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,

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Slovakia;

11

3 I.I.Schmalhausen Institute of Zoology of NASU, B. Khmelnytskogo 15, 01601 Kiev,

12

Ukraine.

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M

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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]

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18

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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.

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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.

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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.

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Page 2 of 21

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Materials and Methods

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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

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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

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Formatted: Font: Not Italic

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Page 3 of 21

followed by 40 cycles, each consisting of a 30-s denaturation at 94°C, a 30-s annealing at

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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.

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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

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

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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

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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.

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

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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

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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.abesia canis canis 18S rRNA nucleotide sequences showed 100% similarity with

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sequences isolated from B. canis canis from naturally infected dogs in Poland (EU622793),

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Slovakia (EU165369), Switzerland (AY648872), Netherlands (AY703073), Croatia

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(FJ209024) and Russia (AY962187).

121

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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 Chernobyl exclusion zoneCEZ, and show the possibility that the

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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.

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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.

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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

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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

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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.

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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

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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

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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

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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

155

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

157

present in the CEZ. Not specified 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

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.,

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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

169

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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References

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Duh, D., Slovák, M., Saksida, A., Strašek, K., Petrovec, M., Avšič-Županc, T., 2006.

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Biologia 61, 231-233.

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Frantsevich, L., 2006. Animal radioecology in the exclusion zone since the Chernobyl

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Grzeszczuk, A., Karbowiak, G., Ziarko, S., Kovalchuk, O., 2006. The root-vole Microtus

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oeconomus (Pallas, 1776): a new potential reservoir of Anaplasma phagocytophilum. Vector

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Borne Zoonotic Dis. 6, 240-243.

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Grzeszczuk, A., Ziarko, S., Prokopowicz, D., Radziwon, P.M., 2004. Zakażenie żubrów z

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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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and farm breeding cervids infections with Anaplasma phagocytophilum. Ann. Agric. Environ.

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Med. 18, 73-77.

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Hulínská, D., Langrová, K., Pejcoch, M., Pavlásek, I., 2004. Detection of Anaplasma

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phagocytophilum in animals by real-time polymerase chain reaction. APMIS 112, 239-247.

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Humair, P.F., Rais, O., Gern, L., 1999. Transmission of Borrelia afzelii from Apodemus mice

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and Clethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and

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overwintering maintenance. Parasitology 118, 33-42.

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Karbowiak, G., Hapunik, J., Miniuk, M., 2008. The case of babesiosis in farmed wolf (Canis

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lupus L.). Wiad. Parazytol. 54, 243.

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Karbowiak, G., Majláthová, V., Hapunik, J., Peťko, B., Wita, I., 2010. Apicomplexan

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parasites of red foxes (Vulpes vulpes) in northeastern Poland. Acta Parasitol. 55, 210-214.

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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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phagocytophilum infection of red foxes (Vulpes vulpes). Ann. Agric. Environ. Med. 16, 299-

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Labetskaya, A.G., Kiriyenko, K.M., Baydakova, I.V., Tishechkina, I.M., 1997. An abundance

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and biodiversity of parasites from micromammalians in the evacuation zone of the Chernobyl

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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

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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

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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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The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma phagocytophilum in the Chernobyl exclusion zone.

Tick occurrence was studied in the Chernobyl exclusion zone (CEZ) during the August-October 2009-2012. Dermacentor reticulatus ticks were collected us...
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