Pathogens of Emerging Tick-Borne Diseases,Anaplasma phagocytophilum,Rickettsia spp.,and Babesia spp.,in Ixodes Ticks Collected from Rodents at Four Sites in Switzerland (Canton of Bern)

Abstract

This study is part of a project that aimed to better understand the role of small mammals in the maintenance of the tick-borne encephalitis virus at four different sites in Switzerland. Here we focused on the detection of three intracellular pathogens, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp., in field-derived ticks that detached from 79 small mammals. We analyzed 465 Ixodes ricinus larvae after their molt and 14 semiengorged I. trianguliceps that were feeding on rodents. No pathogen was detected in I. trianguliceps. In I. ricinus, the most frequently detected pathogen was Rickettsia spp. (7.3%). All Rickettsia spp. identified DNA belonged to R. helvetica except one DNA sample that was identified as R. monacensis. The prevalence of Babesia spp. reached 2.4% and identification at the species level revealed B. venatorum (1.7%) and B. microti (0.4%). A. phagocytophilum was not detected in I. ricinus that detached from rodents. To verify the absence of A. phagocytophilum at the four sites, additional questing nymphs collected at these sites were analyzed for A. phagocytophilum. This pathogen was detected at one site only, where 2% (2/100) of questing ticks were infected. Some of these emerging pathogens are described for the first time in molted larvae that fed on rodents. The presence of medically relevant pathogens, with a global prevalence of 9.9%, highlights the importance to inform the medical corporation on the risk for human health in these areas.

Introduction

I n Europe, the tick Ixodes ricinus is the vector of numerous pathogens such as viruses, bacteria, and protozoa. Some of them, like Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp., are considered as agents of emerging human diseases (Hildebrandt et al. 2010a).

Members of the genera Ehrlichia and Anaplasma are obligate intracellular bacteria that target granulocytes or monocytes according to species. The Ehrlichia species were first recognized as pathogens of veterinary importance. Since the reorganization of the Anaplasmatacea family, E. equi and E. phagocytophila, the recognized agent of the human granulocytic ehrlichiosis, are now described as a unique species named A. phagocytophilum (Dumler et al. 2001). However, not all strains are pathogenic for humans (Massung et al. 2003, de la Fuente et al. 2005), and currently less than 70 human cases have been reported throughout Europe (Hildebrandt et al. 2010b). In Switzerland, A. phagocytophilum was detected in cattle (Pfister et al. 1987), dogs (Pusterla et al. 1997), horses (Pusterla et al. 1998a), and in questing ticks (Pusterla et al. 1998b), and Liz et al. (2000) reported infected rodents and infected I. ricinus on rodents. In addition, serological studies provided evidence of contacts of Swiss residents with the human granulocytic ehrlichiosis agent (Pusterla et al. 1999).

Rickettsia species from spotted fever group are obligate intracellular bacteria, and some species such as R. helvetica cause human diseases in Europe (Brouqui et al. 2007, Nilsson et al. 2010). In Switzerland, R. helvetica and R. monacensis have been described in I. ricinus (Beati et al. 1994, Boretti et al. 2009), but no human case has been reported so far.

The genus Babesia is a protozoan parasite of erythrocytes that was first recognized as an agent of animal disease until the first human case was documented in Yugoslavia in 1957 (Skrabalo and Deanovic 1957). In United States it is B. microti that is mainly associated with human pathogenesis, whereas in Europe, several Babesia species are involved such as B. divergens, a cattle parasite, B. microti associated with rodents, and Babesia sp. EU1 (temporarily named B. venatorum) (Gray et al. 2010). In Switzerland, B. microti, B. divergens, and B. venatorum have been described in questing I. ricinus and in I. ricinus collected from hosts such as dogs, cats, cattle, asses, and goats (Foppa et al. 2002, Casati et al. 2006, Hilpertshauser et al. 2006, Gigandet et al. in press). One human case due to B. microti was reported in Switzerland (Meer-Scherrer et al. 2004) and another confirmed human case due to this species was identified in Germany (Hildebrandt et al. 2007).

