Accepted Manuscript Title: The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma phagocytophilum in the Chernobyl exclusion zone Author: Grzegorz Karbowiak Bronislav´a Vichov´a Kateryna Slivinska Joanna Werszko Julia Didyk Branislav Peˇtko Michal Stanko Igor Akimov PII: DOI: Reference:
S0304-4017(14)00309-4 http://dx.doi.org/doi:10.1016/j.vetpar.2014.05.030 VETPAR 7266
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
4-10-2013 9-5-2014 10-5-2014
Please cite this article as: Karbowiak, G., Vichov´a, B., Slivinska, K., Werszko, J., Didyk, J., Peˇtko, B., Stanko, M., Akimov, I.,The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma phagocytophilum in the Chernobyl exclusion zone., Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.05.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Revised Manuscript with changes marked Click here to view linked References
1
The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma
2
phagocytophilum in the Chernobyl exclusion zone (CEZ).
4
Grzegorz Karbowiak1, Bronislavá Vichová2, Kateryna Slivinska3, Joanna Werszko2, Julia
5
Didyk3, Branislav Peťko2, Michal Stanko2, Igor Akimov3
6
1 W. Stefański Institute of Parasitology of the Polish Academy of Sciences, Twarda 51/55,
8
00-818 Warsaw, Poland;
9
2 Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice,
us
cr
7
Slovakia;
11
3 I.I.Schmalhausen Institute of Zoology of NASU, B. Khmelnytskogo 15, 01601 Kiev,
12
Ukraine.
an
10
M
13
Corresponding author: Grzegorz Karbowiak,
15
W. Stefański Institute of Parasitology of the Polish Academy of Sciences, Twarda 51/55,
16
00-818 Warsaw, Poland;
17
tel. 22 6978975; fax. 22 620-62-27; e-mail:
[email protected] ce pt
ed
14
18
ip t
3
Abstract
20
Tick occurrence was studied in the Chernobyl exclusion zone (CEZ) during the August-
21
October period in the years 2009-2012. Dermacentor reticulatus ticks were collected using
22
the flagging method and then screened for infection with Anaplasma phagocytophilum and
23
Babesia canis by a PCR method incorporating specific primers and sequence analysis. The
24
prevalence of infection with B. canis canis and A. phagocytophilum was found to be 3.41%
25
and 25.36% respectively. The results present the first evidence of B. canis canis and A.
Ac
19
1
Page 1 of 21
phagocytophilum in questing Dermacentor reticulatus ticks from the Chernobyl exclusion
27
zone. They also reveal the presence of tick-borne disease foci in areas with no human activity,
28
and confirm that they can be maintained in areas after a nuclear disaster with radioactive
29
contamination.
30
Key words: Chernobyl exclusion zone; Anaplasma phagocytophilum; Babesia canis;
32
Dermacentor reticulatus.
cr
31
ip t
26
us
33
Introduction
35
In 1986, as a consequence of the disaster at the Chernobyl nuclear power plant, a mass of
36
radioactive material was released into the atmosphere, causing extensive pollution to the
37
neighbouring regions. An Exclusion Zone (CEZ) was established within a 30 – 60 km radius
38
of the explosion point, where human activity was minimized and entrance was restricted. The
39
direct consequences of radioactive pollution, as well as the evacuation of the public and
40
cessation of agriculture and forest management caused significant ecological changes, such as
41
the spontaneous restitution of the original natural habitats, plant and animal populations, as
42
well as their interdependences. Many aspects of the consequences of radionuclide
43
contamination of wild animals have been studied during the short time since the catastrophe.
44
For instance, the study of radionuclide accumulation in the soil and by different species of
45
animals (Frantsevich, 2006), the effects of radiation on cytogenetics and mutation, as well as
46
the frequency of abnormalities potentially caused by pollution (Møller and Mousseau, 2006;
47
Møller et al., 2007). Although parasitological studies are not numerous, the increase and
48
biodiversity of micromammalian parasite complexes have been examined (Labetskaya et al.,
49
1997). This study presents the first evidence of tick-borne pathogen foci in the Chernobyl
50
Exclusion Zone.
Ac
ce pt
ed
M
an
34
2
Page 2 of 21
51
Materials and Methods
53
Study sites and tick collection method.
