Reproducible and Quantitative Model of Infection of Dermacentor variabilis with the Live Vaccine Strain of Francisella tularensis

Abstract

ABSTRACTPathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers ofFrancisella tularensisstrain LVS from mice intoDermacentor variabilisnymphs. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophagesin vitroor in mice to nymphs. Our data show that the LVS ΔpurMCDmutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCDmutant versus ΔpurMCDcomplement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCDmutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identifyF. tularensisgenes that are expressed or required in this unique environment.