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Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia
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Original article
Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia L.D. Shchuchinova a , I.V. Kozlova b,c,∗ , V.I. Zlobin c a b c
Federal Service on Customers’ Rights Protection and Human Well-being Surveillance in the Altai Republic, 649000 Gorno-Altaisk, Russia FSBI “Scientific Health Centre for Family and Human Reproduction” SB RAMS, 664025 Irkutsk, Russia Irkutsk State Medical University of Healthcare of Russian Federation, 664003 Irkutsk, Russia
a r t i c l e
i n f o
Article history: Received 17 June 2014 Received in revised form 20 January 2015 Accepted 12 February 2015 Available online xxx Keywords: Tick-borne encephalitis Altitude above sea level Ticks
a b s t r a c t The Altai Republic is a highly endemic area as far as tick-borne encephalitis (TBE) is concerned. The aim of the research was to study the effect of altitude on the risk of tick-borne encephalitis infection in the Altai Republic. The paper analyzes the following data: the study of ixodid ticks collected from the vegetation in 116 sites at the 200–2383 m elevation above sea level in 2012–2014, TBE virus prevalence of these vectors, tick-bite incidence rate, and TBE incidence rate of the population. Species identification of 4503 specimens has shown that the most common species are Dermacentor nuttalli (45.3%), Ixodes persulcatus (33.1%), Dermacentor silvarum (9.4%), Dermacentor reticulatus (8.9%), and Haemaphysalis concinna (5.0%). A total of 2997 adult ixodid ticks were studied for the presence of the TBE virus; 2163 samples were examined by ELISA, while 834 specimens were tested by PCR. The TBE virus prevalence of Dermacentor spp. ticks in both reactions was significantly higher than of Ixodes persulcatus ticks (p < 0.001). The work shows that the altitude is an important factor in the development of the epidemiological situation of tick-borne encephalitis: the higher the elevation of the area above sea level, the smaller the range of vectors. There is also a change of a leading species: in middle altitude (800–1700 m above sea level) the virus is transmitted by ticks of D. nuttalli along with I. persulcatus, and in high mountains (above 1700 m above sea level) D. nuttalli becomes an absolute dominant species. However, these species of ticks are less effective vectors than I. persulcatus. With the increase of altitude the tick-bite incidence rate decreases (r = −0.78, p < 0.05), and TBE incidence also reduces (r = −0.67, p < 0.05). © 2015 Elsevier GmbH. All rights reserved.
Introduction Russia possesses the most extensive geographic distribution of tick-borne encephalitis. It is believed that the intensity of the natural TBE foci determines the high abundance of the main vector species I. ricinus and I. persulcatus and their infection rate (Korenberg and Kovalevskij, 2000). The distribution of these tick species has been well studied. In recent years, some investigations have been focused on the positive effect of continuous temperature rise and humidity on the improvement of habitat conditions for ticks (Süss, 2008). In mountainous areas of Central Europe, ticks have been recorded at higher altitudes up to 1100 and 1300 m above sea level (a.s.l.) compared to the cases recorded before 1990 (at
∗ Corresponding author at: FSBI “Scientific Health Centre for Family and Human Reproduction” SB RAMS, 664025 Irkutsk, Russia. Tel.: +7 83952333951. E-mail addresses: [email protected] (L.D. Shchuchinova), [email protected] (I.V. Kozlova), [email protected] (V.I. Zlobin).
1000 m a.s.l.) (Danielová et al., 2006, 2008; Materna et al., 2008; Daniel et al., 2009). Moreover, some cases of alimentary TBE infection caused by the consumption of nonpasteurized milk of goats, which were grazing in pastures at 1500 m a.s.l., were recorded in Austria (Holzmann et al., 2009). In Russia, I. persulcatus was recorded at 1400 m a.s.l. on the northern slopes of the Western Sayan, in the Central and Eastern Altai (at 1900 m a.s.l.) and Tian Shan (at 3000 m a.s.l.) (Rubina, 1969; Sapegina, 1980; Grebenyuk, 1966). Besides I. ricinus and I. persulcatus, other tick species can be additional vectors for tick-borne encephalitis virus (TBEV) in some areas (Danchinova et al., 2006; Verhozina et al., 2008; Zlobin, 2010). Their role in the natural TBE foci is still unclear, as well as the altitude influence on their density. The Altai Republic is one of the areas where along with I. persulcatus as a dominant vector at the natural foci of TBE, there are other species of ixodid ticks. This is a mountain terrain in the south of Western Siberia, located on a junction of the Siberian taiga, Kazakh steppes, and semi-deserts of Mongolia that determines a
http://dx.doi.org/10.1016/j.ttbdis.2015.02.005 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005
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surprising diversity of landscapes. Settlements are situated at 200–2200 m a.s.l. The size of the republic area is 92,903 km2 . The population is 210,344 people. The length of the terrain from the north to the south is 400 km, and from the west to the east is 360 km. Average annual temperatures in Gorny Altai fluctuate within the limits of +4◦ (in northern and western suburbs) to −7◦ (in highmountainous regions). The fauna of the Altai Republic includes 93 species of mammals and 312 species of birds. The abundance of ixodid ticks and their widespread presence are attributed to a large number of hosts and diversity of landscapes in this territory. Along with natural factors, social factors contribute to the spread of the TBE virus. The mountain landscape predetermines cattlebreeding in the Altai Republic. Crop lands occupy only 1.5% of the republic, whereas the largest part of agricultural lands belongs to grazing – 84.7%. Cattle breeding industry is one of the primary sources of income for the agricultural population. According to the report of the Territorial Agency of the Federal Service of State Statistics on January, 1st, 2014 in the Altai Republic the farms of all categories comprised 245,000 heads of cattle, 11,900 pigs, 477,500 sheep, 149,400 goats and 136,200 horses. Domestic animals are active hosts for ixodid ticks, sustaining a high number of vectors, and also carry ticks into settlements. The average yearly TBE incidence of the Altai Republic between 2004 and 2013 was 22.6 per 100,000 inhabitants which is almost 10 times higher than TBE incidence of Russia. However, disease levels in different districts of the Altai Republic differ considerably. The aim of this work was to assess the influence of altitude on a tick-borne encephalitis infection risk in the natural foci of the Altai Republic.
Materials and methods
In addition, 834 questing adult ticks collected from 27 sites were tested by PCR. Total RNA was extracted from ixodid ticks using the “AmpliPrime® RIBO-prep-100” kit (“AmpliPrime”, Russia). The reverse transcription was performed with “RevertaL-100” kit containing random hexanucleotides (“Amplisence”, Russia). PCR reaction was performed in 20 l of the reaction mixture containing 67 mM Tris–HCl (pH 8.9), 16.6 mM (NH4 )2 SO4 , 2 mM MgCl2 , 0.01% Tween 20, 200 M of each dNTP, 5% glycerol, 0.5 M primers, 2 U of Taq DNA polymerase (“BioSan”, Russia) and 3 l of cDNA for primary reactions or 1 l of the primary PCR products for nested reaction. The PCR conditions comprised initial denaturation for 3 min at 94 ◦ C followed by 35 cycles of denaturation for 0.5 min at 94 ◦ C, annealing for 0.5 min at 48 ◦ C and elongation for 1 min at 72 ◦ C. The primers corresponding to positions 2199–2219 and 2517–2539 nr or positions 2214–2238 and 2402–2424 nr of TBEV genome were used for primary or nested reactions, respectively. The length of final PCR-products was 211 bp. Epidemiological data The analysis of the TBE incidence rate of the population in the Altai Republic (during 2004–2013) was done according to the state statistical reporting: Form # 2 “Information on Infectious and Parasitic Diseases”, Form # 60 “Register of Infectious Patients”, Form # 003/U “Medical Record of the Inpatient”, Form # 025/U “Medical Record of the Outpatient”. We also analyzed TBE incidence using data from epidemiological records (n = 2183). All cases of tick bites have been registered in the Russian Federation. We analyzed the tick-bite incidence rate per 100,000 inhabitants in the Altai Republic for the period of 2013. The information on a number of the livestock was obtained from the reports of the Territorial Agency of the Federal State Statistics Service in the Altai Republic (http://statra.gks.ru).
Tick collection Statistic analysis Questing adult ticks were collected by flagging the vegetation every 8–12 km along the highways of the Altai Republic during the peak of tick activity (from mid April till early June of 2012–2014). The sampling sites were located 30–300 m from the motorways. The preference was given to the places where earlier there had been cases of TBE infections, that is, to the tourist camps, popular vacation and recreation spots in the vicinities of settlements, and to the scenic view points at mountain passes. A total of 4503 ixodid ticks were collected in 116 sites in all districts of the Altai Republic. Ticks were identified using a light microscope MBS-10 (LOMO, St. Petersburg) and Identification Keys (Filippova, 1977, 1997). The abundance of ixodid ticks (an average number of ticks collected by 1 person for 1 h) was defined for each place, for each district, and for the Altai Republic as a whole. The areas, where ticks were collected, were situated at the 200–2383 m elevation above sea level. They are considered to be lowlands (< 800 m a.s.l.), midlands (800–1700 m a.s.l.), and highlands (>1700 m a.s.l.).
