Borrelia and Other Zoonotic Pathogens in Ixodes ricinus and Dermacentor reticulatus Ticks Collected from the Chernobyl Exclusion Zone on the 30th Anniversary of the Nuclear Disaster

Abstract

Background:

The 26th of April 2016 marked 30 years since the Chernobyl accident has occurred in Ukraine. As a result, the uninhabited Chernobyl region has been directly exposed to ionizing radiation for >30 years. Most work has focused on identifying associations between levels of radiation and the abundance, distribution, and mutation rates of plants and animals. Much less, however, is known about microbial communities in this affected region. To date, there are no reports on the prevalence of any tick-borne pathogens in Ixodes ricinus ticks from the Chernobyl exclusion zone (CEZ). The objective of our study was to examine the abundance of I. ricinus and Dermacentor reticulatus ticks in the CEZ and to investigate the prevalence of Borrelia burgdorferi sensu lato (s.l.) and other zoonotic agents in these ixodid ticks.

Methods:

A total of 260 questing I. ricinus and 100 D. reticulatus adult ticks were individually polymerase chain reaction analyzed for the presence of Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Borrelia burgdorferi s.l., Francisella tularensis, and/or Rickettsia spp.

Results:

The respective infections rates were identified and compared with those of ixodid ticks that were concurrently collected from Kyiv. The significant differences between the infection rates of the CEZ and Kyiv ticks were observed for Rickettsia raoultii in D. reticulatus ticks (53.0% vs. 35.7%, respectively; p < 0.05) and Bartonella spp. (8.1% vs. 2.7%; P < 0.05) in I. ricinus ticks.

Conclusions:

Although the current data clearly demonstrated that the prevalence of some zoonotic pathogens were significantly higher in the ixodid ticks from the CEZ, a more comprehensive systematic approach is required to examine the causal effect of long-term ionizing radiation on adaptive changes of tick-borne pathogens.

Introduction

Numerous bacterial, viral, and protozoan pathogens of public health significance are carried and transmitted by tick vectors (Day 2011, Brites-Neto et al. 2015, Pettersson et al. 2017). The most medically important American and European ticks belong to the genus Ixodes (Latreille 1795) (Brites-Neto et al. 2015). Ixodes ricinus Linnaeus, 1758 ticks harbor spirochete bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex. B. burgdorferi s.l. causes Lyme borreliosis (LB), one of the most common tick-borne diseases in both Europe and North America (Rizzoli et al. 2011, Hinckley et al. 2014).

In the United States, there are ∼30,000 confirmed cases of human LB each year (Maxam and Gilbert 1980), although a recent study suggests that the actual incidence is about 10 times higher (Hinckley et al. 2014). In European countries, it is estimated that from 65,500 to 85,000 LB cases occur each year (Lindgren and Jaenson 2006, Hubálek 2009, van den Wijngaard et al. 2017). The B. burgdorferi s.l. complex consists of >22 genospecies (Stanek et al. 2011), of which B. afzelii, B. garinii, and B. burgdorferi sensu stricto (hereafter referred to as B. burgdorferi) are the main pathogenic genospecies for humans in Europe.

In Ukraine, 4597 human cases were officially reported between 2000 and 2010. In 2015 and 2016, a total of 3413 and 2758 cases, respectively, were recorded according to the Ministry of Health of Ukraine (Nebogatkin et al. 2017). In Ukraine, most cases are reported in May through September (81.7–85.6%). LB cases have been recorded in all 25 regions of Ukraine, including the Autonomous Republic of Crimea (Biletska 2011).

Despite the fact that LB is the most prevalent vector-borne disease in Ukraine, studies on the prevalence of B. burgdorferi s.l. in I. ricinus ticks in this country are very limited. The most comprehensive study included 2811 I. ricinus adults collected in 12 regions of Ukraine between 2003 and 2006. The dark-field microscopy approach demonstrated that 9.5% I. ricinus ticks were positive for Borrelia spirochetes (Biletska et al. 2008). The prevalence of B. burgdorferi s.l. varied from 5.6% to 25.0% between different biotopes. A more recent study demonstrated that 4.0% of I. ricinus ticks collected in Kyiv between 2013 and 2014 carried B. burgdorferi s.l. (Didyk et al. 2017). The latest data showed even higher prevalence: 10.4% and 80.6% of I. ricinus adults and nymphs collected from Kyiv tested positive for B. burgdorferi s.l., respectively (Rogovskyy et al. 2018). To date, however, the prevalence of B. burgdorferi s.l. and other zoonotic tick-borne pathogens in ixodid ticks from other regions of Ukraine remains unknown.

