a total of 109 i. ricinus specimens were collected in northern and central italy ( figure 1 ) , identified by using standard taxonomic keys , and stored at 20c . specifically , 89 ticks ( 70 adults and 19 nymphs ) were collected by dragging vegetation in different parts of trentino province in april october 1997 and 1999 , and 10 ticks ( 7 adults and 3 nymphs ) by dragging in feltre ( veneto region ) in march 2000 . ten more ticks ( 7 adults and 3 nymphs ) were collected from a patient at the ospedale di careggi in firenze in may 1997 . the man had been bitten in parco nazionale delle foreste casentinesi ( toscana region ; see figure 1 ) a number of hours earlier but did not display any illness . mif tests with r. conorii antigens were performed on his arrival at the hospital and again 4 weeks later ; results were negative in each instance . tick samples were placed in 50 l of 10 mm trishcl ( ph 8.0 ) , heated at 90c for 10 min , crushed with a sterile plastic homogenizer , and treated with 10 g of proteinase k at 50c for 3 h. polymerase chain reaction ( pcr ) of a 341-bp portion of glta was performed by using the primers rp cs.877p and rp cs.1258n under conditions previously described ( 10 ) . these primers were chosen for an initial screening because they are known to amplify all rickettsiae ( 11 ) . dots represent places where ticks were not found to have rickettsiae ; different shapes represent the places where irita1 ( terlago , denno , verv ) , irita2 ( molina di ledro , drena ) and irita3 ( drena ) were detected . in feltre ( 2 ; veneto region ) only irita1 was detected , while in parco nazionale delle foreste casentinesi ( 3 ; toscana region ) only irita2 was detected . one hundred nine pcrs were performed , and nine positives ( two adult females , three adult males , and four nymphs ) were found . an initial estimate of the overall prevalence in italian i. ricinus is thus 8.25% . to better establish intrageneric relationships , the nine positive samples were subjected to further pcr analysis with the primer pairs rr 17.61p / rr 17.492n and rr 190.70p / rr 190.602n ( 10 ) , which amplified 394-bp and 488-bp portions of 17kda and ompa , respectively . pcr bands for all three genes were then sequenced directly by using abi prism sequencer ( perkin - elmer , foster city , ca ) . to compare the sequences obtained during this study with those of other rickettsiae , sequences present in genbank were selected by means of blast as well as on the basis of previous reports ( 1,12 ) . sequences were converted to their putative amino acid sequences and aligned by using the program clustal x ( ftp://ftp-igbmc.u-strasbg.fr/pub/clustalx/ ) . based on these alignments , nucleotide alignments were performed manually , and phylogenetic relationships were inferred by maximum likelihood ( ml ) . the appropriate model of sequence evolution was determined by modeltest 3.06 ( http://zoology.byu.edu/crandall_lab/modeltest.htm ) , and trees were produced using the program treepuzzle 5.0 ( www.tree-puzzle.de ) , which provides branch lengths as well as quartet puzzling support values at each node with > 50% support . comparisons of the sequences identified with those from closely related sfg rickettsia spp . are shown in the table ; figure 2 shows the results of phylogenetic analysis . glta - based results ( figure 2a ) show that all strains detected are sfg rickettsiae . for 17kda ( figure 2b ) , no identical sequences for irita2 and irita3 were present in genbank , and they clustered with r. cooleyi ( isolated from i. scapularis in texas ) . ompa was the most variable of the three genes analyzed ( figure 2c ) and could only be amplified from irita2 and irita3 . consistent with the results from glta , ompa from irita2 was 100% identical to irr / munich ; however , two substitutions were found between these two sequences and that of irs4 . notably , for ompa , the cluster to which irita2 and irita3 belong also contains a strain detected in spain ( 14 ) . this finding suggests that these bacteria may be widespread in europe . on the basis of ompa ( and 17kda ) sequences , the clade containing irita2 and irita3 was closest to a clade containing r. 
 cooleyi and an endosymbiont ( 10 ) , both hosted by i. scapularis . all previous attempts to amplify ompa from r. helvetica by using various primers have failed , which suggests that the gene is either absent or too variable to work with primers designed from other sfg bacteria ( 12 ) . taken together , the results from the three genes indicate that the clade containing irita2 and irita3 represents a lineage divergent from the seven described previously ( 1 ) . the number of ticks examined from trento , feltre , and toscana were 89 , 10 , and 10 respectively . phylogenetic analyses of rickettsiae based on glta ( a ) , 17 kda ( b ) and ompa ( c ) . based on analyses in modeltest 3.06 , the models of substitution chosen for analysis in treepuzzle 5.0 were trn+g for glta and 17kda , and hky+g for ompa . genbank accession numbers for each sequence , including those found in this study ( irita1 - 3 ) , are shown adjacent to each strain . our results represent the first demonstration of rickettsiae in italian i. ricinus and the first use of molecular - sequence  based methods to identify rickettsiae in italy . one bacterium , r. helvetica , occurs in several parts of europe and has been implicated as a human pathogen . the other two strains have only recently been discovered in i. 
 ricinus from slovakia and germany . whether they are pathogenic is not known , but since other rickettsiae of previously unknown pathogenicity have subsequently been shown to be associated with disease ( r. helvetica and r. slovaca ) , these new strains warrant attention . several studies on rickettsioses in italy have been published in the last two decades , and they all report r. conorii as the causative agent . as msf is the only known rickettsiosis in italy , diagnostic tests use r. conorii as the only antigen for serologic assays ( 16,17 ) . however , since sfg rickettsiae cause cross - reactions , confusion about the source of the illness may occur . although antibiotic therapy is generally effective for all sfg - related diseases , a better understanding of how different rickettsiae cause different symptoms will only come with their correct identification . during 19961999 in the regions we sampled , 23 rickettsioses ( assumed to be msf ) were reported from veneto , 42 from toscana , and 3 from trentino province ( italian ministry of health , unpub . data ) . while many were likely to be msf cases , the possibility exists that some were caused by other sfg ( perhaps r. helvetica ) . unlike most studies , one serosurvey in northeastern italy ( 18 ) used the complement - fixation test , which is less prone to cross - reactions ( 19 ) ; none of the sera tested was found positive for antibodies to rickettsiae . this finding may be explained by the use of r. conorii , r. rickettsii , r. typhi , and r. akari as the only antigens . serosurveys such as these could therefore benefit from the use of antigens from the bacteria identified in our study . i. ricinus is one of the most abundant tick species in italy , having a very low host specificity and a record of attaching to large numbers of humans ( 8) . the results reported here add sfg rickettsiae to the list of potentially dangerous pathogens that italian i. ricinus carry .