serum specimens from the patients were tested at the centers for disease control and prevention ( cdc ) in serial fourfold dilutions by indirect fluorescent antibody ( ifa ) testing for reactivity to b. microti ( 11 ) , wa1 ( 8) , and b. divergens antigens . the antigen sources were human isolates of b. microti and wa1 and a bovine isolate of b. divergens ( the purnell strain from the republic of ireland ) that had been passaged in gerbils ( mongolian jirds ; meriones unguiculatus ) and adapted to culture in bovine erythrocytes . the serum specimens were also tested at the clinical institute of hygiene of the university of vienna by ifa for reactivity to b. divergens antigens ( from a bovine isolate from hanover , germany , that had been passaged in jirds ) ; the dilutions of serum that were tested were 1:16 , 1:64 , 1:256 , 1:1,000 , and 1:4,000 . five jirds , which are competent hosts for b. divergens ( 13 ) , were injected intraperitoneally with 0.5 ml of 1-day old , refrigerated , pretreatment blood from the austrian patient . the jirds were monitored periodically ( at least weekly ; 26 times in 17.5 weeks ) for parasitemia by examination of diff quik - stained ( dade ag , ddingen , switzerland ) smears of blood obtained either by tail snip or , at the end of the monitoring period , by cardiac puncture after anesthesia with ketamine . the blood obtained by cardiac puncture was also examined by polymerase chain reaction ( pcr ) ( see below ) . dna was extracted from edta - stabilized whole blood from the two patients by using the qiaamp dna blood mini kit ( qiagen inc . , the complete 18s ribosomal rna ( 18s rrna ) gene was amplified by pcr , with a pair of generic apicomplexan 18s rrna - specific primers : cryptof , the forward primer ( 5-aacctggttgatcctgccagt-3 ) , and cryptor , the reverse primer ( 5-gcttgatccttctgcaggttcacctac-3 ) . pcr was conducted with the amplitaq gold dna polymerase ( applied biosystems , foster city , ca ) . the conditions for pcr included 95c for 15 min , followed by 45 cycles of denaturation at 94c for 30 s , annealing at 65c for 30 s , and extension at 72c for 1.5 min . final extension was done at 72c for 9 min , followed by a hold step at 4c . amplification products were purified by using the strataprep dna purification kit ( stratagene , la jolla , ca ) . in addition , dna provided us that had been extracted from two isolates of b. odocoilei ( i.e. , the brushy creek and engeling isolates ) , a parasite of white - tailed deer ( odocoileus virginianus ) ( 15,16 ) , and from b. divergens [ purnell strain ] ) was analyzed . both strands of the pcr products were sequenced by using a set of internal primers . sequencing reactions were conducted with the abi prism bigdye terminator cycle sequencing kit ( applied biosystems ) , and reactions were analyzed on the abi 377 or abi 3100 automatic dna sequencer ( applied biosystems ) . the resulting sequences were assembled by using the program seqman ii ( dnastar , inc . the genbank accession numbers for the complete sequences we generated of the 18s rrna gene for the various organisms are as follows : b. odocoilei , ay046577 ; b. divergens , ay046576 ; and eu1 , ay046575 . the complete sequences of the 18s rrna genes for b. bigemina , b. bovis , b. caballi , b. divergens , b. gibsoni , b. odocoilei , and babesia sp . ( isolated from bos taurus ) were retrieved from the genbank database ( see figure 1 legend for genbank accession numbers ) and aligned with the sequence for eu1 by using the program clustal w version 1.83 ( 17 ) . the 18s rrna sequence for theileria annulata was included as the outgroup for the phylogenetic analysis . this analysis was performed with the following programs : the phylip package , which includes versions 3.5 of consense , dnadist , dnaml , neighbor , and seqboot ( 18 ) ; and version 5.73c of tree - puzzle ( 19 ) . the phylogenetic trees inferred by these programs were drawn by using the program treeview , version 1.6.6 ( 20 ) . the trees were statistically evaluated by using bootstrap ( 18 ) and quartet puzzling methods ( 19 ) . the tree was computed by using the quarter puzzling maximum likelihood method of the tree - puzzle program and was oriented by using theileria annulata as the outgroup . the genbank accession numbers for the sequences used in the analysis are as follows : babesia bigemina a , x59604 ; b. bovis , l19077 ; b. caballi , z15104 ; b. divergens ( purnell isolate ) , ay046576 ; b. gibsoni ( genotype asia 1 ) , af175300 ; b. odocoilei ( brushy creek and engeling isolates ) , ay046577 ; babesia sp . ( isolated from bos taurus ) , u09834 ; eu1 ( the etiologic agent of infection in the two cases described here ) , ay046575 ; and