Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

Abstract

Background: Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella 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 was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results.

Objective: The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus.

Results: Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes.

Conclusions: 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 that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.

Fig 1. Experimental design.

Guinea pig infection and tick blood meal.

Fig 2. Experimental design: moulting and testing samples.

Real-time PCR and culture: □; Transmission Electron Microscopy: ●; Fluorescence In Situ Hybridization: ▲.

Fig 3. TEM micrographs of Ixodes ricinus pre-vitellogenic oocyte after feeding on guinea pigs infected with F. tularensis subsp. holarctica.

The morphology of some inclusions is typical of a Gram-negative bacterium, and their size is congruent with that of bacteria from the Francisella genus. m: mithocondrium, b: bacterium. Scale bar: A 0.22 μm, B 1.1 μm.

Fig 4. TEM micrographs of Ixodes ricinus pre-vitellogenic oocyte after feeding on guinea pigs infected with F. tularensis subsp. holarctica.

Note the presence of residual bodies into the large, phagocytic vacuole. Oo: oocyte, mv: microvilli, Tp: tunica propria. Scale bar: 0.37 μm.

Fig 5. FISH staining with a probe specific for Francisella tularensis in Dermacentor reticulatus oocytes after feeding on guinea pigs infected with F. tularensis subsp. holarctica.

(A) Image obtained from light transmission; (B) Overlay image with blue signal for F. tularensis (23S rRNA probe for F. tularensis labelled with the fluorochrome Cy5) and red signal for universal eubacterial probe EUB338; (C) 23S rRNA probe for all F. tularensis; (D) universal eubacterial probe EUB338. Scale bar: 200 μm.

Fig 6. FISH staining with a probe specific for Francisella tularensis in Ixodes ricinus salivary glands from female after feeding on guinea pigs infected with F. tularensis subsp. holarctica.

(A) Image obtained from light transmission; (B) Overlay image with blue signal for F. tularensis (23S rRNA probe for F. tularensis labelled with the fluorochrome Cy5) and red signal for universal eubacterial probe EUB338; (C) 23S rRNA probe for F. tularensis; (D) universal eubacterial probe EUB338. Scale bar: 100 μm.