: USA Jake2 isolate, BRZ: Brazilian Sao Paulo isolate, ISR: Israeli 611 isolate, TWN: Taiwanese sample.

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  Fig. 4 Phylogenetic tree based on deduced amino acid sequences of the E. canis gp200. To root the tree, the sequence of an ortholog (ankyrin protein 200) in E. chaffeensis was used as an outgroup. Accession numbers for E. canis isolates and the outgroup species E. chaffeensis are given in parentheses. The lengths of the lines are proportional to the number of amino acid changes. The scale bar at the lower left indicates the number of substitutions per sequence position. The numbers at the nodes represent the percentage of 1,000 bootstrap resamplings.

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  Fig. 5 The specificity and detection limit of the gp200-targeted PCR amplification using the primer set EC200-F3/R3. (A) The DNA extracted from blood samples of dogs infected by different rickettsia (Lane 1: E. canis, Lane 2: E. chaffeensis, Lane 3: E. ewingii, Lane 4: A. platys, Lane 5: B. canis vogeli, Lane 6: B. gibsoni) and a healthy dog (Lane 7) were subjected to PCR. The products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. Lane M, 100-bp DNA ladder marker; Lane 8: no template DNA. (B) The blood sample with 5% parasitemia was serially diluted 10-fold from 100 to 10-7 (Lanes 1-8, respectively) with blood obtained from a healthy dog. Lane M, 100-bp DNA ladder marker; Lane 9: no template DNA.

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