in july 2007 , an outbreak of ulceroglandular tularemia occurred in utah among visitors to the southwest shore of utah lake ; an epidemiologic investigation implicated deer fly bites as the source of infection ( 8) .
molecular subtyping of isolates was performed by using pmei pulsed - field gel electrophoresis ( pfge ) as previously described ( 2 ) .
pfge gels were normalized by comparison to the salmonella enterica serotype braenderup ( h9812 ) reference strain by using bionumerics software ( v. 4.0 , applied maths bvba , sint - martens - latem , belgium ) ( 2 ) .
a dendrogram was constructed by comparison with pmei pfge patterns for a1 ( schu s4 , ma002972 ) and a2 ( atcc 6223 , wy963418 ) control strains ( 1,2,47,9 ) ( figure 1 ) .
the 5 clinical isolates fell into the 2 major type a clades ; 2 isolates were identified as a2 ( ut074632 , ut074633 ) , and 3 were identified as a1 ( ut074262 , ut074263 , ut074265 ) ( figure 1 ) .
pfge patterns for the 3 a1 isolates were indistinguishable from each other , as were patterns for the 2 a2 isolates ( figure 1 ) .
dendrogram based on pmei pulsed - field gel electrophoresis ( pfge ) patterns of francisella tularensis type a isolates .
the dendrogram was constructed by using dice similarity coefficients ( 1.5% optimization and 1.5% tolerance ) and unweighted pair group method with averages .
strains wy963418 , atcc 6223 , schu s4 , and ma002972 were included as known a1 and a2 controls for creation of the dendrogram .
control strains were previously identified as either a1 or a2 by multiple methods including multilocus variable number tandem repeat analysis , pfge , housekeeping gene sequence analysis , whole genome sequencing , and indel analysis ( 1,2,47,9 ) . a brief search of the exposure area yielded desiccated carcasses of 10 black - tailed jackrabbits ( lepus californicus ) and 2 desert cottontail rabbits ( sylvilagus audubonii ) .
the carcasses were found within a few hundred meters of each other , in an overall area < 0.8 km across . living deer flies ( chrysops spp . )
dna was extracted from lagomorph bone marrow and flies by using the qiaamp dna minikit ( qiagen , valencia , ca , usa ) and tested with real - time pcr f. tularensis multitarget , type a and type b assays ( 10,11 ) . although all deer fly samples were negative , 11 of 12 lagomorph carcasses tested positive for f. tularensis by the multitarget assay ( 3 of 3 targets positive ; crossing threshold ( ct ) range 1338 ) . among the infected samples , 9 tested positive for type a ( ct range 1336 ) and
the subtyping results were verified by sequencing of the succinate dehydrogenase gene ( sdha ) , which distinguishes type a and type b strains on the basis of a single nucleotide polymorphism ( 12 ) .
type b strains have a g at nt 465 of the sdha gene sequence , whereas type a strains have an a at this position . to further distinguish the type a samples between clade a1 or clade a2 , conventional pcrs were used ( 13 ) .
sufficient f. tularensis dna was present to type infections for 5 of the 9 type a  positive lagomorph carcasses ; 4 yielded a pcr product consistent with the a1 clade ( 570 bp ) , and 1 yielded a product consistent with the a2 clade ( 396 bp ) ( figure 2 ) ( 13 ) .
pcr typing of francisella tularensis , clades a1 and a2 , in dessicated lagomorph carcasses .
lane 1 , 100-bp ladder ( bio - rad , hercules , ca , usa ) ; lane 2 , a1 positive control ( schu s4 ) ; lane 3 , a1 negative control ( nm99 ) ; lane 4 , ut075156 ( a1 ) ; lane 5 , ut075152 ( a1 ) ; lane 6 , ut075157 ( a1 ) , lane 7 , ut075159 ( a1 ) , lane 8 , a2 positive control ( nm99 ) ; lane 9 , a2 negative control ( schu s4 ) ; lane 10 , ut075161 ( a2 ) .
few studies have reported on the diversity of f. tularensis subsp . or clades present during outbreaks of tularemia , in part because molecular methods for strain discrimination have only recently been described ( 1,2,47 ) . in this discrete deer fly 
associated outbreak , we found human infections caused by both a1 and a2 strains , and evidence that a1 , a2 , and type b strains were circulating among lagomorphs in the exposure area .
these findings demonstrate that multiple subspecies and clades can cause disease in a localized outbreak of tularemia and that deer - flies are associated with transmission of a1 strains .
published reports indicate that a1 and a2 strains are generally segregated into areas east and west of the rocky mountains , respectively , with some overlap in coastal california ( 1,2 ) . in contrast , our results demonstrate that a1 strains are present in areas of the intermountain west and at elevations > 1,200 m. this finding is supported by identification of an additional case of human tularemia in utah caused by an a1 strain in 1998 ( centers for disease control and prevention , unpub
our results indicate that a1 , a2 , and type b strains can coexist naturally within the same ecosystem , a paradox when compared with the segregation that appears to exist on a larger scale .
overall , these observations underscore the need for future studies to define the ecologic and evolutionary factors underlying the distributions of f. tularensis strains in north america .
although the role of deer flies as vectors of f. tularensis is well established , the dynamics of deer fly  associated outbreaks have not been well researched .
transmission of f. tularensis by deer flies is believed to be entirely mechanical , through contamination of the mouthparts .
long - term maintenance of f. tularensis has not been shown to occur in deer flies , and it is therefore not surprising that the deer flies we collected 3 weeks after the outbreak tested negative for this organism .
our findings suggest that deer flies nonselectively acquire and transmit whatever strains are circulating in enzootic hosts .
we postulate that , in this instance , an abundance of deer flies led to extensive feeding on many hosts , resulting in the simultaneous transmission of multiple strains .
high mortality rates among lagomorphs may have forced deer flies to seek alternate hosts , specifically muskrats , which are associated with type b strains and have been linked to outbreaks among trappers at utah lake ( 3 ) .
the co - occurrence of multiple subspecies and clades may be unique to arthropod - associated outbreaks of tularemia and not characteristic of outbreaks resulting from other modes of f. tularensis transmission , such as contaminated water .
further work is needed to determine whether our findings will apply to other deer fly  associated outbreaks or for outbreaks of tularemia associated with ticks , which are known to maintain as well as transmit f. tularensis .
notably , while investigating a tick - borne outbreak of presumed type b infections in south dakota , markowitz and colleagues found evidence of both type a and type b strains in dermacentor variabilis ticks collected from dogs ( 14 ) .
outbreaks involving multiple serotypes have been observed with other vector - borne pathogens , including dengue virus ( 15 ) , which suggests that amplification and transmission of multiple strains in a focal area may represent a general feature of some vector - borne diseases .
 in july 2007 , a deer fly  associated outbreak of tularemia occurred in utah . 
 human infections were caused by 2 clades ( a1 and a2 ) of francisella tularensis subsp . 
 tularensis . 
 lagomorph carcasses from the area yielded evidence of infection with a1 and a2 , as well as f. tularensis subsp . 
 holarctica . 
 these findings indicate that multiple subspecies and clades can cause disease in a localized outbreak of tularemia . 
The Outbreak
Conclusions