54
Tick occurrence in the Chernobyl Exclusion ZoneCEZ was studied during the August-
55
October period each year from 2009 to 2012. The study area was located in preventive zone
56
B, within a 10-20 km radius of the disaster point. The flagging method was used to collect the
57
ticks from areas where they were known to appear in the surroundings of the former villages
58
of Korogod (51°16'02"N; 30°01'04"E) and Cherevach (51°12'44"N; 30°07'45"E). The
59
investigated habitats included open areas and the remnants of farmlands. The vegetation in the
60
tick collection sites included grass and ruderal plants, and some bushes. The collected
61
Dermacentor reticulatus ticks, mainly Dermacentor reticulatus, were fixed in ethanol and
62
transported to the laboratory for further analysis.
M
63
an
us
cr
ip t
52
Molecular detection of Babesia spp. and Anaplasma phagocytophilum.
65
The DNA extraction was performed using the ammonium hydroxide method (Rijpkema et al.,
66
1996). After washing in PBS/EDTA 0.5 %, all D. reticulatus ticks were cut and crushed
67
separately in Eppendorf tubes. Genomic DNA isolation was performed by boiling in 200 μL
68
of 0.7 M NH4OH for 30 min. The remaining tick tissue and chitin exoskeleton were removed
69
by a short spin, following which, the supernatant containing the DNA was collected and the
70
pellet discarded.
71
All ticks were screened by nested PCR amplification of the 16S rRNA gene. In the first round
72
of amplification, primers ge3a and ge10r were used to detect all members of the
73
Anaplasmataceae. For the second round, one microlitre of PCR product from the first round
74
was used as a template to detect A. phagocytophilum, using primers ge9f and ge2 (Massung et
75
al., 1998). Cycling conditions in the first round involved an initial 2-min denaturation at 95°C,
Ac
ce pt
ed
64
Formatted: Font: Not Italic
3
Page 3 of 21
followed by 40 cycles, each consisting of a 30-s denaturation at 94°C, a 30-s annealing at
77
55°C, and a 1-min extension at 72°C. These 40 cycles were followed by a 5-min extension at
78
72°C. The second round was performed under the same conditions, although 30 cycles were
79
used.
80
DNA samples were also tested for the presence of protozoan pathogens from the genus
81
Babesia by using genus-specific BJ1 and BN2 primers, which amplify a portion of 18S rRNA
82
(Casati et al., 2006), and by genus-specific BJ1 and BN2 primers, which amplify a portion of
83
18S rRNA (Casati et al., 2006). All PCR amplifications were performed in a total volume of
84
25 μl of reaction mixture, containing 7.6 μl of deionized sterile water, 12.5 μl of 2
85
DyNAzyme II Master Mix (Finnzymes, Espoo, Finland), 1.2 μl of each primer (10 pmol/μl)
86
and 2.5 μl of DNA template. Nuclease-free water was used as a negative control. A positive
87
control with previously sequenced DNA of the given pathogen was used in each PCR assay.
88
All PCR reactions were performed in a personal thermal cycler (MyCycler, Bio-Rad). The
89
PCR products were analyzed electrophoretically in 1.5% agarose gels stained with GelRed
90
stain (Roche Diagnostics) and visualized under UV light.
91
Four randomly-chosen Babesia-positive amplicons and four Anaplasma-positive samples
92
were used for sequence analysis. The randomly-selected amplicons were purified using a
93
QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions.
94
Sequence analyses were performed using internal primers specific for the 16S rRNA of A.
95
phagocytophilum, and primers used for the PCR detection of Babesia species, at the
96
Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine
97
and Pharmacy, Košice, Slovakia. The complementary strands of sequenced products were
98
manually assembled. Electropherograms were evaluated and the results were exported to
99
Alignment Explorer in MEGA 5, where they were compared to each other. Sequences were
100
compared with GenBank entries by Blast N 2.2.13. Obtained sequences were submitted to the
Ac
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Page 4 of 21
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GenBank database under accession numbers: KF381412 (443 bp portion of 18S rRNA of
102
Babesia canis canis) and KF381413 (497 bp portion of 16S rRNA gene of A.
103
phagocytophilum).
ip t
104
Results
106
A total of 205 Dermacentor reticulatus ticks were screened. The prevalence of infection with
107
B. canis canis was 3.41%, with four males and two females infected. Infection with A.
108
phagocytophilum was detected in 25.36% of ticks, with 21 males and 31 females infected.
