1) The study detected Anaplasma phagocytophilum and Anaplasma platys in ticks collected from Mongolia through PCR and sequencing of the groEL gene. 2) A. phagocytophilum was found in 6.14% of Ixodes persulcatus ticks and 20% of Dermacentor nuttalli ticks. A. platys was found in 1.75% of I. persulcatus and 10% of D. nuttalli. 3) Sequence analysis showed the A. phagocytophilum strain from Mongolia is most similar to strains found in Russia. This research provides new information on tick-borne pathogens in Mongolia.

1. 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

2. Introduction
Anaplasma phagocytophilum is a Gram-negative
obligate intracellular bacterium, which have long
been recognized as worldwide tick-borne agents for
several species of wild and domesticated mammals,
and human.
The disease usually presents as an acute febrile
illness characterized by headache, chill, myalgias,
arthralgia, malaise, and hematological abnormalities,
such as thrombocytopenia, leukopenia, and elevated
hepatic aminotransferase levels.

3. 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.

4. Aim of the study
- Identification of collected tick species from study area
- Detection Anaplasma phagocytophilum infection in
tick vector using specific gene fragments by molecular
biological assay
-Sequencing and analyzing A.phagocytophilum groEL
gene fragment partially

5. Methods and Results
Methods and results

6. 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 nuttalli
A B
Female Male Male Female

7. 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

8. 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 .

9. Samples for detect Anaplasma phagocytophilum
№ Tick species Sum, province Tick gender Number of
sample
1 Dermacentor nuttalli Altanbulag, Selenge F 6
M 4
2 Khuder, Selenge F 6
M 4
total F 12
M 8
Net 20
3 Ixodes persulcatus Altanbulag, Selenge F 23
M 26
4 Khuder, Selenge F 88
M 77
total F 111
M 103
5 Biotop-3 F 3
M 2
6 Biotop-1 M 1
7 unknown unknown 8
Net 228

10. Extract tick genomic DNA
G-spin genomic DNA extraction kit
Extract DNA from ticks after ticks were
freezed and mashed by liquid nitrogen are
performed through G-spin genomic DNA
extraction kit protocol.

11. Polymerase chain reaction
Maxime PCR PreMix in one tube 95 0 С – 15’
kit 94 0 С – 30’’
Sterilized distilled 16 µl 55 0 С – 30’’ 35 cycle
water 72 0 С – 45’’
Primer - forward 1 µl 72 0 С – 7’
4 0 С - ∞
Primer - reverse 1 µl
Sample 2 µl
Total 20 µl

12. 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.

13. Results of polymerase chain reaction
M 1 2 3 4 5 6 7 8
M : 100bp marker DNA
1-8: samples
2 : positive for
500bp A.phagocytophilum
200bp
100bp 1, 7, 8: positive for A.platys
A.phagocytophilum positive control was chosen after verified
PCR results by sequencing.

14. 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%) 0
2 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 0
6 Biotop-1 M 1 0 0
7 unknown unknown 8 0 0
Net 228 14 (6,14%) 4 (1,75%)

15. 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.

16. Sequence of Anaplasma phagocytophilum groEL gene,
partial
CGAGCGTCTT GCATGCTCCG GCCGCCATGG CCGCGGGATT ATGGTATGCA GTTTGATCGC GGATATCTTT
CGCCTTACTT TGTTACAAAT GCTGAAAAAA TGCTGGTGGA ATTTGAAAAT CCATACATAT TCCTTACTGA
AAAGAAGATT AATCTTGTAC AAAGCATTTT ACCAATATTA GAAAACGTTG CGAGAGCTGG CAGACCATTG
CTCATCATAG CTGAAGATGT TGAAGGTGAA GCTCTGAGCA CGCTTGTACT CAATAAGCTC CGTGGTGGGC
TCCAAGTTGC TGCTGTAAAG GCGCCTGGTT TCGGTGACAG AAGAAAAGAC ATGCTAGGCG ATATTGCCGT
AATAGTAGGC GCTAAGTATG TAGTAAATGA CGAGCTTGCT GTTAAGATGG AAGATATCGC TCTAAGCGAT
CTGGGTACTG CTAAGAGCGT GCGCATCACA AAAGACGCAA CTACTATCAT AGGTAGCGTT GATAGCAGTT
CTGAAAGCAT AGCTAGCAGG ACTAATCAAA TCAAAGCTCA GATAGAAAAC TCTAGTTCTG ATTATGACAA
GGAAAAGCTT AGAGAACGTT TAGCGAAGCT TTCCGGTGGC GTTGCTGTAC TCAAGGTTGG TGGATCCAGC
GAAGTTGAGG TGAAGGAACG CAAAGACAGA GTAGAAATCA CTAGTGCGGC CGCCTGCAGG TCGACCATAT
GGGAGAGCTC CCAACGCGTT GGATGCATAG CTTGAGTATT CTATAGTGTC ACCTAAATAG CTTGGCGTAA
TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA
GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC
CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGG
TTTGCGTATT GGGCGCTCTT
Insert (red): 625 bp

17. 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.

18. Europe I
Russia
Europe II
USA

19. Conclusion
l 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.

20. Conclusion
4. 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.

21. Aknowledgements
• Organizers of International Conference “Tick-Borne Encephalitis
and Other Tick-Borne Infections” Dedicated to the 75th Anniversary
of 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 of
Veterinary Medicine, Mongolia
•Other researchers and organizations

22. THANK YOU FOR YOUR
ATTENTION
e_mail: bata07@gmail.com
Laboratory of Molecular Genetics,
Institute of Veterinary Medicine,
Zaisan-210153, Ulaanbaatar, Mongolia