Francisella tularensis subspecies holarctica occurs in Swedish mosquitoes, persists through the developmental stages of laboratory-infected mosquitoes and is transmissible during blood feeding

Abstract

In Sweden, mosquitoes are considered the major vectors of the bacterium Francisella tularensis subsp. holarctica, which causes tularaemia. The aim of this study was to investigate whether mosquitoes acquire the bacterium as aquatic larvae and transmit the disease as adults. Mosquitoes sampled in a Swedish area where tularaemia is endemic (Örebro) were positive for the presence of F. tularensis deoxyribonucleic acid throughout the summer. Presence of the clinically relevant F. tularensis subsp. holarctica was confirmed in 11 out of the 14 mosquito species sampled. Experiments performed using laboratory-reared Aedes aegypti confirmed that F. tularensis subsp. holarctica was transstadially maintained from orally infected larvae to adult mosquitoes and that 25% of the adults exposed as larvae were positive for the presence of F. tularensis-specific sequences for at least 2 weeks. In addition, we found that F. tularensis subsp. holarctica was transmitted to 58% of the adult mosquitoes feeding on diseased mice. In a small-scale in vivo transmission experiment with F. tularensis subsp. holarctica-positive adult mosquitoes and susceptible mice, none of the animals developed tularaemia. However, we confirmed that there was transmission of the bacterium to blood vials by mosquitoes that had been exposed to the bacterium in the larval stage. Taken together, these results provide evidence that mosquitoes play a role in disease transmission in part of Sweden where tularaemia recurs.

Fig. 1

Experimental set-up for investigating the uptake and transstadial maintenance of F. tularensis subsp. holarctica in the mosquito A. aegypti. Percent of F. tularensis-positive mosquitoes during different developmental stages (fourth instar larva, pupa and adult mosquito). Data are summarized from three separate experiments involving between 59 and 68 mosquitoes in each run. Transmission of the transstadially maintained F. tularensis subsp. holarctica was investigated after mosquito feeding from an artificial source of blood. The mosquitoes that were exposed to F. tularensis subsp. holarctica as larvae were allowed to feed from blood vials. The results are presented as percent of blood vials positive for F. tularensis DNA after mosquito feeding

Fig. 2

Experiment to investigate the ability of the mosquito A. aegypti to transstadially and directly transmit F. tularensis subsp. holarctica to susceptible hosts. a Transstadial transmission of F. tularensis subsp. holarctica: mosquitoes exposed to the bacterium as larvae were allowed to feed on the blood of naïve mice. Chance of transmission is the number of mosquitoes that took a blood meal and were later identified as positive for F. tularensis. Transmission is the number of mice positive for F. tularensis at the end of the experiment. b Mosquito-mediated direct transmission of F. tularensis subsp. holarctica between hosts: mosquitoes were allowed to feed on the blood of mice infected with F. tularensis subsp. holarctica. Five days later, the mosquitoes were allowed to feed on blood of naïve mice in order to establish whether there was direct transmission. Uptake is the number of mosquitoes that took blood from infected mice and were later identified as positive for F. tularensis. In both experiments, mice were monitored for signs of disease for up to 25 days, and no traces of F. tularensis were detected in mouse spleen or blood samples analyzed using real-time PCR and culture methods (transmission). Mosquitoes were analyzed for the presence of F. tularensis and mouse DNA using PCR (uptake and chance of transmission)

Fig. 3

The first group of the mosquitoes collected in the field that was subjected to an initial PCR screening for the presence of F. tularensis. The number of mosquitoes caught on each sampling occasion in relation to the estimated IR per 1,000 mosquitoes collected. Dotted lines represent the calculated upper and lower IR limits. The F. tularensis infection rate were highest at 2 to 6 weeks after the peak in total number of mosquitoes

Fig. 4

Bacterial counts of F. tularensis subsp. holarctica in blood during mouse infection. Colony-forming units (cfu) of the bacterium F. tularensis subsp. holarctica in the mouse blood following subcutaneous injection (infectious dose of 27 cfu/ml)