theileria annulata , m64243 . the italian and austrian patients were 55- and 56-year - old men , respectively , who had undergone splenectomy in the 1980s because of stage ia hodgkin s disease . the italian patient had recently begun chemotherapy ( table footnote ) for stage iiia diffuse large b - cell lymphoma , which had been diagnosed in june 1998 . both men lived in small towns and hunted avocationally ( table ) ; neither had pets . only the austrian patient recalled tick exposure  a tick bite while hunting about 2 weeks before he noticed his urine was dark . neither patient had traveled extensively : the italian patient had never left italy , and the austrian patient had been in barbados ( 1998 ) and turkey ( 1999 ) . ifa , indirect fluorescent antibody ; pcr , polymerase chain reaction ; cdc , centers for disease control and prevention . non - hodgkin s lymphoma developed in the italian patient ( diagnosis : june 1998 ) . chemotherapy , begun on september 23 , 1998 , was stopped prematurely on october 14 , after he became febrile . his chemotherapeutic regimen included daily prednisone ( 75 mg ) and weekly administration of various drugs in rotation . he received 4 of the intended 12 weeks of therapy , which included doxorubicin and cyclophosphamide during odd - numbered weeks ( weeks 1 and 3 ) and vincristine and either methotrexate ( week 2 ) or bleomycin ( week 4 ) during even - numbered weeks . although the possibility that he became infected by blood transfusion could not be excluded because he had been transfused before blood smears were examined , his febrile illness and hemolytic anemia preceded the transfusions . laboratory values were from hospital admission ( october 18 , 1998 , for the italian patient , and july 25 , 2000 , for the austrian patient ) , unless otherwise specified . values for the austrian patient are from testing performed at the hospital to which he was transferred after a brief ( < 24-hour ) stay at a local hospital . earlier on july 25 , at a local hospital , his hemoglobin value was 16.2 g / dl , which had been his approximate baseline value during the previous 10 months . ifa testing of serum specimens from both patients was negative for antibodies to b. microti . a specimen from the italian patient ( february 15 , 1999 ) was negative for antibodies to wa1 . plasma exchange was performed on october 23 , when he mistakenly was thought to have thrombotic th the two cases ranged in severity from quite mild ( austrian case ) to moderately severe ( italian case ) . the salient clinical details of their cases and the relevant laboratory values are provided in the table . fever occurred only in the italian patient ( maximum of 39c ) , which initially was considered a reaction to one of his chemotherapeutic agents ( i.e. , bleomycin ) . he also had marked anemia , for which he received blood transfusions ( table ) . both patients had thrombocytopenia , elevated serum lactate dehydrogenase and bilirubin values , and dark urine from hemoglobinuria . babesiosis was diagnosed by noting parasitic inclusions in erythrocytes on peripheral blood smears ( table ; figure 2 ) . the intervals between onset of the symptoms that ultimately were attributed to babesiosis and confirmation of the diagnosis ranged from 2 days ( austrian case ) to 10 days ( italian case ) . subsequent testing of serum specimens from both patients showed ifa reactivity to b. divergens but not to b. microti antigens ; serum from the italian patient was also tested for reactivity to wa1 antigens and was negative . attempts to obtain an isolate of the parasite that infected the austrian patient , by injecting specimens of his blood into jirds , were unsuccessful ; the smears of blood from periodic tail snips and pcr analysis of blood obtained by cardiac puncture of the jirds were negative . both patients responded to antimicrobial therapy for babesiosis : the austrian patient was treated with clindamycin , and the italian patient was treated with both clindamycin and quinine ( table ) . panel of computer - generated electronic images of photomicrographs of babesia - infected erythrocytes on a giemsa - stained smear of peripheral blood from the patient who became infected in austria . the electronic images were edited for uniformity of color , without changing the form or size of the organisms . the image on the far right shows a tetrad ( maltese - cross form ) . three glass slides of the actual blood films have been deposited in the obersterreichisches landesmuseum , biologiezentrum , linz ( i.e. , biology center of the upper austrian museum , linz ) , with the accession number 2002/9 . ( eu1 ) , patient 001 , austria , krems land , july 25 , 2000 . amplification