Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulatus Ticks from Different Geographical Locations in Belarus

Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.


Introduction
Throughout the world, ticks are important vectors of human and animal pathogens. In Eastern Europe, Lyme Borreliosis (LB) is a major public health threat with annual incidence rates ranging from 4.8 (Poland) to 35 (Lithuania) cases per 100.000 population [1]. In Belarus, 1.0 to 9.1 cases per 100.000 have been reported over a ten year period [2]. Although early manifestations of the disease usually can be diagnosed and treated successfully, late manifestations such as Lyme arthritis, Acrodermatitis chronica atrophicans and neuroborreliosis can be severe. The causative agents are members of the Borrelia burgdorferi sensu lato (s.l.) complex, of which at least Borrelia burgdorferi sensu stricto (s.s.), B. afzelii and B. garinii are known to be human pathogens, whereas pathogenicity has not been confirmed for B. spielmanii, B. lusitaniae and B. valaisiana. In the neighbouring countries of Belarus, the prevalence of Borrelia species in questing Ixodes ticks ranges from 6.2% in Poland, 11% in Lithuania to 40.7% in Russia and 18-51% in Latvia [3][4][5][6]. No such prevalence data are available for Belarus, but the three human pathogenic Borrelia species have been isolated from ticks before [7].
Six other tick-borne bacterial and protozoal pathogen genera are of medical interest in Europe. Rickettsia species of the spotted fever group (SFG) can cause rickettsioses in humans, which are mainly characterized by feverish infections. Little is known about the incidence of spotted fever rickettsioses in Eastern Europe, but surveillance studies revealed that Rickettsia prevalence in ticks ranges from about 3% in Poland and 6% in the Ukraine and Slovakia to 15% in Russia [8][9][10][11].
Anaplasma phagocytophilum is another rickettsial agent pathogenic for humans, causing human granulocytic anaplasmosis. Although the incidence of anaplasmosis in Europe is not well documented, clinical cases seem to be rare. In ticks, a prevalence of 2.9% in Lithuania, 2.9-8.7% in Poland, 3.6% in the Ukraine and 5.0% in Russia have been observed [3,4,10,12,13].
Coxiella burnetii is the causative agent of Q fever in humans, but it also affects domestic and wild ruminants, leading to increased rates of abortion. Although ticks are competent vectors of this pathogen, so far Q fever outbreaks have been linked to exposure to infected animals and their products. Despite only limited studies the prevalence of C. burnetii in European ticks, does not seem to exceed 2.6% [9,[14][15][16].
Another tick-borne pathogen that can also be transmitted by aerosol is Francisella tularensis, causing tularaemia in humans. Prevalence rates in ticks are at least in France and Germany below 1.6% [17,18] and like Q fever, tularaemia outbreaks are usually linked to the exposure to contaminated biological matter (faeces, blood, milk, etc.) rather than tick bites.
Bartonella species can be transmitted to humans by various arthropods including ticks and cause diseases like trench fever and cat scratch disease. The prevalence of these bacteria in ticks can vary as much as from 3.7% in Poland to 40% in Russia and France [19][20][21].
Besides these bacterial agents, also human pathogenic protozoans can be transmitted by tick bites. At least two Babesia species, namely B. microti and B. divergens, are known to cause babesiosis in humans, whereas for B. venatorum (previously Babesia sp. EU1) human pathogenicity is suspected. In Poland, 3.5% to 5.4% of ticks were found to be infected with B. microti [22,23], whereas 1.6% of ticks from Russia were infected with B. venatorum [8].
The wide distribution of bacterial and protozoal agents of human pathogenicity and the extreme range of their prevalence in ticks indicate the need for comprehensive studies on tick-borne pathogens in particular in Eastern Europe. So far, comparatively few studies on the prevalence of tick-borne pathogens in ticks from Belarus have been published in peer-reviewed journals [24,25]. However, official data on LB incidence and limited information on Borrelia burgdorferi s.l. prevalence in ticks exist [2]. In this study, we investigated the prevalence of seven tick-borne pathogen genera of medical interest in questing and feeding Ixodes ricinus and Dermacentor reticulatus ticks in Belarus.

