For International Conference “Tick-Borne Encephalitis and Other Tick-Borne Infections”Dedicated to the 75th Anniversary of the Tick-Borne Encephalitis Virus Discovery MOLECULAR DETECTION OF ZOONOTIC ANAPLASMA IN VECTOR TICK IN MONGOLIA Javkhlan G.1, Enkhtaivan B1., Baigalmaa B.2 , Enkhtogtoh B. 1, Bolorchimeg B.3, Battur B. 1, Tuvshintulga B. 1, Undraa B2., Battsetseg B. 1 Irkutsk, Russia-2012 1 Laboratory of Molecular Genetics, IVM, 2 National Center for Infectious Diseases with Natural Foci , 3 National Center for Infectious Diseases with Natural Foci, Selenge province
IntroductionAnaplasma phagocytophilum is a Gram-negativeobligate intracellular bacterium, which have longbeen recognized as worldwide tick-borne agents forseveral species of wild and domesticated mammals,and human.The disease usually presents as an acute febrileillness characterized by headache, chill, myalgias,arthralgia, malaise, and hematological abnormalities,such as thrombocytopenia, leukopenia, and elevatedhepatic aminotransferase levels.
Basis of the study In Mongolia: Anaplasma ovis and Anaplasma marginale detected in reindeer by microscopic examination (Purevsuren, 1981) and PCR (Nansalmaa, 2012). Anaplasma phagocytophilum antibody found in human by IFAT (Walder et al., 2006). The tick vectors unknown. Therefore, we completed this study for detect Anaplasma phagocytophilum in tick vectors.
Aim of the study- Identification of collected tick species from study area- Detection Anaplasma phagocytophilum infection intick vector using specific gene fragments by molecularbiological assay-Sequencing and analyzing A.phagocytophilum groELgene fragment partially
Sample for study Totally about 1300 ticks were collected from forest area of Selenge province. Ticks were identified as I.persulcatus and D.nuttalli by using identification key. Ixodes persulcatus Dermacentor nuttalliA B Female Male Male Female
Distribution of Ixodes genus• Ixodes crenulatus Koch 1935• Ixodes laguri ol 1928• Ixodes lividus Koch 1844• Ixodes arboricola Schulze et Schlottke 1929• Ixodes passericola Schulze 1933• Ixodes persulcatus P.Sch 1930• Ixodes prokopheri Jem Jesse I. Goodman et al, 2005 Ixodes persulcatus is main species for transmit infectious diseases to human and animals Dash Mo., 1986
Distribution of Dermacentor genus• Dermacentor nuttalli ol 1929• Dermacentor silvarum ol 1929• Dermacentor daghestanicus ol 1929• Dermacentor asiaticum Jemelyanova et kozlovskaya 1967 Dermacentor nuttalli is widely distributed tick and the most important in veterinary .
Samples for detect Anaplasma phagocytophilum№ Tick species Sum, province Tick gender Number of sample1 Dermacentor nuttalli Altanbulag, Selenge F 6 M 42 Khuder, Selenge F 6 M 4 total F 12 M 8 Net 203 Ixodes persulcatus Altanbulag, Selenge F 23 M 264 Khuder, Selenge F 88 M 77 total F 111 M 1035 Biotop-3 F 3 M 26 Biotop-1 M 17 unknown unknown 8 Net 228
Extract tick genomic DNA G-spin genomic DNA extraction kitExtract DNA from ticks after ticks werefreezed and mashed by liquid nitrogen areperformed through G-spin genomic DNAextraction kit protocol.
Polymerase chain reactionMaxime PCR PreMix in one tube 95 0 С – 15’kit 94 0 С – 30’’Sterilized distilled 16 µl 55 0 С – 30’’ 35 cyclewater 72 0 С – 45’’Primer - forward 1 µl 72 0 С – 7’ 4 0 С - ∞Primer - reverse 1 µlSample 2 µl Total 20 µl
Primers Primers: - EphplgroEL(569)F (5’-ATGGTATGCAGTTTGATCGC-3’) - EphplgroEL(1193)R (5’-TCTACTCTGTCTTTGCGTTC-3’) The primers anneal to nucleotide strings conserved in A. phagocytophilum and A. platys and were designed to selectively amplify, 624 bp of the groEL gene of both species from samples.
Results of polymerase chain reaction M 1 2 3 4 5 6 7 8 M : 100bp marker DNA 1-8: samples 2 : positive for500bp A.phagocytophilum200bp100bp 1, 7, 8: positive for A.platys A.phagocytophilum positive control was chosen after verified PCR results by sequencing.
Results№ Tick species Sum, province Tick Number of Positive for Positive for gender sample A.phagocytophilum (%) A.platys (%)1 Altanbulag, F 6 1 (16,66%) 0 Selenge M 4 1 (25%) 02 D.nutalli Khuder, Selenge F 6 1 (16,66%) 2 (33,33%) M 4 1 (25%) 0 total F 12 2 (16,66) 2 (16,66%) M 8 2 (25%) 0 Net 20 4 (20%) 2 (10%)3 Altanbulag, F 23 3 (13,04%) 1 (4,35%) Selenge M 26 2 (7,69%) 1 (3,84%)4 Khuder, Selenge F 88 5 (5,68%) 2 (2,27%) M 77 4 (5,19%) 0 I.persulcatus total F 111 8 (7,2%) 3 (2,7%) M 103 6 (5,82%) 1 (0,97%)5 Biotop-3 F 3 0 0 M 2 0 06 Biotop-1 M 1 0 07 unknown unknown 8 0 0 Net 228 14 (6,14%) 4 (1,75%)
Sequencing Direct DNA sequencing method was basically performed using the same PCR primers in the present study. The amplicon was cloned into a plasmid vector using a TOPO TA cloning kit (Invitrogen), and then sequenced using the primers provided with the kit.
Phylogenetic analyses Nucleotide sequences were initially checked using a BLAST search hosted by the NCBI for the comparison with other known nucleotide sequences. The multiple alignment analysis was performed using the ClustalW online server. Phylogenetic analysis was performed by UPGMA method using ClustalW online server.
Conclusionl All samples were studied by using tick identification key, and identified as Dermacentor nuttalli and Ixodes persulcatus. Ixodes persulcatus (91.9%) is dominant tick species in this local area.l groEL gene fragments amplified in 26.14% of all tick DNA samples for Anaplasma phagocytophilium and in 11.75% for Аnaplasma platys.l In this study, 6.14% of I.persulcatus was positive for A. phagocytophilium and 1.75% was positive for A.platys. 20% of D.nuttalli was positive for A. phagocytophilium and 10% was positive for A.platys. These ticks play role to transmit the agents in nature.
Conclusion4. А.platys groEL gene fragment is detected in this study, and this become new information in Mongolia for further study.5. Detected Anaplasma phagocytophilium and Аnaplasma platys groEL gene fragments were sequenced and analyzed for verification.6. A. phagocytophilum 625bp of groEL gene partially sequenced and performed phylogenetic analysis. Mongolian A. phagocytophilum groEL gene is in Russian group.7. Further studies for understanding of the basic molecular mechanisms of transmission mechanism, particularly the process of stored in tick organs, is required for the development of preventive measure against anaplasmosis.
Aknowledgements• Organizers of International Conference “Tick-Borne Encephalitisand Other Tick-Borne Infections” Dedicated to the 75th Anniversaryof the Tick-Borne Encephalitis Virus Discovery• WHO Representative Office in Mongolia• Workers of local stations for infectious diseases with Natural Foci• Researchers of Laboratory of Molecular Genetics, Institute ofVeterinary Medicine, Mongolia•Other researchers and organizations
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