Prevalence of Three Zoonotic Babesia Species in Ixodes ricinus (Linné,1758) Nymphs in a Suburban Forest in Switzerland

Introduction

T he tick Ixodes ricinus (Linné, 1758), which is known as one of the most important blood-sucking parasites in Europe, harbors a great diversity of microorganisms potentially pathogenic for humans, among them Protozoan belonging to the genus Babesia. Most of the Babesia species are inducing disease in animals, but human babesiosis cases have also been documented and increasing attention has been focused on these parasites. Most human cases occur in North America and are caused principally by Babesia microti. However, some human cases have also been reported in Europe. European cases are mainly due to Babesia divergens (Gray et al. 2010), but recently Babesia venatorum, also known as Babesia EU1, was described in patients in Austria and Italy (Herwaldt et al. 2003), and in Germany (Häselbarth et al. 2007). In Europe, a third species, B. microti, has also been recently incriminated in Germany (Hildebrandt et al. 2007). The disease may appear as mild or severe depending mainly on the Babesia species (Gray et al. 2010). Patients infected by Babesia may develop haemoglobinuria, jaundice, and respiratory, renal, hepatic, and cardiac failure. Additional clinical manifestations like high fever, chills, sweat, headache, myalgia, lumbar, and abdominal pains may also be present.

In Switzerland, these three human pathogenic species have been reported in I. ricinus ticks from various areas (Gern et al. 1982, Gern and Aeschlimann 1986, Foppa et al. 2002, Casati et al. 2006, Hilpertshauser et al. 2006). However, to our knowledge, only two cases of human babesiosis have been reported in Switzerland, one due to B. microti (Meer-Scherrer et al. 2004) and another case due to B. divergens, which was an imported babesiosis (Loutan et al. 1993). In addition, a serologic study measured a seroprevalence of 1.5% of immunoglobulin G against B. microti in 396 residents living nearby a site where 4% of I. ricinus ticks were infected by B. microti (Foppa et al. 2002).

In the present study, we examined I. ricinus nymphs collected from vegetation in a Lyme borreliosis-endemic area, where B. venatorum has previously been reported (Casati et al. 2006). The aim of this study was to determine how frequently B. venatorum, B. divergens, and B. microti infect I. ricinus and to determine their prevalence over 3 years along a pathway delimited in four different sampling sections.

Materials and Methods

Nymphal I. ricinus ticks were collected by flagging the low vegetation with a 1 m² flag along a 1049 m long path divided into four consecutive sections (section 1: 47°00′16.52′′ N; 6°57′8.42′′ E, 437 m²; section 2: 47°00′13.98′′ N; 6°56′51.07′′ E, 210 m²; section 3: 47°00′17.82′′ N; 6°56′56.43′′ E, 245 m² and section 4: 47°00′19.00′′ N; 6°57′6.41′′ E, 157 m²) in a suburban forest, the Bois de l'Hôpital, close to Neuchâtel, Switzerland. Sections 1 and 3 are horizontal, whereas sections 2 and 4 are on a slope. The pathway is located at altitudes that range between 520 m and 585 m above sea level. Samplings were conducted each month from March to June in 2006, 2007, and 2008, as well as in September and October 2008. Ticks were stored at −80°C until processed.

DNA extraction was carried out by the ammonium hydroxide method as described in Morán Cadenas et al. (2007b). Briefly, nymphs were first soaked in 70% ethanol and put individually in tubes containing 100 μL of 0.7M ammonium hydroxide solution. Tubes were boiled at 100°C two times for 15 min, the second time with opened cap to concentrate the solution at 50 μL. Extracted DNA was then stored at −20°C for further investigations. Primers Reverse Line Blotting (RLB)-F2 and RLB-R2 (forward primer RLB-F2, 5′-GAC ACA GGG AGG TAG TGA CAA G; reverse primer RLB-R2, biotin-5′-CTA AGA ATT TCA CCT CTG ACA GT) were used to amplify a fragment of ∼400 bp of the 18S SSU rRNA gene spanning the V4 region of Babesia spp. (Georges et al. 2001). Polymerase chain reactions (PCRs) were performed in a Whatman Biometra H Tgradient basic thermocycler 96 (Göttingen, Germany) by using a touchdown PCR program described in Tonetti et al. (2009). Positive controls (B. divergens kindly provided by Casati S. from the Istituto di Microbiologia, Ticino, Switzerland) and negative controls (for extraction and PCR) were included in each run.