Materials and Methods
Belarus is a landlocked country in North-Eastern Europe with a population of 9.4 million, 70% of which reside in urban areas. Its terrain is generally flat, with an average elevation of 160m above sea level and about 40% of the country is covered by forest. The most common tick species in Belarus are Ixodes ricinus and Dermacentor reticulatus [2].
In April and May 2009, ticks were collected from the vegetation and from cows at 32 collection sites in the administrative regions of Brest (n = 8), Gomel (n = 7), Grodno (n = 2), Minsk (n = 3), Mogilev (n = 7) and Vitebsk (n = 5) (Figure 1). One tick was removed from a dog in Minsk region. Verbal authorisation for collection of feeding ticks was obtained from farmers and animal owners. Collection of questing ticks was performed with the cloth-dragging method; feeding ticks were removed with forceps. Ticks were identified to species level using standard morphological identification keys [26] and stored in 70% ethanol at 4uC. Ticks were washed three times in 1x phosphate buffered saline, rinsed with distilled water and dried on sterile filter paper prior to DNA extraction. Disruption and homogenization was performed in lysis buffer of the InviMag Tissue DNA Mini kit (Invitek, Berlin, Germany) using the TissueLyser II (Qiagen, Venlo, Netherlands) and 5 mm stainless steel beads. The KingFisher 96 Magnetic Particle Processor (Thermo Scientific, Waltham, Massachusetts, USA) was used for the extraction of DNA according to the manufacturers' instructions. Detection PCRs for A. phagocytophilum, Bartonella sp., Babesia sp., Borrelia sp., C. burnetii, Francisella sp. and Rickettsia sp. were carried out using 5ml of raw DNA extract and 1ml of first round products. In addition to the 17kDa PCR used for detection of Rickettsia species, a second PCR assay based on the ompA gene was performed for better species discrimination. Primer sequences and PCR conditions are specified in Table 1. In each PCR run, cloned fragments of target genes of the respective pathogens were included as specific positive controls while distilled water was used as negative control. Spike experiments with culture extract from Borrelia garinii showed a similar PCR sensitivity in DNA extracts derived from feeding and questing ticks.

Pathogens in questing and feeding ticks
Overall, the pathogen species composition was significantly more diverse in questing than in feeding ticks (14 vs. 4 species; p,0.05). Questing I. ricinus ticks were infected with more pathogen species than questing D. reticulatus (13 vs. 5 species; not significant) ( Table 4). Interestingly, the pathogen prevalence in I. ricinus was significantly lower in feeding than in questing ticks (13.2% vs. 32.9%; p,0.01), whereas it was similar in D. reticulatus ticks (42.6% vs. 46.3%; not significant). Mixed infections were detected in 2.5% (14/553) of ticks, the majority of which were formed between members of the two most prevalent pathogen genera Rickettsia and Borrelia (57.1%; 8/14) (Table 5). Also, mixed infections occurred more often in I. ricinus than in D. reticulatus ticks (3.4% vs. 1.3%; not significant). Gender specific differences in the pathogen prevalence in ticks could not be evaluated due to the low number of feeding male ticks (n = 5).

