Sample Collection

Mosquito larvae were sampled on August 28, 2008, in Örebro, an area where tularemia is endemic. Nine human tularemia cases (3.24/100,000 persons) were reported from Örebro County in June–September 2008 (www.smi.se/statistik/harpest). Two sampling locations were selected, Ormesta (WGS84; 59°16′12′′N, 15°16′48′′E) and Vattenparken (WGS84; 59°16′55′′N, 15°15′00′′E), on the basis of a geographic distribution study of human tularemia cases in the area (13). Both locations are situated at Lake Hjälmaren near the city of Örebro and are characterized by lush vegetation of reed belts and deciduous trees and bushes. Using a standard dipper, we collected mosquito larvae from shallow temporary water bodies in the transition zone between reed and willow bush habitats (Ormesta 1), in the deciduous forest (Ormesta 2), and in a ditch covered by bush and grass (Vattenparken).

Mosquito larvae from each water body were reared to adults in their original pond water (Ormesta 1, containers A and B; Ormesta 2, containers C, D, E, F, and G; and Vattenparken, container H). At the start of this study, time-zero samples of the original water from each container were collected and stored at –20°C. Emerging adult mosquitoes were collected by a mechanical aspirator, killed by freezing, and stored at –80ºC until species was identified. During identification, adult mosquitoes were kept cold on a chill table, illuminated by a cold light lamp, and identified to species based on morphologic features. Identified mosquitoes were sorted by area, species, and sex in pools of 1–50 specimens and returned to the –80ºC freezer.

DNA Extraction from Mosquitoes and Water Samples

For DNA extraction, 10 μL of 2.8 M NH₄OH solution and 450 mg each of 1.0-mm and 0.1-mm silica beads were added to each pooled mosquito sample. Samples were homogenized for 60 s (BeadBeater FastPrep; BioSpec Products, Inc., Bartlesville, OK, USA). The homogenized samples were incubated at room temperature for 15 min, and 60 μL of sterile water was added. DNA extraction was performed by using SoilMaster DNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions. The resulting DNA pellet was resuspended in 60 μL Tris EDTA buffer.

DNA extraction from water samples was performed as previously described (14). Two milliliters of each water sample was centrifuged at 16,000 × g for 1 h, 1.9 mL of the resulting supernatant was discarded, and DNA was extracted from the remaining volume by using a SoilMaster DNA Extraction Kit (Epicentre Biotechnologies).

PCR Analysis of Mosquitoes and Water

Mosquitoes and water samples were screened for F. tularensis by using a modified real-time PCR SYBR-based assay (Quanta BioSciences, Gaithersburg, MD, USA) for detection of the F. tularensis-specific lpnA gene. The PCR assay was modified from the methods of Thelaus et al. (11). Each reaction consisted of 1 µL template, 1× Quanta PerfeCTa SYBR Green FastMix (Quanta BioSciences), 400 nmol/L for each of the lpnA2F/R-primers (5′-CGCAGGTTTAGCGAGCTGTT-3′ and 5′-GAGCAGCAGCAGTATCTTTAGC-3′), and Milli-Q (Millipore, Billerica, MA, USA) up to 20 µL. An initial denaturation at 95°C for 5 min was followed by 40 cycles at 95°C for 10 s, 60°C for 30 s, and a melt curve 60°C–95°C on Mastercycler (Eppendorf, Hamburg, Germany).

Mosquitoes and water samples then underwent a F. tularensis holarctica–specific PCR, based on the 30-bp–deletion region FtM19 and using the FtM19InDelF/R primer pair, and modified from PCR (14). Each reaction consisted of 1–3 µL templates, 1x SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) 400 nmol/L for each of the primers FtM19Indel F/R (5′-GAATTACATAAAGTTCATGGTCCAGTAC-3′ and 5′-GTTTCAGAATTCATTTTTGTCCGTAA-3′) and Milli-Q (Millipore) water to give a final volume of 20 µL. An initial denaturation at 98°C for 2 min was followed by 50 cycles at 98°C for 5 s, 60°C for 5 s, and a melt curve 65°C–95°C on a Bio-Rad CFX96. Positive control mixtures, using DNA from F. tularensis holarctica and negative control mixtures without a template, were included in each PCR run.

Sequencing

The lpnA gene and FtM19InDel amplicons were cloned with TOPO TA cloning kit PCR4 (Invitrogen, Carlsbad, CA, USA) and sequenced. The acquired sequences were deposited in GenBank under accession nos. GY97987–GU97997 and HQ289871–HQ289876 (lpnA and FtM19InDel, respectively).

F. tularensis in Mosquitoes Hatched from Field-collected Larvae

Figure 1

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...

The 334 adult mosquitoes of 9 species hatched from mosquito larvae collected in the tularemia-endemic Örebro area were analyzed in 48 pools; 14 pools (29%) were positive for the F. tularensis lpnA gene (Table 1). Eleven of the 14 lpnA-positive samples were possible to sequence (Figure 1). All obtained sequences showed high sequence similarity (>97%) with F. tularensis in alignment with published sequences from representatives of subspecies of F. tularensis and their closest known relatives (i.e., Francisella-like endosymbionts).

We observed no difference in the F. tularensis detection rate between male (6/20 pools, 30%) and female (8/28 pools, 29%) mosquitoes (Table 1). Using the definitions in Schäfer et al. (15), we determined that the mosquito species belonged to 4 of 10 defined mosquito functional groups. Functional group 2a (snow-pool mosquitoes) and functional group 2b (floodwater mosquitoes) constitute most of the mosquitoes tested. Notably, the F. tularensis detection rate in female floodwater mosquitoes (6/13 pools, 46%) was higher than in female snow-pool mosquitoes (2/12 pools, 17%).

F. tularensis in Water

Water samples from 5 of the 8 water containers used for rearing were positive for the F. tularensis lpnA gene (Table 2). Four of these containers yielded mosquitoes that were positive for the F. tularensis lpnA gene. However, 3 water containers used for rearing that were negative for the F. tularensis lpnA gene, all yielded adult mosquitoes positive for the F. tularensis lpnA gene. Thus, there was no correlation between detection of F. tularensis in water from a specific container used for rearing and detection of F. tularensis in adult mosquitoes hatched from the container (Table 2).

Detection of Francisella tularenis spp. holarctica

Figure 2

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...

Using the FtM19InDel primers, we detected sequences specific for F. tularensis holarctica in 2 of the 8 water samples analyzed and 4 of the 48 mosquito pools (Figure 2; Table 2). Mosquito species positive for F. tularensis holarctica sequences were A. punctor (snow-pool mosquito), Ae. vexans, Ae. sticticus (both floodwater mosquitoes), and Culex pipens/torrentium (Table 1).