University Hospital of Frankfurt - Mosquito Study

By Ticks & Tick-Borne Diseases Journal & ScienceDirect

INTRODUCTION: Occurrence of Borrelia burgdorferi s.l. in different genera of mosquitoes (Culicidae) in Central Europe

In  the second half of the 20th century, infectious diseases became increasingly important. About 15 million (>25%) of 57 mil- lion  annual deaths worldwide are  estimated to be directly related to  emerging and re-emerging infectious diseases (Morens et al., 2004). Of these, an accelerating number is transmitted by obligate haematophagous arthropods with mosquitoes and ticks as the most crucial vectors. Lyme  borreliosis is  a multisystemic infectious disease caused by distinct spirochetes belonging to  the Borrelia burgdorferi sensu lato (s.l.)  complex, which are   transmitted by  hard ticks (Ixodi- dae).

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Fig. 1. Distribution of the detected Borrelia species. Borrelia afzelii: blue circles, B. bavariensis: green circles, B. garinii: red circles. The species detection was based on the detection of fragments of the flaB and ospA genes. The circle size varies for better illustration. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Positive tested mosquito species and detected Borrelia species. The corre- sponding (reservoir-) hosts of borreliae are shown as shadows of the vertebrate class; feeding habits of the mosquito species, as far as known, are shown behind each species as crosses.

Early signs of the disease often include unspecific symptoms such as  headache, fever and fatigue. After 3–30 days, 70–80% of infected individuals developed a ring-like skin rash at the ticks bite site (erythema migrans). If left untreated, the pathogens can disseminate, leading to more severe symptoms involving diverse organs, e.g. the heart, the joints, and the central nervous system.

The  B. burgdorferi s.l. complex comprises a group of at least 20 genospecies worldwide (Casjens et al., 2011; Skuballa et al., 2012; Ivanova et al., 2014). Regarding their potential to cause the disease, the 20 genospecies can be divided into two groups of which the first group consists of five genospecies frequently isolated from patient samples while the second group comprises genospecies with either uncertain pathogenic potential or that have never been detected in human samples (Rudenko et al., 2011).

Among human pathogenic genospecies, Borrelia garinii, B. burgdorferi, and Borrelia afzelii, the three main European ones are  maintained by birds or small mammals. Borrelia bavariensis, a serotype formerly regarded as B. garinii OspA type 4 is also adapted to rodents (Skuballa et al., 2012; Margos et al., 2013). Of these, different genospecies can  be present simultaneously in  a  single tick  (Rauter and Hartung, 2005; Wodecka et al.,  2010).

This  is  of  particular interest as  genospecies can be associated with different clinical manifestations, e.g.  B. burgdorferi sensu stricto (s.s.)  is often associated with arthritis, B. garinii with neurological manifestation, B. afzelii almost always induces acrodermatitis chronica atrophicans (Skotarczak, 2014) and  B. bavariensis has been linked to neuroborreliosis (Margos et al., 2013).

Therefore, an infection with various genospecies may result in the manifestation of multiple disease symptoms. Although ticks are the prominent transmission vectors of these particular pathogens, borreliae have also been detected in other arthropods, especially in  mosquitoes.

Mosquitoes have been known since the late 19th century to be the vectors for a number of several human diseases, such as malaria, dengue, chikungunya or yellow fever. Recently, it has been demonstrated that human cases of tularemia caused by  Francisella tularensis holarctica are transmitted by mosquitoes (Lundström et al., 2011; Thelaus et al., 2014).

Especially in Germany, there is a lack of knowledge which pathogens can be successfully transmitted by diverse mosquito species. In mosquitoes, Spirochetes were found for the first time, in a Culex species in  1907 (Jaffé,  1907). Concerning spirochetes of the B. burgdorferi s.l. complex, different genospecies could be identified in mosquitoes already (Halouzka et al., 1999; Zˇ ákovska et al., 2004).

So far the maximal periods of survival for borreliae in naturally infected mosquitoes are still unknown (Magnarelli and Anderson, 1988). In experimentally fed mosquitoes borreliae could be detected up to 14 days after a blood meal in two species (Aedes aegypti and Aedes triseriatus) (Magnarelli et al., 1987). Additionally, there is one report of a patient who developed erythema migrans after a mosquito bite (Hård, 1966).

Owing to the very different feeding time and frequency of mosquitoes and ticks, Matuschka and Richter (2002) postulated that transmission of Borrelia species by mosquitoes appears to be impossible, with the result that research on this topic stopped nearly completely for over a decade. This  is  astonishing, as it is still unknown whether these insects may be  able to play a small role influencing the ecology and epidemiology of Lyme borreliosis.

Aim of this study is to check whether borreliae can be found in different species or even genera of mosquitoes with different feeding habits. Therefore, anthropophilic, mammalophilic as well as ornithophilic species have been screened for the presence of borreliae.

In addition, we  sought to  examine, whether borreliae can also be detected directly after the metamorphosis in mosquitoes, as  transstadial transmission is known in  ticks, which is a crucial step for a vector-borne pathogen to be transmitted later on.

2. Material and methods

2.1.  Collection of samples

Mosquitoes were sampled from April to October 2013 at 42 sampling sites in Germany during a  mosquito monitoring program, which included the detection of possible pathogens. Most of these sites were in Saxony (n = 9) followed by Brandenburg (n = 6) and Hesse (n = 15)  (SI 1).

Individuals  were collected  using EVS-traps with dry ice, BG-SentinelTM traps (Biogents AG, Regensburg, Germany) with CO2 as an attractant, hand nets and by human bait (Supplemental Table  1).

Larvae were collected in natural (e.g. puddles, ponds, tree holes) as well as in artificial (e.g. vases, rain barrels) water pools using hand nets. After  capture, larvae were kept alive and raised to adults. For this study, no specific permissions were required and no endangered or protected species are  involved.

Supplementary Table 1 related to this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.10.018.

2.2.  Morphological identification

Morphological identification of mosquito species was carried out with a stereomicroscope. The morphological characters of each specimen were analyzed using two identification keys (Mohrig, 1969; Becker et al., 2010).

2.3.  DNA-extraction

For DNA-extraction, morphologically identified mosquitoes were pooled by  species, collection date and collection site,  each pool contained up  to  ten individuals. Samples were homogenized individually with a tissue mill  (MM400, Retsch GmbH, Germany) and 2  stainless steel beads (3 mm, VWR, Germany) for 2 min at 25 Hz. Subsequent DNA-extraction was carried out with glass fiber plates (Pall  GmBH,  Dreieich, Germany) following the protocol of Ivanova et al. (2006).

2.4.  Detection of borrelial DNA by nested PCR

Nested PCR was applied to detect two different borrelial genes, flaB and ospA in  the collected samples. The flaB fragments were amplified using primers FL3/5 and FL6/7and primer pairs N1/C1 and N2/C2  were used to identify the ospA sequences (Table 1) (Picken, 1992; Rijpkema et al., 1997; Zˇ ákovska et al., 2004). The reaction mixtures for primers FL3/5 as well as N1/C1  (both 25    l) consisted of  12.5   l  Master Mix  (Peqlab  Biotechnology GmbH, Erlangen, Germany) containing 0.4 mM dNTP, 4 mM MgCl2, 40 mM Tris–HCl, 32 mM  (NH4 )2 SO4 , 0.02%  Tween 20  and 1.25 U Taq-Polymerase, 1    l of each primer (10 pmol  l−1 ) 9    l ddH2 O and 1.5    l genomic DNA. The  reactions for  the FL6/7  nested PCR contained 12.5    l Master Mix,  1    l of each primer (10 pmol  l−1 ) 5.5    l ddH2 O and 5   l PCR-product of  FL3/5.  The  amplification of  N2/C2  was per- formed with 12.5    l Master Mix, 1    l of each primer (10 pmol   l−1 ) 9    l ddH2 O and 1.5    l PCR-product of N1/C1.  For FL6/7  the cycle parameters followed the protocol of Picken et al. (1996): FL3/5 = 1 cycle  of 94 ◦ C, 12 min; 30  cycles of 94 ◦ C, 1 min; 70 ◦ C, 2 min and 72 ◦ C, 3 min followed by terminal extension of 72 ◦ C, 7 min and a final ramping to  8 ◦ C; FL6/7 = 1  cycle  of  94 ◦ C, 12 min; 30  cycles of  94 ◦ C, 1 min; 54 ◦ C, 2 min and 72 ◦ C, 3 min, final extension at 72 ◦ C, 7 min and a final ramping to  8 ◦ C; N1/C1 = 1 cycle  of 37 ◦ C, 5 min and 94 ◦ C for  10 min; 30  cycles of 94 ◦ C, 1 min; 45 ◦ C, 1 min and 72 ◦ C (extended for  5 s with each cycle), 1 min, final exten- sion at 72 ◦ C, 5 min and a  final ramping to  8 ◦ C; N2/C2 = 1  cycle of 94 ◦ C, 10 min; 25  cycles of 94 ◦ C, 1 min; 43 ◦ C, 1 min and 72 ◦ C, 1 min, and a final extension at 72 ◦ C for  5 min and a final ramp- ing  to 8 ◦ C (slightly modified after Rijpkema et al. (1997)).

In each PCR attempt, a positive as well a negative control were performed to rule out the possibility of  laboratory contamination.  Quality and yield of PCR products was analyzed by Midori Green (Nippon Genetic EUROPE GmbH) staining and agarose gel-electrophoresis. For subsequent Sanger-sequencing reactions, with product specific  forward primers, positive samples were purified using the peqGOLD  Cycle-Pure Kit (Peqlab Biotechnology GmbH, Erlangen, Germany).

Each obtained sequence was edited using BioEdit (Hall, 1999) and compared with sequences deposit in GenBank using the BLAST algorithm (Altschul et al., 1997). The sequences obtained were deposited in EMBL Nucleotide Sequence Database under accession numbers LN650604–LN650631.

3. Results

In total 3615 adult mosquitoes were morphologically identified (Supplemental Table  2)  and analyzed, including 74  adults reared from field-caught larvae in the laboratory. Specimens which could not be identified morphologically to species level because of their poor state of preservation were analyzed individually.

Supplementary Table  2 related to  this article can  be  found, in the online version, at doi:10.1016/j.ttbdis.2015.10.018.

For  all  analyses, no  distinction was made between Culex pipiens  and Culex torrentium and specimens were treated as  pools of “Cx. pipiens/torrentium” as well as between Ochlerotatus annulipes and  Ochlerotatus cantans, which were treated as pools of  “Oc. annulipes/cantans”. The analyzed adult mosquitoes belong to the genera Aedes  (npools = 265), Anopheles (npools = 12), Coquillettidia

Table 1

Primers used for  the detection of Borrelia species.

Primer Target gene Primer sequence (5   → 3 ) Fragment length [bp]

(npools = 12), Culex (npools = 116), Culiseta (npools  = 7) and Ochlerotatus

(npools = 270).

All pools were analyzed for  the presence of borrelial DNA targeting the flaB and ospA genes. Of the 682 tested pools, seventeen pools were positive for flaB and eleven for ospA.

Sequence analysis and comparison with those deposited in GenBank revealed positive results for B. afzelii, B. bavariensis as well as B. garinii. borreliae were detected in eight out of 265  Aedes spp. pools, with a total of 1861 specimens of wild-caught adults as well as in eighteen of 270 Ochlerotatus spp. pools, with a  total of 865 individuals. We also found borrelial DNA in  one of 116  Culex spp. pools, consisting of 796 specimens, and in one of twelve specimens of Culiseta spp.

In total, detection of borrelial DNA was successful in 25 of 682 adult field-caught mosquito pools and three pools from laboratory-raised specimens, which evaluated the nested PCR method positively for the detection of  borreliae in mosquitoes. Out of the laboratory- raised pools, three samples of Ochlerotatus (two single individuals and one two specimen pool) were positive for B. burgdorferi s.l.

The most frequent species was B. garinii, which was found in twelve mosquito pools; B. afzelii could be detected in six mosquito pools consisting of four  species and in all three positive laboratory-raised pools. B. bavariensis DNA could be  detected in seven pools. Table 2 shows the occurrence of the different Borrelia species in the respective mosquito species.

The distribution pattern of the detected Borrelia species (B. afzelii, B. bavariensis, and B. garinii) is summarized in Fig. 1. B. afzelii was more frequently found in the southern and western collection sites of Germany, while B. bavariensis appears to be the dominant species at the eastern sampling sites. A diverse distribution could be suggested for B. garinii which was found in southwestern, southern as well as in eastern localities. The hosts of the identified Borrelia and mosquito species are  shown in Fig. 2.

4. Discussion

About 51 mosquito species are known in Germany of which 24 were detected during the present study. Differences in the number of species identified at the distinct collection sites may be attributed to differences in  mosquito abundance. The  observed differences can most likely be connected to  sampling at altered time points, variation of species activity or various collection methods used.

Previously published  epidemiological data indicate that haematophagous insects could be involved in the transmission of borreliae in some areas (Magnarelli et al.,  1986; Kosik-Bogacka et al., 2002). But so far the role of  arthropods other than ticks in  the transmission cycle of borreliae remains unclear, although a  few  reports have been published on cases of Lyme borreliosis following an insect bite (Doby et al.,  1986; Hård, 1966; Luger, 1990).