109
The sequencing of randomly-chosen positive PCR products confirmed the identity of A.
110
phagocytophilum and B. canis canis in the screened samples of genomic DNA isolated from
111
the D. reticulatus ticks.
112
The sequences obtained above were 100% identical respectively. Blast analysis of the
113
obtained 16S rRNA sequence of A. phagocytophilum revealed 99% similarity of the
114
overlapping region with A. phagocytophilum nucleotide sequences isolated from D.
115
reticulatus and Ixodes ricinus ticks in Lithuania, Belarus and Russia (Acc. Nos. JN181063;
116
JN181064; HQ629915; HQ629911).
117
The B.abesia canis canis 18S rRNA nucleotide sequences showed 100% similarity with
118
sequences isolated from B. canis canis from naturally infected dogs in Poland (EU622793),
119
Slovakia (EU165369), Switzerland (AY648872), Netherlands (AY703073), Croatia
120
(FJ209024) and Russia (AY962187).
121
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122
Discussion
123
The results present the first evidence of B. canis canis and A. phagocytophilum in questing D.
124
reticulatus ticks from the Chernobyl exclusion zoneCEZ, and show the possibility that the
5
Page 5 of 21
foci of tick-borne diseases can be maintained as one of the integral elements of the natural
126
environment in areas with no human activity.
127
The presence of tick-borne pathogens in the environment depends on favourable
128
environmental conditions and the simultaneous presence of pathogens, hosts and vector ticks.
129
Altogether, these factors constitute the zoonotic foci and the source of infection (Humair et
130
al., 1999; Siński, 1999; Tälleklint and Jaenson, 1993). The presence of A. phagocytophilum in
131
the CEZ is easy to explain. The reservoir hosts of the bacteria include a wide range of wild
132
mammals: small rodents (Grzeszczuk et al., 2006), medium-sized mammals such as hares and
133
foxes (Hulínská et al., 2004; Karbowiak et al.; 2009) and large ruminants (Grzeszczuk et al.;
134
2004; Hulínská et al.; 2004; Skotarczak et al., 2008; Hapunik et al., 2011), all of which occur
135
in the investigated areas of the CEZ. The main vector of A. phagocytophilum is the I.xodes
136
ricinus tick, which is known to be present in the CEZ, albeit in small numbers (Labetskaya et
137
al. 1997, Karbowiak et al., 2013), as confirmed by the present study. The prevalence of A.
138
phagocytophilum infection in questing I. ricinus ticks studied in other European countries is
139
variable, ranging from 3 to 9 % and closely depends on the presence of reservoir and
140
susceptible hosts (Hulínská et al., 2004; Radzijewskaya et al., 2008; Rosef et al., 2009). As
141
the records of D. reticulatus infection with A. phagocytophilum are rare, and concern mostly
142
adult engorged ticks collected from animals (Duh et al., 2006; Wirtgen et al., 2011), the
143
ability of this tick species to transmit these bacteria is still not clearly understood. The
144
presence of a pathogen in ticks collected from animal hosts gives no information whether the
145
source of infection is the blood of the host, or whether the pathogen was present in the tick
146
before feeding. The presence of A. phagocytophilum in D. reticulatus ticks collected in the
147
CEZ is also the first molecular evidence of this pathogen in questing tick species. Moreover,
148
the percentage of infected D. reticulatus ticks is unusually high in comparison to previous
Ac
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Page 6 of 21
studies, and further research on their presence in I. ricinus collected from the same area and
150
mammal hosts is required.
151
The presence of B. canis canis in the CEZ also requires further investigation. The main
152
vector, Dermacentor reticulatus, is commonly distributed in the CEZ, but the main host of
153
these blood parasites, the dog, is rare in locations inhabited by humans and entirely absent in
154
abandoned areas. Although the wild reservoir of B. canis in natural environments has not been
155
confirmed so far, potential reservoirs could possibly be wild Canidae: red foxes (Vulpes
156
vulpes), wolves (Canis lupus) and raccoon dogs (Nyctereutes procyonoides), which are all
157
present in the CEZ. Not specified Babesia canis infection in foxes was reported in 1938
158
(Schoop and Dedié), however, this cannot be verified using present-day molecular tools.
