Transstadial transmission of Francisella tularensis holarctica in mosquitoes, Sweden

Abstract

In Sweden, human cases of tularemia caused by Francisella tularensis holarctica are assumed to be transmitted by mosquitoes, but how mosquito vectors acquire and transmit the bacterium is not clear. To determine how transmission of this bacterium occurs, mosquito larvae were collected in an area where tularemia is endemic, brought to the laboratory, and reared to adults in their original pond water. Screening of adult mosquitoes by real-time PCR demonstrated F. tularensis lpnA sequences in 14 of the 48 mosquito pools tested; lpnA sequences were demonstrated in 6 of 9 identified mosquito species. Further analysis confirmed the presence of F. tularensis holarctica-specific 30-bp deletion region sequences (FtM19inDel) in water from breeding containers and in 3 mosquito species (Aedes sticticus, Ae. vexans, and Ae. punctor) known to take blood from humans. Our results suggest that the mosquitoes that transmit F. tularensis holarctica during tularemia outbreaks acquire the bacterium already as larvae.

Figure 1

Multiple alignment of the 11 Francisella lpnA sequences obtained from mosquitoes in Sweden (hatched from field-collected larvae) with previously published sequences of Francisella species and subspecies, and Francisella-like endosymbionts (FLE). Boxed nucleotides represent target sequences of lpnA primers. The nucleotide positions 592–674 refer to F. tularensis holarctica live vaccine strain (LVS). Colors indicate individual nucleotides to clearly delineate those diverging from the F. tularensis holarctica LVS sequence. Reference sequences from GenBank; F. tularensis holarctica LVS (M32059), F. tularensis tularensis strain WY96-3418 (CP000608), F. tularensis tularensis strain Schu S4 (NC_006570), F. tularensis mediasiatica strain FSC147 (NC_010677), F. tularensis novicida strain U112 (CP000439), Amblyomma maculatum FLE (AY375422), Dermacentor hunteri FLE (AY375417), D. andersonii FLE (AY375413), D. variabilis FLE (AY375420), D. variabilis FLE (AY375421), F. philomiragia (AY243030), and F. piscicida (DQ825765).

Figure 2

Multiple alignments of the 11 Francisella lpnA sequences (designated OMA_xxx) obtained from mosquitoes and water samples with previously published sequences of Francisella species and subspecies. Boxed nucleotides represent target sequences of FtM19InDel primers. In this alignment the F. tularensis subsp. holarctica specific deletion is located from position 57 to 87. Colors indicate individual nucleotides. Reference sequences from GenBank: F. tularensis holarctica LVS(M32059), F. tularensis subsp. tularensis strain WY96-3418 (CP000608), F. tularensis subsp. tularensis strain Schu S4 (NC_006570), F. tularensis mediasiatica strain FSC147 (NC_010677), F. tularensis novicida strain U112 (CP000439), and F. philomiragia (AY243030).