This work is part of a project conducted at four sites in Switzerland that aimed to better understand the maintenance of tick-borne encephalitis virus (TBEV) in endemic areas and more specifically the role of rodents as reservoirs for TBEV (Burri et al. in press). To gain additional information on other pathogens considered as agents of emerging human diseases in Europe, we analyzed part of field-derived ticks (I. ricinus and I. trianguliceps) infesting the rodents captured at these four sites for A. phagocytophilum, Rickettsia spp., and Babesia spp.

Materials and Methods

Study sites

This study was carried out in Switzerland in the canton of Bern from May 2006 to April 2009 (Burri et al. in press). Four sites were chosen: Thun (46°43′N, 7°36′E, 642 m above sea level), Belp (46°52′N, 7°30′E, 687 m above sea level), Kiesen (46°48′N, 7°34′E, 566 m above sea level), and Trimstein (46°53′N, 7°34′E, 620 m above sea level).

Tick sampling on rodents and vegetation

Rodents were trapped once a month from May 2006 in Belp and Thun, and from June 2006 in Kiesen and Trimstein to April 2009. Briefly, 50 traps were set at each study site. Captured rodents were released at the point of capture a few days after ticks dropped off. Engorged ticks were identified to species according to Cordas et al. (1993). Because I. trianguliceps ticks are difficult to maintain in the laboratory, they were stored as engorged ticks at −20°C, whereas I. ricinus were stored in tubes at 98% relative humidity and room temperature until their molt. Two months after their molt, live ticks were washed in 70% ethanol and maintained at −20°C until DNA extraction. The low number of ticks analyzed per rodent was because ticks collected from rodents were used as a priority to detect TBEV in ticks.

In addition, questing nymphs collected in 2008 by flagging vegetation at the four study sites and maintained in the laboratory as described above. One hundred ticks per site were screened for blood meal analysis (Burri et al. in press) and tested for A. phagocytophilum detection.

DNA extraction

DNA from I. ricinus nymphs that fed as larvae on rodents and DNA of engorged larvae, nymphs, and one female of I. trianguliceps (as well as one egg laying) was isolated in a final volume of 85μL using the kit Qiasymphony virus/bacteria (Qiagen) with a robot Qiasymphony^® SP system (Qiagen) according to the manufacture and were stored at −80°C.

Detection and identification of pathogens

A real-time polymerase chain reaction (PCR) modified from Courtney et al. (2004) was used to amplify and detect a 77 bp fragment of the msp2 gene of A. phagocytophilum. Primers ApMSP2f and ApMSP2r with the probe ApMSP2-FAM (0.72 μM each) (Table 1), KapaTaq hotstart (Kapabiosystems by Labgene Scientific) (0.75 U per test), dNTPs (200 μM each), MgCl₂ (6 mM), and 2 μL of DNA sample were used for the reaction. The amplification was performed in an iCycler (Biorad) in a final volume of 25 μL with 2 μL of A. phagocytophilum as positive control (Webster strain, kindly provided by Ana Sofia Santos, CEVDI). Amplification started with an initial activation at 95°C for 15 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.

Table 1.

Primers and Probes Used for the Amplification and Detection of the Different Pathogens in Ticks

Target organism	Name	Design	Sequence (5′ → 3′)	Concentration (pmol) for RLB	References
Babesia spp.	B-R2	Primer	biotin-CTA AGA ATT TCA CCT CTG ACA GT	—	Georges et al. (2001)
Babesia spp.	F2	Primer	GAC ACA GGG AGG TAG TGA CAA G	—	Georges et al. (2001)
Babesia/Theileria	catch-All B/T	Probe	amino-TAA TGG TTA ATA GGA RCR GTT G	50	Georges et al. (2001)
B. venatorum	B.EU1	Probe	amino-GAG TTA TTG ACT CTT GTC TTT AA	500	Gigandet et al. (in press)
B. microti	B. microti	Probe	amino-GCT TCC GAG CGT TTT TTT AT	500	Gigandet et al. (in press)
B. divergens	B. divergens	Probe	amino-GTT AAT ATT GAC TAA TGT CGA G	500	Gubbels et al. (1999)
Rickettsia spp.	RCK/23-5-F	Primer	biotin-GAT AGG TCR GRT GTG GAA GCA C	—	Jado et al. (2006)
Rickettsia spp.	RCK/23-5-R	Primer	TCG GGA YGG GAT CGT GTG TTT C	—	Jado et al. (2006)
Rickettsiales	Generic	Probe	amino-TAG CTC GAT TGR TTT ACT TTG	100	Jado et al. (2006)
R. helvetica	R. helvetica	Probe	amino-CAT GGC TTG ATC CAC GGT A	100	Jado et al. (2006)
Anaplasma phagocytophilum	AmSp2f	Primer	ATG GAA GGT AGT GTT GGT TAT GGT ATT	—	Courtney et al. (2004)
A. phagocytophilum	AmSp2r	Primer	TTG GTC TTG AAG CGC TCG TA	—	Courtney et al. (2004)
A. phagocytophilum	APMSp2-FAM	Probe	FAM-TGG TGC CAG GGT TGA GCT TGA GAT TG-TAMRA	—	Courtney et al. (2004)

