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 . distance between sampling points is 30 m. the letters and solid arrows denote the sections used for prevalence estimation . sampling lines that connect points not belonging to a section are not included in that section ( e.g. , line b4c2 is not part of section b ) . to ensure the quality of tick - derived dna , pools of 211 nymphal ticks were transferred to 0.5-ml microcentrifuge tubes containing 50 l 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 60c for 2 h. dna was extracted from the tick homogenate by the phenol - chloroform method . the resulting dna pellet was suspended in 30 l 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 94c , 40 cycles were performed , with 45 sec at 94c , 45 sec at 55c , and 45 sec at 72c , 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 . , 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 . 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 . 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 ) . 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 . 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 . distance between sampling points is 30 m. the letters and solid arrows denote the sections used for prevalence estimation . sampling lines that connect points not belonging to a section are not included in that section ( e.g. , line b4c2 is not part of section b ) . to ensure the quality of tick - derived dna , pools of 211 nymphal ticks were transferred to 0.5-ml microcentrifuge tubes containing 50 l 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 60c for 2 h. dna was extracted from the tick homogenate by the phenol - chloroform method . the resulting dna pellet was suspended in 30 l 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 94c , 40 cycles were performed , with 45 sec at 94c , 45 sec at 55c , and 45 sec at 72c , 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 . , 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 . 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 . 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 ) . 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 . 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 . 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 . : babesia microti - slovenia af373332 ; b. microti - switzerland af494286 ; b. microti- nantucket af231348 ; " toxoplasma annae " af188001 ; b. rodhaini ab049999 ; wa1 af158700 ; b. gibsoni 1 af158702 ; b. divergens 1 u07885 ; b. divergens 2 u16370 ; b. odocoilei u16369 ; b. gibsoni 2 af175300 ; b. gibsoni 3 af175301 ; and toxoplasma gondii x68523 . 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 . thus , b. microti infection appears to be common in human - biting ticks at this central european study site . pools in bold are positive ; dashes represent the absence of further pools . to determine whether the prevalence of b. microti is distributed homogeneously within the study site , prevalence of infection in ticks 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 ) . mle ( maximum likelihood estimate ) of the point estimates are shown with 95% bootstrap confidence limits . 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 1:64 . 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 ) . 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 . 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 . 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 . : babesia microti - slovenia af373332 ; b. microti - switzerland af494286 ; b. microti- nantucket af231348 ; " toxoplasma annae " af188001 ; b. rodhaini ab049999 ; wa1 af158700 ; b. gibsoni 1 af158702 ; b. divergens 1 u07885 ; b. divergens 2 u16370 ; b. odocoilei u16369 ; b. gibsoni 2 af175300 ; b. gibsoni 3 af175301 ; and toxoplasma gondii x68523 . 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 . thus , b. microti infection appears to be common in human - biting ticks at this central european study site . pools in bold are positive ; dashes represent the absence of further pools . to determine whether the prevalence of b. microti is distributed homogeneously within the study site , prevalence of infection in ticks 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 ) . mle ( maximum likelihood estimate ) of the point estimates are shown with 95% bootstrap confidence limits . 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 1:64 . 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 ) . 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 . b. microti  infected nymphal i. ricinus ticks are present at swiss study site , and human residents of the area appear to be exposed to this agent . however , the presence of b. microti in i. ricinus ticks has been reported only once before ( 5 ) . a phylogenetic analysis clearly demonstrated that the piroplasms found in our study site belong to b. microti , rather than to other babesia or theileria species ( figure 2 ) . the detection of b. microti dna in host - seeking nymphal i. ricinus ticks may , however , simply reflect  spill - over  from enzootic transmission by the accepted maintenance vector for b. microti in eurasia , the tick i. trianguliceps ( 15 ) . this tick , which does not bite humans , infests small rodents that also are abundantly parasitized by i. ricinus ( 3,16 ) . the prevalence of infection in ticks may be underestimated as only one genome copy is present per parasite in unfed ticks before sporogony . therefore , low parasite loads may escape detection , thus increasing the specificity of the assay . in our study , maximizing specificity was desirable because infection of i. ricinus with b. microti was a priori assumed to be rare , and underestimation of prevalence therefore is conservative . regardless , the proportion of ticks that appear to be infected by b. microti is similar to that in coastal new england ( s. telford , unpub . data ) . recently , duh et al . reported a similarly high prevalence of b. microti infection in nymphal i. ricinus ticks collected in slovenia ( 7 of 69 ticks tested by pcr ) ( 17 ) . in combination with our findings , this report suggests that b. microti infection in i. ricinus ticks is far more common than traditionally thought . in addition , vector competence of i. ricinus for b. microti has been demonstrated experimentally ( 7,18 ) . the frequent infection of i. ricinus with this piroplasm therefore implies zoonotic relevance of this vector - pathogen association in switzerland and possibly in other parts of europe . similar to tick - borne encephalitis virus , b. microti seems to be maintained in small focal areas . the risk of human infection therefore is spatially highly variable and may be conditional on tick density . preliminary analysis of tick infection data from the study site over a 3-year period ( foppa , unpub . data ) suggests that b. microti is locally maintained , especially in the lower portion of that site , while the exact location of the maximum risk changes over the years . residents of this site in eastern switzerland appear to be exposed to bites by ticks infected with b. microti . the serologic result is unlikely to reflect low specificity of the assay , as previous evaluation of this ifa has demonstrated high specificity ( 19,20 ) . as part of this evaluation , we tested 50 sera from residents of iceland , where ticks capable of transmitting b. microti are absent ; none of the sera reacted with b. microti antigen ( 19 ) . this finding suggests a satisfactory positive predictive value of the serologic test even in settings of low prevalence . in the northeastern united states , b. microti seroprevalence has varied in endemic regions , from 3.7% in red cross blood donors on cape cod , massachusetts ( 21 ) , to 2.5% and 9.5% in connecticut residents who were seronegative and seroreactive , respectively , to borrelia burgdorferi ( 22 ) . our findings of high local prevalence of b. microti infection in i. ricinus ticks may seem counterintuitive given the lower seroprevalence in residents of our study area . the findings may , however , reflect a high degree of spatial clustering of transmission with a low average risk ( 18 ) . alternatively , the low seroprevalence may be the result of low test sensitivity resulting from antigenic differences between north american b. microti strains , which were used for the serologic testing , and the european strains to which our study population had been exposed . at least locally , the potential for zoonotic transmission of b. microti by i. ricinus is considerable , which explains serologic evidence in human beings of exposure to this agent in parts of europe . the lack of recognized human pathology associated with european strains of b. microti , despite exposure to infectious tick bites , may be a consequence of a lower virulence of european strains than those of north american . disease episodes due to b. microti , on the other hand , may be overlooked because of the relative nonspecificity of signs and symptoms and the presumption that this agent rarely infects i. ricinus .