Investigation of spatial and temporal distribution in abundance of Ixodes ricinus and prevalence of
tick-borne pathogens in different habitat types of Slovakia in frame of the EDENext project
M. Kazimírová1, Z. Svitálková1, L. Mahríková1, E. Špitalská2, L. Berthová2, L. Blaňarová3, M. Bona4, J. Kraljik3,5, D. Miklisová3, M. Mojšová5, L. Mošanský3, M. Slovák1, E. Kocianová2 & M. Stanko1,3
Slovak Academy of Sciences: 1Institute of Zoology, 2Institute of Virology, 3Parasitological Institute; 4Department of Anatomy, Faculty of Medicine, Pavol Jozef Šafárik University, Košice; 5 Department of Zoology, Faculty of Science,
Comenius University
Introduction
Tick-borne diseases have (re)emerged and spread in Europe as a result of global climatic and socio-economic changes. Ixodes ricinus is the principal vector of a number of microbial pathogens of medical and veterinary importance. Shift in the
distribution of I. ricinus northward and to higher altitudes was observed and ticks became more abundant in city and periurban forest parks and gardens, which leads to increasing risk of exposure of humans and domestic animals to potentially infected
ticks. In frame of the EDENext project, spatial and temporal changes in prevalence of I. ricinus ticks infected with tick-borne micro-organisms (Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia of the spotted fever group,
Borrelia burgdorferi s.l., Babesia spp. were studied in three habitat types of Slovakia with different level of anthropogenic impact.
Study sites
Urban/suburban site: Bratislava – Bratislava forest park is located in the NW part of Bratislava (capital of Slovakia) on the foothills of the Small Carpathian Mts. The site is influenced by
recreational activities (hiking, jogging, dog-walking, etc.).
Natural site: Fugelka, a non-fragmented woodland area located in the Small Carpathian Mts
Agricultural site: Rozhanovce, a fragmented habitat comprising patches of oak-hornbeam forest and a few cultivated fields and meadows.
Material and methods
Bratislava Forest Park
Fugelka
Three 100 m transects were selected across each habitat. Ticks were dragged with a 1 m2 blanket along transects in monthly intervals during the season of their highest questing activity
(April-June) and in September-October 2011-2013. In addition to transects, random tick collections were also carried out in each habitat to increase tick numbers in the collections. Ticks
were stored in 70% ethanol and genomic DNA was extracted from individual ticks by the NucleoSpin® Tissue Macherey-Nagel kit. DNA samples were screened by PCR based methods for
A. phagocytophilum (real-time PCR amplifying the msp2 gene - Courtney et al. 2004), Cand. N. mikurensis (real-time PCR amplifying the groEL gene - Jahfari et al. 2012, modified by
Silaghi et al. 2012), Rickettsia spp. (PCR amplifying partial regions of the gltA gene - Regnery et al. 1991, and sequencing), B. burgdorferi s.l. (PCR amplifying the fragment of 5S-23S rRNA
gene, identification of genospecies by RFLP analysis – Derdáková et al. 2003, Ondrisková & Derdáková 2013) and Babesia spp. (PCR amplifying the 18S rRNA gene - Casati et al. 2006,
and sequencing).
Rozhanovce
Natural site - Fugelka
Urban/suburban site - Bratislava forest park)
Total numbers and species representation in questing tick collections (Slovakia, 2011-2013)
Site / species
Urban/Suburban
Agricultural
Natural
Ixodes ricinus: co-infection rates with tick-borne microorganisms
%
N
%
N
%
N
Ixodes ricinus
Haemaphysalis concinna
Dermacenor reticulatus
4204
61
98.57
1.43
3655
64
98.28
1.72
2195
99.91
10054
125
2
TOTAL
4265
3719
0.09
2197
Site
TOTAL
N
2
Agricultural site - Rozhanovce
TOTAL
Nymphs
Adults
1012
527
1539
1400
440
1840
Co-infected (N)
16
25
41
40
13
Co-infected (%)
1.58
4.74
2.66
2.86
2.95
Urban/Suburban
25
Rickettsia spp.
Urban
Natural
Natural
14
Agricultural
20
Urban
16
Agricultural
Prevalence (%)
Prevalence (%)
15
10
100%
100%
90%
90%
6
TOTAL
853
201
1054
53
20
8
28
2.88
2.34
3.98
2.66
Ba/R/CNm
Bb/CNm
Bb/CNm
80%
R/CNm
70%
R/CNm
70%
R/Bb
60%
Ba/CNm
50%
Ba/Bb
60%
R/Bb
50%
40%
Ap/Bb
30%
20%
Ap/Ba
20%
10%
Ap/R
10%
30%
8
Adults
80%
40%
10
TOTAL Nymphs
Natural
Ba/Bb
12
Agricultural
Adults
Examined (N)
10181
Natural
Nymphs
Ixodes ricinus
Pathogen prevalence in questing Ixodes ricinus
Anaplasma phagocytophilum
Urban
Ap/CNm
Ap/Bb
Ap/Ba
Ap/R
0%
0%
Nymphs
Adults
Nymphs
Total
Adults
Total
4
5
Agricultural
2
100%
0
0
2011
2011
2012
2012
2013
2013
Nymphs Adults Nymphs Adults Nymphs Adults
90%
2011
2011
2012
2012
2013
2013
Nymphs Adults Nymphs Adults Nymphs Adults
Overall
Overall
80%
70%
R/Bb/CNm
60%
Bb/CNm
50%
5
Cand. Neoehrlichia mikurensis
Prevalence (%)
3
2.5
2
1.5
Urban
Natural
4
Agricultural
3.5
Piroplasms (Babesia, Hepatozoon, Theileria)
4.5
Natural
4
Prevalence (%)
5
Urban
4.5
Agricultural
3.5
2011
2011
2012
2012
2013
2013
Nymphs Adults Nymphs Adults Nymphs Adults
Overall
10%
0%
2
25