159
Since then, only a few cases of fox infections with Babesia microti-like protozoans have been
160
reported, however, B. canis canis has been not detected (Karbowiak et al., 2010). The role of
161
wolves is not clear; the single record of wolf infection with Babesia canis is based on clinical
162
symptoms and direct observation of the parasite is yet to be confirmed (Karbowiak et al.,
163
2008).
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164
Acknowledgements
166
The study was supported by a Polish-Ukrainian joint research project for the years 2012-2014,
167
and partially from the projects NCN 2011/01/B/NZ7/03574, ”Centre of Excellence for
168
Parasitology“ (Code ITMS: 26220120022) based on the support of the Operational
169
Programme “Research & Development” funded from the European Regional Development
170
Fund (rate 0.2), the Slovak Agency for Research and Development APVV project 0267-10,
171
and the Scientific Grant Agency of the Ministry of Education SR and Slovak Academy of
172
Sciences VEGA project 2/0113/12.
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We would like to thank Alexander Borovsky from the fire station of Chernobyl town for his
174
great help during the study.
175
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Puszczy Białowieskiej bakteriami Anaplasma phagocytophilum. Med. Wet. 60, 600-601. (in
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Hapunik, J., Víchová, B., Karbowiak, G., Wita, I., Bogdaszewski, M., Peťko, B., 2011. Wild
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Hulínská, D., Langrová, K., Pejcoch, M., Pavlásek, I., 2004. Detection of Anaplasma
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Humair, P.F., Rais, O., Gern, L., 1999. Transmission of Borrelia afzelii from Apodemus mice
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Karbowiak, G., Majláthová, V., Hapunik, J., Peťko, B., Wita, I., 2010. Apicomplexan
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Karbowiak, G., Slivinska, K., Werszko, J., Didyk, J., 2013. The occurrence of hard ticks in
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Chernobyl Exclusion Zone. The 15th International Symposium “Parasitic and allergic
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arthropods – Medical and Sanitary Significance. Kazimierz Dolny, 3-5.06.2013: 48-49.
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Karbowiak, G., Víchová, B., Majláthová, V., Hapunik, J., Peťko, B., 2009. Anaplasma
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Labetskaya, A.G., Kiriyenko, K.M., Baydakova, I.V., Tishechkina, I.M., 1997. An abundance
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Chernobyl'skoi atomnoi stantsii. Parasitologiya 31, 391-396. (in Russian)
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Møller, A.P., Mousseau, T.A., 2006. Biological consequences of Chernobyl: 20 years on.
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Trends Ecol. Evol. 21, 200-207.
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Møller, A.P., Mousseau, T.A., de Lope, F., Saino, N., 2007. Elevated frequency of
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abnormalities in barn swallows from Chernobyl. Biol. Lett. 3, 414-417.
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Radzijevskaja, J., Paulauskas, A., Rosef, O., 2008. Prevalence of Anaplasma
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phagocytophilum and Babesia divergens in Ixodes ricinus ticks from Lithuania and Norway.
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Int. J. Med. Microbiol. 298, S1, 218–221.
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Rijpkema, S., Golubić, D., Molkenboer, M., Verbeek-De Kruif, N., Schellekens, J., 1996.
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Identification of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus
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ticks collected in a Lyme borreliosis endemic region of northern Croatia. Exp. Apel. Acar. 20,
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Rosef, O., Paulauskas, A., Radzijevskaja, J., 2009. Prevalence of Borrelia burgdorferi sensu
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lato and Anaplasma phagocytophilum in questing Ixodes ricinus ticks in relation to the
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density of wild cervids. Acta Vet. Scand. 51, 47.
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Schoop, G., Dedié, K., 1938. Uebertragung von Babesia canis auf Füchse. Dtsch. Tierärztl.
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Wochenschr. 46, 88-90.
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Siński, E., 1999. Enzootic reservoir for new Ixodes ricinus –transmitted
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infectionsEnzootyczne źródła nowych infekcji przenoszonych przez kleszcze Ixodes ricinus.
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Wiad. Parazytol. 45, 135-142. (in Polish)
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Skotarczak, B., Adamska, M., Sawczuk, M., Maciejewska, A., Wodecka, B., Rymaszewska,
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A., 2008. Coexistence of tick-borne pathogens in game animals and ticks in western Poland.
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Vet. Med. 53, 668–675.
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Tälleklint, L., Jaenson, T.G.T., 1993. Maintenance by hares of european Borrelia burgdorferi
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in ecosystems without rodents. J. Med. Entomol. 30, 273-276.