Detection of TBEV Two methods – ELISA and PCR were analyzed the ixodid ticks for TBEV presence individually. ELISA was used to study 2163 questing adult ticks collected from 116 sites. TBEV antigens were detected with the help of the test systems produced by CJSC “Vector-Best” (Novosibirsk city, Russia) according to the manufacturer’s recommendations. The absorbency measurement was carried out on a plan-table photometer “Uniplan” (manufactured by the company of “Pikon”, Russia) the length of a wave is 450 nm.
For the graphic illustrations of the material and statistical data processing the following Microsoft programs were applied: STATISTICA-6.1 and the systems of spreadsheets of Microsoft Excel. The analysis was carried out according to the standard methods of biological statistics (mean value and standard error, regression and correlation analyses. The difference between the two groups for mean values was assessed with the help of the Student’s t-test. To identify the relations between variables, the Pearson’s coefficients of linear correlation were used. The critical level of significance when testing statistical hypotheses, p = 0.05. Results Sampling of ixodid ticks performed at 116 sites in 2012–2014 showed that in the Altai Republic there are 8 species. The most common species in this area are Dermacentor nuttalli – 45.3%, Ixodes persulcatus – 33.1%, Dermacentor silvarum – 9.4%, Dermacentor reticulatus – 6.9%, Haemaphysalis concinna – 5.0%. Other tick species occurred more rarely: Ixodes pavlovskyi – 0.09%, Dermacentor marginatus – 0.18%, Haemaphysalis pospelovashtromae – 0.02%. The tick collection has revealed a wide distribution of ixodid ticks: they have been found in all of 10 districts of the Altai Republic and even within the boundaries of Gorno-Altaisk city. The biotopes, where ticks were collected, are classified according to elevation into 3 groups: lowlands (<800 m a.s.l.), midlands (800–1700 m a.s.l.), and highlands (>1700 m a.s.l.). The obtained information showed that the range of species was the most diverse in lowlands (Table 1). The division of districts into 3 groups according to the altitude is a little conditional. For example, the level of elevation in the Shebalinsky district varies from 400 m up to 1100 m a.s.l. Hence,
Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005
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Species of ixodid ticks
Altitude
Turochaksky district
800–1700 m a.s.l. Choisky district
Maiminsky district
Gorno-Altaisk
Chemalsky district
Shebalinsky district
>1700 m a.s.l. Ust-Kansky district
Ongudaisky district
Ust-Koksinsky district
Ulagansky district
Kosh-Agachsky district
I. persulcatus (n = 1492)
♂♂ ♀♀
263 245
95 74
170 152
1 2
11 14
28 26
5 6
122 102
99 76
1 0
0 0
I. pavlovskyi (n = 4)
♂♂ ♀♀
0 1
1 0
0 1
0 0
0 1
0 0
0 0
0 0
0 0
0 0
0 0
H. concinna (n = 226)
♂♂ ♀♀
1 0
29 41
56 47
4 2
1 2
21 22
0 0
0 0
0 0
0 0
0 0
H. pospelovashtromae (n = 1) D. marginatus (n = 8)
♂♂ ♀♀
0 0
0 0
0 0
0 0
0 0
0 1
0 0
0 0
0 0
0 0
0 0
♂♂ ♀♀
0 0
0 0
0 1
0 0
1 0
0 2
0 0
2 2
0 0
0 0
0 0
D. silvarum (n = 423)
♂♂ ♀♀
0 0
17 18
77 79
4 9
1 3
60 96
0 8
14 14
5 15
1 2
0 0
D. reticulatus (n = 310)
♂♂ ♀♀
0 0
16 16
122 138
0 1
1 0
10 6
0 0
0 0
0 0
0 0
0 0
D. nuttalli (n = 2039)
♂♂ ♀♀
0 0
0 0
0 0
0 0
30 60
17 20
174 181
152 142
26 24
11 22
588 592
510
307
843
23
125
309
374
550
245
37
1180
Total (n = 4503)
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Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005
Table 1 Fauna of ixodid ticks and its vertical distribution in the Altai Republic.
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Fig. 1. Species diversity of ixodid ticks in the Altai Republic. Altitude 250–800 m a.s.l.: Turochaksky district (1), Choisky district (2), Maiminsky district (3), Gorno-Altaisk (4), Chemalsky district (5). Altitude 800–1700 m a.s.l.: Shebalinsky district (6), Ust-Kansky district (7), Ongudaisky district (8), Ust-Koksinsky district (9). Altitude >1700 m a.s.l.: Ulagansky district (10), Kosh-Agachsky district (11).
this district of middle altitude contains areas which are below the marked range (800–1700 m a.s.l.). Besides, specifically in these areas there are ticks of H. concinna and D. reticulatus which cannot be found in midlands. A similar situation develops in the Ulagansky district where the scope of altitudes is even wider – from 450 m up to 2000 m above sea level. However, on the whole this classification is convenient and reflects the situation rather reliably. It shows that in the direction from the north to the south-east, that is, from foothills to high mountains, the increase of altitudes leads to a gradual reduction of the tick fauna (Fig. 1). The research has revealed that for the ticks of H. concinna and D. reticulatus a critical level is 560 m a.s.l. (51◦ 28 42 N, 85◦ 33 55 E), for D. silvarum – 1321 m a.s.l. (51◦ 05 32 N, 85◦ 35 21 E), for I. persulcatus – 1500 m a.s.l. (50◦ 18 40 N, 87◦ 37 12 E) and D. nuttalli ticks prefer to be settled in a range of 630–2200 m above sea level. The highest point where ixodid ticks were found (D. nuttalli) is 2383 m a.s.l. (49◦ 41 17 N, 89◦ 08 18 E). The principal vector of the TBE virus, I. persulcatus, has an extensive geographic range. The ticks of this species inhabit almost all the area of the Altai Republic except for the Kosh-Agachsky district. For I. persulcatus a necessary requirement for the habitat is wood. In the republic from the north to the south forests are replaced by turns with forest-steppe, mountain steppe and high-mountainous desert. In the high-mountainous southeast area with no large forests, the tick of I. persulcatus is absent, and the absolute dominant species becomes the D. nuttalli (Fig. 1). And though in the Altai Republic woods reach a mark of 1800–2000 m a.s.l., in our findings there were no I. persulcatus ticks above 1500 m above sea level. The abundance of ticks varied in the sites from 0.5 to 350.0 specimens/person/1 h of collection averaging 36.2 specimens/ person/1 h of collection across the republic (Table 2). The dependence of the abundance of ixodid ticks on the elevation of the area was not determined. A substantially large number of ticks were registered at the cattle grazing lands – up to 300–350 specimens per person/1 h of collection. Such an influence of livestock on the population density of ixodid ticks has been also noted by other researchers (Danchinova et al., 2006).
Thus, the epidemiological features of the Altai Republic are the following: a diversity of species which quite often are located in one terrain, and prevalence of Dermacentor spp. in the tick collection sites. Dermacentor comprised 61.7% (2780 specimens), whereas the main vectors of the TBE virus (I. persulcatus) comprised only 33.1%. A portion of the collected ticks were examined for the presence of the TBEV by the ELISA (2163 specimens) and by the PCR (834 specimens). The analysis of ixodid ticks for the antigen presence of TBE by the ELISA has shown that all species, including Dermacentor ticks, are vectors of the TBE virus (Table 3). ELISA revealed that TBEV prevalence in tick species were various: I. persulcatus – 2.6%, H. concinna – 0.8%, D. silvarum – 16.7%, D. reticulatus – 10.6% and D. nuttalli – 26.5%. The lowest level was in H. concinna, and the highest one was in D. nuttalli. Generally the prevalence rate of Dermacentor spp. analyzed by the ELISA made up 20.9% (237 positive specimens of 1134 tested) and was significantly higher than that of the main vector I. persulcatus (p < 0.001). The difference of the virus prevalence of infective males and females from each species was not statistically significant. In addition, the analysis has shown the drop in the infection rate at the rise of elevation for ticks D. silvarum from 21.6% to 12.8% (p < 0.05) and for D. nuttalli from 34.8% to 3.7% (p < 0.001) (Fig. 2). Taking into account that the ELISA can show false-positive reactions because of nonspecific records of other flaviviruses and microorganisms identified in ticks (Holodilov et al., 2013), there
Fig. 2. Vertical distribution of infected ixodid ticks in the Altai Republic (TBEV detection by ELISA).
Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005
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Table 2 The abundance of collected ixodid ticks in the Altai Republic. Administrative divisions
Altitude (m a.s.l.)
Number of collected ticks
1. Turochaksky district 2. Choisky district 3. Maiminsky district 4. Gorno-Altaisk 5. Chemalsky district 6. Shebalinsky district 7. Ust-Kansky district 8. Ongudaisky district 9. Ust-Koksinsky district 10. Ulagansky district 11. Kosh-Agachsky district
313–363 350–600 284–640 290–310 400–700 460–1200 1000–1200 800–1758 1000–1220 1100–1790 1740–2390
510 307 843 23 125 309 374 550 245 37 1180
10.0 6.5 9.5 11.0 5.5 6.0 6.5 45.5 10.0 10.0 4.0
51.0 47.2 88.7 2.1 22.7 51.5 57.5 12.1 24.5 3.7 295.0
284–2390
4503
124.5
36.2
Total
Number of flag/h
Tick number (specimens/1 h of collection)
Table 3 TBEV detection by ELISA in questing ticks collected in the Altai Republic. m a.s.l.