In addition to B. burgdorferi s.l., I. ricinus ticks may also be carriers of other zoonotic pathogens, such as Anaplasma phagocytophilum, Bartonella spp., Francisella tularensis, Rickettsia spp., and Babesia spp. (Brites-Neto et al. 2015). Dermacentor reticulatus is another significant ixodid tick vector of zoonotic pathogens in Europe (Rubel et al. 2014). D. reticulatus ticks are the main vector for Babesia canis and Ba. caballi as well as Rickettsia slovaca and R. raoultii (Rubel et al. 2016). Furthermore, questing D. reticulatus adults could also carry A. phagocytophilum, Bartonella henselae, and F. tularensis, although the vector function for these pathogens has not been proven (Rubel et al. 2016).

A. phagocytophilum is an emerging pathogen responsible for human granulocytic anaplasmosis (Jin et al. 2012, Bakken and Dumler 2015). Bartonella spp., a causative agent of bartonellosis, may establish persistent bloodstream infections in both humans and animals (Florin et al. 2008, Day 2011, Nelson et al. 2017). Although cat fleas are the primary vectors for B. henselae, transmission by ticks was previously suggested (Angelakis et al. 2010).

F. tularensis causes tularemia as a result of exposure to arthropods, aerosolized bacteria, or contaminated food of animal origin (Staples et al. 2006), and I. ricinus ticks are significant vectors of this disease (Gyuranecz et al. 2011). Rickettsia spp. belonging to the spotted fever group can cause potentially fatal diseases and are transmitted to humans through ixodid ticks (Wood and Artsob 2012, Parola et al. 2013). Lastly, pathogenic protozoa in the genus Babesia cause babesiosis in vertebrate hosts, including humans (Hunfeld et al. 2008, Colwell et al. 2011, Vannier and Krause 2012).

The 26th of April 2016 marked 30 years since the Chernobyl accident has occurred in Ukraine. As a result of the nuclear meltdown, the uninhabited Chernobyl region has been directly exposed to ionizing radiation for >30 years. Most work has focused on identifying associations between levels of radiation and the abundance, distribution, life history, and mutation rates of plants and animals (Moller and Mousseau 2006, Yablokov 2009, Yablokov et al. 2009, Mietelski et al. 2010, Oskolkov et al. 2011, Moller et al. 2014, Mousseau and Moller 2014). Much less, however, is known about microbial communities in this affected region.

To date, there are no reports on the prevalence of any tick-borne pathogens in I. ricinus ticks from the Chernobyl exclusion zone (CEZ). Thus, the objective of our study was twofold: to examine the abundance of I. ricinus and D. reticulatus ticks in the CEZ and to investigate the prevalence of the aforementioned zoonotic agents in I. ricinus and D. reticulatus ticks from the CEZ.

Materials and Methods

Tick collection

On May 18th and 27th, 2016, a total of 367 adult and 5 nymphal I. ricinus ticks and 129 adult D. reticulatus ticks were collected in the CEZ. Ticks were collected from five collection sites located in the zones that were within 10 and 30 km of the Chernobyl nuclear power station (Table 1). A map of the sampling sites is shown in Fig. 1.

FIG. 1.

Tick collection area in the Chernobyl exclusion zone, Ukraine. The ixodid ticks were collected at five sites within 10- and 30-km Chernobyl exclusion zones. The tick collection area is indicated as a black oval.

Table 1.

Ixodid Ticks Collected in the Chernobyl Exclusion Zone (May 2016) and Their Indices of Abundance

	Geo-references		Ixodes ricinus					Dermacentor reticulatus
Collection sites (radiation level, mR/h)	Latitude	Longitude	M	F	IA ^a	N	IA ^b	M	F	IA ^a
10-km zone
Site 1 (0.025–0.040)	N51° 16.627′	E30° 09.915′	0	4	1.33	0	n/a	15	35	16.67
Site 2 (0.025–0.040)	N51° 16.490′	E30° 09.539′	1	7	6.40	0	n/a	3	38	32.80
Site 3 (0.025–0.040)	N51° 16.627′	E30° 09.914′	17	8	14.29	0	n/a	2	8	5.71
Site 4 (200–330)	n/p	n/p	0	0	n/a	1	0.50	6	0	3.00
30-km zone
Site 5 (0.015–0.025)	N51° 16.118′	E30° 14.840′	153	177	25.48	4	0.28	7	15	2.38
Total			171	196	11.87^c	5	0.39^c	33	96	12.11^c

Index of abundance (IA) is provided for adult ticks (both males and females) for each collection site.

IA values are provided for nymphal ticks.

Mean of IA indices.

M, males; F, females; N, nymphs; n/a, not applicable; n/p, not provided.

Ticks were collected by flagging over and through the vegetation and at ground level with a cotton cloth (100 × 60 cm) between 8:00 am and 6:00 pm. Collection time varied between the collection sites. Each site was only visited once. The minimum and maximum sampling efforts included 4 and 8 h of collection, respectively. All the ticks were identified to the species level according to their morphology (Filippova 1997) by using a stereoscopic microscope at the Schmalhausen Institute of Zoology of National Academy of Sciences of Ukraine. Index of abundance (IA) was determined as described (Vershinin and Vershinin 2009, Rogovskyy et al. 2017): IA = Q (total number of ticks)/(t₁ + t₂ + … + t_n) (the sum of time spent by all collectors in minutes) × 60 (1 h reduction index).

DNA extraction

Individual ticks (260 I. ricinus and 100 D. reticulatus adult ticks) were frozen in liquid nitrogen and crushed in a bead beater for 60 s (TisueLyser II; Qiagen, Inc.). A total of 360 DNA samples were extracted by utilizing the DNeasy Minikit and QIAamp DNA Mini Kit (Qiagen, Inc.) according to the manufacturer's protocols with the following modification as described (Rogovskyy et al. 2018). Extracted DNA was stored at −20°C until use.

Borrelia-specific PCR

To detect B. burgdorferi s.l., individual DNA samples of adult I. ricinus were subjected to Borrelia-specific nested PCR that amplified a 587-bp fragment of the 16S–23S intergenic spacer (IGS) gene (Bunikis et al. 2004, Scott 2005). The master mix consisted of 0.2 mM of each dNTP, 0.5 μM of the forward and reverse primers, 1 unit of Taq DNA Polymerase with ThermoPol^® Buffer (New England BioLabs, Inc.), and 1.5 mM of MgCl₂ (Life Technologies). Each PCR reaction contained 5 μL of DNA extract in a 50-μL PCR reaction. The previously developed primers for the first stage, rrs and rrl (5′-GTATGTTTAGTGAGGGGGGTG-3′ and 5′-GGATCATAGCTCAGGTGGTTAG-3′, respectively) and the second stage, Fn and Rn (5′-AGGGGGGTGAAGTCGTAACAAG-3′ and 5′-GTCTGATAAACCTGAGGTCGGA-3′, respectively) were used (Scott 2005).

The thermocycler conditions for the nested PCR included the following steps: denaturation at 94°C for 4 min, 35 cycles of amplification at 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, followed by a 10-min extension phase at 72°C for both stages of the nested PCR reaction. PCR amplicons were visualized on ethidium bromide-stained 1% agarose gels. Nested PCR amplicons from the second stage of the IGS set were sequenced by MCLAB (Molecular Cloning Laboratories). For each PCR run and cocktail, Borrelia-positive (DNA extracted from in vitro-grown B. burgdorferi B31-A3 strain) and nuclease-free water (Life Technologies) were utilized as positive and negative controls, respectively. PCR results were acceptable only when the positive and negative controls worked: band of expected size was present and absent, respectively.

Conventional PCRs for other tick-borne pathogens

DNAs from 260 I. ricinus and 100 D. reticulatus ticks were subjected to conventional PCRs for A. phagocytophilum, Bartonella spp., Babesia spp., F. tularensis, and/or Rickettsia spp. as previously described (Roux et al. 1997, Reye et al. 2013). The following primers EL(569)F and EL(1193)R for A. phagocytophilum groEL gene (Alberti et al. 2005); BJ1 and BN2 for Babesia spp. 18S rRNA gene (Casati et al. 2006); BH1, BH4, and HSPps1 for Bartonella spp. groEL gene (Zeaiter et al. 2002, Rar et al. 2005); Fr153F0.1 and Fr1281R0.1 for F. tularensis spp. 16S rRNA gene (Barns et al. 2005); and Rp1258n and CS409d for Rickettsia spp. gltA gene (Roux et al. 1997), and all the protocols used in this study were previously published (Reye et al. 2013).

To confirm Rickettsia raoultii in D. reticulatus ticks, an additional PCR using published ompA primers and protocol was performed (Phan et al. 2011). PCR reactions contained 5 μL of DNA extract in a 50-μL PCR reaction. Cloned fragments of target genes of the respective pathogens and nuclease-free water (Life Technologies) were used for each PCR run as positive and negative controls, respectively. PCR results were acceptable only when the positive and negative controls worked: band of expected size was present and absent, respectively.

Sequencing

PCR amplicons were subjected to sequencing (Molecular Cloning Laboratories, San Francisco) and analyzed by applying Mac Vector 14 software. The assembled sequences were analyzed through BLASTn (Camacho et al. 2009). The obtained nucleotide sequences are provided as Supplementary Data.

Statistics

The “N-1” chi-squared test was used for comparison of proportions (Campbell 2007, Richardson 2011). A p value of <0.05 was considered significant. To directly compare the prevalence rates of the tick-borne pathogens in questing adult ticks of I. ricinus and D. reticulatus collected from Ukraine and neighboring Belarus, the pertinent data from this and most recent studies are summarized in Tables 2 and 3. Table 3 also shows which statistical test was applied for each comparison and the respective p values.

Table 2.

The Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulatus Adults Collected in the Chernobyl Exclusion Zone (May 2016)

Tick-borne pathogen	No. of I. ricinus ticks positive/total no. tested	No. of D. reticulatus ticks positive/total no. tested
Anaplasma phagocytophilum	1/260	0/100
Babesia spp.	0/260	0/100
Bartonella spp.	21/260	3/100
B. henselae	21/21	3/3
Borrelia spp.	51^a/260	nt
B. burgdorferi s.l.	35/35	nt
Francisella tularensis spp.	0/260	0/100
Rickettsia spp.	30/260	53/100

The sequencing of some amplicons repeatedly failed.

nt, not tested.

Table 3.

The Prevalence of Tick-Borne Pathogens, Derived from the Present and Most Recently Published Studies, in Questing Ixodes Ricinus and Dermacentor Reticulatus Adults Collected from Ukraine and Neighboring Belarus