of the complete 18s rrna gene , by using generic protozoan primers and the babesia dna extracted from the patients as the templates , yielded a specific product of approximately 1,700 base pairs for each patient . sequence analysis showed that the 18s rrna gene was 1,727 bases long and that the pcr products from the two patients had identical sequences . blast ( available from : url : http://www.ncbi.nlm.nih.gov/blast/ ) search showed that the sequence , although clearly from a babesia sp . in phylogenetic analysis , eu1 clusters together with b. odocoilei , and these two organisms form a sister group with b. divergens ( figure 1 ) . the clustering of these organisms was identical , regardless of which phylogenetic method was used . support for the internal branch leading to the b. divergens , b. odocoilei , and eu1 group was 100% with both quartet puzzling and bootstrap distance analysis ; for the internal branch separating b. divergens from b. odocoilei and eu1 , the support was 100% for quartet puzzling and 88% for bootstrapped distance analysis . the alignment of the sequences used to construct the phylogenetic tree ( figure 1 ) is available from the authors upon request . because the complete 18s rrna sequences in genbank that were previously determined for various bovine isolates of b. divergens were not identical , we reanalyzed the complete 18s rrna gene from isolates ( cultures or dna ) from ireland ( purnell ; genbank accession no . u16370 ) , germany ( u07885 ) , and northern ireland ( z48751 ) that were provided to us . the sequences of the 18s rrna gene we obtained for these isolates were identical , which suggests that no variability is present in this gene among geographically distinct bovine isolates of b. divergens ( slemenda et al . , data ) . in contrast , the eu1 and b. divergens 18s rrna sequences differed by 31 bases . similarly , our sequences of the 18s rrna gene for both isolates of b. odocoilei ( i.e. , brushy creek and engeling isolates ) were identical to each other ( genbank accession no . ay046577 ) and to the b. odocoilei sequence with the genbank accession no . u16369 ( 14 ) . serum specimens from the patients were tested at the centers for disease control and prevention ( cdc ) in serial fourfold dilutions by indirect fluorescent antibody ( ifa ) testing for reactivity to b. microti ( 11 ) , wa1 ( 8) , and b. divergens antigens . the antigen sources were human isolates of b. microti and wa1 and a bovine isolate of b. divergens ( the purnell strain from the republic of ireland ) that had been passaged in gerbils ( mongolian jirds ; meriones unguiculatus ) and adapted to culture in bovine erythrocytes . the serum specimens were also tested at the clinical institute of hygiene of the university of vienna by ifa for reactivity to b. divergens antigens ( from a bovine isolate from hanover , germany , that had been passaged in jirds ) ; the dilutions of serum that were tested were 1:16 , 1:64 , 1:256 , 1:1,000 , and 1:4,000 . five jirds , which are competent hosts for b. divergens ( 13 ) , were injected intraperitoneally with 0.5 ml of 1-day old , refrigerated , pretreatment blood from the austrian patient . the jirds were monitored periodically ( at least weekly ; 26 times in 17.5 weeks ) for parasitemia by examination of diff quik - stained ( dade ag , ddingen , switzerland ) smears of blood obtained either by tail snip or , at the end of the monitoring period , by cardiac puncture after anesthesia with ketamine . the blood obtained by cardiac puncture was also examined by polymerase chain reaction ( pcr ) ( see below ) . dna was extracted from edta - stabilized whole blood from the two patients by using the qiaamp dna blood mini kit ( qiagen inc . , valencia , ca ) ; the dna was stored at 4c . the complete 18s ribosomal rna ( 18s rrna ) gene was amplified by pcr , with a pair of generic apicomplexan 18s rrna - specific primers : cryptof , the forward primer ( 5-aacctggttgatcctgccagt-3 ) , and cryptor , the reverse primer ( 5-gcttgatccttctgcaggttcacctac-3 ) . pcr was conducted with the amplitaq gold dna polymerase ( applied biosystems , foster city , ca ) . the conditions for pcr included 95c for 15 min , followed by 45 cycles of denaturation at 94c for 30 s , annealing at 65c for 30 s , and extension at 72c for 1.5 min . final extension was done at 72c for 9 min , followed by a hold step at 4c . amplification products were purified by using the strataprep dna purification kit ( stratagene , la jolla , ca ) . in addition , dna provided us that had been extracted from two isolates of b. odocoilei ( i.e. , the brushy creek and engeling isolates ) , a parasite of white - tailed deer ( odocoileus virginianus ) ( 