For the detection of Babesia spp. a fragment of 450 bp of the 18S gene was targeted (Georges et al. 2001). Amplifications using primers B-R2 and F2 (0.2 μM each) (Table 1) with Taq (Qiagen) (0.63 U per test), dNTPs (200 μM each), MgCl₂ (1 mM), and 10 μL of DNA were performed in a single reaction volume of 25 μL with 1 μL of B. divergens as positive control (kindly provided by Simona Casati, Istituto Cantonale di Microbiologia) in a Whatman Biometra Tgradient Thermocycler 96 (Göttingen Deutschland). A touchdown PCR was used according to Bekker et al. (2002) with an initial temperature of 94°C for 3 min, followed by a denaturation step at 94°C for 20 s, annealing at 67°C for 30 s, and extension for 72°C for 30 s. Then, during the subsequent cycle set, the annealing temperature was lowered by 1°C during 11 cycles until it reached 57°C. Additional 40 cycles followed with 20 s at 94°C, 30 s at 57°C, 30 s at 72°C, and as a final extension, 10 min at 72°C.

For the detection of Rickettsia spp., DNA from the intergenic spacer 23S-5S was amplified by a single PCR. This method described by Jado et al. (2006) amplified a 345 bp fragment. A volume of 50 μL with primers RCK/23-5F, RCK/23-5R (0.5μM each) (Table 1), Taq (Qiagen) (1.5 U per test), and dNTPs (200 μM each), and 10 μL of DNA was used. The positive control consisted of 1 μL of R. conorii (kindly provided by Simona Casati, Istituto Cantonale di Microbiologia). PCR was used with an initial temperature of 94°C for 9 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, and 65°C for 4 min with a final extension at 65°C for 7 min.

RLB technique was used for the identification at the species level of Babesia spp. and Rickettsia spp. (Bekker et al. 2002, Gigandet et al. in press). The amplified products were hybridized to four probes for Babesia spp. and two probes for Rickettsia spp. (Table 1). Hybridization conditions were 42°C for Babesia spp. and 48°C for Rickettsia spp. for 1 h followed by two washings at 52°C for 10 min. Samples that could not be identified at the species level were purified with a purification kit (Promega) and sent for sequencing to Microsynth AG.

Statistical analysis

The χ ²-test was used for statistical analysis, and a p-value of ≤0.05 was considered statistically significant.

Results

In total, 479 ticks fed on 90 rodents captured in 2006 (n = 23), 2007 (n = 55), and in 2008 (n = 12) were analyzed. Among them, 465 I. ricinus larvae (analyzed after their molt as nymphs) fed on 79 rodents (51 Apodemus spp. and 28 Myodes glareolus) (Table 2) and 14 partially engorged I. trianguliceps (12 larvae, 1 nymph, and 1 female) feeding on 12 rodents (10 M. glareolus and 2 A. sylvaticus), and one egg laying were tested. No pathogen was detected in I. trianguliceps, but 9.9% (45/465) of I. ricinus were infected by at least one pathogen. These infected ticks were recovered from one-third (26/79) of hosts that were infested by I. ricinus (Table 2). In 2006 more rodents (43.5%, 10/23) were carrying infected ticks than in 2007 (30.8%, 16/52), but the difference was not significant (p > 0.05). For 2008, not enough data were available for comparison.

