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Cent Eur J Public Health 2018 Dec; 26 (Suppl): S56–S60
SEROSTATUS AND RISK FACTORS OF TICK-BORNE
ENCEPHALITIS
Andrea Bušová1, Erik Dorko1, Eva Feketeová2, Matúš Bereš3, Kvetoslava Rimárová1, Jana Diabelková1, Tímea
Rovenská1, Róbert Čellár3, Tomáš Csank4
1Department of Public Health and Hygiene, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Košice, Slovak Republic
2Department of Neurology, Faculty of Medicine, Pavol Jozef Šafárik University in Košice and Louis Pasteur University Hospital, Košice, Slovak
Republic
3Department of Orthopaedics and Traumatology of Locomotory Apparatus, Faculty of Medicine, Pavol Jozef Šafárik University in Košice and
Louis Pasteur University Hospital, Košice, Slovak Republic
4Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Košice, Slovak Republic
SUMMARY
Objective: The aim of this seroepidemiological study was to determine the current prevalence of antibodies against tick-borne encephalitis
virus (TBEV) in the representative group of Slovak population with included potential risk factors for TBEV.
Methods: Representative group consisted of 428 persons (also with possible exposure to risk factors for tick bite or raw milk consumption).
Serum samples were screened by commercial enzyme-linked immunosorbent assay (ELISA). The persons involved in the study completed ques-
tionnaires with general demographic, epidemiological and clinical data. During the analysis, we used linear regression to interpret the influence
between selected variables.
Results: We detected 1.2% prevalence of positive IgG and 1.6% prevalence of positive IgM antibodies in all tested groups. Our results also
confirmed that the following risk factors such as tourism, hunting, fishing, and consumption of raw milk are significantly associated with the preva-
lence of specific antibodies against TBEV.
Conclusion: The results of seroprevalence obtained by this study confirm the possibility of infection with TBEV among respondents exposed
to possible contact with ticks.
Key words: tick-borne encephalitis, risk factor, seroprevalence
Address for correspondence: E. Dorko, Department of Public Health and Hygiene, Faculty of Medicine, Pavol Jozef Šafárik University in Košice,
Šrobárova 2, 041 80 Košice, Slovak Republic. E-mail: erik.dorko@upjs.sk
https://doi.org/10.21101/cejph.a5289
INTRODUCTION
Tick-borne encephalitis virus (TBEV) is one of the most fre-
quent causes of the central nervous system infections in Europe,
especially in endemic areas of Central and Eastern Europe and
Russia (1, 2). The epidemiology of TBE depends on various fac-
tors, such as increased recreational activities in areas infested by
ticks, and climatic changes affecting tick habitats and in addition,
anthropomorphic changes in agriculture and outdoor and free-time
activities, increase the risk that humans will be infected TBE
(2, 6). The virus can be transmitted by bites of infectious ticks or
alimentary infections caused by consumption of raw milk (3, 4).
Increase in traveling, access to high-risk areas, and the free-time
activities within TBE-endemic areas are puts more people at
risk of exposition to TBE (5). With the favoured consumption of
unpasteurized milk and dairy products of goat, sheep and cow
milk in Slovakia, outbreaks of alimentary infection of TBE are
common. Therefore, alimentary cases are responsible for almost
23% of all TBE cases in Slovakia. Food-borne transmission of
TBE was first reported in Czechoslovakia in 1954 (9).
This was finded by following the TBE epidemic of 1951
in the east Slovak town of Rožňava where 660 persons were
infected. The source of infection was contaminated goat milk
which had been mixed into cow milk at the local dairy factory
and distributed without pasteurization (10). Human behavior is
an important factor and should always be considered in studies
of TBE (12).
The main aim of this study was to detect the TBE seropreva-
lence in a selected group risk group of the population and to
confirm the association with the risk factors common for Slovakia.
MATERIALS AND METHODS
Study Design
Study Design: Cross-sectional Study. Approval of the study
protocol has been approved by the Ethics Committee of the Uni-
versity Hospital of Košice and informed consents were signed
by all the participants.
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Data Collection
Blood samples with questionnaires were collected in eastern
Slovakia from July 2014 to December 2016. The group consisted
of 428 persons (examined in the Department of Neurology, De-
partment of Dermatovenerology, Department of Orthopedics and
Traumatology of Locomotory Apparatus and from Department
of Rheumatology without TBE suspection and random persons
who should be exposed to ticks from Zlatá Idka, Hrabušice and
Jánovce). People involved in this study declared that they had
not been vaccinated against tick-borne encephalitis, Japanese
encephalitis or yellow fever.