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Wirtgen, M., Nahayo, A., Linden, A., Garigliany, M., Desmechtl, D., 2011. Detection of
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Anaplasma phagocytophilum in Dermacentor reticulatus ticks. Vet. Rec. 168, 195.
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Formatted: Font: Italic
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Abbreviations
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Figure 1. The map of the sampling points in the Chernobyl EZexclusion zone.
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*Revised Manuscript with NO changes marked (clean) Click here to view linked References
1
The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma
2
phagocytophilum in the Chernobyl exclusion zone.
3
Grzegorz Karbowiak1, Bronislavá Vichová2, Kateryna Slivinska3, Joanna Werszko2, Julia
5
Didyk3, Branislav Peťko2, Michal Stanko2, Igor Akimov3
ip t
4
6
1 W. Stefański Institute of Parasitology of the Polish Academy of Sciences, Twarda 51/55,
8
00-818 Warsaw, Poland;
9
2 Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice,
us
cr
7
Slovakia;
11
3 I.I.Schmalhausen Institute of Zoology of NASU, B. Khmelnytskogo 15, 01601 Kiev,
12
Ukraine.
M
an
10
13
Corresponding author: Grzegorz Karbowiak,
15
W. Stefański Institute of Parasitology of the Polish Academy of Sciences, Twarda 51/55,
16
00-818 Warsaw, Poland;
17
tel. 22 6978975; fax. 22 620-62-27; e-mail:
[email protected] ce pt
18
ed
14
Abstract
20
Tick occurrence was studied in the Chernobyl exclusion zone (CEZ) during the August-
21
October 2009-2012. Dermacentor reticulatus ticks were collected using the flagging method
22
and then screened for infection with Anaplasma phagocytophilum and Babesia canis by a
23
PCR method incorporating specific primers and sequence analysis. The prevalence of
24
infection with B. canis canis and A. phagocytophilum was found to be 3.41% and 25.36%
25
respectively. The results present the first evidence of B. canis canis and A. phagocytophilum
Ac
19
1 Page 11 of 21
26
in questing Dermacentor reticulatus ticks from the Chernobyl exclusion zone. They also
27
reveal the presence of tick-borne disease foci in areas with no human activity, and confirm
28
that they can be maintained in areas after a nuclear disaster with radioactive contamination.
29
Key words: Chernobyl exclusion zone; Anaplasma phagocytophilum; Babesia canis;
31
Dermacentor reticulatus.
ip t
30
cr
32
Introduction
34
In 1986, as a consequence of the disaster at the Chernobyl nuclear power plant, a mass of
35
radioactive material was released into the atmosphere, causing extensive pollution to the
36
neighbouring regions. An Exclusion Zone (CEZ) was established within a 30 – 60 km radius
37
of the explosion point, where human activity was minimized and entrance was restricted. The
38
direct consequences of radioactive pollution, as well as the evacuation of the public and
39
cessation of agriculture and forest management caused significant ecological changes, such as
40
the spontaneous restitution of the original natural habitats, plant and animal populations, as
41
well as their interdependences. Many aspects of the consequences of radionuclide
42
contamination of wild animals have been studied during the short time since the catastrophe.
43
For instance, the study of radionuclide accumulation in the soil and by different species of
44
animals (Frantsevich, 2006), the effects of radiation on cytogenetics and mutation, as well as
45
the frequency of abnormalities potentially caused by pollution (Møller and Mousseau, 2006;
46
Møller et al., 2007). Although parasitological studies are not numerous, the increase and
47
biodiversity of micromammalian parasite complexes have been examined (Labetskaya et al.,
48
1997). This study presents the first evidence of tick-borne pathogen foci in the Chernobyl
49
Exclusion Zone.
Ac
ce pt
ed
M
an
us
33
50
2 Page 12 of 21
Materials and Methods
52
Study sites and tick collection method.
53
Tick occurrence in the CEZ was studied during the August-October period each year from
54
2009 to 2012. The study area was located in preventive zone B, within a 10-20 km radius of
55
the disaster point. The flagging method was used to collect the ticks from areas where they
56
were known to appear in the surroundings of the former villages of Korogod (51°16'02"N;
57
30°01'04"E) and Cherevach (51°12'44"N; 30°07'45"E). The investigated habitats included
58
open areas and the remnants of farmlands. The vegetation in the tick collection sites included
59
grass and ruderal plants, and some bushes. The collected Dermacentor reticulatus ticks were
60
fixed in ethanol and transported to the laboratory for further analysis.
an
us
cr
ip t
51
61
Molecular detection of Babesia spp. and Anaplasma phagocytophilum.