Administrative divisions
TBEV prevalence of ixodid species (adults positive/exam) I. persulcatus (23/897)
H. concinna (1/132)
D. silvarum (57/342)
D. reticulatus (20/189)
D. nuttalli (160/603)
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
<800 m
Turochaksky district Choisky district Maiminsky district Gorno-Altaisk Chemalsky district
1/112 2/95 8/118 0/1 0/11
3/74 3/74 2/131 0/2 0/14
0 0/29 0/21 0/4 0
0 1/41 0/22 0/2 0
0 2/7 13/57 0/4 0/1
0 3/8 14/59 0/9 0/3
0 0/6 8/52 0 0
0 2/16 9/98 0/1 0
0 0 0 0 10/30
0 0 0 0 15/60
800–1700 m
Shebalinsky district Ust-Kansky district Ongudaisky district Ust-Koksinsky
0/15 0/5 2/79 0/31
0/16 0/6 0/72 2/40
0/10 0 0 0
0/3 0 0 0
6/53 0 1/9 0/5
13/90 0/8 2/14 3/15
0/10 0 0 0
1/6 0 0 0
10/17 28/58 24/82 5/13
5/20 15/47 22/69 8/13
>1700 m
Ulagansky district Kosh-Agachsky Total Prevalence rate (%)
0/1 0 13/468 2.8
0 0 10/429 2.3
0 0 0/64 0
0 0 1/68 1.5
0 0 22/136 16.2
0 0 35/206 16.9
0 0 8/68 11.8
0 0 12/121 9.9
5/11 2/80 84/291 28.9
7/22 4/81 76/312 24.4
were tests run simultaneously for revealing the RNA of the TBE virus by the PCR (Table 4). Thus the RNA of the TBE virus was found in 12.3% of Dermacentor ticks (48 out of 389 specimens) and in 1.7% of I. persulcatus ticks – (in 6 out of 361 specimens). The D. nuttalli had the highest prevalence rate (14.9%), as it had been identified by the ELISA. The ticks of H. concinna showed a low infection rate (1.2%). The comparison of the infection rate of the species in both reactions (ELISA and PCR) is shown in Fig. 3. The difference in the TBEV prevalence in Dermacentor ticks and I. persulcatus was also confirmed by PCR: it was statistically significant (p < 0.001). Despite the fact that virus prevalence of ixodid ticks at PCR was lower than at ELISA, the infection rate was recorded as one of the highest in Russia.
We also studied the dependence of TBEV prevalence in ticks on an altitude factor. This regularity was revealed for I. persulcatus, whereas the infection rate for D. nuttalli (PCR) decreased with altitude: Ust-Kan neighborhood – 23.4% (1059 m), Elo neighborhood – 18.9% (1156 m), Kosh-Agach – 3.3% (1758 m), and the Ulandryk River valley (Kosh-Agach district) – 0.42% (2155 m). In midlands the average infection rate for D. nuttalli was 20.0%, whereas in highlands it was 3.3%. The difference was statistically significant (p < 0.001). The high threshold of the TBEV detection for I. persulcatus ticks was at 1295 m a.s.l. (50◦ 38 41 N, 86◦ 18 44 E), for D. reticulatus – 440 m a.s.l. (52◦ 02 30 N, 86◦ 52 11 E), for H. concinna – 440 m a.s.l. (52◦ 02 30 N, 86◦ 52 11 E), for D. silvarum – 1282 m a.s.l. (50◦ 58 04 N, 85◦ 43 11 E), and for D. nuttalli – 2155 m a.s.l.
Table 4 TBEV detection by PCR in questing ticks collected in the Altai Republic. m a.s.l.
Administrative divisions
TBEV prevalence of ixodid species (adults positive/exam.) I. persulcatus (6/361)
H. concinna (1/84)
D. silvarum (4/53)
D. reticulatus (3/60)
D. nuttalli (41/276)
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
<800 m
Turochaksky district Maiminsky district
2/120 0/24
2/100 0/21
0 1/35
0 0/25
0 2/20
0 2/20
0 1/20
0 2/40
0 0
0 0
800–1700 m
Shebalinsky district Ust-Kansky district Ongudaisky district Ust-Koksinsky
0/13 0 1/43 0
0/10 0 1/30 0
0/11 0 0 0
0/13 0 0 0
0/7 0 0 0
0/6 0 0 0
0 0 0 0
0 0 0 0
0 5/22 15/70 0/13
0 6/25 13/73 0/11
>1700 m
Kosh-Agachsky district Total Prevalence rate (%)
0 3/200 1.5
0 3/161 1.9
0 1/46 2.2
0 0/38 0
0 2/27 7.4
0 2/26 7.7
0 1/20 5.0
0 2/40 5.0
1/30 21/135 15.6
1/32 20/141 14.2
Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005
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Fig. 3. Comparison of TBEV detection by ELISA and PCR in questing ticks collected in the Altai Republic.
Fig. 4. Tick-bite incidence rate in different landscape areas of the Altai Republic (2013).
(50◦ 01 01 N, 88◦ 40 58 E), which is slightly lower than the elevation limits for the ticks of these species. The study has shown that the intensity of the TBE foci in the Altai Republic is caused by a wide distribution, a large population, and a high TBEV prevalence in ticks. Annually from 1500 to 3300 people seek medical treatment due to tick bites. In 2013, 3186 people were registered with ixodid tick bites: the incidence rate was 1511.9 per 100,000 inhabitants. Correlation analysis showed that there is statistically significant strong reverse interconnection between altitude and tick-bite incidence (Pearson’s coefficient r = −0.78, p < 0.05). Permanent high values of tick-bite incidences are recorded in the lowlands and low values in the highlands (Fig. 4). This is attributed to gradual decrease of I. persulcatus in nature from north to south and increase of the less aggressive D. nuttalli that attacks people more rarely: people have time to notice these large and less mobile ticks and to remove them before they attach themselves. The tick-bite incidence is the highest in the I. persulcatus dominance zone (lowlands) (Fig. 5). On average, the TBE season in the Altai Republic lasts from early March to late October. In 2013, the first tick-bite was registered on March 11, and the last one on October 29. From the end of August till the end of October only the bites of Dermacentor spp. are registered; however, there are usually only a few visits to the hospital (single instances). Collectively, there are 81.0% of cases of visits to the hospital after tick bites in the lowlands. Therefore, people in these areas have a higher risk of getting a TBE infection than the population of the midland (15.6%) and of the highlands (3.4%). If
Fig. 5. Seasonal dynamics of tick bite cases in lowland, midland, and highland areas of the Altai Republic (2013). The figure shows the seasonal dynamics of tick bite cases in three districts: Turochaksky (lowland), Ongudaisky (midland), and KoshAgachsky (highland). The influence of D. nuttalli is noticeable in the midlands and highlands: the two latter graphics show a second peak caused by the abundance increase of D. nuttalli in August.
calculated as a population size, in the lowland areas 2 out of 100 inhabitants have tick bites, in the midland – 1 out of 120, and in high mountains – 1 out of 250 inhabitants. The average yearly TBE incidence rate of the Altai Republic between 2004 and 2013 was 22.6 per 100,000 inhabitants. That is 10 times higher than the rate of Russia. Territory ranking according to the TBE infection shows that the population incidence is associated with the altitude. There is statistically significant strong reverse interconnection between elevation above sea level and TBE incidence (Pearson’s coefficient r = −0.67, p < 0.05). The highest TBE infection risk is recorded in the Turochaksky district (lowland), where the majority of settlements are located at 300–400 m a.s.l.: I. persulcatus is the dominant species here (dominance index 99.3%) and the average yearly TBE incidence is 50.0 per 100,000 inhabitants. The least risk of infection is recorded in the highland Kosh-Agachsky district (1700–2200 m a.s.l.): D. nuttalli transmits TBEV at this altitude and average yearly TBE incidence is 7.4 per 100,000 inhabitants. The rest regions of the Altai Republic (400–1700 m a.s.l.) have TBE foci with mixed ixodid fauna, which belong to the group of mean risk: average yearly TBE incidence is 15.9–28.1 per 100,000 inhabitants (Fig. 6). Among the districts which are part of the midlands, the Ongudaisky district is notable for high TBE incidence. The analysis of the epidemiological records shows that 26 out of 42 patients registered in 2004–2013 were infected in the terrains which are at 800–860 m a.s.l., and 16 people at the altitude 1000–1240 m a.s.l. An unusual situation of the tick-borne encephalitis disease is marked in the Ulagansky district. Officially it belongs to the regions of the Far North because of a severe climate and a location of villages at the elevation exceeding 1500 m above sea level. However, 5 of 15 settlements in this district are situated at a much lower altitude. The analysis shows that the most troubled village, in terms of the TBE disease, is Balykcha (51◦ 17 05 N, 87◦ 42 30 E). Here 8 people (out of 25 patients registered in the Ulagansky district) became sick in the described season. This village is located in a deep hollow (the Chulushman valley) at the elevation of only 450 m above sea level. There are boreal woods (called taiga) around Balykcha, where I. persulcatus ticks are widely spread. Other cases of infection in the Ulagansky district occurred at the heights of 1450 m a.s.l., that is, in the middle altitude, not in highlands. Therefore, we can trace the effect of the altitude on the intensity of the foci and the TBE incidence rate in the Ongudaisky and Ulagansky districts.
