thirteen f. tularensis isolates collected over the past 10 years in switzerland ( figure 1 ) were subjected to extensive genetic characterization .
the species and subspecies designations of all strains were confirmed by real - time pcr that targeted the fopa gene and by amplification of the rd1 region ( 8) , which showed that all strains were f. tularensis subsp .
holarctica strains ( 7 ) and the genome sequence of the strain isolated in france , ftnf002 ( genbank accession no .
nc_009749 ) , were included in the study to represent the currently known genetic subpopulations within the subspecies .
all strains from switzerland were genetically characterized at 6 highly variable loci ( by mlva ) and 14 more stable loci that indicate the classification f. tularensis subsp .
the rd analysis was also performed because a 1.59-kb deletion marker , rd23 , was reported to be restricted to strains from france and spain ( 3 ) .
the mlva markers ( m3 , m6 , m20 , m21 , m22 , and m24 ) and ftind markers ( ftind 2538 ) were amplified by pcr and then sequenced with an abi prism 3100 genetic analyzer ( applied biosystems , foster city , ca , usa ) and the bigdye terminator cycle sequencing kit ( applied biosystems ) .
dna fragment sizes were calculated from the nucleotide sequences of the mlva and ftind markers and used to compare the isolates with previously analyzed strains from the united states , japan , france , and russia ( 3,7 ) .
the rd23 marker was assayed by using standard pcr and agarose gel methods as previously described ( 3 ) . a cluster analysis based on the mlva and indel size data
was performed by using bionumerics version 3.5 ( applied maths , kortrjik , belgium ) .
the unweighted pair group method with arithmetic mean phylogram is based on the combined ftind and multiple - locus variable - number tandem repeat analysis .
bootstrap values > 80% are given at the respective nodes and were calculated by using 10,000 iterations .
as expected , the indel markers served to place each strain into major branches of the cluster tree , and the more variable mlva markers provided the fine resolution at the tips of the tree .
the switzerland strains belonged to the same genetic cluster as the f. tularensis strain ftnf002 from france ( figure 1 ) that in a previous work clustered with strains from spain ( 3,9 ) .
moreover , all the swiss strains exhibited the 1.59-kb genomic deletion at the rd23 locus and the unique 464-bp size at mlva marker m24 , which confirmed their close relationship to the french strain ftnf002 as well as to other strains from france and spain ( 3 ) .
the finding of f. tularensis strains in switzerland represent sporadic occurrences of tularemia without any obvious epidemiologic connection .
the strains originated from 6 hares , 3 monkeys , and 4 persons and were collected at different locations in switzerland over a period of 10 years ( figure 2 ) .
the human infections most likely occurred through direct contact with wild animals , through rodent bites , and through consumption of a hare cooked at low temperature .
four swiss strains displayed a genetic profile identical to that of the representative french strain ftnf002 ( 3 ) .
the other 6 genotypes were closely related to ftnf002 , and all corresponded to the subclade b.br:ftnf00200 as defined by canonical single nucleotide polymorphisms by vogler et al .
this cluster , which also contained the strains from the iberian peninsula , seems to have spread throughout central and western europe .
moreover , all the swiss strains were susceptible to erythromycin ( mics 0.25 g / ml to 1 g / ml ) , which is a phenotypic marker that has previously been suggested to divide f. tularensis subsp .
strains of f. tularensis from switzerland ( central europe ) genetically clustered with strains from france and spain ( western europe ) as determined by the unique 464-bp genetic marker m24 and a specific deletion at marker rd23 .
furthermore , strains within the cluster differed at only 2 mlva markers , and 4 other mlva and 14 ftind markers were identical . in a previous study that included strains from the 19971998 tularemia outbreak in spain , the specific m24 allele and the rd23 deletion
were found in 49 of 49 strains from spain and france but in only 1 of 189 strains from 7 northern and eastern european countries and japan ( 3 ) .
the tularemia outbreak of 19971998 in spain , which resulted in > 500 human cases ( 5 ) , was thus caused by f. tularensis strains that were genetically closely related to strains recovered in switzerland from 1996 onwards , before the beginning of the outbreaks in spain .
this genetic relationship shows that factors other than the presence or introduction of a specific clone of the infectious agent per se determined the magnitude of the tularemia outbreaks in spain . for epidemiologists to understand the distribution of f. tularensis ( and other rare disease agents ) in the environment and their propagation across national and geographic borders , surveillance programs that include molecular analyses of these agents
should be undertaken in multiple countries , and the resulting data should be shared internationally .
 we conducted a molecular analysis of francisella tularensis strains isolated in switzerland and identified a specific subpopulation belonging to a cluster of f. tularensis subsp . 
 holarctica that is widely dispersed in central and western continental europe . 
 this subpopulation was present before the tularemia epidemics on the iberian peninsula . 
The Study
Conclusions