Species and Genetic Diversity of Representatives of the Anaplasmataceae Family Found in the Sympatry Zone of the Ixodes, Dermacentor and Haemaphysalis Genera Ticks

Introduction.On the territory of the Ekhirit-Bulagatsky district of the Irkutsk region zones of sympatry of four Ixodes ticks species are found, where the species and genetic diversity of infectious agents transmitted through tick bites may be more pronounced than in foci with a mono-dominant type of ticks’ population. In this connection, the study of the species and genetic diversity of representatives of the Anaplasmataceae family in the sympatry zone of the Ixodes ticks of closely related species was of scientific interest.

Objective: To study the species and genetic diversity of members of the Anaplasmataceae family in the zones of sympatry of Ixodes ticks Ixodes persulcatus, Dermacentor silvarum, D. nuttalli and Haemaphysalis concinna, to identify the main carriers and potential reservoir hosts of ehrlichia and anaplasma.

Methods. In the course of the study, 1106 specimens of adult ticks and 49 samples of small mammalian livers from the Ekhirit-Bulagatsky area were analyzed. Anaplasma and ehrlichia DNA were detected by two-round PCR in the presence of genus- and species-specific primers from the 16S rRNA gene region. The nucleotide sequences of the 16S rRNA gene and the fragment of the groESL operon were identified in some samples. Sequencing was carried out according to the Sanger method. Comparative analysis was performed using the BLASTN program and ClustalW method. Epidemiological data analysis was performed using parametric methods of statistical processing of the material.

Results. The DNA of Ehrlichia muris and Anaplasma phagocytophilum were detected in all studied species of ticks in their sympatry area. However, the rate of infection of taiga ticks was significantly higher than that of H. concinna and Dermacentor spp. Potential reservoir hosts of the Anaplasmataceae family members can be classified as Microtus oeconomus, M. gregalis, Myodes rutilus and Sorex spp. When analyzing the nucleotide sequences of the 16S rRNA gene, three genetic variants of anaplasma were detected. The nucleotide sequences of the A. phagocytophilum groESL operon belonged to two genetic groups.

1. Filippova NA. Sympathy of closely related species of ixodic ticks and its possible role in the parasitic systems of natural foci of vector-borne diseases. Parazitologiya. 1999; 33(3): 223-241. (In Russ.)

2. Kozlova IV, Zlobin VI, Verkhozina MM, Rar VA, Lisak OV, Doroshchenko EK, et al. The results of reconnaissance studies on monocytic ehrlichiosis and human granulocytic anaplasmosis in the Baikal region.Acta biomedica scientifica. 2007; 3(55): 112-116. (In Russ.)

3. Kozlova IV, Verkhozina MM, Dyomina TV, Dzhioev YuP, Doroshchenko EK, Lisak OV, et al. Combined foci of transmissible tick-borne infections in the Baikal region. Epidemiologiya i vaktsin­ oprofilaktika. 2010; 4(53): 40-46. (In Russ.)

4. Livanova NN, Rar VA, Livanov SG, Igolkina YaP. Diversity of parasitic systems involving small mammals and Ixodes persulcatus Schulze in the Northern Urals. Sibirskiy ehkologicheskiy zhurnal. 2005; (6): 1079-1084 (In Russ.)

5. Rar VA, Pukhovskaya NM, Vysochina NP, Zaynulina ZU, Gulyako LF, Ivanov LI. Distribution and genetic diversity of Ehrlichia and Anaplasma in taiga ticks and small mammals on the territory of the Khabarovsk Territory. Acta biomedica scientifica. 2007; 3 (Supplement): 156-159 (In Russ.)

6. Rar VA, Livanova NN, Panov VV, Kozlova IV, Pukhovskaya NM, Vysochina NP, et al. Prevalence of Anaplasmaand Ehrlichia species in Ixodes persulcatus ticks and small mammals from different regions of Asian part of Russia. Int J Med Microbiol. 2008; 298(1): 222-230. doi: 10.1016/j.ijmm.2008.01.001

7. Rar VA, Fomenko NV, Dobrotvorsky AK, Livanova NN, Rudakova SA, Fedorov EG, et al. Tick-borne pathogen detection, Western Sibiria, Russia. Emerg Infect Dis. 2005; 11: 1708-1715.