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Altai Republic, including the highland territory, is an endemic TBE area. Conclusion
Fig. 6. Territory ranking according to TBE infection risk in the Altai Republic (2004–2013). Lowlands: Turochaksky district (1), Choysky district (2), Mayminsky district (3), Gorno-Altaysk (4), Chemalsky district (5). Midlands: Shebalinsky district (6), Ust-Kansky district (7), Ongudaysky district (8), Ust-Koksinsky district (9). Highlands: Ulagansky district (10), Kosh-Agachsky district (11).
Discussion We have found out several epidemiological features of TBE in the Altai Republic, which are the following: a specific diversity and a large population of ixodid ticks, their infection with the TBE virus, close contacts of the population with vectors, and an involvement into the circulation of the TBE virus of not only the main vector, I. persulcatus, but also other species. In spite of the fact that the infection rate with the TBE virus of Haemaphysalis spp. and Dermacentor spp. ticks was proved a long time ago (Ryzhov and Skrynnik, 1941), the issue of their role in TBE foci remains debatable (Korenberg et al., 2013). The laboratory research shows that in the experiments the ticks of genus Dermacentor are capable to accept, keep, and transmit the TBE virus to susceptible animals: however, the efficacy of this process is lower than for I. persulcatus ticks (Alekseev and Chunikhin, 1990, 1992; Belova et al., 2013). Meanwhile, in the Altai Republic the virus in D. nuttalli ticks, a reservoir host and vector of the TBEV in the high-mountainous Kosh-Agachsky district, was found in 1954 (Ljubushkina and Jugova, 1963). The TBE cases among the population in this highland area have been registered since 1984. The Kosh-Agachsky district is the coldest area in Gorny Altai where the winter lasts more than 7 months, the outside temperature reaches −62 ◦ C, and the absolute value of a vegetation period according to the sum of mean daily air temperature above +5 ◦ C makes up only 1300–1400 ◦ C (whereas for I. persulcatus ticks it should not be lower than 1600 ◦ C). The annual precipitation amount in this area is small and makes up about 116 mm; with the highest amount of precipitation falling in summer. However, cold and dry winters do not exterminate D. nuttalli. The abundance of ticks in the vicinities of the settlements in the Kosh-Agachsky district (Kokorya, Tashanta, Zhana-Aul, and Telengit-Sortogoy) is especially high in April – 300–350 specimens/person/1 h of collection. This is due to biological characteristics of D. nuttalli, able to resist to low temperatures, and the great abundance of its hosts. Although the TBE incidence rate is the lowest in the highland Kosh-Agachsky district (7.4 per 100,000 inhabitants), it is 3 times higher than the all-Russian TBE incidence value; i.e. the whole the
This study showed that the altitude results in the development of the epidemiological TBE situation. The highest species diversity of ixodid ticks is recorded at 280–560 m a.s.l. The spectrum of vector species decreases with altitude and the leading species changes: besides I. persulcatus, D. nuttalli also transmits TBEV in the midlands, whereas in the highlands the latter is an absolute dominant species. However, these ticks are less effective vector species than I. persulcatus. The tick-bite incidence decreases with altitude (r = −0.78, p < 0.05) and the TBE incidence also decreases (r = −0.67, p < 0.05). Thus, the highest risk of TBE infection is recorded in the lowland Turochaksky district (300–400 m a.s.l.) where I. persulcatus is a dominant species, whereas the lowest risk is registered in the highland foci of the Altai Republic (1700–2200 m a.s.l.): D. nuttalli is a vector species in this place. Other areas with mixed ixodid fauna are included in the zone of a mean tick-borne encephalitis infection risk. References Alekseev, A.N., Chunikhin, S.P., 1990. The experimental transmission of the tickborne encephalitis virus by ixodid ticks (the mechanisms, time periods, species and sex differences). Parazitologiia 24, 177–185 (in Russian). Alekseev, A.N., Chunikhin, S.P., 1992. Difference in distant transmission ability of tick-borne encephalitis virus by Ixodid ticks belonging to different subfamilies. Parazitologia 26, 506–515 (in Russian). Belova, O.A., Brisker, S.A., Burebkova, L.A., Karganova, G.G., 2013. Ticks of genus Dermacentor Koch., 1844 (Acari: Ixodidae) as possible vectors of tick-borne encephalitis virus. In: Fundamental and Applied Aspects of the Study of Parasitic Arthropods in the XXI Century: International Conference in Memoriam of Prof. Yuri S. Balashov, a Corresponding Member of the RAS, St. Petersburg, Russia, pp. 167–168 (in Russian). Danchinova, G.A., Hasnatinov, M.A., Zlobin, V.I., et al., 2006. Iksodid ticks of Eastern Siberia and Mongolia and their spontaneous pathogen contamination in natural focal transmissible infections. Bjul. sib. med. T.5. (Pril.1), 137–143 (in Russian). Daniel, M., Materna, J., Honig, V., Metelka, L., Danielová, V., Harcarik, J., Kliegrová, S., Grubhoffer, L., 2009. Vertical distribution of the tick Ixodes ricinus and tickborne pathogens in the northern Moravian mountains correlated with climate warming (Jeseníky Mts., Czech Republic). Cent. Eur. J. Public Health 17 (3), 139– 145. Danielová, V., Rudenko, N., Daniel, M., Holubová, J., Materna, J., Golovchenko, M., Schwarzová, L., 2006. Extension of Ixodes ricinus ticks and agents of tick-borne diseases to mountain areas in the Czech Republic. Int. J. Med. Microbiol. 296 (Suppl. 40), 48–53. Danielová, V., Schwarzová, L., Materna, J., Daniel, M., Metelka, L., Holubová, J., Kˇríˇz, B., 2008. Tick-borne encephalitis virus expansion to higher altitudes correlated with climate warming. Int. J. Med. Microbiol. 298 (Suppl. 1), 68–72. Filippova, N.A., 1977. Ixodid ticks of the subfamily Ixodinae. Fauna S.S.S.R., Paukoobraznye 4 (1), 393 (in Russian). Filippova, N.A., 1997. Ixodid ticks of subfamily Amblyomminae. Arachnoidea 5 (1), 436 (in Russian). Grebenyuk, R.V., 1966. Ixodid Ticks of Kirgizia, Frunze. Ilim, Russian. Holodilov, I.S., Motuzova, O.V., Belova, O.A., Orlova, O.E., Karganova, G.G., 2013. Problem of identification of tick-borne encephalitis virus in ticks. In: Fundamental and Applied Aspects of the Study of Parasitic Arthropods in the XXI Century: International Conference in Memoriam of Prof. Yuri S. Balashov, a Corresponding Member of the RAS, St. Petersburg, Russia, pp. 167–168 (in Russian). Holzmann, H., Aberle, S.W., Stiasny, K., Werner, P., Mischak, A., Zainer, B., Netzer, M., Koppi, S., Bechter, E., Heinz, F.X., 2009. Tick-borne encephalitis from eating goat cheese in a mountain region of Austria. Emerg. Infect. Dis. 15 (10), 1671–1673. Korenberg, Je.I., Kovalevskij, Ju.V., 2000. Main Features of Tick-Borne Encephalitis Eco-epidemiology in Russia. Problems of Tick-Borne and Parasitic Diseases. St. Petersburg., pp. 13–20 (in Russian). Korenberg, E., Pomelova, V., Osin, N., 2013. Infections with Natural Focality Transmitted by Ticks. Moscow, 463 pp. (in Russian). Ljubushkina, V.M., Jugova, L.A., 1963. Tick-Borne Encephalitis in the GornoAltaysk Autonomous Region. Tick-Borne Encephalitis in the Altai Krai. Barnaul., pp. 30–41 (in Russian). Materna, J., Daniel, M., Metelka, L., Harcarik, J., 2008. The vertical distribution, density and the development of the tick Ixodes ricinus in mountain areas influenced by climate changes (The Krkonose Mts., Czech Republic). Int. J. Med. Microbiol. 298 (Suppl. 1), 25–37.
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Rubina, M.A., 1969. Activity and life expectancy of a taiga tick I. persulcatus in Western Sayan Mountains. Natural Focal diseases of the Urals, Siberia and Far East. Sverdlovsk., pp. 80–81. Ryzhov, N.V., Skrynnik, A.N., 1941. On the natural infection of ticks of the family Ixodldae with the virus of spring-summer encephalitis. Trud. voenn. med. Akad. krasn. Armi. 25, 27–33 (in Russian). Sapegina, V.F., 1980. Distribution of Iksodid Ticks in a Forest Zone of the Western and Central Siberia. Problems of Tick-Borne and Parasitic Diseases. Novosibirsk., pp. 67–76 (in Russian).
Süss, J., 2008. Tick-borne encephalitis in Europe and beyond – the epidemiological situation as of 2007. Euro Surveill. 13 (26). Verhozina, M.M., Zlobin, V.I., Kozlova, I.V., Demina, T.V., Dzhioev, Ju.P., Lisak, O.V., Doroshhenko, E.K., Protasova, E.G., 2008. Epidemioligical and molecular genetic analysis of tick-borne encephalitis virus population in the Irkutsk region. Jepidemiologija i vakcinoprofilaktika 38, 12–18 (in Russian). Zlobin, V.I., 2010. Tick-borne encephalitis in the Russian Federation: etiology, epidemiology and prevention strategy. Terra Medica 2, 13–21 (in Russian).