Discussion
This is the first comprehensive study on tick-borne bacterial and protozoan pathogens of human and veterinary interest in Eastern Europe. In this study, the ticks collected both from the vegetation and from hosts were mostly adults. This finding is not surprising for ticks removed from cows, as larger mammals serve as the main blood meal host for adult ticks, whereas immature ticks preferably feed on smaller vertebrates. In contrast, on vegetation immature tick stages are normally more abundant than adults due to high interstadial mortality rates [27]. Although cloth dragging has been described as a suitable collection method also for nymphs [28], our results nevertheless suggest that our collection was biased towards adult ticks.
In questing I. ricinus ticks, we observed a prevalence of Rickettsia species of 11.7%, which was similar to that reported from questing adult I. ricinus ticks from Poland (7.8-11.1%) and Slovakia (6.1%) [9,11,29]. Also, the prevalence of Rickettsia in questing D. reticulatus (44.5%) was comparable to reports from Poland (40.7%) [30]. Although not all 100 RRG samples were positive in the discriminatory ompA PCR, all amplified sequences allowed the identification of R. raoultii in at least one tick from each RRG positive collection site. R. raoultii and RRG were almost exclusively detected in D. reticulatus, only two feeding I. ricinus ticks were found to be infected by these rickettsiae. This suggests a high adaptation of these bacteria to the former tick species and indeed, high rates of transovarial transmission of 90% to 100% have been reported for R. raoultii in D. reticulatus and D. marginatus ticks [31].
The observed prevalence of R. raoultii of 22.6% in questing D. reticulatus was comparable to the 22.3% reported from Slovakia [32]. Although R. raoultii is endemic in Poland and Russia [31,33], the prevalence in ticks is not known. R. raoultii is a member of the spotted fever group rickettsiae and is suspected to cause tick-borne lymphadenopathy (TIBOLA) in humans [34]. Despite the high prevalence of infected D. reticulatus and their implication in human TIBOLA infections, there are no such reports from Belarus available.
The overall prevalence of Borrelia infected I. ricinus collected from the vegetation was 12.5%, which is in the lower range of the prevalences of 10.1-32.7% reported for questing adult I. ricinus from Eastern Europe [35]. For questing D. reticulatus, the prevalence of B. burgdorferi s.l. (1.8%) was also lower than that reported for questing adult D. reticulatus from Russia (3.6%) [21]. On a regional level, the highest Borrelia prevalence in ticks of 15.9% was observed in Brest region, which seems to be in line with reports of the highest LB incidence in this region [2]. B. afzelii and B. garinii, the most prevalent Borrelia species in this study, have been previously reported to be predominant in Belarus [25]. However, this is the first report on B. burgdorferi s.s. and B. lusitaniae prevalence in ticks from Belarus.
Interestingly, the Borrelia prevalence was significantly higher in I. ricinus ticks collected from the vegetation than in those from cattle. This is surprising, as feeding adult ticks were removed during their third blood meal, whereas questing adults only fed twice. Here, however, the prevalence of feeding and questing adult ticks should be similar as cattle are not considered a competent reservoir host for B. burgdorferi s.l. due to the bactericidal activity of bovine serum which results in the complement-mediated lysis of Borrelia [36]. Sensitivity testing of our PCR on DNA extracts from feeding and questing ticks spiked with B. garinii excluded a difference in detection sensitivity. This suggests that the reduced Borrelia prevalence in feeding ticks was not an artifact due to PCR inhibition. Cattle on pastures may serve as an easier blood meal host than wild animals and thus act as a dilution factor for Borrelia prevalence in ticks. This inhibitory influence of less competent  I. ricinus  40  168  44  37  21  17  327   Vegetation  T  40  163  44  37  -5  289   Vegetation  F  18  82  30  16  -1  147   Vegetation  M  22  81  14  19  -4  140   Vegetation  N  ---2  --2   Host  T  -5  -21  12  38   Host  F  -5  --18  12  35 Host reservoir hosts on an enzootic cycle has been modeled before [37,38]. Pathogens of the genera Anaplasma, Babesia, Bartonella, Coxiella and Francisella were exclusively found in questing ticks from Belarus with low prevalences comparable to reports from questing adult ticks from other Eastern European countries [3,8,9,12,21,22,39], although occasionally also higher prevalences have been reported for these pathogens [3,21,40]. The only study from Belarus reports a prevalence of A. phagocytophilum in questing I. ricinus from Minsk region of 4.2% [24], which is significantly lower than our findings (13.5%; 5/37; p,0.05).
These observed low prevalences in Belarusian ticks are in line with low incidence reports of these diseases in Eastern Europe. However, so far no such incidence data is available for Belarus and there are limited reports from the neighbouring countries, e.g. a single case report of arthropod-borne tularaemia in Poland has been published [41]. Serological studies from Poland and Russia reveal antibody positivities in humans of 5.1 to 11.8% for Anaplasma phagocytophilum and 2.6 to 9.0% for Babesia microti [42,43]. Interestingly, a high seroprevalence was reported for Bartonella henselae from Poland (23.1 to 37.5%); however, this may be due to other routes of transmission like contact to infected cats rather than to tick bites. Interestingly, hotspots of infection were discovered at sites in Minsk and Gomel region for A. phagocytophilum (12.5-17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%). The focality of prevalence rates suggests that zoonotic cycles of these pathogens are well established at least at these sites. Since the inhalation of contaminated aerosols (e.g. dried faeces) is an important route of transmission of F. tularensis ssp. and C. burnetii [44,45], these sites may represent a significant threat to human health independent of tick exposure. Therefore, studies on the seroprevalence and incidence of these diseases in humans as well as the surveillance of these pathogens not only in ticks but also in reservoir hosts (e.g. members of the Leporidae for F. tularensis ssp. and of the Bovidae for C. burnetii) at the identified hotspots and neighbouring regions are warranted in order to predict and avoid outbreaks of tularaemia and Q fever.
Gomel region displayed the highest pathogen diversity in the country and is also the region with the largest numbers of ticks, suggesting that it may best reflect the infection status of ticks in Belarus at least for the low prevalent pathogens Anaplasma, Bartonella, Babesia, Coxiella and Francisella. Nevertheless, the prevalence of Borrelia infected ticks in this region seemed to be lower than in the western regions Grodno and Brest, even when only I. ricinus, a competent vector of Borrelia species, is considered. These differences may be due to habitat differences rather than the geographic location.
We found that pathogen diversity was higher in questing I. ricinus than in questing D. reticulatus ticks, suggesting that the latter species serves as reservoir for fewer pathogens than I. ricinus, at least in Belarus. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. The pathogenic potential of RRG and the role of D. reticulatus as its arthropod vector require further attention. Identified hotspots of infected ticks, especially when F. tularensis ssp. and C. burnetii are involved, should be included in future surveillance studies. In addition, the impact of the highly