To identify pathogens at the species level a RLB method was used as described in Tonetti et al. (2009). Four probes, one for each species (500 pmol) and one probe (50 pmol) for the genus Babesia (catch-All probe), were used to detect and identify pathogen DNA (Table 1). All probes were obtained from Microsynth AG (Balgach, Switzerland). New probes designed to detect B. venatorum and B. microti were tested with extracted DNA from B. venatorum strains Babesia sp. EU1 strain 519; Babesia sp. EU1 strain 532 provided by Casati S. (Istituto di Microbiologia, Ticino, Switzerland); Babesia odocoilei strain RDWI obtained from Holman P. (University of Texas); and B. microti strains HK and GI obtained from Gray J. (University College Dublin, Ireland). No cross-reaction was observed with any other tested DNA.

Amplicons from some positive samples detected by RLB were purified using a DNA extraction kit (QIAquick PCR Purification Kit; Qiagen, Hombrechtikon, Switzerland) and purified DNA was then sent to Microsynth AG for DNA sequencing. Each obtained sequence was compared with available sequences from the international data bank (NCBI BLAST) with the use of a software package (Bioedit, Tom Hall Ibis Biosciences, Carlsbad, California).

Due to the small number of positive samples, χ ²-test and Fisher's test could not be used to compare infection prevalence between the sampling sections and between the sampling months. Therefore, we performed a test using trusted intervals of percentages (Scherrer 2008) with the statistics program S-PLUS. The value of the trusted interval was 95%.

Results

Babesia spp. were detected and identified in 44/2568 (1.7%) I. ricinus nymphs using RLB. The annual prevalence of Babesia spp. ranged between 1.6% (14/891) in 2006, 1.7% (5/292) in 2007, and 1.8% (25/1385) in 2008. Ticks questing on vegetation showed infection rates ranging between 2.2% (20/896) on section 1, 1.1% (6/545) on section 2, 2% (17/857) on section 3, and 0.4% (1/270) on section 4 (Table 2).

Three Babesia species were identified in ticks. B. venatorum (EU1) was infecting 1.1% (27/2568) of nymphs, B. divergens 0.2% (4/2568), and B. microti 0.7% (13/1908) (Table 2). Due to technical problems and lack of DNA, the number of samples tested for the identification of B. microti in ticks is reduced compared to other Babesia species. Differences of infection rate between years were not significant, except for B. microti, which was significantly less frequent in ticks in 2008 than in 2006 and 2007 according to the test using trusted intervals of percentages (Scherrer 2008) (Table 2). B. microti is the Babesia species displaying the greater differences of prevalence among sampling sections, ranging from 1.6% in section 1 to 0% in section 4 (Table 2).

To confirm the identification of a subset of our samples, six amplicons that reacted with probes B. venatorum and B. divergens were sequenced. The sequences obtained were 100% identical to the 18S rRNA gene fragments of EU1 (B. venatorum) (FJ215873.1) for 2 samples, of B. divergens (AY098643.2) for 3 samples, and of B. divergens (DQ866844.1) for the last sample.

Discussion

Here we examined more than 2500 I. ricinus nymphal ticks for Babesia spp. These ticks were collected along a 1046 m pathway in Neuchâtel (Switzerland) in 2006, 2007, and 2008. A global infection rate by Babesia spp. of 1.7% was obtained. This infection rate is included in the range described in most studies in Europe. In fact, 0.8% of I. ricinus nymphs were found infected by these parasites in Switzerland (Casati et al. 2006), whereas Babesia spp. were reported in 49% of examined nymphs in Austria (Blaschitz et al. 2008). In this last study, infection rates were highly variable from collection site to collection site ranging from 0% to 100%. Blaschitz et al. (2008) considered tick density as probably being the factor determining the infection rate. In fact, at sampling sites where high tick densities were present, either all individuals or no individuals were infected with Babesia spp., whereas in sites with medium or low tick densities, the infection rates were more variable. Here, we observed that ticks questing on vegetation on sections 1 (2.2%) and 3 (2%) tended to display higher infection rates than ticks from the two other sections (1.1% and 0.4%). Whether there is a link between this observation and the fact that in sections 1 and 3, tick density is higher than in sections 2 and 4 (unpublished data) remains to be clarified. A follow-up of the present study is underway to survey variation in the prevalence of Babesia spp. in this forest.