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Original article
Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia L.D. Shchuchinova a , I.V. Kozlova b,c,∗ , V.I. Zlobin c a b c
Federal Service on Customers’ Rights Protection and Human Well-being Surveillance in the Altai Republic, 649000 Gorno-Altaisk, Russia FSBI “Scientific Health Centre for Family and Human Reproduction” SB RAMS, 664025 Irkutsk, Russia Irkutsk State Medical University of Healthcare of Russian Federation, 664003 Irkutsk, Russia
a r t i c l e
i n f o
Article history: Received 17 June 2014 Received in revised form 20 January 2015 Accepted 12 February 2015 Available online xxx Keywords: Tick-borne encephalitis Altitude above sea level Ticks
a b s t r a c t The Altai Republic is a highly endemic area as far as tick-borne encephalitis (TBE) is concerned. The aim of the research was to study the effect of altitude on the risk of tick-borne encephalitis infection in the Altai Republic. The paper analyzes the following data: the study of ixodid ticks collected from the vegetation in 116 sites at the 200–2383 m elevation above sea level in 2012–2014, TBE virus prevalence of these vectors, tick-bite incidence rate, and TBE incidence rate of the population. Species identification of 4503 specimens has shown that the most common species are Dermacentor nuttalli (45.3%), Ixodes persulcatus (33.1%), Dermacentor silvarum (9.4%), Dermacentor reticulatus (8.9%), and Haemaphysalis concinna (5.0%). A total of 2997 adult ixodid ticks were studied for the presence of the TBE virus; 2163 samples were examined by ELISA, while 834 specimens were tested by PCR. The TBE virus prevalence of Dermacentor spp. ticks in both reactions was significantly higher than of Ixodes persulcatus ticks (p < 0.001). The work shows that the altitude is an important factor in the development of the epidemiological situation of tick-borne encephalitis: the higher the elevation of the area above sea level, the smaller the range of vectors. There is also a change of a leading species: in middle altitude (800–1700 m above sea level) the virus is transmitted by ticks of D. nuttalli along with I. persulcatus, and in high mountains (above 1700 m above sea level) D. nuttalli becomes an absolute dominant species. However, these species of ticks are less effective vectors than I. persulcatus. With the increase of altitude the tick-bite incidence rate decreases (r = −0.78, p < 0.05), and TBE incidence also reduces (r = −0.67, p < 0.05). © 2015 Elsevier GmbH. All rights reserved.
Introduction Russia possesses the most extensive geographic distribution of tick-borne encephalitis. It is believed that the intensity of the natural TBE foci determines the high abundance of the main vector species I. ricinus and I. persulcatus and their infection rate (Korenberg and Kovalevskij, 2000). The distribution of these tick species has been well studied. In recent years, some investigations have been focused on the positive effect of continuous temperature rise and humidity on the improvement of habitat conditions for ticks (Süss, 2008). In mountainous areas of Central Europe, ticks have been recorded at higher altitudes up to 1100 and 1300 m above sea level (a.s.l.) compared to the cases recorded before 1990 (at
∗ Corresponding author at: FSBI “Scientific Health Centre for Family and Human Reproduction” SB RAMS, 664025 Irkutsk, Russia. Tel.: +7 83952333951. E-mail addresses: [email protected] (L.D. Shchuchinova), [email protected] (I.V. Kozlova), [email protected] (V.I. Zlobin).
1000 m a.s.l.) (Danielová et al., 2006, 2008; Materna et al., 2008; Daniel et al., 2009). Moreover, some cases of alimentary TBE infection caused by the consumption of nonpasteurized milk of goats, which were grazing in pastures at 1500 m a.s.l., were recorded in Austria (Holzmann et al., 2009). In Russia, I. persulcatus was recorded at 1400 m a.s.l. on the northern slopes of the Western Sayan, in the Central and Eastern Altai (at 1900 m a.s.l.) and Tian Shan (at 3000 m a.s.l.) (Rubina, 1969; Sapegina, 1980; Grebenyuk, 1966). Besides I. ricinus and I. persulcatus, other tick species can be additional vectors for tick-borne encephalitis virus (TBEV) in some areas (Danchinova et al., 2006; Verhozina et al., 2008; Zlobin, 2010). Their role in the natural TBE foci is still unclear, as well as the altitude influence on their density. The Altai Republic is one of the areas where along with I. persulcatus as a dominant vector at the natural foci of TBE, there are other species of ixodid ticks. This is a mountain terrain in the south of Western Siberia, located on a junction of the Siberian taiga, Kazakh steppes, and semi-deserts of Mongolia that determines a
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surprising diversity of landscapes. Settlements are situated at 200–2200 m a.s.l. The size of the republic area is 92,903 km2 . The population is 210,344 people. The length of the terrain from the north to the south is 400 km, and from the west to the east is 360 km. Average annual temperatures in Gorny Altai fluctuate within the limits of +4◦ (in northern and western suburbs) to −7◦ (in highmountainous regions). The fauna of the Altai Republic includes 93 species of mammals and 312 species of birds. The abundance of ixodid ticks and their widespread presence are attributed to a large number of hosts and diversity of landscapes in this territory. Along with natural factors, social factors contribute to the spread of the TBE virus. The mountain landscape predetermines cattlebreeding in the Altai Republic. Crop lands occupy only 1.5% of the republic, whereas the largest part of agricultural lands belongs to grazing – 84.7%. Cattle breeding industry is one of the primary sources of income for the agricultural population. According to the report of the Territorial Agency of the Federal Service of State Statistics on January, 1st, 2014 in the Altai Republic the farms of all categories comprised 245,000 heads of cattle, 11,900 pigs, 477,500 sheep, 149,400 goats and 136,200 horses. Domestic animals are active hosts for ixodid ticks, sustaining a high number of vectors, and also carry ticks into settlements. The average yearly TBE incidence of the Altai Republic between 2004 and 2013 was 22.6 per 100,000 inhabitants which is almost 10 times higher than TBE incidence of Russia. However, disease levels in different districts of the Altai Republic differ considerably. The aim of this work was to assess the influence of altitude on a tick-borne encephalitis infection risk in the natural foci of the Altai Republic.
Materials and methods
In addition, 834 questing adult ticks collected from 27 sites were tested by PCR. Total RNA was extracted from ixodid ticks using the “AmpliPrime® RIBO-prep-100” kit (“AmpliPrime”, Russia). The reverse transcription was performed with “RevertaL-100” kit containing random hexanucleotides (“Amplisence”, Russia). PCR reaction was performed in 20 l of the reaction mixture containing 67 mM Tris–HCl (pH 8.9), 16.6 mM (NH4 )2 SO4 , 2 mM MgCl2 , 0.01% Tween 20, 200 M of each dNTP, 5% glycerol, 0.5 M primers, 2 U of Taq DNA polymerase (“BioSan”, Russia) and 3 l of cDNA for primary reactions or 1 l of the primary PCR products for nested reaction. The PCR conditions comprised initial denaturation for 3 min at 94 ◦ C followed by 35 cycles of denaturation for 0.5 min at 94 ◦ C, annealing for 0.5 min at 48 ◦ C and elongation for 1 min at 72 ◦ C. The primers corresponding to positions 2199–2219 and 2517–2539 nr or positions 2214–2238 and 2402–2424 nr of TBEV genome were used for primary or nested reactions, respectively. The length of final PCR-products was 211 bp. Epidemiological data The analysis of the TBE incidence rate of the population in the Altai Republic (during 2004–2013) was done according to the state statistical reporting: Form # 2 “Information on Infectious and Parasitic Diseases”, Form # 60 “Register of Infectious Patients”, Form # 003/U “Medical Record of the Inpatient”, Form # 025/U “Medical Record of the Outpatient”. We also analyzed TBE incidence using data from epidemiological records (n = 2183). All cases of tick bites have been registered in the Russian Federation. We analyzed the tick-bite incidence rate per 100,000 inhabitants in the Altai Republic for the period of 2013. The information on a number of the livestock was obtained from the reports of the Territorial Agency of the Federal State Statistics Service in the Altai Republic (http://statra.gks.ru).
Tick collection Statistic analysis Questing adult ticks were collected by flagging the vegetation every 8–12 km along the highways of the Altai Republic during the peak of tick activity (from mid April till early June of 2012–2014). The sampling sites were located 30–300 m from the motorways. The preference was given to the places where earlier there had been cases of TBE infections, that is, to the tourist camps, popular vacation and recreation spots in the vicinities of settlements, and to the scenic view points at mountain passes. A total of 4503 ixodid ticks were collected in 116 sites in all districts of the Altai Republic. Ticks were identified using a light microscope MBS-10 (LOMO, St. Petersburg) and Identification Keys (Filippova, 1977, 1997). The abundance of ixodid ticks (an average number of ticks collected by 1 person for 1 h) was defined for each place, for each district, and for the Altai Republic as a whole. The areas, where ticks were collected, were situated at the 200–2383 m elevation above sea level. They are considered to be lowlands (< 800 m a.s.l.), midlands (800–1700 m a.s.l.), and highlands (>1700 m a.s.l.).