63
The DNA extraction was performed using the ammonium hydroxide method (Rijpkema et al.,
64
1996). After washing in PBS/EDTA 0.5 %, all D. reticulatus ticks were cut and crushed
65
separately in Eppendorf tubes. Genomic DNA isolation was performed by boiling in 200 μL
66
of 0.7 M NH4OH for 30 min. The remaining tick tissue and chitin exoskeleton were removed
67
by a short spin, following which, the supernatant containing the DNA was collected and the
68
pellet discarded.
69
All ticks were screened by nested PCR amplification of the 16S rRNA gene. In the first round
70
of amplification, primers ge3a and ge10r were used to detect all members of the
71
Anaplasmataceae. For the second round, one microlitre of PCR product from the first round
72
was used as a template to detect A. phagocytophilum, using primers ge9f and ge2 (Massung et
73
al., 1998). Cycling conditions in the first round involved an initial 2-min denaturation at 95°C,
74
followed by 40 cycles, each consisting of a 30-s denaturation at 94°C, a 30-s annealing at
75
55°C, and a 1-min extension at 72°C. These 40 cycles were followed by a 5-min extension at
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72°C. The second round was performed under the same conditions, although 30 cycles were
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used.
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DNA samples were also tested for the presence of protozoan pathogens from the genus
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Babesia by using genus-specific BJ1 and BN2 primers, which amplify a portion of 18S rRNA
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(Casati et al., 2006), and by genus-specific BJ1 and BN2 primers, which amplify a portion of
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18S rRNA (Casati et al., 2006). All PCR amplifications were performed in a total volume of
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25 μl of reaction mixture, containing 7.6 μl of deionized sterile water, 12.5 μl of 2
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DyNAzyme II Master Mix (Finnzymes, Espoo, Finland), 1.2 μl of each primer (10 pmol/μl)
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and 2.5 μl of DNA template. Nuclease-free water was used as a negative control. A positive
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control with previously sequenced DNA of the given pathogen was used in each PCR assay.
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All PCR reactions were performed in a personal thermal cycler (MyCycler, Bio-Rad). The
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PCR products were analyzed electrophoretically in 1.5% agarose gels stained with GelRed
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stain (Roche Diagnostics) and visualized under UV light.
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Four randomly-chosen Babesia-positive amplicons and four Anaplasma-positive samples
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were used for sequence analysis. The randomly-selected amplicons were purified using a
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QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions.
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Sequence analyses were performed using internal primers specific for the 16S rRNA of A.
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phagocytophilum, and primers used for the PCR detection of Babesia species, at the
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Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine
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and Pharmacy, Košice, Slovakia. The complementary strands of sequenced products were
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manually assembled. Electropherograms were evaluated and the results were exported to
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Alignment Explorer in MEGA 5, where they were compared to each other. Sequences were
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compared with GenBank entries by Blast N 2.2.13. Obtained sequences were submitted to the
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GenBank database under accession numbers: KF381412 (443 bp portion of 18S rRNA of
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Babesia canis canis) and KF381413 (497 bp portion of 16S rRNA gene of A.
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phagocytophilum).
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Results
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A total of 205 Dermacentor reticulatus ticks were screened. The prevalence of infection with
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B. canis canis was 3.41%, with four males and two females infected. Infection with A.
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phagocytophilum was detected in 25.36% of ticks, with 21 males and 31 females infected.
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The sequencing of randomly-chosen positive PCR products confirmed the identity of A.
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phagocytophilum and B. canis canis in the screened samples of genomic DNA isolated from
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the D. reticulatus ticks.
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The sequences obtained above were 100% identical respectively. Blast analysis of the
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obtained 16S rRNA sequence of A. phagocytophilum revealed 99% similarity of the
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overlapping region with A. phagocytophilum nucleotide sequences isolated from D.
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reticulatus and Ixodes ricinus ticks in Lithuania, Belarus and Russia (Acc. Nos. JN181063;
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JN181064; HQ629915; HQ629911).
