Detection and genotyping of Anaplasma phagocytophilum in I. persulcatus and D. reticulatus ticks collected in Tomsk (Western Siberia) in 2015–2016

Introduction. The detection of the first cases of tick-borne human granulocytic anaplasmosis in Russia, discovery of genetic markers for Anaplasma spp. in ixodid ticks and reporting of a significant number of cases of tick-borne infections in the southern part of Western Siberia give reason to suppose that causative agents of tick-borne anaplasmosis may be transmitted in Tomsk and its suburbs.
Objective. To study the distribution and species biodiversity of A. phagocytophilum in ixodid ticks in Tomsk Region.
Materials and methods. The analysis of 690 individual ixodid ticks (larvae and adults) was carried out for Ixodes persulcatus (n = 530) and Dermacentor reticulatus (n = 160) ticks collected in 2015–2016 on the territory of urban and suburban biotopes of Tomsk. Primary screening of ticks for the presence of genetic material of A. phagocytophilum was conducted using two-round PCR with species-specific primers for the 16S rRNA gene. The amplification (1,220 kB) of the groESL fragment of the heat shock protein operon was performed for positive isolates with subsequent determination of the nucleotide sequence in the gene fragment for phylogenetic analysis.
Results. The number of A. phagocytophilum positive samples for I. persulcatus (larvae) was 1.2 ± 0.6%, I. persulcatus (adult) was 1.8 ± 0.7%; and D. reticulatus (adult) was 0.6 ± 0.3%. Analysis of the nucleotide sequence of the gene fragments in groESL operon for nine isolates confirmed that the genetic material of the granulocytic anaplasmosis was detected. Phylogenetic analysis showed that all the isolates belonged to the first group of the “new cluster” of A. phagocytophilum.
Conclusion. The causative agent of human granulocytic anaplasmosis has been newly detected in I. persulcatus ticks collected in urban and suburban biotopes of Tomsk and in D. reticulatus from urban foci.

1. Atif F.A. Anaplasma marginale and Anaplasma phagocytophilum: Rickettsiales pathogens of veterinary and public health significance. Parasitol. Res. 2015; 114 (11): 3941–3957. DOI: 10.1007/s00436-015-4698-2.

2. Stuen S. Anaplasma phagocytophilum – the most widespread tick-borne infection in animals in Europe. Vet. Res. Commun. 2007; 31 Suppl. 1: 79–84. DOI: 10.1007/s11259-007-0071-y.

3. Grova L., Olesen I., Steinshamn H., Stuen S. Prevalence of Anaplasma phagocytophilum infection and effect on lamb growth. Acta Vet. Scand. 2011; 53: 30. DOI: 10.1186/1751-0147-53-30.

4. Truchan H.K., Seidman D., Carlyon J.A. Breaking in and grabbing a meal: Anaplasma phagocytophilum cellular invasion, nutrient acquisition, and promising tools for their study. Microbes Infect. 2013; 15 (14–15): 1017–1025. DOI: 10.1016/j.micinf.2013.10.010.

5. Schaff U.Y., Trott K.A., Chase S., Tam K., Johns J.L. Neutrophils exposed to A. phagocytophilum under shear stress fail to fully activate, polarize, and transmigrate across inflamed endothelium. Am. J. Physiol. Cell Physiol. 2010; 299 (1): 87–96. DOI: 10.1152/ajpcell.00165.2009.

6. Loewenich F.D., Scorpio D.G., Reischl U., Dumler J.S., Bogdan C. Frontline: control of Anaplasma phagocytophilum, an obligate intracellular pathogen, in the absence of inducible nitric oxide synthase, phagocyte NADPH oxidase, tumor necrosis factor, Toll-like receptor (TLR) 2 and TLR4, or TLR adaptor molecule MyD 88. Eur. J. Immunol. 2000; 34 (7): 1789–1797.

7. Ohashi N., Gaowa W., Kawamori F., Wu D., Yoshikawa Y. Human granulocytic Anaplasmosis, Japan. Emerg. Infect. Dis. 2013; 19 (2): 289–292. DOI: 10.3201/eid1902.120855.

8. Brouqui P., Bacellar F., Baranton G. Guidelines for the diagnosisof tick-borne bacterial diseases in Europe. Clin. Microbiol. Infect. 2004; 10 (12): 1108–1132. DOI: 10.1111/j.1469-0691.2004.01019.x

9. Strle F. Human granulocytic ehrlichiosis in Europe. Int. J. Med. Microbiol. 2004; 293 Suppl. 37: 27–35. DOI: 10.1016/s1433-1128(04)80006-8.

10. Sidel’nikov Yu.N., Medyannikov O.Yu., Ivanov L.I., Zdanovskaya N.I. The first case of granulocyte erlichiosis in the Far East of the Russian Federation. Clinical Мedicine. 2003; 81 (2): 67–68 (in Russ.)].

11. Leonova G.N., Yastrebov V.K., Khazova T.G., Sherstneva M.B., Shpynov S.N., Egorova N.V., Rudakov I.V., Fedyanin A.P. New data on the detection of Ehrlichia and Anapalasma in ixodid ticks in Russia and Kazakhstan. Medical Рarasitology. 2004; 2: 10–14 (in Russ.)].

12. Chausov E.V., Ternovoy V.A., Protopopova E.V., Konovalova S.N., Kononova Yu.V. The genetic diversity of infectious agents, transmitted by ixodid ticks in Tomsk and its suburbs. Parasitology. 2009; 43 (5): 374–388 (in Russ.)].

13. Rar V.A., Livanova N.N., Panov V.V., Doroschenko E.K. Genetic diversity of Anaplasma and Ehrlichia in Asian part of Russia. Ticks Tick Borne Dis. 2010; 1 (1): 57–65. DOI: 10.1016/j.ttbdis.2010.01.002.

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

15. Liz J.S., Anderes L., Sumner J.W., Massung R.F. PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland. J. Clin. Microbiol. 2000; 38 (3): 1002–1007.

16. Burland T.G. DNASTAR’s Lasergene sequence analysis software. Methods Mol. Biol. 2000; 132: 71–91. DOI: 10.1385/1-59259-192-2:71.

17. Okonechnikov K., Golosova O., Fursov M. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012; 28 (8): 1166–1167. DOI: 10.1093/bioinformatics/bts091.

18. Tamura K., Dudley J., Nei M., Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 2007; 24 (8): 1596–1599. DOI: 10.1093/molbev/msm092.

19. Romanenko V.N. Monitoring of species composition and numbers of ixodid ticks (Parasitiformes: Ixodidae) in anthropurgic biotopes. Journal of Tomsk State University. 2009; 324: 376–379 (in Russ.)].

20. Pankina T.M., Romanenko V.N., Istratkina S.V., Shikhin A.V., Poltoratskaya T.N. Acarological situation in the south of Tomsk region. Journal of Tomsk State University. 2013; 4 (24): 67–76 (in Russ.).

21. Rymaszewska A. Divergence within the marker region of the groESL operon in Anaplasma phagocytophilum. Eu. J. Clin. Microbiol. Infect. Dis. 2008; 27 (11): 1025–1036. DOI: 10.1007/s10096-008-0539-x.

22. Rar V.A., Epikhina T.I., Livanova N.N., Panov V.V., Doroschenko E.K., Pukhovskaya N.M., Vysochina N.P., Ivanov L.I. Genetic variability of Anaplasma phagocytophilum in Ixodes persulcatus ticks and small mammals in the Asian part of Russia. Vector Borne Zoonotic Dis. 2011; 11 (8): 1013–1021. DOI: 10.1089/vbz.2010.0266.