15,16 ) , and from b. divergens [ purnell strain ] ) was analyzed . both strands of the pcr products were sequenced by using a set of internal primers . sequencing reactions were conducted with the abi prism bigdye terminator cycle sequencing kit ( applied biosystems ) , and reactions were analyzed on the abi 377 or abi 3100 automatic dna sequencer ( applied biosystems ) . the resulting sequences were assembled by using the program seqman ii ( dnastar , inc . the genbank accession numbers for the complete sequences we generated of the 18s rrna gene for the various organisms are as follows : b. odocoilei , ay046577 ; b. divergens , ay046576 ; and eu1 , ay046575 . the complete sequences of the 18s rrna genes for b. bigemina , b. bovis , b. caballi , b. divergens , b. gibsoni , b. odocoilei , and babesia sp . ( isolated from bos taurus ) were retrieved from the genbank database ( see figure 1 legend for genbank accession numbers ) and aligned with the sequence for eu1 by using the program clustal w version 1.83 ( 17 ) . the 18s rrna sequence for theileria annulata was included as the outgroup for the phylogenetic analysis . this analysis was performed with the following programs : the phylip package , which includes versions 3.5 of consense , dnadist , dnaml , neighbor , and seqboot ( 18 ) ; and version 5.73c of tree - puzzle ( 19 ) . the phylogenetic trees inferred by these programs were drawn by using the program treeview , version 1.6.6 ( 20 ) . the trees were statistically evaluated by using bootstrap ( 18 ) and quartet puzzling methods ( 19 ) . the tree was computed by using the quarter puzzling maximum likelihood method of the tree - puzzle program and was oriented by using theileria annulata as the outgroup . the genbank accession numbers for the sequences used in the analysis are as follows : babesia bigemina a , x59604 ; b. bovis , l19077 ; b. caballi , z15104 ; b. divergens ( purnell isolate ) , ay046576 ; b. gibsoni ( genotype asia 1 ) , af175300 ; b. odocoilei ( brushy creek and engeling isolates ) , ay046577 ; babesia sp . ( isolated from bos taurus ) , u09834 ; eu1 ( the etiologic agent of infection in the two cases described here ) , ay046575 ; and theileria annulata , m64243 . the italian and austrian patients were 55- and 56-year - old men , respectively , who had undergone splenectomy in the 1980s because of stage ia hodgkin s disease . the italian patient had recently begun chemotherapy ( table footnote ) for stage iiia diffuse large b - cell lymphoma , which had been diagnosed in june 1998 . both men lived in small towns and hunted avocationally ( table ) ; neither had pets . only the austrian patient recalled tick exposure  a tick bite while hunting about 2 weeks before he noticed his urine was dark . neither patient had traveled extensively : the italian patient had never left italy , and the austrian patient had been in barbados ( 1998 ) and turkey ( 1999 ) . ifa , indirect fluorescent antibody ; pcr , polymerase chain reaction ; cdc , centers for disease control and prevention . non - hodgkin s lymphoma developed in the italian patient ( diagnosis : june 1998 ) . chemotherapy , begun on september 23 , 1998 , was stopped prematurely on october 14 , after he became febrile . his chemotherapeutic regimen included daily prednisone ( 75 mg ) and weekly administration of various drugs in rotation . he received 4 of the intended 12 weeks of therapy , which included doxorubicin and cyclophosphamide during odd - numbered weeks ( weeks 1 and 3 ) and vincristine and either methotrexate ( week 2 ) or bleomycin ( week 4 ) during even - numbered weeks . although the possibility that he became infected by blood transfusion could not be excluded because he had been transfused before blood smears were examined , his febrile illness and hemolytic anemia preceded the transfusions . laboratory values were from hospital admission ( october 18 , 1998 , for the italian patient , and july 25 , 2000 , for the austrian patient ) , unless otherwise specified . values for the austrian patient are from testing performed at the hospital to which he was transferred after a brief ( < 24-hour ) stay at a local hospital . earlier on july 25 , at a local hospital , his hemoglobin value was 16.2 g / dl , which had been his approximate baseline value during the previous 10 months . ifa testing of serum specimens from both patients was negative for antibodies to b. microti . a specimen from the italian patient ( february 15 , 1999 ) was negative for antibodies to wa1 . plasma exchange was performed on october 23 , when he mistakenly was thought to have thrombotic th the two cases ranged in severity from quite mild ( austrian case ) to moderately severe ( italian case ) . the salient clinical details of their cases and the relevant laboratory values are provided in the table . fever occurred only in the italian patient ( maximum of 39c ) , which initially was considered a reaction to one of his chemotherapeutic agents ( i.e. , bleomycin ) . he also had marked anemia , for which he received blood transfusions ( table ) . both patients had thrombocytopenia , elevated serum lactate dehydrogenase and bilirubin values , and dark urine from hemoglobinuria . babesiosis was diagnosed by noting parasitic inclusions in erythrocytes on peripheral blood smears ( table ; figure 2 ) . the intervals between onset of the symptoms that ultimately were attributed to babesiosis and confirmation of the diagnosis ranged from 2 days ( austrian case ) to 10 days ( italian case ) . subsequent testing of serum specimens from both patients showed ifa reactivity to b. divergens but not to b. microti antigens ; serum from the italian patient was also tested for reactivity to wa1 antigens and was negative . attempts to obtain an isolate of the parasite that infected the austrian patient , by injecting specimens of his blood into jirds , were unsuccessful ; the smears of blood from periodic tail snips and pcr analysis of blood obtained by cardiac puncture of the jirds were negative . both patients responded to antimicrobial therapy for babesiosis : the austrian patient was treated with clindamycin , and the italian patient was treated with both clindamycin and quinine ( table ) . panel of computer - generated electronic images of photomicrographs of babesia - infected erythrocytes on a giemsa - stained smear of peripheral blood from the patient who became infected in austria . the electronic images were edited for uniformity of color , without changing the form or size of the organisms . the image on the far right shows a tetrad ( maltese - cross form ) . three glass slides of the actual blood films have been deposited in the obersterreichisches landesmuseum , biologiezentrum , linz ( i.e. , biology center of the upper austrian museum , linz ) , with the accession number 2002/9 . ( eu1 ) , patient 001 , austria , krems land , july 25 , 2000 . amplification of the complete 18s rrna gene , by using generic protozoan primers and the babesia dna extracted from the patients as the templates , yielded a specific product of approximately 1,700 base pairs for each patient . sequence analysis showed that the 18s rrna gene was 1,727 bases long and that the pcr products from the two patients had identical sequences . blast ( available from : url : http://www.ncbi.nlm.nih.gov/blast/ ) search showed that the sequence , although clearly from a babesia sp . , was not identical to any complete 18s rrna sequences in the genbank database . in phylogenetic analysis , eu1 clusters together with b. odocoilei , and these two organisms form a sister group with b. divergens ( figure 1 ) . the clustering of these organisms was identical , regardless of which phylogenetic method was used . support for the internal branch leading to the b. divergens , b. odocoilei , and eu1 group was 100% with both quartet puzzling and bootstrap distance analysis ; for the internal branch separating b. divergens from b. odocoilei and eu1 , the support was 100% for quartet puzzling and 88% for bootstrapped distance analysis . the alignment of the sequences used to construct the phylogenetic tree ( figure 1 ) is available from the authors upon request . because the complete 18s rrna sequences in genbank that were previously determined for various bovine isolates of b. divergens were not identical , we reanalyzed the complete 18s rrna gene from isolates ( cultures or dna ) from ireland ( purnell ; genbank accession no . u16370 ) , germany ( u07885 ) , and northern ireland ( z48751 ) that were provided to us . the sequences of the 18s rrna gene we obtained for these isolates were identical , which suggests that no variability is present in this gene among geographically distinct bovine isolates of b. divergens ( slemenda et al . in contrast , the eu1 and b. divergens 18s rrna sequences differed by 31 bases . similarly , our sequences of the 18s rrna gene for both isolates of b. odocoilei ( i.e. , brushy creek and engeling isolates ) were identical to each other ( genbank accession no . ay046577 ) and to the b. odocoilei sequence with the genbank accession no . u16369 ( 14 ) . we investigated the first reported human cases of babesiosis in italy and austria and have provided molecular evidence that the etiologic agent was a previously uncharacterized babesia organism , which we refer to here as eu1 . the organism was found in countries in europe not previously known to have zoonotic