Location and time of tick collection (reference)	Tick-borne pathogen	No. of I. ricinus ticks positive/total no. tested (% positive ticks)	No. of D. reticulatus ticks positive/total no. tested (% positive ticks)	No. of I. ricinus and D. reticulatus ticks positive/total no. tested (% positive ticks)
Chernobyl exclusion zone, Ukraine, May 2016 (this study)	B. burgdorferi s.l.	35/260 (13.5%)^{&^}	nt	nt^#
Kyiv, Ukraine, May 2016 (Rogovskyy et al. 2017)		19/182 (10.4%)^&	nt	nt
Kyiv, Ukraine, September–October 2013 and March–May 2014 (Didyk et al. 2016)		27/524 (5.15%)^{^}	nt	nt
P value (Chi-squared test)		^& P = 0.3280 ^{^} P < 0.0001	na	na
Chernobyl exclusion zone, Ukraine, May 2016 (this study)	Anaplasma phagocytophilum	1/260 (0.4%)^*&	0/100 (0%)	1/360 (0.3%)^{^&}
Kyiv, Ukraine, May 2016 (Rogovskyy et al. 2017)		5/182 (2.7%)^*	1/98 (1.0%)^{^}	6/280 (2.1%)^&
Chernobyl exclusion zone, Ukraine, August–October 2009–2012 (Karbowiak et al. 2014)		nt	52/205 (25.4%)^{^}	nt
Various regions, Belarus, April–May 2009 (Reye et al. 2013)		12/289^$ (4.1%)^&	0/164 (0%)	12/453^$ (2.7%)^{^}
P value (Chi-squared test)		^* P = 0.0392 ^& P = 0.0043	^{^} P < 0.0001	^{^} P = 0.0074 ^& P = 0.0295
Chernobyl exclusion zone, Ukraine, May 2016 (this study)	Babesia spp.	0/260 (0%)	0/100 (0%)	0/360 (0%)
Kyiv, Ukraine, May 2016 (Rogovskyy et al. 2017)	Babesia spp.	1/182 (0.5%)	5/98 (5.1%)^*	6/280 (2.1%)^&
Chernobyl exclusion zone, Ukraine, August–October 2009–2012 (Karbowiak et al. 2014)	Babesia canis	nt	6/205 (2.9%)^*	nt
Various regions, Belarus, April–May 2009 (Reye et al. 2013)	Babesia microti and Babesia venatorum	5/289^$ (1.7%)	0/164 (0%)	5/453^$ (1.1%)^&
P value (Chi-squared test)		na	^* P = 0.3378	^& P = 0.2766
Chernobyl exclusion zone, Ukraine, May 2016 (this study)	Bartonella spp.	21/260 (8.1%)^*	3/100 (3.0%)^{^}	24/360 (6.7%)^&
Kyiv, Ukraine, May 2016 (Rogovskyy et al. 2017)		5/182 (2.7%)^*	1/98 (1.0%)^{^}	6/280 (2.1%)^&
P value (Chi-squared test)		^* P = 0.0176	^{^} P = 0.3173	^& P = 0.0064
Chernobyl exclusion zone, Ukraine, May 2016 (this study)	Francisella tularensis spp.	0/260 (0%)	0/100 (0%)	0/360 (0%)
Kyiv, Ukraine, May 2016 (Rogovskyy et al. 2017)		4/182 (2.2%)	2/98 (2.0%)	6/280 (2.1%)
P value (Chi-squared test)		na	na	na
Chernobyl exclusion zone, Ukraine, May 2016 (this study)	Rickettsia spp.	30/260 (11.5%)^**	53⁺/100 (53.0%)^*&	84/360 (23.3%)^***
Kyiv, Ukraine, May 2016 (Rogovskyy et al. 2017)		23/182 (12.6%)^**	35⁺/98 (35.7%)^{*^}	58/280 (20.7%)^***
Chernobyl exclusion zone, Ukraine, September 2010 (Karbowiak et al. 2016)		nt	146⁺/201 (72.6%)^{^}	nt
Kyiv, Ukraine, September–October 2013 and March–May 2014 (Didyk et al. 2016)		nt	7⁺/69 (10.1%)^&	nt
P value (Chi-squared test)		^** P = 0.7260	^* P = 0.0146 ^{^} P < 0.0001 ^& P < 0.0001	^*** P = 0.4325

, ^&, or ^{^} indicates which values within a given section of each column are statistically compared and the respective P values.

nt and na denote not tested and nonapplicable, respectively.

indicates that the data analysis, in addition to questing tick adults, included two questing nymphs.

indicates Rickettsia raoultii

Results

A total of 501 questing ixodid ticks, specifically 367 adults and 5 nymphs of I. ricinus (74.2%) and 129 adults of D. reticulatus (25.8%) were collected during the 2-day period in May 2016 within the 10- and 30-km zones of the CEZ (Fig. 1). The georeferences of the five collection sites are provided in Table 1. The IA varied considerably between sampling sites for both I. ricinus (1.33–25.48 ticks collected per hour of sampling effort) and D. reticulatus (3.00–32.80) adults. Overall, the mean IA values for I. ricinus and D. reticulatus adults were almost the same, 11.87 and 12.11, respectively. No Ixodes larvae were sampled during the collection period (Table 1).