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The B. canis 18S rRNA nucleotide sequences showed 100% similarity with sequences
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isolated from B. canis canis from naturally infected dogs in Poland (EU622793), Slovakia
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(EU165369), Switzerland (AY648872), Netherlands (AY703073), Croatia (FJ209024) and
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Russia (AY962187).
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Discussion
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The results present the first evidence of B. canis canis and A. phagocytophilum in questing D.
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reticulatus ticks from the CEZ, and show the possibility that the foci of tick-borne diseases
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can be maintained as one of the integral elements of the natural environment in areas with no
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human activity.
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The presence of tick-borne pathogens in the environment depends on favourable
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environmental conditions and the simultaneous presence of pathogens, hosts and vector ticks.
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Altogether, these factors constitute the zoonotic foci and the source of infection (Humair et
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al., 1999; Siński, 1999; Tälleklint and Jaenson, 1993). The presence of A. phagocytophilum in
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the CEZ is easy to explain. The reservoir hosts of the bacteria include a wide range of wild
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mammals: small rodents (Grzeszczuk et al., 2006), medium-sized mammals such as hares and
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foxes (Hulínská et al., 2004; Karbowiak et al.; 2009) and large ruminants (Grzeszczuk et al.;
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2004; Hulínská et al.; 2004; Skotarczak et al., 2008; Hapunik et al., 2011), all of which occur
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in the investigated areas of the CEZ. The main vector of A. phagocytophilum is the I. ricinus
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tick, which is known to be present in the CEZ, albeit in small numbers (Labetskaya et al.
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1997, Karbowiak et al., 2013), as confirmed by the present study. The prevalence of A.
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phagocytophilum infection in questing I. ricinus ticks studied in other European countries is
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variable, ranging from 3 to 9 % and closely depends on the presence of reservoir and
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susceptible hosts (Hulínská et al., 2004; Radzijewskaya et al., 2008; Rosef et al., 2009). As
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the records of D. reticulatus infection with A. phagocytophilum are rare, and concern mostly
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adult engorged ticks collected from animals (Duh et al., 2006; Wirtgen et al., 2011), the
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ability of this tick species to transmit these bacteria is still not clearly understood. The
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presence of a pathogen in ticks collected from animal hosts gives no information whether the
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source of infection is the blood of the host, or whether the pathogen was present in the tick
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before feeding. The presence of A. phagocytophilum in D. reticulatus ticks collected in the
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CEZ is also the first molecular evidence of this pathogen in questing tick species. Moreover,
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the percentage of infected D. reticulatus ticks is unusually high in comparison to previous
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studies, and further research on their presence in I. ricinus collected from the same area and
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mammal hosts is required.
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The presence of B. canis canis in the CEZ also requires further investigation. The main
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vector, Dermacentor reticulatus, is commonly distributed in the CEZ, but the main host of
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these blood parasites, the dog, is rare in locations inhabited by humans and entirely absent in
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abandoned areas. Although the wild reservoir of B. canis in natural environments has not been
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confirmed so far, potential reservoirs could possibly be wild Canidae: red foxes (Vulpes
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vulpes), wolves (Canis lupus) and raccoon dogs (Nyctereutes procyonoides), which are all
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present in the CEZ. Nonspecified Babesia canis infection in foxes was reported in 1938
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(Schoop and Dedié), however, this cannot be verified using present-day molecular tools.
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Since then, only a few cases of fox infections with Babesia microti-like protozoans have been
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reported, however, B. canis has been not detected (Karbowiak et al., 2010). The role of
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wolves is not clear; the single record of wolf infection with Babesia canis is based on clinical
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symptoms and direct observation of the parasite is yet to be confirmed (Karbowiak et al.,
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2008).
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Acknowledgements
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The study was supported by a Polish-Ukrainian joint research project for the years 2012-2014,
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and partially from the projects NCN 2011/01/B/NZ7/03574, ”Centre of Excellence for
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Parasitology“ (Code ITMS: 26220120022) based on the support of the Operational
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Programme “Research & Development” funded from the European Regional Development
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Fund (rate 0.2), the Slovak Agency for Research and Development APVV project 0267-10,
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and the Scientific Grant Agency of the Ministry of Education SR and Slovak Academy of
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Sciences VEGA project 2/0113/12.
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We would like to thank Alexander Borovsky from the fire station of Chernobyl town for his
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great help during the study.
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Figure 1. The map of the sampling points in the Chernobyl exclusion zone.
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Figure Click here to download high resolution image
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