Table 2.

Prevalence of Rodents Infested with Ixodes ricinus Ticks Infected by At Least One Pathogen and Prevalence of Rickettsia spp., Babesia spp. and Anaplasma phagocytophilum in Ixodes ricinus Ticks After Feeding on Rodents

		Nb infected ticks/tested ticks (%)
Sites	Nb hosts with infected ticks/nb hosts with ticks (%)	Rickettsia spp.	Babesia spp.	A. phagocytophilum
Thun	13/33 (39)	19/173 (11)	6/173 (3.5)	0/173 (0)
Belp	5/11 (46)	7/71 (9.9)	3/71 (4.2)	0/71 (0)
Kiesen	6/15 (40)	7/118 (5.9)	1/118 (0.8)	0/118 (0)
Trimstein	2/20 (10)	1/103 (1)	1/103 (1)	0/103 (0)
Total	26/79 (33)	34/465 (7.3)	11/465 (2.4)	0/465 (0)

Rickettsia spp. were the pathogens the most frequently detected in ticks from rodents with a global prevalence of 7.3% (34/465, p < 0.001) (Table 2). Ticks infected with Rickettsia spp. were recovered from 26.5% (21/79) rodents. All identified Rickettsia spp. DNA belonged to R. helvetica (7.1%, 33/465) except one sample from Trimstein that was showing 100% homology with Rickettsia spp. 362 (R. monacensis) (GenBank accession number DQ139797). Two ticks from two rodents captured in Thun were coinfected with R. helvetica and Babesia spp.: one with B. microti and one with B. venatorum.

The total prevalence of Babesia spp. in I. ricinus from rodents was 2.4% (11/465) (Table 2) and the frequency of Babesia spp. tended to be higher at Thun and Belp. B. venatorum was identified in 1.7% (8/465) examined ticks and B. microti in 0.4% (2/465). Babesia spp. were detected in ticks recovered from 11.4% (9/79) of rodents. Two M. glareolus were infested by ticks in which B. microti was identified, whereas B. venatorum was detected in ticks that were attached on four A. sylvaticus, one A. flavicollis and one M. glareolus. One sample from a tick feeding on one M. glareolus from Trimstein could be identified at the genus level only and sequencing result was not conclusive.

A. phagocytophilum was not detected in the 465 I. ricinus infesting the rodents (n = 79). Therefore, in addition, questing nymphs were screened to verify the absence of A. phagocytophilum at the four sites. A. phagocytophilum was detected in 2% (2/100) of questing nymphs at one site (Kiesen).

Discussion

In Switzerland I. ricinus is the most important vector of pathogens of medical and veterinary importance. In a previous study, we investigated the ecological factors determining the presence of TBEV at four different sites and more specifically the role of rodents as reservoirs for TBEV (Burri et al. in press). To gain more information on the presence of other emerging tick-borne pathogens at these sites, we screened I. ricinus larvae (analyzed as nymphs) that detached from small mammals for A. phagocytophilum, Rickettsia spp., and Babesia spp.

Rickettsia spp. and Babesia spp. were detected in ticks collected from rodents at each site. Although it has been shown that rodents can be reservoir hosts for A. phagocytophilum (Liz et al. 2000, Bown et al. 2008), we did not detect this organism in ticks feeding on rodents. A coexisting cycle between I. ricinus and I. trianguliceps as it has been demonstrated in United Kingdom for A. phagocytophilum might explain the absence of the pathogen in I. ricinus feeding on rodents, suggesting that the pathogen cycle might be supported by I. trianguliceps (Bown et al. 2008). At our study sites, few I. trianguliceps were infesting rodents, only a dozen of ticks could be tested, and none was infected. Since Liz et al. (2000) reported infected rodents and infected I. ricinus on rodents in Switzerland, the absence of A. phagocytophilum observed here could also be due to the low number of examined hosts in each site. Therefore, we analyzed questing ticks to confirm the absence of A. phagocytophilum at the sites. A. phagocytophilum was identified in questing nymphs (2%) at one site only (Kiesen) and one infected nymph showed that its previous host as larva was S. scrofa (data not shown). Thus, other reservoir hosts like wild boar and wild cervids could play a more important role than rodents in the maintenance of this pathogen, at least at some sites (Rosef et al. 2009, Portillo et al. 2011).