Detection of TBEV Two methods – ELISA and PCR were analyzed the ixodid ticks for TBEV presence individually. ELISA was used to study 2163 questing adult ticks collected from 116 sites. TBEV antigens were detected with the help of the test systems produced by CJSC “Vector-Best” (Novosibirsk city, Russia) according to the manufacturer’s recommendations. The absorbency measurement was carried out on a plan-table photometer “Uniplan” (manufactured by the company of “Pikon”, Russia) the length of a wave is 450 nm.
For the graphic illustrations of the material and statistical data processing the following Microsoft programs were applied: STATISTICA-6.1 and the systems of spreadsheets of Microsoft Excel. The analysis was carried out according to the standard methods of biological statistics (mean value and standard error, regression and correlation analyses. The difference between the two groups for mean values was assessed with the help of the Student’s t-test. To identify the relations between variables, the Pearson’s coefficients of linear correlation were used. The critical level of significance when testing statistical hypotheses, p = 0.05. Results Sampling of ixodid ticks performed at 116 sites in 2012–2014 showed that in the Altai Republic there are 8 species. The most common species in this area are Dermacentor nuttalli – 45.3%, Ixodes persulcatus – 33.1%, Dermacentor silvarum – 9.4%, Dermacentor reticulatus – 6.9%, Haemaphysalis concinna – 5.0%. Other tick species occurred more rarely: Ixodes pavlovskyi – 0.09%, Dermacentor marginatus – 0.18%, Haemaphysalis pospelovashtromae – 0.02%. The tick collection has revealed a wide distribution of ixodid ticks: they have been found in all of 10 districts of the Altai Republic and even within the boundaries of Gorno-Altaisk city. The biotopes, where ticks were collected, are classified according to elevation into 3 groups: lowlands (<800 m a.s.l.), midlands (800–1700 m a.s.l.), and highlands (>1700 m a.s.l.). The obtained information showed that the range of species was the most diverse in lowlands (Table 1). The division of districts into 3 groups according to the altitude is a little conditional. For example, the level of elevation in the Shebalinsky district varies from 400 m up to 1100 m a.s.l. Hence,
Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005
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Species of ixodid ticks
Altitude
Turochaksky district
800–1700 m a.s.l. Choisky district
Maiminsky district
Gorno-Altaisk
Chemalsky district
Shebalinsky district
>1700 m a.s.l. Ust-Kansky district
Ongudaisky district
Ust-Koksinsky district
Ulagansky district
Kosh-Agachsky district
I. persulcatus (n = 1492)
♂♂ ♀♀
263 245
95 74
170 152
1 2
11 14
28 26
5 6
122 102
99 76
1 0
0 0
I. pavlovskyi (n = 4)
♂♂ ♀♀
0 1
1 0
0 1
0 0
0 1
0 0
0 0
0 0
0 0
0 0
0 0
H. concinna (n = 226)
♂♂ ♀♀
1 0
29 41
56 47
4 2
1 2
21 22
0 0
0 0
0 0
0 0
0 0
H. pospelovashtromae (n = 1) D. marginatus (n = 8)
♂♂ ♀♀
0 0
0 0
0 0
0 0
0 0
0 1
0 0
0 0
0 0
0 0
0 0
♂♂ ♀♀
0 0
0 0
0 1
0 0
1 0
0 2
0 0
2 2
0 0
0 0
0 0
D. silvarum (n = 423)
♂♂ ♀♀
0 0
17 18
77 79
4 9
1 3
60 96
0 8
14 14
5 15
1 2
0 0
D. reticulatus (n = 310)
♂♂ ♀♀
0 0
16 16
122 138
0 1
1 0
10 6
0 0
0 0
0 0
0 0
0 0
D. nuttalli (n = 2039)
♂♂ ♀♀
0 0
0 0
0 0
0 0
30 60
17 20
174 181
152 142
26 24
11 22
588 592
510
307
843
23
125
309
374
550
245
37
1180
Total (n = 4503)
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<800 m a.s.l.
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Table 1 Fauna of ixodid ticks and its vertical distribution in the Altai Republic.
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Fig. 1. Species diversity of ixodid ticks in the Altai Republic. Altitude 250–800 m a.s.l.: Turochaksky district (1), Choisky district (2), Maiminsky district (3), Gorno-Altaisk (4), Chemalsky district (5). Altitude 800–1700 m a.s.l.: Shebalinsky district (6), Ust-Kansky district (7), Ongudaisky district (8), Ust-Koksinsky district (9). Altitude >1700 m a.s.l.: Ulagansky district (10), Kosh-Agachsky district (11).
this district of middle altitude contains areas which are below the marked range (800–1700 m a.s.l.). Besides, specifically in these areas there are ticks of H. concinna and D. reticulatus which cannot be found in midlands. A similar situation develops in the Ulagansky district where the scope of altitudes is even wider – from 450 m up to 2000 m above sea level. However, on the whole this classification is convenient and reflects the situation rather reliably. It shows that in the direction from the north to the south-east, that is, from foothills to high mountains, the increase of altitudes leads to a gradual reduction of the tick fauna (Fig. 1). The research has revealed that for the ticks of H. concinna and D. reticulatus a critical level is 560 m a.s.l. (51◦ 28 42 N, 85◦ 33 55 E), for D. silvarum – 1321 m a.s.l. (51◦ 05 32 N, 85◦ 35 21 E), for I. persulcatus – 1500 m a.s.l. (50◦ 18 40 N, 87◦ 37 12 E) and D. nuttalli ticks prefer to be settled in a range of 630–2200 m above sea level. The highest point where ixodid ticks were found (D. nuttalli) is 2383 m a.s.l. (49◦ 41 17 N, 89◦ 08 18 E). The principal vector of the TBE virus, I. persulcatus, has an extensive geographic range. The ticks of this species inhabit almost all the area of the Altai Republic except for the Kosh-Agachsky district. For I. persulcatus a necessary requirement for the habitat is wood. In the republic from the north to the south forests are replaced by turns with forest-steppe, mountain steppe and high-mountainous desert. In the high-mountainous southeast area with no large forests, the tick of I. persulcatus is absent, and the absolute dominant species becomes the D. nuttalli (Fig. 1). And though in the Altai Republic woods reach a mark of 1800–2000 m a.s.l., in our findings there were no I. persulcatus ticks above 1500 m above sea level. The abundance of ticks varied in the sites from 0.5 to 350.0 specimens/person/1 h of collection averaging 36.2 specimens/ person/1 h of collection across the republic (Table 2). The dependence of the abundance of ixodid ticks on the elevation of the area was not determined. A substantially large number of ticks were registered at the cattle grazing lands – up to 300–350 specimens per person/1 h of collection. Such an influence of livestock on the population density of ixodid ticks has been also noted by other researchers (Danchinova et al., 2006).
Thus, the epidemiological features of the Altai Republic are the following: a diversity of species which quite often are located in one terrain, and prevalence of Dermacentor spp. in the tick collection sites. Dermacentor comprised 61.7% (2780 specimens), whereas the main vectors of the TBE virus (I. persulcatus) comprised only 33.1%. A portion of the collected ticks were examined for the presence of the TBEV by the ELISA (2163 specimens) and by the PCR (834 specimens). The analysis of ixodid ticks for the antigen presence of TBE by the ELISA has shown that all species, including Dermacentor ticks, are vectors of the TBE virus (Table 3). ELISA revealed that TBEV prevalence in tick species were various: I. persulcatus – 2.6%, H. concinna – 0.8%, D. silvarum – 16.7%, D. reticulatus – 10.6% and D. nuttalli – 26.5%. The lowest level was in H. concinna, and the highest one was in D. nuttalli. Generally the prevalence rate of Dermacentor spp. analyzed by the ELISA made up 20.9% (237 positive specimens of 1134 tested) and was significantly higher than that of the main vector I. persulcatus (p < 0.001). The difference of the virus prevalence of infective males and females from each species was not statistically significant. In addition, the analysis has shown the drop in the infection rate at the rise of elevation for ticks D. silvarum from 21.6% to 12.8% (p < 0.05) and for D. nuttalli from 34.8% to 3.7% (p < 0.001) (Fig. 2). Taking into account that the ELISA can show false-positive reactions because of nonspecific records of other flaviviruses and microorganisms identified in ticks (Holodilov et al., 2013), there
Fig. 2. Vertical distribution of infected ixodid ticks in the Altai Republic (TBEV detection by ELISA).
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Table 2 The abundance of collected ixodid ticks in the Altai Republic. Administrative divisions
Altitude (m a.s.l.)
Number of collected ticks
1. Turochaksky district 2. Choisky district 3. Maiminsky district 4. Gorno-Altaisk 5. Chemalsky district 6. Shebalinsky district 7. Ust-Kansky district 8. Ongudaisky district 9. Ust-Koksinsky district 10. Ulagansky district 11. Kosh-Agachsky district
313–363 350–600 284–640 290–310 400–700 460–1200 1000–1200 800–1758 1000–1220 1100–1790 1740–2390
510 307 843 23 125 309 374 550 245 37 1180
10.0 6.5 9.5 11.0 5.5 6.0 6.5 45.5 10.0 10.0 4.0
51.0 47.2 88.7 2.1 22.7 51.5 57.5 12.1 24.5 3.7 295.0
284–2390
4503
124.5
36.2
Total
Number of flag/h
Tick number (specimens/1 h of collection)
Table 3 TBEV detection by ELISA in questing ticks collected in the Altai Republic. m a.s.l.