Serum Samples
The serum was acquired from blood samples by the cen-
trifugation (2,500 rpm/10 min) and stored at −80 °C until
serological testing. The serum samples were processed in the
Laboratory of the Department of Microbiology and Immunol-
ogy of the University of Veterinary Medicine and Pharmacy
in Košice.
Questionnaire
The questionnaires consist of demographic data (gender,
age, residence) and epidemiological data (tick bite, contact with
animals, frequent outdoor activities such nature walks, hunting,
fishing, tourism and consumption of raw milk).
Laboratory Analysis
All serological assays were carried out and results were inter-
preted according to the manufacturers’ instructions and the optical
density was measured with a spectrophotometer at 450 nm using
an ELISA reader (Synergy HT BioTek). The ELISA test (TBE/
FSME IgG and IgM-ELISA NovaLisaTM, NovaTec, Germany)
was applied for general screening of specific TBE antibodies.
Serum IgM levels equal to 11 NTU/ml and above were con-
sidered as positive, those between 9 and 10 NTU/ml were consid-
ered as borderline, and those below 9 NTU/ml were considered
as negative. Serum IgG levels equal to 110 NTU/ml and above
were considered as positive, those between 55–110 NTU/ml were
borderline, and those below 55 were considered as negative.
Table 1. General characteristics of examined subjects (N = 428)
Risk factors/characteristics
Investigated groups
Men Women Total
n % n % n %
Age – Mean (SD) – 50.78 (16.45) – 55.87 (17.20) – 53.57 (17.04)
Residence urban 93 47.9 127 54.3 220 51.4
rural 101 52.1 107 45.7 208 48.6
Nature walks yes 73 37.6 103 44.0 176 41.1
no 121 62.4 131 56.0 252 58.9
Tourism yes 53 27.3 56 23.9 109 25.5
no 141 72.7 178 76.1 319 74.5
Hunting yes 9 4.6 2 0.9 11 2.6
no 185 95.4 232 99.1 417 97.4
Fishing yes 12 6.2 6 2.6 18 4.2
no 182 93.8 228 97.4 410 95.8
Contact with animals yes 89 45.9 99 42.3 188 43.9
no 105 54.1 135 57.7 240 56.1
Tick bite yes 120 61.9 128 54.7 248 57.9
no 74 38.1 106 45.3 180 42.1
Consumption of raw milk yes 70 36.1 67 28.6 137 32.0
no 124 63.9 167 71.4 291 68.0
Cow milk yes 42 21.6 41 17.5 83 19.4
no 152 78.4 193 82.5 345 80.6
Sheep milk yes 46 23.7 41 17.5 87 20.3
no 148 76.3 193 82.5 341 79.7
Goat milk yes 24 12.4 22 9.4 46 10.7
no 170 87.6 212 90.6 382 89.3
Total number of subjects 194 45.3 234 54.7 428 100.0
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Statistical Analysis
The data were analyzed statistically using IBM SPSS 21.0
statistical program. Demographic data were interpreted by
descriptive statistics as averages with standard deviations
and as percentages for categorical variables. During the
analysis, we used linear regression to interpret the influence
between selected variables. For depended variable, we used
sero prevalence of IgG/IgM antibodies. For independent vari-
able, we use gender, age, residence, nature walks, tourism,
hunting, fishing, contact with animals, consumption of raw
milk and tick bite. Considered value for statistical signifi-
cance was p ≤ 0.05.
RESULTS
File Characteristics
The tested group consisted of 428 persons aged between 12.0
and 93.0 yr (average age 53.57 yr, SD 17.04), which included
194 men (45.3%) with average age 50.78 yr, SD 16.45 and 234
women (54.7%) with average age 55.87 yr, SD 17.2. Urban
residents created 51.4% of the sample, 48.6% subjects resided
in rural areas.
