To assess local prevalence of B. microti in host-seeking nymphal I. ricinus ticks, we developed a tick-sampling procedure with high spatial resolution (Figure 1). The roughly rectangular, 0.7-ha field site, Ruetiwis (9° 38´ E, 46° 59´ N), is located on a steep southwesterly slope near Seewis in the lower Praettigau Valley of eastern Switzerland at an approximate mean altitude of 850 m above sea level. The site is characterized by abandoned pastures that are partly overgrown by young stands of deciduous and coniferous trees and bushes, as well as by mature mixed forest. All ticks were collected by flagging in July 1997.

To ensure the quality of tick-derived DNA, pools of 2–11 nymphal ticks were transferred to 0.5-mL microcentrifuge tubes containing 50 µL guanidium thiocyanate solution and stored at room temperature until further processing. Before homogenization with a glass pestle, the solution containing the ticks was incubated at 60°C for 2 h. DNA was extracted from the tick homogenate by the phenol-chloroform method. The resulting DNA pellet was suspended in 30 µL DNAse-free H₂0.

To determine whether B. microti–specific DNA was present, the extracted DNA was subjected to polymerase chain reaction (PCR) with the primer pair Bab-1 (5´-ttagtataagcttttatacagc-3´) and Bab-4 (5´-ataggtcagaaacttgaatgataca-3´) (10), which targets a 250-bp fragment of the 18s rRNA gene of B. microti. After denaturation for 2 min at 94°C, 40 cycles were performed, with 45 sec at 94°C, 45 sec at 55°C, and 45 sec at 72°C, followed by a 7-min final extension. Amplification products were separated on 2% agarose gel in Tris-borate-EDTA buffer, stained with ethidium bromide, and visualized under UV light. To differentiate the sequence of interest from a frequently observed, slightly smaller fragment, purified PCR products were digested with XhoI (Life Technologies, Invitrogen Corp., Gaithersburg, MD). The resulting two fragments specific for B. microti migrate in one band on the 2% agarose gel. The corresponding sequence of B. divergens lacks the restriction site.

To verify the taxonomic status of the Babesia spp. detected in these ticks, we conducted a phylogenetic analysis on a representative sample from which DNA had been amplified (Bab-1/Bab-4). For this purpose we used the primer pair PIRO A and PIRO B, which also targets 18s rDNA but is less specific for B. microti; the resulting sequence is longer (400 bp) than the Bab-1/Bab-4 amplicon (11). After PCR amplification, the respective band was excised from the agarose gel, purified with spin columns (Qiagen Inc., Valencia, CA), and sent to the University of Maine sequencing facility for sequence analysis. The resulting sequence was aligned against other Babesia spp. listed in GenBank by using Clustal X and consecutive adjustment visually. Phylogenetic analysis was performed by both maximum parsimony (Swofford D. Phylogenetic analysis using parsimony, PAUP*4b61; Sinauer Associates, Inc., Sunderland, MA) and neighbor-joining analyses (12) with Toxoplasma gondii (GenBank accession no. X68523) as outgroup. Robustness of the nodes was assessed by bootstrap analysis with 500 bootstrap replicates.

To accurately estimate the local prevalence of B. microti infection in I. ricinus ticks, we developed a maximum likelihood method for point estimation as well as a method for calculating confidence intervals. Briefly, the maximum likelihood estimate (MLE) of the prevalence p is based on the likelihood

To determine whether humans may be exposed to B. microti in the study area, we recruited 400 blood donors living within 10 km of the field site for a serologic survey of tick-borne zoonoses. Volunteers were recruited for this cross-sectional seroprevalence study during blood drives from December 1997 to May 1998 in towns within a 10-km radius of the study site. This protocol was approved by the Human Subjects Committee of the Harvard School of Public Health (protocol number 9712THEE). The sera of participants who gave their written informed consent were tested by indirect immunofluorescence assay (IFA) as described (14). Antigen slides were prepared from erythrocytes of B. microti–infected hamsters (the GI strain, originally derived from a Nantucket Island patient). Sera were first screened by IFA at 1:64 dilution. A panel of sera included all samples reactive in the screening test for which enough serum was available, including samples with borderline reactivity and representative controls. This panel was coded and blindly retested for B. microti IFA at the University of Connecticut laboratories, which specialize in Babesia serology. An IFA titer >1:64 was considered reactive. All reactive sera were titrated to endpoint.

To verify the identity of the amplified DNA, a phylogenetic analysis was performed. The sequence amplified from our I. ricinus ticks, which was deposited in GenBank (accession no. AF494286), differed by l bp from the North American B. microti sequence (GenBank accession no. AF231348) and was identical with that of B. microti from Slovenia (GenBank accession no. AF373332). Accordingly, our sequence clearly clustered with the European and the American strain of B. microti (Figure 2), with concordant results from both maximum parsimony and neighbor-joining analyses. Therefore, the piroplasms detected in I. ricinus ticks from Switzerland must be considered B. microti.

We then determined the prevalence of B. microti infection in nymphal I. ricinus ticks from the study site, on the basis of PCR amplification of B. microti in DNA tick pools. Overall, we analyzed 408 ticks in 64 pools. We detected B. microti–specific DNA in 14 pools (Table 1). Thus, B. microti infection appears to be common in human-biting ticks at this central European study site.

To determine whether the prevalence of B. microti is distributed homogeneously within the study site, prevalence of infection in ticks was estimated for selected segments of the sampling grid. The overall prevalence of B. microti infection in ticks, as estimated by MLE, was close to 4%. More detailed spatial analysis, however, indicates that the distribution of babesial infection at the site is heterogeneous (Table 2). In the lower portion of the site (section D), >10% of all ticks were infected, while in the upper portion (B–C), prevalence was <1%. Prevalence of infection in ticks was similar in sections B and C (p=0.161), but greater in section D than in sections B and C (p=0.003). Thus, B. microti transmission is focal at this site.

To determine how frequently humans are exposed to B. microti infection in the region around the study site, sera of local residents were screened by IFA for the presence of antibodies to B. microti antigen. Most sera (80%) were collected from December through March, when I. ricinus is not active. Five (1.5%) of 396 human sera were immunoglobulin (Ig) G-reactive against B. microti antigen at a titer ³1:64. All the samples positive on initial testing and one with a borderline result were confirmed on retesting to be reactive at a titer >1:64, while none of the samples with a negative screening result reacted (Table 3). B. microti–specific IgM seroreactivity was not found in any of the sera, a finding compatible with the time of the year when most of the sera had been collected. These results indicate that residents of our central European study site are exposed to bites of ticks infected with B. microti.