Administrative divisions
TBEV prevalence of ixodid species (adults positive/exam) I. persulcatus (23/897)
H. concinna (1/132)
D. silvarum (57/342)
D. reticulatus (20/189)
D. nuttalli (160/603)
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
<800 m
Turochaksky district Choisky district Maiminsky district Gorno-Altaisk Chemalsky district
1/112 2/95 8/118 0/1 0/11
3/74 3/74 2/131 0/2 0/14
0 0/29 0/21 0/4 0
0 1/41 0/22 0/2 0
0 2/7 13/57 0/4 0/1
0 3/8 14/59 0/9 0/3
0 0/6 8/52 0 0
0 2/16 9/98 0/1 0
0 0 0 0 10/30
0 0 0 0 15/60
800–1700 m
Shebalinsky district Ust-Kansky district Ongudaisky district Ust-Koksinsky
0/15 0/5 2/79 0/31
0/16 0/6 0/72 2/40
0/10 0 0 0
0/3 0 0 0
6/53 0 1/9 0/5
13/90 0/8 2/14 3/15
0/10 0 0 0
1/6 0 0 0
10/17 28/58 24/82 5/13
5/20 15/47 22/69 8/13
>1700 m
Ulagansky district Kosh-Agachsky Total Prevalence rate (%)
0/1 0 13/468 2.8
0 0 10/429 2.3
0 0 0/64 0
0 0 1/68 1.5
0 0 22/136 16.2
0 0 35/206 16.9
0 0 8/68 11.8
0 0 12/121 9.9
5/11 2/80 84/291 28.9
7/22 4/81 76/312 24.4
were tests run simultaneously for revealing the RNA of the TBE virus by the PCR (Table 4). Thus the RNA of the TBE virus was found in 12.3% of Dermacentor ticks (48 out of 389 specimens) and in 1.7% of I. persulcatus ticks – (in 6 out of 361 specimens). The D. nuttalli had the highest prevalence rate (14.9%), as it had been identified by the ELISA. The ticks of H. concinna showed a low infection rate (1.2%). The comparison of the infection rate of the species in both reactions (ELISA and PCR) is shown in Fig. 3. The difference in the TBEV prevalence in Dermacentor ticks and I. persulcatus was also confirmed by PCR: it was statistically significant (p < 0.001). Despite the fact that virus prevalence of ixodid ticks at PCR was lower than at ELISA, the infection rate was recorded as one of the highest in Russia.
We also studied the dependence of TBEV prevalence in ticks on an altitude factor. This regularity was revealed for I. persulcatus, whereas the infection rate for D. nuttalli (PCR) decreased with altitude: Ust-Kan neighborhood – 23.4% (1059 m), Elo neighborhood – 18.9% (1156 m), Kosh-Agach – 3.3% (1758 m), and the Ulandryk River valley (Kosh-Agach district) – 0.42% (2155 m). In midlands the average infection rate for D. nuttalli was 20.0%, whereas in highlands it was 3.3%. The difference was statistically significant (p < 0.001). The high threshold of the TBEV detection for I. persulcatus ticks was at 1295 m a.s.l. (50◦ 38 41 N, 86◦ 18 44 E), for D. reticulatus – 440 m a.s.l. (52◦ 02 30 N, 86◦ 52 11 E), for H. concinna – 440 m a.s.l. (52◦ 02 30 N, 86◦ 52 11 E), for D. silvarum – 1282 m a.s.l. (50◦ 58 04 N, 85◦ 43 11 E), and for D. nuttalli – 2155 m a.s.l.
Table 4 TBEV detection by PCR in questing ticks collected in the Altai Republic. m a.s.l.
Administrative divisions
TBEV prevalence of ixodid species (adults positive/exam.) I. persulcatus (6/361)
H. concinna (1/84)
D. silvarum (4/53)
D. reticulatus (3/60)
D. nuttalli (41/276)
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
♂♂
♀♀
<800 m
Turochaksky district Maiminsky district
2/120 0/24
2/100 0/21
0 1/35
0 0/25
0 2/20
0 2/20
0 1/20
0 2/40
0 0
0 0
800–1700 m
Shebalinsky district Ust-Kansky district Ongudaisky district Ust-Koksinsky
0/13 0 1/43 0
0/10 0 1/30 0
0/11 0 0 0
0/13 0 0 0
0/7 0 0 0
0/6 0 0 0
0 0 0 0
0 0 0 0
0 5/22 15/70 0/13
0 6/25 13/73 0/11
>1700 m
Kosh-Agachsky district Total Prevalence rate (%)
0 3/200 1.5
0 3/161 1.9
0 1/46 2.2
0 0/38 0
0 2/27 7.4
0 2/26 7.7
0 1/20 5.0
0 2/40 5.0
1/30 21/135 15.6
1/32 20/141 14.2
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Fig. 3. Comparison of TBEV detection by ELISA and PCR in questing ticks collected in the Altai Republic.
Fig. 4. Tick-bite incidence rate in different landscape areas of the Altai Republic (2013).
(50◦ 01 01 N, 88◦ 40 58 E), which is slightly lower than the elevation limits for the ticks of these species. The study has shown that the intensity of the TBE foci in the Altai Republic is caused by a wide distribution, a large population, and a high TBEV prevalence in ticks. Annually from 1500 to 3300 people seek medical treatment due to tick bites. In 2013, 3186 people were registered with ixodid tick bites: the incidence rate was 1511.9 per 100,000 inhabitants. Correlation analysis showed that there is statistically significant strong reverse interconnection between altitude and tick-bite incidence (Pearson’s coefficient r = −0.78, p < 0.05). Permanent high values of tick-bite incidences are recorded in the lowlands and low values in the highlands (Fig. 4). This is attributed to gradual decrease of I. persulcatus in nature from north to south and increase of the less aggressive D. nuttalli that attacks people more rarely: people have time to notice these large and less mobile ticks and to remove them before they attach themselves. The tick-bite incidence is the highest in the I. persulcatus dominance zone (lowlands) (Fig. 5). On average, the TBE season in the Altai Republic lasts from early March to late October. In 2013, the first tick-bite was registered on March 11, and the last one on October 29. From the end of August till the end of October only the bites of Dermacentor spp. are registered; however, there are usually only a few visits to the hospital (single instances). Collectively, there are 81.0% of cases of visits to the hospital after tick bites in the lowlands. Therefore, people in these areas have a higher risk of getting a TBE infection than the population of the midland (15.6%) and of the highlands (3.4%). If
Fig. 5. Seasonal dynamics of tick bite cases in lowland, midland, and highland areas of the Altai Republic (2013). The figure shows the seasonal dynamics of tick bite cases in three districts: Turochaksky (lowland), Ongudaisky (midland), and KoshAgachsky (highland). The influence of D. nuttalli is noticeable in the midlands and highlands: the two latter graphics show a second peak caused by the abundance increase of D. nuttalli in August.
calculated as a population size, in the lowland areas 2 out of 100 inhabitants have tick bites, in the midland – 1 out of 120, and in high mountains – 1 out of 250 inhabitants. The average yearly TBE incidence rate of the Altai Republic between 2004 and 2013 was 22.6 per 100,000 inhabitants. That is 10 times higher than the rate of Russia. Territory ranking according to the TBE infection shows that the population incidence is associated with the altitude. There is statistically significant strong reverse interconnection between elevation above sea level and TBE incidence (Pearson’s coefficient r = −0.67, p < 0.05). The highest TBE infection risk is recorded in the Turochaksky district (lowland), where the majority of settlements are located at 300–400 m a.s.l.: I. persulcatus is the dominant species here (dominance index 99.3%) and the average yearly TBE incidence is 50.0 per 100,000 inhabitants. The least risk of infection is recorded in the highland Kosh-Agachsky district (1700–2200 m a.s.l.): D. nuttalli transmits TBEV at this altitude and average yearly TBE incidence is 7.4 per 100,000 inhabitants. The rest regions of the Altai Republic (400–1700 m a.s.l.) have TBE foci with mixed ixodid fauna, which belong to the group of mean risk: average yearly TBE incidence is 15.9–28.1 per 100,000 inhabitants (Fig. 6). Among the districts which are part of the midlands, the Ongudaisky district is notable for high TBE incidence. The analysis of the epidemiological records shows that 26 out of 42 patients registered in 2004–2013 were infected in the terrains which are at 800–860 m a.s.l., and 16 people at the altitude 1000–1240 m a.s.l. An unusual situation of the tick-borne encephalitis disease is marked in the Ulagansky district. Officially it belongs to the regions of the Far North because of a severe climate and a location of villages at the elevation exceeding 1500 m above sea level. However, 5 of 15 settlements in this district are situated at a much lower altitude. The analysis shows that the most troubled village, in terms of the TBE disease, is Balykcha (51◦ 17 05 N, 87◦ 42 30 E). Here 8 people (out of 25 patients registered in the Ulagansky district) became sick in the described season. This village is located in a deep hollow (the Chulushman valley) at the elevation of only 450 m above sea level. There are boreal woods (called taiga) around Balykcha, where I. persulcatus ticks are widely spread. Other cases of infection in the Ulagansky district occurred at the heights of 1450 m a.s.l., that is, in the middle altitude, not in highlands. Therefore, we can trace the effect of the altitude on the intensity of the foci and the TBE incidence rate in the Ongudaisky and Ulagansky districts.