According to the questionnaire results, 176 (41.1%) persons
spend their free-time by walking in nature, nature walk, 109
IgG IgM
n%n%
Negative 415 97.0 403 94.2
Borderline 8 1.9 18 4.2
Positive 5 1.2 7 1.6
Table 2. Analysis of anti-TBEV antibodies IgG/IgM detected by ELISA (N = 428)
Table 3. Associations between risk factors and seroprevalence of anti-TBEV antibodies IgM/IgG detected by ELISA
Linear regression, B – linear regression coefcients, CI 95% – condence interval, statistically signicant values are marked in bold
Risk factors
Seroprevalence IgM Seroprevalence IgG
B
(95% CI) p-value B
(95% CI) p-value
Gender woman/man (ref.) 0.086
(−0.585–0.756) 0.802 −2.672
(−11.356–6.617) 0.572
Age↑ −0.003
(−0.23–0.017) 0.755 0.118
(−0.156–0.391) 0.397
Residence rural/urban (ref.) −0.577
(−1.242–0.089) 0.089 −3.601
(−12.871–5.669) 0.446
Nature walks yes/no (ref.) −0.649
(−1.363–0.065) 0.075 6.670
(−3.246–16.585) 0.187
Tourism yes/no (ref.) 0.708
(0.034–1.383) 0.040 -3.241
(−12.637–6.156) 0.498
Hunting yes/no (ref.) 1.619
(−0.464–3.723) 0.131 110.042
(82.751–137.334) < 0.001
Fishing yes/no (ref.). 1.701
(0.046–3.356) 0.044 28.607
(5.720–51.493) 0.014
Consumption of raw milk 0.942
(0.232–1.652) 0.009 2.521
(−7.392–12.435) 0.617
Cow milk –0.005
(−0.849–0.839) 0.991 −3.934
(−15.629–7.761) 0.509
Sheep milk 1.351
(0.531–2.170) 0.001 7.698
(−3.774–19.170) 0.188
Goat milk 0.244
(−0.834–1.321) 0.657 2.724
(−12.210–17.658) 0.720
Contact with animals yes/no (ref.) 0.509
(−0.159–1.177) 0.135 8.341
(−0.873–17.55) 0.076
Tick bite yes/no (ref.) 0.354
(−0.321–1.029) 0.303 3.366
(−6.0–12.732) 0.480
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(25.5%) by tourism, 11 (2.6%) by hunting and 18 (4.2%) by fish-
ing. Two hundred and forty-eight (57.9%) of asked had a history
of tick bites and 188 (43.9%) had a history contact with animal.
One hundred and thirty-seven (32%) of asked had a history of con-
sumption of raw milk. The detailed results can be seen in Table 1.
General Screening
An ELISA test revealed 415 (97.0%) negative samples for
anti-TBEV IgG, whereas 8 (1.9%) samples were borderline and
5 (1.2%) were positive. The results for IgM were as follows:
negative 403 (94.2%), 18 (4.2%) were borderline and only 7
(1.6%) were confirmed as positive results. The detailed results
of the detected antibodies prevalence are presented in Table 2.
Association of IgG and IgM Antibodies Prevalence
with Gender, Age and Residence
By gender, age and residence parameters we did not record
any statistically significant effect on seroprevalence of IgM/IgG
class antibodies (Table 3).
Association of IgG and IgM Antibodies Prevalence
with External Activities
Results of the linear regression for seroprevalence indicated
that tourism (B, 0.708, 95% CI, 0.034–1.383), fishing (B, 1.701,
95% CI, 0.046–3.356) were significantly associated with sero-
prevalence IgM. The analysis revealed a significant association
between hunting (B, 110.042, CI 95%, 82.751–137.334), fishing
(B, 28.607, CI 95%, 5.720– 51.493) and seroprevalence IgG. No
significant difference in seroprevalence was observed stratified
outdoor activities as nature walk (Table 3).
Association of IgG and IgM Antibodies Prevalence
with Animal Contact Associated to Tick Bite
No significant difference in seroprevalence was observed for
contact with animals and tick bite as a risk factor (Table 3).
Association of IgG and IgM Antibodies Prevalence
with Consumption of Raw Milk
Consumption of raw milk was reported in 32% of respondents
and this factor was significant (B, 0.942, CI 95%, 0.232–1.652)
and consumption of sheep´s milk and milk products (B, 1.351,
CI 95%, 0.531–2.170) was also significant for seroprevalence
IgM antibodies. No relationship between seroprevalence of
consumption of raw cow and goat milk and dairy products was
demonstrated (Table 1, 3).
DISCUSSION
Over the last four decades, TBE has become a growing public-
health challenge in Europe and parts of Asia (9). The aim of this
seroepidemiological study was to determine the seroprevalence
of TBEV and to reevaluate out the possible risk factors associated
with the disease in the selected population group in Slovakia.
After exclusion of participants who reported vaccination
against TBEV, Japanese encephalitis or yellow fever (n = 102),
5 of the 428 examined serum samples were positive for IgG TBEV
(seroprevalence 1.2%) and 7 for IgM TBEV (seroprevalence
1.6%). Differences between the prevalence of IgG and IgM anti-
bodies can be explained in the acute stage of the disease, mainly
by serum screening. The TBEV seroprevalence observed in our
study is similar to that reported by French study conducted among
forestry workers of northeastern France where ELISA testing
confirmed 1.7% seroprevalence (16).
