Detection of Ehrlichia spp. and Theileria spp. in Hyalomma anatolicum ticks collected in Tajikistan

The objectives of our study were to survey the prevalence of genetic markers for Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. in Hyalomma anatolicum ticks collected in southwestern Tajikistan and to perform sequencing and phylogenetic analysis of fragments of the 16S rRNA gene and groESL ope-ron from Ehrlichia spp. and fragments of the 18S rRNA gene of Theileria spp. detected in H. anatolicum ticks. Hyalomma anatolicum ticks collected in the Tursunzade and Rudaki districts of Tajikistan were tested for DNA of Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. by PCR with specific primers. The amplified fragments were sequenced and analyzed. DNA of Ehrlichia spp. (3.3 %) and Theileria spp. (3.3 %) was detected only in H. anatolicum ticks collected from the Rudaki district, and DNA of Ehrlichia spp. (0.7 %) was found in H. anatolicum ticks from the Tursunzade district. Sequence analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. revealed high similarity to Ehrlichia spp. The Tajik isolates of Theileria spp. were genotyped as Theileriaannulata based on the analysis of 18S rRNA gene sequences. The phylogenetic analysis demonstrates that Ehrlichia spp. isolates are highly similar to Ehrlichia spp. circulating in China and Brazil. The isolate Tajikistan-5 is closely related to the putative novel species Ehrlichia mineirensis. The Tajik isolates of Theileria spp. were clustered with T. annulata isolates from Turkey, Iran, Pakistan, and China by phylogenetic analyses.

1. Aguiar D.M., Ziliani T.F., Zhang X., Melo A.L., Braga I.A., Witter R. A novel Ehrlichia genotype strain distinguished by the TRP36 gene naturally infects cattle in Brazil and causes clinical manifestations associated with ehrlichiosis. Ticks Tick Borne Dis. 2014;5(5):537-544. DOI 10.1016/j.ttbdis.2014.03.010.

2. Begum F., Wisseman C.J., Casals J. Tick-borne viruses of West Pakistan: IV. Viruses similar to or identical with, Crimean hemorrhagic fever (Congo-Semunya), Wad Medani and Pak Argas 461 isolated from ticks of the Changa Manga Forest, Lahore District, and of Hunza, Gilgit Agency, W. Pakistan. Am. J. Epidemiol. 1970;92(3): 197-202.

3. Cabezas-Cruz A., Valdes J.J., de la Fuente J. 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. Parasit. Vectors. 2014;7:584. DOI 10.1186/s13071-014-0584-5.

4. Cruz A.C., Zweygarth E., Ribeiro M.F., da Silveira J.A., de la Fuente J., Grubhoffer L., Valdes J.J., Passos L.M. New species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus shows an ortholog of the E. canis major immunogenic glycoprotein gp36 with a new sequence of tandem repeats. Parasit. Vectors. 2012;11(5):291. DOI 10.1186/1756-3305-5-291.

5. Gajadhar A.A., Lobanov V., Scandrett W.B., Campbell J., Al-Adhami B. A novel Ehrlichia genotype detected in naturally infected cattle in North America. Vet. Parasitol. 2010;173(3-4):324-329. DOI 10.1016/j.vetpar.2010.06.034.

6. Gresikova M., Sekeyova M., Tempera G., Guglielmino S., Castro A. Identification of a Sindbis virus strain isolated from Hyaloma marginatum ticks in Sicily. Acta Virol. 1978;22(3):231-232.

7. Parola P., Inokuma H., Camicas J.L., Brouqui P., Raoult D. Detection and identification of spotted fever group Rickettsiae and Ehrlichiae in African ticks. Emerg. Infect. Dis. 2001;7(6):1014-1017. DOI 10.3201/eid0706.010616.

8. Petrova I.D., Kononova Iu.V., Chausov E.V., Shestopalov A.M., Tishkova F.Kh. Genetic variants of the Crimean-Congo hemorrhagic fever virus circulating in endemic areas of Southern Tajikistan in 2009. Molecular Genetics, Microbiology, and Virology. 2013;(3):29-36. DOI 10.3103/S0891416813030063.

9. Rar V.A., Livanova N.N., Panov V.V., Doroschenko E.K., Pukhov-skaya N.M., Vysochina N.P., Ivanov L.I. Genetic diversity of Ana-plasma and Ehrlichia in the Asian part of Russia. Ticks Tick Borne Dis. 2010;1(1):57-65. DOI 10.1016/j.ttbdis.2010.01.002.

10. Rar V.A., Maksimova T.G., Zakharenko L.P., Bolykhina S.A., Dobrotvorsky A.K., Morozova O.V. Babesia DNA detection in canine blood and Dermacentor reticulatus ticks in southwestern Siberia, Russia. Vector-Borne Zoonotic Dis. 2005;5(3):285-287. DOI 10.1089/vbz.2005.5.285.

11. Rasulov I. Ticks status in Central Asia with a special emphasis on Uzbekistan. Parasitol. Res. 2007;101(Suppl.2):183-186.

12. Robinson P.M. Theileriosis annulata and its transmission - a review. Trop. Anim. Health Prod. 1982;14(1):3-12.

13. Roux V., Rydkina E., Eremeeva M., Raoult D. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the Rickettsiae. Int. J. Syst. Bacteriol. 1997;47(2):252-261. DOI 10.1099/00207713-47-2-252.

14. Sumner J.W., Nicholson W.L., Massung R.F. PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species. J. Clin. Microbiol. 1997;35(8):2087-2092.

15. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011;28(10):2731-2739. DOI 10.1093/molbev/msr121.

16. Tishkova F.H., Belobrova E.A., Valikhodzhaeva M., Atkinson B., Hewson R., Mullojonova M. Crimean-Congo hemorrhagic fever in Tajikistan. Vector-Borne Zoonotic Dis. 2012;12(9):722-726. DOI 10.1089/vbz.2011.0769.

17. 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.

18. Wu X.B., Na R.H., Wei S.S., Zhu J.S., Peng H.J. Distribution of tick-borne diseases in China. Parasit. Vectors. 2013;6:119. DOI 10.1186/1756-3305-6-119.