babesiosis and had novel molecular characteristics for the genetic marker we analyzed , the complete 18s rrna gene . sequence analysis of this gene provides an objective and precise means of species identification and phylogenetic classification . the dna sequences of the 18s rrna gene were identical for the babesia organisms from the two patients , which indicates that they were infected with the same organism . each of the organisms was sequenced in a different country , which indicates that the findings were not artifactual . the phylogenetic analysis ( figure 1 ) indicates that eu1 is most closely related to but distinct from b. odocoilei , which infects white - tailed deer ( 15,16 ) and is not known to infect humans . eu1 and b. odocoilei form a sister group to b. divergens , a bovine parasite that has been considered the main babesia pathogen of humans in europe . we have demonstrated that no variability exists in the 18s rrna sequences among several geographically distinct bovine isolates of b. divergens ( slemenda et al . data ) , which is the organism to which the name b. divergens legitimately applies , and showed that eu1 clearly is not b. divergens . eu1 is also distinct from the mo1 parasite , which caused a fatal human case of babesiosis in missouri in 1992 and was thought then by the investigators to be b. divergens  like but distinct from it ( 10 ) . the sequence provided in the publication about mo1 ( 10 ) was for only a 128base pair fragment ; in that region , the eu1 and mo1 sequences differ by four bases , and three positions in the mo1 sequence were unresolved . the dna sequences available in genbank for b. divergens in europe are from cattle not humans . to our knowledge , molecular data have been reported for only one of the purported human cases of b. divergens infection in europe , a case on the canary islands ( 22,23 ) . however , the data were for an incomplete 18s rrna sequence ( genbank accession no . af435415 ) , and therefore were not suitable for the phylogenetic analysis we performed of complete 18s rrna sequences . nevertheless , the sequence for the case on the canary islands differs by 18 bases with the sequence for eu1 and by 1 base with the b. divergens sequence from cattle ( ay046576 ) in the 369-base - long region of the gene that could be compared . in the absence of molecular data , we are not certain which organisms have caused the human cases of babesiosis in europe that have been attributed to b. divergens . the evidence that particular human cases were caused by b. divergens has varied in quantity , quality , and type . the evidence typically has included various combinations of morphologic data , from examination of blood smears ; serologic data ( usually , but not always , from ifa testing ) ; and data concerning whether jirds or cattle injected with the patient s blood become parasitemic . although these techniques are useful for detecting babesia infection , they do not necessarily provide reliable species identification ( e.g. , because of serologic cross - reactivity between eu1 and b. divergens in ifa testing [ table ] ) . although some of the human cases attributed to b. divergens may truly have been caused by the bovine b. divergens , others might have been caused by eu1 . the cases of eu1 infection we reported likely would have been attributed to b. divergens had only the traditional methods of characterization , without molecular analysis , been used . our molecular characterization also showed that eu1 is not closely related to the other babesia ( or babesia - like ) agents known to have infected humans ( most notably , b. microti and the wa1- and ca1-type parasites ) . b. microti , together with b. rodhaini , cytauxzoon felis , and b. equi , is ancestral to the theileria spp . and perhaps also to the babesia sensu stricto group ( depending on which tree topology is used ) ( 24 ) . reclassification of the b. microti group to a new family has been proposed ( 24 ) . the wa1- and ca1-type parasites , which have caused human cases of babesiosis in the western united states ( 8,9 ) , also form a well - defined group , whose position in the phylogeny of the piroplasms is uncertain ( 25 ) . although eu1 represents a zoonotic pathogen with previously unreported molecular characteristics , whether it represents a new species per se awaits further evidence . eu1 might constitute a new species in the sense that it was never previously recognized or characterized in any way or one that was characterized but not with molecular data ( e.g. , was misnamed b. divergens or some other babesia sp . ) . because dna sequence data are not available for most of the babesia spp . found over the past century in