Many studies conducted on a group of exposed forestry workers
shows the presence of antibodies against TBEV: 19.8% to 81.3% in
eastern Poland (19, 20), 0% to 5.7% in Italy (21, 22), and 7.3% in
southwestern Germany with variations according to district ranging
from 0% to 43% (23). In the study conducted by Christova et al.,
the examined group consist 1,451 residents from all 28 districts in
Bulgaria the authors detected 0.6% of IgG antibodies that shows
slightly lower prevalence than in our group (24).
Identification and evaluation of risk factors of TBE is im-
portant to understand the risk of contracting TBE in those who
travel to industrialized European countries, work in endemic
afforested areas and consume of raw milk. Tick habitats are
mainly forested and rural areas up to ∼1,500 m of altitude but
ticks may also be found in gardens. In Eastern Europe, TBE
endemics in urban parks has been described and transmission
in Europe is usually limited to the period between April and
November (8, 11). Human behavior affects TBE incidence, as
it can trigger actions that increase the contact of people with
infected ticks. One example is nice weather which increases the
time spent outdoor activities including: camping, nature walks,
biking, fishing, hunting, professional sports and activities of
Scouts and Guides (12). Among professional exposure, the
highest risk is encountered in woodcutting, forestry, farming,
and military activities (7). In our study, we find out up to 41.1%
of nature walks, 25.5% tourism, 2.6% hunting, 4.2% fishing.
Significance in a relationship between seroprevalence IgM and
tourism (B, 0.708, 95% CI, 0.034–1.383), fishing (B, 1.701,
95% CI, 0.046–3.356) was confirmed. Similar, was confirmed
significance in a relationship between seroprevalence IgG and
hunting (B, 110.042, CI 95%, 82.751–137.334) and fishing
(B, 28.607, CI 95% 5.720–51.493). In the study carried on by
Blasko-Markic and Socan, high-risk behavior in Slovenia has
been shown to include free-time activities, mushroom and berry
picking, and farming (13). A recent study in Lithuania carried
on by Juceviciene et al. demonstrated that antibodies to TBEV
were often in people who frequently spent time in the nature or
who had consumed unpasteurized goat
’
s milk (14).
Data from Latvia confirm that people who visit forests (for
work, food collection or leisure) are four to five times more likely
to encounter ticks than people who do not visit forests (15).
TBE is an acute febrile disease with neurological manifes-
tations and transmitted by ticks and rarely by unpasteurized
milk and dairy products (7). The number of cases infected via
unpasteurized milk has been estimated to 9 % in Slovakia (17)
and 0.9 % in Czech Republic from 1997 to 2008 (18). The Study
carried by Cisak et al. found that more than 20% of goat and sheep
milk samples and 11% of cow milk samples tested were positive
for TBEV (4). In our study, we find out that up to32 % cases of
consumption of raw milk is in association with seropositivity
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and it was proven as statistically significant (B, 0.942, CI 95%,
0.232–1.652). However only consumption of raw sheep´s milk
was significantly associated with serological status (B, 1.351, CI
95%, 0.531–2.170).
Anthropomorphic changes in agriculture and the growing
popularity of spending free-time in the countryside and outdoor
activities influenced the risk of human infection with TBE (6).
As there is no effective treatment for TBE, prevention such as
tick infestation control at endemic habitats is only possible op-
tion alongside with vaccination, and could be observed in order
to prevent infection through a tick bite.
CONCLUSION
High-risk factors have been confirmed including leisure, tour-
ism, hunting and fishing. Unpasteurized milk and dairy products
may also be high risk factor as source of infection, as we con-
firmed by the statistical significance of antibody seroprevalence.
The number of TBE cases caused by consumption of raw milk
suggests certain fact that need to be addressed. The threat of
infectious diseases due to changing dietary habits, especially the
consumption of raw foods, implies the need to better educate
consumers about the risks of infection by certain pathogens,
including TBEV, in endemic areas. It is important to educate
the public through educational campaigns about the diseases
and their symptoms, using of protective repellents and appropri-
ate clothing for work in nature and recreation. Considering the
present trend of TBEV in the last decade, more clinical case and
a higher seroprevalence rate in Slovakia are likely to be expected
in the next future.
Acknowledgements
This work was supported by Grants VEGA No. 1/0198/13, 1/0011/14 of
the Ministry of Education, Science, Research and Sport of the Slovak
Republic and project VVGS – 3/GSD/2016. We thank also Directory
board of University Hospital in Košice for assistance with organization
of samples collection at the hospital wards.
Conflict of Interests
None declared
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Received December 19, 2017
Accepted in revised form August 28, 2018