8. Rar VA, Epikhina TI, Livanova NN, Panov VV, Doroshchenko EK, Pukhovskaya NM, et al. The study of heterogeneity of 16S rRNA gene and groESL operone in DNA samples of Anaplasma phagocytophilum, Ehrlichia muris, and “Candidatus Neoehrlichia mikurensis” determined in Ixodes persulcatus ticks on the territory of Ural, Siberia and Far East of Russia. Mol Gen Microbiol Virol. 2011; 26(2): 66-73. doi: 10.3103/S0891416811020091

9. Sumner JW, Nicholson WL, Massung RF. PCR amplification and comparison of nucleotide sequences from the groESLheat shock operon of Ehrlichiaspecies. J Clin Microbiol. 1997; 35(8): 2087-2092.

10. Liz JS, Anderes L, Sumner JW, Massung RF, Gern L, Rutti B, et al. PCR detection of granulocytic ehrlichia in Ixodes ricinusticks and wild small mammals in western Switzerland. J Clin Microbiol. 2000; 38(3): 1002-1007.

11. Savilov ED, Astafyev VA, Zhdanova SN, Zarudnev EA. Epidemiological analysis: Methods of the statistical processing of materials. Novosibirsk: Nauka-Tsentr; 2011. (In Russian)

12. Rar VA, Livanova NN, Panov VV, Doroshchenko EK, Pukhovskaya NM, Vysochina NP, et al. Genetic diversity of Anaplasma and Ehrlichiain Asian part of Russia. Ticks Tick­Borne Dis. 2010; 1(1): 57-65. doi: 10.1016/j.ttbdis.2010.01.002

13. Rar VA, Epikhina TI, Livanova NN, Panov VV, Doroschenko EK, Pukhovskaya NM, et al. Genetic variability of Anaplasma phagocytophilumin Ixodes persulcatusticks and small mammals in the Asian part of Russia. Vector­Borne Zoonot Dis. 2011; 11(8): 1013-1021. doi: 10.1089/vbz.2010.0266

14. Kawahara M, Suto C, Rikihisa Y, Yamamoto S, Tsuboi Y. Characterization of ehrlichial organisms isolated from a wild mouse. J Clin Microbiol. 1993; (31): 89-96.

15. Wen B, Rikihisa Y, Mott J, Fuerst PA, Kawahara M, Suto C. Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological and biological characteristics. Int J Syst Bacteriol. 1995: (45): 250-254. doi: 10.1099/00207713-45-2-250

16. Kawahara M, Ito T, Suto C, Shibata S, Rikihisa Y, Hata K, et al. Comparison of Ehrlichia murisstrains isolated from wild mice and ticks and serologic evidence of humans and animals with E. murisas antigen. J Clin Microbiol. 1999: 37(4): 1123-1129.

17. Sumner JW, Storch GA, Buller RS, Liddell AM, Stockham SL, Rikihisa Y, et al. PCR amplification and phylogenetic analysis of groESL operon sequences from Ehrlichia ewingii and Ehrlichia muris. J Clin Microbiol. 2000; 38(7): 2746-2749.

18. Von Loewenich FD, Stumpf G, Baumgarten BU, Rollinghoff M, Dumler JS, Bogdan C. A case of equine granulocytic ehrlichiosis provides molecular evidence for the presence of pathogenic Anaplasma phagocytophilum(HGE agent) in Germany. Eur J Clin Microbiol Infect Dis. 2003; 22(5): 303-305. doi: 10.1007/s10096-003-0935-1

19. Cao WC, Zhan L, He J, Foley J, De Vlas SJ, Wu XM, Yang H, Richardus JH, Habbema JD. Natural Anaplasma phagocytophilum infection of ticks and rodents from a forest area of Jilin Province, China. Am J Trop Med Hyg. 2006; 75(4): 664-668.

20. Cao WC, Zhao QM, Zhang PH, Dumler JS, Zhang XT, Fang LQ, et al. Granulocytic ehrlichia in Ixodes persulcatusticks from an area in China where Lyme disease is endemic. J Clin Microbiol. 2000; 38(11): 4208-4210.

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

22. Rar VA, Epikhina TI, Tikunova NV, Yakimenko VV, Malkova MG, Tancev AK, et al. Genetic variability of Anaplasma phagocy­ tophilum in ticks and voles from Ixodes persulcatus/ Ixodes triangu­liceps sympatric areas from Western Siberia, Russia. Ticks Tick­Borne Dis. 2014; 5(6): 854-863. doi: 10.1016/j.ttbdis.2014.07.008

23. Blaňarová L, Stanko M, Carpi G, Miklisová D, Víchová B, Mošanský L, et al. Distinct Anaplasma phagocytophilumgenotypes associated with Ixodes triangulicepsticks and rodents in Central Europe. Ticks Tick­Borne Dis. 2014; 5(6): 928-38. doi: 10.1016/j.ttbdis.2014.07.012.