To examine the prevalence of tick-borne pathogens in the collected ixodid ticks, a total of 260 and 100 questing I. ricinus and D. reticulatus adults, respectively, were individually PCR analyzed for B. burgdorferi s.l. and other zoonotic agents, specifically A. phagocytophilum, Babesia spp., Bartonella spp., Borrelia spp., F. tularensis, and/or Rickettsia spp.. The results demonstrated that 19.6% of I. ricinus adult ticks carried Borrelia spp., of which at least 13.5% ticks were specifically positive for B. burgdorferi s.l. (Table 2). The sequence analysis of amplified 16S–23S IGS suggested that the most prevalent genospecies of the B. burgdorferi s.l. complex were B. burgdorferi (21 sequences out of 35 analyzed [21/35]) followed by B. afzelii (9/35), B. garinii (3/35), and B. valaisiana (2/35). The results also showed that A. phagocytophilum DNA was only detected in a single I. ricinus adult out of 360 ixodid ticks analyzed (Table 2).

All the questing I. ricinus and D. reticulatus ticks examined were negative for Babesia spp. and F. tularensis DNA (Table 2). The infection rate of I. ricinus ticks for Bartonella spp. was 8.1% and sequence analysis of amplicons (n = 21) showed 99–100% identity with a fragment of groEL gene of B. henselae. Bartonella DNA was detected in 3 out of 100 D. reticulatus ticks analyzed. Only 11.5% of I. ricinus adult ticks tested positive for Rickettsia DNA, whereas the infection rate of Rickettsia spp. was significantly higher for D. reticulatus ticks (53%; p < 0.0001; Tables 2 and 3). Owing to limited amount of DNA obtained from I. ricinus ticks, species determination of Rickettsia spp. was not achieved. However, sequencing of both gltA and ompA amplicons from D. reticulatus ticks revealed that all the 53 sequences represented R. raoultii DNA.

Discussion

To date, very few studies have examined the prevalence of tick-borne pathogens in ticks collected from the areas that have been subjected to decades of ionizing radiation (Movila et al. 2012, 2013). Even fewer investigated the diversity of tick microflora in this affected ecosystem (Karbowiak et al. 2014, 2016). This study has examined the abundance of ixodid ticks in the CEZ and the prevalence of some medically important tick-born pathogens.

Our study has shown that I. ricinus and D. reticulatus adults were equally abundant in the CEZ. This is in contrast to previous studies that consistently found D. reticulatus to be the most abundant tick species in the CEZ (Movila et al. 2012, Karbowiak et al. 2016). In contrast to 74.2% of I. ricinus ticks collected in this study, only one out of 122 questing ticks collected from the CEZ by dragging in Spring 2010 was I. ricinus and the other 99.2% of ticks were D. reticulatus (Movila et al. 2012) (p < 0.0001). The discrepancy between the present and prior findings may be explained by different sampling sites and times of tick collection.

The present data also showed that gender distribution among I. ricinus adults were almost equal, 47% and 53% for male and female I. ricinus ticks, respectively, which is consistent with the data of the concurrent study performed in Kyiv (Rogovskyy et al. 2018). In contrast, D. reticulatus ticks were predominantly represented by female ticks (74%), the findings that have been consistently supported by prior studies (Movila et al. 2012, Akimov and Nebogatkin 2013, Rogovskyy et al. 2018).

This study is the first to examine the prevalence of B. burgdorferi s.l. in I. ricinus ticks from the CEZ. Overall, the prevalence of B. burgdorferi s.l. for I. ricinus ticks from the CEZ was not significantly higher (13.5%) than that of I. ricinus adults concurrently collected from Kyiv (10.4%) (Table 3) (Rogovskyy et al. 2018). Similar to our prior study (Rogovskyy et al. 2018), the current findings suggest that B. burgdorferi and B. afzelii were the most common genospecies of B. burgdorferi s.l. complex.

One of the limitations of our two concurrent studies is that the approach taken did not allow us to detect coinfection of ticks by multiple genospecies of B. burgdorferi s.l. A number of prior studies have shown that I. ricinus ticks are often coinfected with multiple genospecies of B. burgdorferi s.l. (Kurtenbach et al. 2001, Rauter and Hartung 2005, Herrmann et al. 2013). The fact that two or more genospecies could have been present in the ticks analyzed may explain why Sanger sequencing repeatedly failed to sequence a third of the B. burgdorferi s.l. 16S–23S IGS amplicons in this study.