In contrast, with a global prevalence of 7.1%, R. helvetica was the pathogen the most frequently detected in ticks fed on rodents. Another study, recently conducted in Switzerland, showed a similar prevalence (11.7%) in questing tick population (Boretti et al. 2009). In addition to R. helvetica, one sample was identified, after sequencing, as Rickettsia sp. 362, a species first reported in the blood of two patients with clinically diagnosed Mediterranean spotted fever (Jado et al. 2006) and later identified as R. monacensis (Jado et al. 2007). This species was recently reported in questing ticks in Switzerland (Boretti et al. 2009), but reservoir hosts for R. monacensis have not yet been identified. Here, one tick that detached from one rodent was infected by R. monacensis. Because nothing is known on the transovarial transmission of this pathogen, the question on the role of rodents as reservoirs remains open.

Concerning Babesia spp., two species (B. microti and B. venatorum) were identified in ticks fed on rodents with prevalences ranging between 0.8% and 4.2% according to sites. B. microti, with a prevalence of 0.5%, was found in one site only (Thun), confirming previous observation at this site (Gern and Aeschlimann 1986). For B. venatorum, the total prevalence was 1.7% and this pathogen was recorded at sites where TBEV is present (Thun, Belp, Kiesen) (Burri et al. in press). This species was first recognized in human (Herwaldt et al. 2003) before being detected in roe deer in Slovenia (Duh et al. 2005) and France (Bonnet et al. 2007). It was also reported in I. ricinus collected from sheep and goats in various European countries, including Switzerland (Casati et al. 2006, Hilpertshauser et al. 2006) and in questing I. ricinus in Switzerland (Casati et al. 2006, Gigandet et al. in press). Here, to our knowledge, it is the first report of B. venatorum in ticks that fed on A. sylvaticus, A. flavicollis, and M. glareolus. As Bonnet et al. (2007) showed that transovarial transmission exists for this pathogen, the question of the role of the rodents as reservoirs remains open. B. microti–infected ticks were recovered only from M. glareolus, but other small rodents such as A. flavicollis are known as reservoirs for B. microti (Gern and Aeschlimann 1986, Duh et al. 2003, Beck et al. 2010). Because transovarial transmission does not occur for B. microti (Gray et al. 2002, Beck et al. 2011), our results confirm that M. glareolus voles act as reservoirs for this Babesia species.

This study showed the presence of R. helvetica, B. venatorum, and B. microti in ticks fed on rodents, but we have no clear information on the infectivity of the rodents because rodent blood was not analyzed for these pathogens. However, a transmission from rodents to ticks of these emerging pathogens is not excluded, as mentioned above for B. microti. For R. helvetica and B. venatorum other modes of transmission might be involved like transovarial and transstadial transmissions (Bonnet et al. 2007, Brouqui et al. 2007), and therefore the role played by rodents cannot be elucidated here. Cofeeding transmission between infected nymphs and uninfected larvae, as it has been demonstrated for TBEV (Labuda et al. 1993), for example, may also be considered (45.5% of examined rodents were infested by larvae and nymphs, data not shown). However, we observed an infection prevalence that was not significantly higher in larvae feeding together with nymphs on rodents than in larvae feeding without nymphs (data not shown); therefore, further studies are needed to explore this transmission way also because we lacked information on infection in cofeeding nymphs.

The presence of emerging pathogens that are of medical relevance, with a global prevalence of 9.9% as observed here, highlights the importance to inform the medical corporation on the risk for emerging tick-borne diseases. This implies that additional investigations on the geographic distribution of ticks infected by these zoonotic pathogens are required in Switzerland.

Footnotes

Acknowledgments

We would like to thank C. Beuret for DNA extraction, S. Casati and A. S. Santos for positive controls, and E. Lommano for her help with the real-time PCR for A. phagocytophilum. This work was financially supported by the Swiss National Scientific Foundation (no 320000-113936/1) and is part of the PhD thesis of one of the authors (C.B.).

Disclosure Statement

No competing financial interests exist.

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