BACKGROUND: Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses that inflict serious and fatal infections in companion animals and livestock. The aim of this paper was to phylogeneticaly characterise a new species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus from Minas Gerais, Brazil. METHODS: The agent was isolated from the hemolymph of Rhipicephalus (B.) microplus engorged females that had been collected from naturally infested cattle in a farm in the state of Minas Gerais, Brazil. This agent was then established and cultured in IDE8 tick cells. The molecular and phylogenetic analysis was based on 16S rRNA, groEL, dsb, gltA and gp36 genes. We used the maximum likelihood method to construct the phylogenetic trees. RESULTS: The phylogenetic trees based on 16S rRNA, groEL, dsb and gltA showed that the Ehrlichia spp isolated in this study falls in a clade separated from any previously reported Ehrlichia spp. The molecular analysis of the ortholog of gp36, the major immunoreactive glycoproteins in E. canis and ortholog of the E. chaffeensis gp47, showed a unique tandem repeat of 9 amino acids (VPAASGDAQ) when compared with those reported for E. canis, E. chaffeensis and the related mucin-like protein in E. ruminantium. CONCLUSIONS: Based on the molecular and phylogenetic analysis of the 16S rRNA, groEL, dsb and gltA genes we concluded that this tick-derived microorganism isolated in Brazil is a new species, named E. mineirensis (UFMG-EV), with predicted novel antigenic properties in the gp36 ortholog glycoprotein. Further studies on this new Ehrlichia spp should address questions about its transmissibility by ticks and its pathogenicity for mammalian hosts.

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Zobrazit více v PubMed

Doyle KC, Labruna MB, Breitschwerdt EB, Tang YW, Corstvet RE, Hegarty BC, Bloch KC, Li P, Walker DH, McBride JW. Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. J Mol Diagn. 2005;7(4):504–510. doi: 10.1016/S1525-1578(10)60581-8. PubMed DOI PMC

Rikihisa Y. Anaplasma phagocytophilum and Ehrlichia chaffeensis: subversive manipulators of host cells. Nat Rev Microbiol. 2010;8:328–339. doi: 10.1038/nrmicro2318. PubMed DOI

Telford SR III, Goethert HK, Cunningham JA. Prevalence of Ehrlichia muris in Wisconsin deer ticks collected during the mid 1990s. The Open Microbiol J. 2011;5:18–20. doi: 10.2174/1874285801105010018. PubMed DOI PMC

Allsopp MTEP, Louw M, Meyer EC. Ehrlichia ruminantium: an emerging human pathogen? Ann NY Acad Sci. 2005;1063:358–360. doi: 10.1196/annals.1355.060. PubMed DOI

Da Costa VRF, Biondo AW, Sá Guimarăes AM, Dos Santos AP, Dos Santos RP, Dutra LH, De Paiva DPPV, De Morais HA, Messick JB, Labruna MB, Vidotto O. Ehrlichiosis in Brazil. Rev Bras Parasitol Vet Jaboticabal. 2011;20(1):1–12. doi: 10.1590/S1984-29612011000100002. PubMed DOI

Rar V, Golovljova I. Anaplasma, Ehrlichia, and “Candidatus Neoehrlichia” bacteria: pathogenicity, biodiversity, and molecular genetic characteristics, a review. Infect Gen and Evol. 2011;11(8):1842–1861. doi: 10.1016/j.meegid.2011.09.019. PubMed DOI

Inokuma H, Brouqui P, Drancourt M, Raoult D. Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia. J Clin Microbiol. 2001;39(9):3031–3039. doi: 10.1128/JCM.39.9.3031-3039.2001. PubMed DOI PMC

Wen B, Jian R, Zhang Y, Chen R. Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet. J Clin Microbiol. 2002;40(9):3286–3290. doi: 10.1128/JCM.40.9.3286-3290.2002. PubMed DOI PMC

Warner CK, Dawson JE. In: PCR protocols for emerging infectious diseases. Persing DH, editor. Washington DC: ASM Press; 1996. Genus- and species-level identification of Ehrlichia species by PCR and sequencing; pp. 100–105.

Sumner JW, Nicholson WL, Massung RF. PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species. J Clin Microbiol. 1997;35(8):2087–2092. PubMed PMC

Yu XJ, Zhang XF, McBride JW, Zhang Y, Walker DH. Phylogenetic relationships of Anaplasma marginale and ‘Ehrlichia platys’ to other Ehrlichia species determined by GroEL aa sequences. Int J Syst Evol Microbiol. 2001;51(3):1143–1146. doi: 10.1099/00207713-51-3-1143. PubMed DOI

Sacchi ABV, Duarte JMB, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus) Comp Immun Microbiol and Inf Dis. 2012;35(4):325–334. doi: 10.1016/j.cimid.2012.02.001. PubMed DOI

Hsieh YC, Lee CC, Tsang CL, Chung YT. Detection and characterization of four novel genotypes of Ehrlichia canis from dogs. Vet Microbiol. 2010;146(1–2):70–75. PubMed

Doyle CK, Nethery KA, Popov VL, McBride JW. Differentially expressed and secreted major immunoreactive protein orthologs of Ehrlichia canis and E. chaffeensis elicit early antibody responses to epitopes on glycosylated tandem repeats. Infect Immun. 2006;74(1):711–720. doi: 10.1128/IAI.74.1.711-720.2006. PubMed DOI PMC

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 and Vacc Immun. 2008;15(7):1080–1088. doi: 10.1128/CVI.00482-07. PubMed DOI PMC

Zweygrath E, Schöl H, Lis K, Cabezas Cruz A, Thiel C, Silaghi C, Ribeiro MFB, Passos LMF. In vitro culture of a new genotype of Ehrlichia sp. from Brazil. Orvieto: Joint Conference on Emerging and Re-emerging Epidemics Affecting Global Health 2012; 2012. Proceedings.