2011
2011
2012
2012
2013
2013 Overall
Nymphs Adults Nymphs Adults Nymphs Adults


B. burgdorferi s.l.
Natural
Prevalence (%)
Agricultural
Identified species of microorganisms in I. ricinus:

Rickettsia: R. helvetica, R. monacensis, Rickettsia sp.

Piroplasms: Babesia microti, B. venatorum, B. capreoli, B.
odocoilei, Hepatozoon canis, Theilleria sp.

20
15
Borrelia burgdorferi s.l. genospecies: B. burgdorferi
sensu stricto, B. afzelii, B. garinii, B. valaisiana, B.
lusitaniae , B. spielmanii
10
5
For more details, see posters 1.4, 3.6, 3.8
0
2011
2011
2012
2012
2013
2013
Nymphs Adults Nymphs Adults Nymphs Adults
Total
Conclusions
1.5

Urban
Adults
2.5
0
0
Ap/R
20%
Nymphs
0.5
0.5
Ba/Bb
30%
3
1
1
30
R/Bb
40%
Co-infections in I. ricinus:
Ap/R – A. phagocytophilum + Rickettsia, Ap/Ba – A.
phagocytophilum + Babesia, Ap/Bb – A. phagocytophilum +
Borrelia, Ap/CNm – A. phagocytophilum + Cand. N.
mikurensis, Ba/Bb – Babesia + Borrelia, Ba/CNm – Babesia +
Cand. N. mikurensis, R/Bb – Rickettsia + Borrelia, R/CNm –
Rickettsia + Cand. N. mikurensis, Bb/CNm – Borrelia + Cand.
N. mikurensis, Ba/R/CNm / Babesia + Rickettsia + Cand. N.
mikurensis, R/Bb/CNm – Rickettsia + Borrelia + Cand. N,.
mikurensis


Overall

The study revealed that abundant I. ricinus populations are present in green urban areas in SW
Slovakia.
Spatial and temporal variations in relative abundance of questing ticks in different habitat types of
Slovakia were observed.
Ixodes ricinus were found to be infected with a variety of microbial pathogens. Presence and prevalence of
individual tick-borne pathogens varied between the different habitats as well as between the years of
investigation.
Prevalence of A. phagocytophilum in questing I. ricinus was found to be highest in the urban/suburban
area, whereas it was lowest in the agricultural site.
Overall prevalence of Rickettsia spp. in I. ricinus was similar in the urban/suburban and agricultural habitat
and was lower in the natural area. Rickettsia helvetica prevailed, but R. monacensis was also present.
Overall prevalence of Cand. N. mikurensis was highest in the agricultural area and lowest in the
urban/suburban habitat.
Overall prevalence of Babesia spp. in I. ricinus was similar in the urban/suburban and woodland habitat,
but it was low in the agricultural site. Babesia microti and B. venatorum prevailed.
Overall prevalence of Borrelia burgdorferi s.l. was highest in the agricultural site and lowest in the
urban/suburban area. Six genospecies were detected: B. burgdorferi s.s., B. afzelii, B. garinii, B. valaisiana,
B. lusitaniae and B. spielmanii. B. afzelii prevailed in the urban and agricultural habitat, B. garinii prevailed in
the natural habitat. However, in 2013 a decrease in the proportion of B. afzelii and an increase in
proportions of B. garinii and B.valaisiana were observed in the agricultural area.
Co-infections with 2-3 different microorganisms were found in 2.66-2.86% of I. ricinus. The representation
of microorgamisms in co-infected ticks depended upon habitat – the broadest spectrum was detected in the
natural habitat.
The results indicate that local abiotic factors and host spectrum probably affect the abundance of I. ricinus
populations and their infection with pathogenic microorganisms in the different habitat types.
Acknowledgement: This work was supported by FP7 project EDENext (No. 261504) and grant APVV DO7RP–0014–11. The technical assistance of M. Onderová and R. Szalayová is acknowledged.