In our study, 1.4% of the examined nymphs were infected by B. venatorum. This species has been recently described in Europe and, to our knowledge, has been poorly investigated in questing I. ricinus ticks. It has been reported in 0.9% of ticks (larvae, nymphs, and adults) collected in different sites in the Netherlands (Wielinga et al. 2009) and in 0.4% of nymphs collected in Switzerland (Casati et al. 2006). We confirm the presence of B. venatorum in the ticks infesting the forest bordering the city of Neuchâtel as already reported by Casati et al. (2006). However, the global prevalence was higher in the present study (27/2568, 1.1% vs. 2/294, 0,7%), and the prevalence in 2008 reached 1.4%. The18S rRNA amplicons of two samples were sequenced successfully and the DNA sequences obtained were proven to be Babesia sequences by a BLAST search. The two obtained sequences showed 100% similarity to Babesia sp. EU1 isolate 7627.

B. divergens was detected in 0.2% (4/2568) of I. ricinus nymphs. Sequences of amplified DNA showed 100% homology with B. divergens sequences that were reported from the spleen of roe deer (García-Sanmartin et al. 2007) for one sample and from the blood of reindeer (Langton et al. 2003) for 3 samples. This suggests a wildlife origin for the B. divergens DNA detected in ticks in the studied site. This is the first detection of B. divergens in the ticks from this forest. In fact, Casati et al. (2006) did not report the presence of this parasite in this area, most probably because of the lower number of ticks examined and the low prevalence of B. divergens in this forest. This is also reflected here by the fact that B. divergens was not detected in 2006 and 2007 when the number of analyzed ticks was lower. Infection rates by this Babesia species in Europe in field-collected I. ricinus ticks range between 0.07% in The Netherlands (Wielinga et al. 2009) and 3% in Poland (Skotarczak and Cichocka 2001). These two studies have also been performed in forested areas, suggesting that reservoir hosts for the detected B. divergens are wild ruminants since bovines are absent. However, according to Gray (2006), some B. divergens strains of wildlife origin show different biology compared to strains of bovine origin (Langdon et al. 2003) and the question of their pathogenicity for humans remains open.

B. microti was identified in 0.7% (13/1908) of examined nymphs. This infection rate is rather low compared to other areas through Europe. In Poland, various studies show that B. microti is present in 1.3% to 11.1% of I. ricinus nymphs (Skotarczak and Cichoka 2001, Stańczak et al. 2004). In Slovenia, Duh et al. (2001) reported an infection rate of 10% in I. ricinus nymphs. In Switzerland, Foppa et al. (2002) and Casati et al. (2006) detected B. microti in 4% and 0.7%, respectively, of nymphs in various areas. These last authors did not detect this Babesia species in the present study area, most probably because of the low observed prevalence of B. microti in ticks and the lower number of ticks they examined. B. microti was the only Babesia species that displayed significant differences in prevalence among the 3 study years. This species displayed a more pronounced focal distribution over the four sections of the collection site than the two other Babesia species. The focal distribution of B. microti probably reflects the way this Babesia species is maintained in nature. In fact, reservoir hosts for B. microti are small mammals and shrews whereas known reservoirs for B. divergens and B. venatorum are large ruminants (see Gray et al. 2010). The smaller home range of small mammals and insectivores may partly explain the higher focality observed for B. microti. Foppa et al. (2002) reported a similar observation for B. microti in Switzerland, suggesting that this Babesia species is maintained in small focal areas and that the risk of human infection is depending on tick density. These authors measured a 1.5% seroprevalence of immunoglobulin G against B. microti in 396 residents living nearby a site where 4% of I. ricinus ticks were infected by B. microti showing a human exposure to this parasite (Foppa et al. 2002).

The extremely low number of clinical cases associated with these three Babesia species in Swiss residents (Meer-Scherrer et al. 2004) despite risk of exposure to infected ticks (Foppa et al. 2002) may reflect either a low virulence or a lack of knowledge on these pathogens and the associated unspecific symptoms. Therefore, the presence of these three Babesia species, which are of medical relevance, in a suburban forest where I. ricinus tick density is high (Morán Cadenas et al. 2007a) requires attention from physicians. In fact, infection by Babesia spp. in patients presenting unspecific symptoms and history of contact with tick biotopes should be seriously taken into consideration and medical awareness should be increased by education on rare tick-borne pathogens. Since Babesia spp. tends to induce more severe manifestations in immunocompromised patients, a special attention should be attributed to these patients.