Munderloh UG, Liu Y, Wang M, Chen C, Kurtti TJ. Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. J Parasitol. 1994;80(4):533–543. doi: 10.2307/3283188. PubMed DOI

Munderloh UG, Kurtti TJ. Formulation of medium for tick cell culture. Exp Appl Acarol. 1989;7(3):219–229. doi: 10.1007/BF01194061. PubMed DOI

Dumler JS. et al.Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol. 2001;51(6):2145–2165. doi: 10.1099/00207713-51-6-2145. PubMed DOI

Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1–2):203–214. PubMed

Thompson JD, Higgins DG, Gibson TJ. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. PubMed DOI PMC

ExPASy Translation Tool. ExPASy Translation Tool. http://expasy.hcuge.ch/tools/dna.html.

Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–1797. doi: 10.1093/nar/gkh340. PubMed DOI PMC

Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17(4):540–552. doi: 10.1093/oxfordjournals.molbev.a026334. PubMed DOI

Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003;52(5):696–704. doi: 10.1080/10635150390235520. PubMed DOI

Anisimova M, Gascuel O. Approximate likelihood ratio test for branchs: a fast, accurate and powerful alternative. Syst Biol. 2006;55(4):539–552. doi: 10.1080/10635150600755453. PubMed DOI

Chevenet F, Brun C, Banuls AL, Jacq B, Chisten R. TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinf. 2006;10(7):439. PubMed PMC

Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of theircleavage sites. Protein Eng. 1997;10(1):1–6. doi: 10.1093/protein/10.1.1. PubMed DOI

Julenius K, Molgaard A, Gupta R, Brunak S. Prediction, conservation analysis, and structural characterization of mammalian mucin type O-glycosylation sites. Glycobiol. 2005;15(2):153–164. PubMed

NetNGlyc 1.0 Server. http://www.cbs.dtu.dk/services/NetNGlyc/.

Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999;27(2):573–580. doi: 10.1093/nar/27.2.573. PubMed DOI PMC

Ponomarenko JV, Bourne PE. Antibody-protein interactions: benchmark datasets and prediction tools evaluation. BMC Struct Biol. 2007;7:64–83. doi: 10.1186/1472-6807-7-64. PubMed DOI PMC

Ponomarenko J, Bui HH, Li W, Fusseder N, Bourne PE, Sette A, Peters B. ElliPro: a new structure-based tool for the prediction of antibody epitopes. BMC Bioinf. 2008;9:514–521. doi: 10.1186/1471-2105-9-514. PubMed DOI PMC

Wood TC, Pearson WR. Evolution of protein sequences and structures. J Mol Biol. 1999;291:977–995. doi: 10.1006/jmbi.1999.2972. PubMed DOI

Woese CR. Bacterial evolution. Microbiol Rev. 1987;51(2):221–271. PubMed PMC

Parola P, Cornet JP, Sanogo YO, Miller RS, Thien HV, Gonzalez JP, Raoult D, Telford SR III, Wongsrichanalai C. Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J Clin Microbiol. 2003;41(4):1600–1608. doi: 10.1128/JCM.41.4.1600-1608.2003. PubMed DOI PMC

Birdsell JA. Integrating genomics, bioinformatics, and classical genetics to study the effects of recombination on genome evolution. Mol Biol Evol. 2002;19(7):1181–1197. doi: 10.1093/oxfordjournals.molbev.a004176. PubMed DOI

Alonso M, Arellano-Sota C, Cereser VH, Cordoves CO, Guglielmone AA, Kessler R, Mangold AJ, Nari A, Patarroyo JH, Solari MA, Vega CA, Vizcaino O, Camus E. Epidemiology of bovine anaplasmosis and babesiosis in Latin America and the Caribbean. Vet Sci Tech Off Int Epiz. 1992;11(3):713–733. PubMed

Pan H. et al.Amplification of 16S rRNA gene fragments of Ehrlichia canis from ticks in southern China. Chin J Zoon. 1999;15(3):3–6.

Jiang BG, Cao WC, Niu JJ, Wang JX, Li HM, Sun Y, Yang H, Richadus JH, Habbema JD. Detection and identification of Ehrlichia species in Rhipicephalus (Boophilus) microplus ticks in cattle from Xiamen China. Vector Borne Zoon Dis. 2011;11(3):325. PubMed

The glycoprotein TRP36 of Ehrlichia sp. UFMG-EV and related cattle pathogen Ehrlichia sp. UFMT-BV evolved from a highly variable clade of E. canis under adaptive diversifying selection

Zobrazit více v PubMed

GENBANK
JX629805, JX629806, JX629807, JX629808, JX629809