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

Affiliations
  • 1Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan. ytchung@dragon.nchu.edu.tw

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

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

  • 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.

  • 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.

  • 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.

  • 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.


Reference

1. de Castro MB, Machado RZ, de Aquino LP, Alessi AC, Costa MT. Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings. Vet Parasitol. 2004. 119:73–86.
Article
2. Groves MG, Dennis GL, Amyx HL, Huxsoll DL. Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus). Am J Vet Res. 1975. 36:937–940.
3. Harrus S, Aroch I, Lavy E, Bark H. Clinical manifestations of infectious canine cyclic thrombocytopenia. Vet Rec. 1997. 141:247–250.
Article
4. Harrus S, Waner T, Bark H, Jongejan F, Cornelissen AWCA. Recent advances in determining the pathogenesis of canine monocytic ehrlichiosis. J Clin Microbiol. 1999. 37:2745–2749.
Article
5. Huxsoll DL, Hildebrandt PK, Nims RM, Walker JS. Tropical canine pancytopenia. J Am Vet Med Assoc. 1970. 157:1627–1632.
6. Komnenou AA, Mylonakis ME, Kouti V, Tendoma L, Leontides L, Skountzou E, Dessiris A, Koutinas AF, Ofri R. Ocular manifestations of natural canine monocytic ehrlichiosis (Ehrlichia canis): a retrospective study of 90 cases. Vet Ophthalmol. 2007. 10:137–142.
Article
7. Luo T, Zhang X, Nicholson WL, Zhu B, McBride JW. Molecular characterization of antibody epitopes of Ehrlichia chaffeensis ankyrin protein 200 and tandem repeat protein 47 and evaluation of synthetic immunodeterminants for serodiagnosis of human monocytotropic ehrlichiosis. Clin Vaccine Immunol. 2010. 17:87–97.
Article
8. Mavromatis K, Doyle CK, Lykidis A, Ivanova N, Francino MP, Chain P, Shin M, Malfatti S, Larimer F, Copeland A, Detter JC, Land M, Richardson PM, Yu XJ, Walker DH, McBride JW, Kyrpides NC. The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies. J Bacteriol. 2006. 188:4015–4023.
Article
9. McBride JW, Comer JE, Walker DH. Novel Immunoreactive glycoprotein orthologs of Ehrlichia spp. Ann N Y Acad Sci. 2003. 990:678–684.
10. McBride JW, Corstvet RE, Gaunt SD, Boudreaux C, Guedry T, Walker DH. Kinetics of antibody response to Ehrlichia canis immunoreactive proteins. Infect Immun. 2003. 71:2516–2524.
Article
11. Mylonakis ME, Koutinas AF, Breitschwerdt EB, Hegarty BC, Billinis CD, Leontides LS, Kontos VS. Chronic canine ehrlichiosis (Ehrlichia canis): a retrospective study of 19 natural cases. J Am Anim Hosp Assoc. 2004. 40:174–184.
Article
12. Neer TM, Breitschwerdt EB, Greene RT, Lappin MR. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. J Vet Intern Med. 2002. 16:309–315.
Article
13. Neer TM, Harrus S. Greene CE, editor. Canine monocytotropic ehrlichiosis and neorickettsiosis (E. canis, E. chaffeensis, E. ruminatium, N. sennetsu and N. risticii infection). Infectious Diseases of the Dog and Cat. 2006. St. Louis: Saunders;203–217.
14. Nethery KA, Doyle CK, Zhang X, McBride JW. Ehrlichia canis gp200 contains dominant species-specific antibody epitopes in terminal acidic domains. Infect Immun. 2007. 75:4900–4908.
Article
15. Pinyoowong D, Jittapalapong S, Suksawat F, Stich RW, Thamchaipenet A. Molecular characterization of Thai Ehrlichia canis and Anaplasma platys strains detected in dogs. Infect Genet Evol. 2008. 8:433–438.
Article
16. Siarkou VI, Mylonakis ME, Bourtzi-Hatzopoulou E, Koutinas AF. Sequence and phylogenetic analysis of the 16S rRNA gene of Ehrlichia canis strains in dogs with clinical monocytic ehrlichiosis. Vet Microbiol. 2007. 125:304–312.
Article
17. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007. 24:1596–1599.
Article
18. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994. 22:4673–4680.
Article
19. Unver A, Huang H, Rikihisa Y. Cytokine gene expression by peripheral blood leukocytes in dogs experimentally infected with a new virulent strain of Ehrlichia canis. Ann N Y Acad Sci. 2006. 1078:482–486.
Article
20. Unver A, Rikihisa Y, Kawahara M, Yamamoto S. Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan. Ann N Y Acad Sci. 2003. 990:692–698.
Article
21. Vinasco J, Li O, Alvarado A, Diaz D, Hoyos L, Tabachi L, Sirigireddy K, Ferguson C, Moro MH. Molecular evidence of a new strain of Ehrlichia canis from South America. J Clin Microbiol. 2007. 45:2716–2719.
Article
22. Walker JS, Rundquist JD, Taylor R, Wilson BL, Andrews MR, Barck J, Hogge AL Jr, Huxsoll DL, Hildebrandt PK, Nims RM. Clinical and clinicopathologic findings in tropical canine pancytopenia. J Am Vet Med Assoc. 1970. 157:43–55.
23. Yu XJ, McBride JW, Walker DH. Restriction and expansion of Ehrlichia strain diversity. Vet Parasitol. 2007. 143:337–346.
24. Zhang X, Luo T, Keysary A, Baneth G, Miyashiro S, Strenger C, Waner T, McBride JW. Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains. Clin Vaccine Immunol. 2008. 15:1080–1088.
Article
Full Text Links
  • JVS
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr