No evidence of transovarial transmission of Francisella tularensis by tick vectors Dermacentor reticulatus and Ixodes ricinus

Due to its easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella tularensis is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis has been recognized since 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, conducted between 1920 and 1960, when molecular techniques had not been developed nor F. tularensis subspecies had been defined yet, have produced conflicting results. The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of fully virulent F. tularensis subsp. holarctica in experimentallyinfected females of Dermacentor reticulatus and Ixodes ricinus. The experimental design was performed in 6 replicates. A total of 150 D. reticulatus and 150 I. ricinus unfed questing adult female ticks were used. For each replicate, 2 guinea pigs were used. On Day -3, all animals were infected with 25 female ticks and 35 male ticks and on Day 0, the tick-infested guinea pigs were inoculated subcutaneously with 500 CFU of the bacterium suspended in 0.3 ml of sterile saline solution. After completion of the tick blood feeding, bacterial culture and real-time PCR confirmed the infection by F. tularensis subsp. holarctica in all animals. All ticks examined during and/or at the end of oviposition were positive by PCR and culture. PCR, culture, transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within tick oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, electron microscopy techniques revealed bacterial degeneration/death in the oocytes. These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but rather various biological niches (e.g. ticks, mosquitoes, rodents, lagomorphs, amoebae) that allow the bacterium to persist in the environment. Our results suggest that ticks are not competent for the vertical transmission of the bacterium and are congruent with this view. However, even in the absence of a transovarial transmission, ticks are able to maintain the infection in the environment during the inter-epizootic period and can be identified as long-term vectors of F. tularensis.