Interestingly, in this study, all of the ixodid ticks analyzed (n = 360) were PCR negative for A. phagocytophilum and Babesia spp. with the exception of one A. phagocytophilum DNA-positive I. ricinus adult. This result is in contrast to a previous study in the CEZ, where sampling occurred from August to October each year from 2009 to 2012 (Karbowiak et al. 2014). That study showed that 25.4% and 2.9% of questing D. reticulatus ticks tested positive for A. phagocytophilum and Babesia canis, respectively (Table 3). Moreover, the infection rates of A. phagocytophilum and Babesia spp. for adult I. ricinus ticks concurrently collected from Kyiv (∼95 km from the CEZ) were 2.7% and 0.5%, respectively (Table 3) (Rogovskyy et al. 2017, 2018). Similarly, in Belarus, the neighboring country that received >70% of the nuclear fallout (Maksimova 2002), the infection rates of A. phagocytophilum and Babesia spp. in questing I. ricinus ticks were, respectively, 4.1% and 1.7% (Reye et al. 2013).

A significance difference between the infection rates of I. ricinus ticks concurrently collected from the CEZ (this study) and Kyiv was observed for Bartonella spp. As opposed to the Kyiv ticks whose infection rate was 2.7% (Rogovskyy et al. 2018), 8.1% of the CEZ I. ricinus ticks were positive for Bartonella DNA (Table 3). Consistently, Bartonella henselae was the only species identified in this and prior studies (Rogovskyy et al. 2018). Finally, in this study, none of ixodid ticks analyzed (n = 360) tested positive for F. tularensis DNA. Low infection rates of F. tularensis were observed for I. ricinus (2.2%) and D. reticulatus (2.0%) adult ticks collected from Kyiv in May 2016 (Rogovskyy et al. 2018). Overall, the aforementioned discrepancies between the present and prior findings could be partially accounted for by potentially existing differences in the abundance and distribution of vertebrate reservoirs at the respective sampling sites and/or times of tick collection.

The present data also showed that 11.5% of I. ricinus ticks tested positive for Rickettsia spp., which is similar to the respective infection rate for I. ricinus adults collected at the same time in Kyiv (12.6%) (Rogovskyy et al. 2018). In contrast, this study showed a significantly higher prevalence of R. raoultii in the D. reticulatus ticks from the CEZ (53.0%) compared with the Kyiv counterparts (Table 3). It was shown that the prevalence of R. raoultii in D. reticulatus ticks collected from Kyiv parks could vary from 10.1% to 35.7% (Didyk et al. 2017, Rogovskyy et al. 2018). The overall high prevalence of R. raoultii in the CEZ is consistent with the prior study, which found that 72.6% of D. reticulatus ticks from the CEZ carried R. raoultii DNA (Table 3) (Karbowiak et al. 2016). Thus, the prevalence of Rickettsia spp. between the two ixodid ticks and various collection sites may significantly vary, which could be partially attributed to a difference in the abundance and distribution of various vertebrate hosts for Rickettsia spp. (Tomassone et al. 2018).

Conclusions

This study has examined the prevalence of various bacterial and protozoan pathogens of public health significance in the questing I. ricinus and D. reticulatus ticks collected from the CEZ. The study demonstrated that the prevalence of some zoonotic pathogens, specifically, R. raoultii in D. reticulatus and Bartonella spp. in I. ricinus was significantly higher in the ticks collected from the CEZ compared with the control area (Kyiv). However, to examine the causal effect of long-term ionizing radiation on potential adaptive changes of tick-borne microorganisms, a more systematic approach is needed. In this regard, the microbiota of tick populations indigenous to the CEZ may well suit the purpose to study eco-evolutionary dynamics that have been driven by decades-long ionizing radiation arising from the nuclear accident.

Footnotes

Acknowledgments

First and foremost, we are deeply indebted to all of those who courageously fought the Chernobyl disaster. A. phagocytophilum, Ba. odocoilei, and R. conorii DNA samples were kind gifts of Drs. Kelly Brayton, Patricia Holman, and Michael Levin, respectively. We also thank Drs. Samiran Sinha and Lan Zhou for consulting with us on the statistical analysis. The study was supported through the department of veterinary pathobiology, Texas A&M College of Veterinary Medicine and Biomedical Sciences to A.S.R.

Author Disclosure Statement

No conflicting financial interests exist.

Supplementary Material

Supplementary Data

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