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Altai Republic, including the highland territory, is an endemic TBE area. Conclusion
Fig. 6. Territory ranking according to TBE infection risk in the Altai Republic (2004–2013). Lowlands: Turochaksky district (1), Choysky district (2), Mayminsky district (3), Gorno-Altaysk (4), Chemalsky district (5). Midlands: Shebalinsky district (6), Ust-Kansky district (7), Ongudaysky district (8), Ust-Koksinsky district (9). Highlands: Ulagansky district (10), Kosh-Agachsky district (11).
Discussion We have found out several epidemiological features of TBE in the Altai Republic, which are the following: a specific diversity and a large population of ixodid ticks, their infection with the TBE virus, close contacts of the population with vectors, and an involvement into the circulation of the TBE virus of not only the main vector, I. persulcatus, but also other species. In spite of the fact that the infection rate with the TBE virus of Haemaphysalis spp. and Dermacentor spp. ticks was proved a long time ago (Ryzhov and Skrynnik, 1941), the issue of their role in TBE foci remains debatable (Korenberg et al., 2013). The laboratory research shows that in the experiments the ticks of genus Dermacentor are capable to accept, keep, and transmit the TBE virus to susceptible animals: however, the efficacy of this process is lower than for I. persulcatus ticks (Alekseev and Chunikhin, 1990, 1992; Belova et al., 2013). Meanwhile, in the Altai Republic the virus in D. nuttalli ticks, a reservoir host and vector of the TBEV in the high-mountainous Kosh-Agachsky district, was found in 1954 (Ljubushkina and Jugova, 1963). The TBE cases among the population in this highland area have been registered since 1984. The Kosh-Agachsky district is the coldest area in Gorny Altai where the winter lasts more than 7 months, the outside temperature reaches −62 ◦ C, and the absolute value of a vegetation period according to the sum of mean daily air temperature above +5 ◦ C makes up only 1300–1400 ◦ C (whereas for I. persulcatus ticks it should not be lower than 1600 ◦ C). The annual precipitation amount in this area is small and makes up about 116 mm; with the highest amount of precipitation falling in summer. However, cold and dry winters do not exterminate D. nuttalli. The abundance of ticks in the vicinities of the settlements in the Kosh-Agachsky district (Kokorya, Tashanta, Zhana-Aul, and Telengit-Sortogoy) is especially high in April – 300–350 specimens/person/1 h of collection. This is due to biological characteristics of D. nuttalli, able to resist to low temperatures, and the great abundance of its hosts. Although the TBE incidence rate is the lowest in the highland Kosh-Agachsky district (7.4 per 100,000 inhabitants), it is 3 times higher than the all-Russian TBE incidence value; i.e. the whole the
This study showed that the altitude results in the development of the epidemiological TBE situation. The highest species diversity of ixodid ticks is recorded at 280–560 m a.s.l. The spectrum of vector species decreases with altitude and the leading species changes: besides I. persulcatus, D. nuttalli also transmits TBEV in the midlands, whereas in the highlands the latter is an absolute dominant species. However, these ticks are less effective vector species than I. persulcatus. The tick-bite incidence decreases with altitude (r = −0.78, p < 0.05) and the TBE incidence also decreases (r = −0.67, p < 0.05). Thus, the highest risk of TBE infection is recorded in the lowland Turochaksky district (300–400 m a.s.l.) where I. persulcatus is a dominant species, whereas the lowest risk is registered in the highland foci of the Altai Republic (1700–2200 m a.s.l.): D. nuttalli is a vector species in this place. Other areas with mixed ixodid fauna are included in the zone of a mean tick-borne encephalitis infection risk. References Alekseev, A.N., Chunikhin, S.P., 1990. The experimental transmission of the tickborne encephalitis virus by ixodid ticks (the mechanisms, time periods, species and sex differences). Parazitologiia 24, 177–185 (in Russian). Alekseev, A.N., Chunikhin, S.P., 1992. Difference in distant transmission ability of tick-borne encephalitis virus by Ixodid ticks belonging to different subfamilies. Parazitologia 26, 506–515 (in Russian). Belova, O.A., Brisker, S.A., Burebkova, L.A., Karganova, G.G., 2013. Ticks of genus Dermacentor Koch., 1844 (Acari: Ixodidae) as possible vectors of tick-borne encephalitis virus. In: Fundamental and Applied Aspects of the Study of Parasitic Arthropods in the XXI Century: International Conference in Memoriam of Prof. Yuri S. Balashov, a Corresponding Member of the RAS, St. Petersburg, Russia, pp. 167–168 (in Russian). Danchinova, G.A., Hasnatinov, M.A., Zlobin, V.I., et al., 2006. Iksodid ticks of Eastern Siberia and Mongolia and their spontaneous pathogen contamination in natural focal transmissible infections. Bjul. sib. med. T.5. (Pril.1), 137–143 (in Russian). Daniel, M., Materna, J., Honig, V., Metelka, L., Danielová, V., Harcarik, J., Kliegrová, S., Grubhoffer, L., 2009. Vertical distribution of the tick Ixodes ricinus and tickborne pathogens in the northern Moravian mountains correlated with climate warming (Jeseníky Mts., Czech Republic). Cent. Eur. J. Public Health 17 (3), 139– 145. Danielová, V., Rudenko, N., Daniel, M., Holubová, J., Materna, J., Golovchenko, M., Schwarzová, L., 2006. Extension of Ixodes ricinus ticks and agents of tick-borne diseases to mountain areas in the Czech Republic. Int. J. Med. Microbiol. 296 (Suppl. 40), 48–53. Danielová, V., Schwarzová, L., Materna, J., Daniel, M., Metelka, L., Holubová, J., Kˇríˇz, B., 2008. Tick-borne encephalitis virus expansion to higher altitudes correlated with climate warming. Int. J. Med. Microbiol. 298 (Suppl. 1), 68–72. Filippova, N.A., 1977. Ixodid ticks of the subfamily Ixodinae. Fauna S.S.S.R., Paukoobraznye 4 (1), 393 (in Russian). Filippova, N.A., 1997. Ixodid ticks of subfamily Amblyomminae. Arachnoidea 5 (1), 436 (in Russian). Grebenyuk, R.V., 1966. Ixodid Ticks of Kirgizia, Frunze. Ilim, Russian. Holodilov, I.S., Motuzova, O.V., Belova, O.A., Orlova, O.E., Karganova, G.G., 2013. Problem of identification of tick-borne encephalitis virus in ticks. In: Fundamental and Applied Aspects of the Study of Parasitic Arthropods in the XXI Century: International Conference in Memoriam of Prof. Yuri S. Balashov, a Corresponding Member of the RAS, St. Petersburg, Russia, pp. 167–168 (in Russian). Holzmann, H., Aberle, S.W., Stiasny, K., Werner, P., Mischak, A., Zainer, B., Netzer, M., Koppi, S., Bechter, E., Heinz, F.X., 2009. Tick-borne encephalitis from eating goat cheese in a mountain region of Austria. Emerg. Infect. Dis. 15 (10), 1671–1673. Korenberg, Je.I., Kovalevskij, Ju.V., 2000. Main Features of Tick-Borne Encephalitis Eco-epidemiology in Russia. Problems of Tick-Borne and Parasitic Diseases. St. Petersburg., pp. 13–20 (in Russian). Korenberg, E., Pomelova, V., Osin, N., 2013. Infections with Natural Focality Transmitted by Ticks. Moscow, 463 pp. (in Russian). Ljubushkina, V.M., Jugova, L.A., 1963. Tick-Borne Encephalitis in the GornoAltaysk Autonomous Region. Tick-Borne Encephalitis in the Altai Krai. Barnaul., pp. 30–41 (in Russian). Materna, J., Daniel, M., Metelka, L., Harcarik, J., 2008. The vertical distribution, density and the development of the tick Ixodes ricinus in mountain areas influenced by climate changes (The Krkonose Mts., Czech Republic). Int. J. Med. Microbiol. 298 (Suppl. 1), 25–37.
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Süss, J., 2008. Tick-borne encephalitis in Europe and beyond – the epidemiological situation as of 2007. Euro Surveill. 13 (26). Verhozina, M.M., Zlobin, V.I., Kozlova, I.V., Demina, T.V., Dzhioev, Ju.P., Lisak, O.V., Doroshhenko, E.K., Protasova, E.G., 2008. Epidemioligical and molecular genetic analysis of tick-borne encephalitis virus population in the Irkutsk region. Jepidemiologija i vakcinoprofilaktika 38, 12–18 (in Russian). Zlobin, V.I., 2010. Tick-borne encephalitis in the Russian Federation: etiology, epidemiology and prevention strategy. Terra Medica 2, 13–21 (in Russian).
Please cite this article in press as: Shchuchinova, L.D., et al., Influence of altitude on tick-borne encephalitis infection risk in the natural foci of the Altai Republic, Southern Siberia. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.02.005