nonhuman animals and because data about the morphologic features and host specificity of a parasite are inadequate for definitive species identification , we can not exclude the possibility that eu1 is one of the many previously described babesia spp . of nonhuman animals , some named and some not , that were not known to be zoonotic . although the serologic cross - reactivity between eu1 and b. divergens could have resulted in diagnostic confusion in the past , cross - reactivity between these two organisms also could be advantageous . the b. divergens ifa could be a useful tool for testing serum from persons who might be infected with eu1 or who participate in serosurveys to determine the prevalence and geographic distribution of eu1 infection . unfortunately , our attempts to obtain an isolate of eu1 by inoculation of jirds were unsuccessful . one consequence is that we did not generate the homologous antigen needed for development of an ifa assay for eu1 . therefore , we could not contrast the degree of reactivity of our patients serum specimens with antigens from eu1 and b. divergens . the importance of determining whether the etiologic agent of a particular case of babesiosis is eu1 rather than b. divergens or some other babesia sp . depends in part on whether the clinical manifestations of infection and the response to antimicrobial therapy differ . however , the range in severity of the two cases , from quite mild ( austrian case ) to moderately severe ( italian case ) , is of interest , particularly because the two patients were similar in some respects ( i.e. , both were asplenic men in their mid - fifties ) . factors that likely placed the italian patient at increased risk for a more severe case included immunosuppressive chemotherapy for lymphoma and the 10-day interval between the onset of fever and the diagnosis of babesiosis ( table ) . largely from data for b. microti infection in the united states , combination therapy with either clindamycin and quinine or atovaquone and azithromycin is recommended for treatment of babesiosis ( 28 ) , with the addition of exchange transfusion in some situations in severely ill patients . some in vitro data and anecdotal clinical data for purported zoonotic cases of b. divergens infection suggest that therapy with clindamycin alone , in combination with exchange transfusion , when indicated , might be effective for treating b. divergens infection ( 4,29,30 ) . however , no clinical trials in humans have evaluated the effectiveness of any antimicrobial regimens for treatment of babesia infection not caused by b. microti . the public health importance of infection with eu1 , including such factors as its biology , geographic distribution , ecology , prevalence , risk factors for infection and disease , clinical manifestations , tick vector , and animal reservoir host(s ) , is not yet known and may take years to determine . the italian patient likely became infected in a garden habitat and the austrian patient in a garden or forest habitat . of interest , an incomplete 18s rrna sequence ( genbank accession no . af373333 ) for a babesia sp . found in i. ricinus ticks was recently reported by investigators in slovenia ( 31 ) , which borders italy and austria . the sequence was reported for only 364 bases and corresponds to positions 433796 of the complete 18s rrna sequence for eu1 . in this part of the gene , however , the relatedness of the two organisms can not be determined without the complete 18s rrna sequence for the organism from the ticks . the occurrence of two identified cases of eu1 infection in humans in different countries ( i.e. , italy and austria ) and years ( i.e. , 1998 and 2000 ) indicates that eu1 is not restricted to one geographic area or time .
in europe , most reported human cases of babesiosis have been attributed , without strong molecular evidence , to infection with the bovine parasite babesia divergens . 
 we investigated the first known human cases of babesiosis in italy and austria , which occurred in two asplenic men . 
 the complete 18s ribosomal rna ( 18s rrna ) gene was amplified from specimens of their whole blood by polymerase chain reaction ( pcr ) . with phylogenetic analysis , we compared the dna sequences of the pcr products with those for other babesia spp . 
 the dna sequences were identical for the organism from the two patients . in phylogenetic analysis , the organism clusters with b. odocoilei , a parasite of white - tailed deer ; these two organisms form a sister group with b. divergens . 
 this evidence indicates the patients were not infected with b. divergens but with an organism with previously unreported molecular characteristics for the 18s rrna gene .