So, borreliae have already been detected in previous studies on mosquitoes in the United States, where Oc. stimulans females from Norwich contained DNA  of  B. burgdorferi (Magnarelli  and Anderson, 1988). In the Czech  Republic, motile borreliae have been isolated from Aedes cinereus, Ae. vexans, Oc. cantans, Ochlerotatus communis and Ochlerotatus sticticus as well as the Cx. pipiens complex (Halouzka, 1998; Halouzka et al., 1998; Zˇ ákovska et al., 2006).

The detection of numerous spirochaete species in salivary glands of Oc. cantans (Zeman, 1998) resembles previous results, which showed spirochetes in British anophelines (Sinton and Shute, 1939). We  found borrelial DNA  in  ten mosquito species comprising four  genera.

In  total in 25 of 682 adult field-caught mosquito pools and three pools from laboratory-raised specimens. Out of the laboratory-raised pools, three samples of  Ochlerotatus (two single individuals and one two specimen pool) were positive for  Borrelia spp.  In total borreliae were detected in  ten mosquito  species of which most  show mainly mammalo-  or anthropophilic feeding habits.

The identification of spirochetes in the salivary glands suggests that mosquitoes may potentially transmit on occasion bacteria. In ixodid ticks, borreliae can be transmitted by different pathways: transstadial, feeding and co- feeding (Vennestrøm et al., 2008). At least feeding and transstadial (although not a  sort of  transmission as just one individual is involved) could be possible pathways along with insects, in particular mosquitoes.

In ticks, the outer surface protein OspA appears to be responsible for the attachment of the spirochetes to the midgut of the ticks by the TROPSA receptor (Li et al., 2007). When the tick begins to feed and the spirochetes in the midgut begin to multiply, most spirochetes cease expressing OspA  on their surfaces while beginning simultaneously expressing OspC and migrate to the salivary gland.

This upregulation of OspC begins during the first day of feeding and peaks 48 h after attachment (Schwan and Piesman, 2000). OspC, which interacts with the salivary protein Salp15 (that way the spirochete gets protected from complement-mediated killing [Schuijt et al., 2008]) plays an essential role during the very early stage of  mammalian infection (Tilly  et al.,  2006) and may also be necessary to allow B. burgdorferi to invade and attach to the salivary gland after leaving the midgut of the tick  (Pal  et al., 2004a; Grimm et al., 2004).

However, neither Salp15 nor the tick gut epithelial cell protein TROSPA (Pal  et al., 2004b), is known to be  present in Culicidae, as well as no homologous proteins have been described so far in Culicidae.  Thus, it is tempting to speculate that other, unrelated proteins might act  as  potential ligands for OspA and OspC.

Most studies on the presence of Borrelia spp. in Culicidae have been done in the Czech Republic. The infection rate in these previous studies was between 0.7% and 7.6% for adults. Nejedlá et al. (2009) reported that 3.3% of the analyzed mosquitoes (Aedes spp., Culex spp.,  Ochlerotatus spp.) were positive for  spirochetes using dark field microscopy (DFM) of which 0.7% being positive for borreliae in a subsequent PCR.

In other studies, the rate ranged from 0.7  to  7.6% (Sanogo et al.,  2000). For northeastern Poland, the rates were 1.1% for  Aedes  spp. and 0.3% for Culex  spp. (Nejedlá et al., 2009) and according to another study, in 1.25% of the Aedes spp. and Culex spp.

In Poland, borrelial DNA was detected (Kosik- Bogacka et al., 2002). In later studies 0.8% of the tested mosquitoes were positive for borreliae (Kosik-Bogacka et al., 2004, 2006, 2007). This is similar to our findings; however, assuming the minimum of one infected specimen per pool would result in the total percent- age within the same range of previous studies. Nevertheless, the number of infected specimens in the recent study might be much higher as we can give only information about the possible range of infected specimens.

Therefore a direct comparison of the values is not possible owing to the design of the present study to analyze pooled samples in contrast to investigate individual mosquitoes. In our investigation no big variation has been shown in the infection rate between the distinct mosquito species.

The calculated infection rates of borreliae seem to largely depend on the method of choice. There are some studies in which the presence of spirochetes was determined using DFM in comparison with the identification of borrelial DNA in the respective sample by PCR (Kosik-Bogacka et al., 2007).

While the percentage of spirochetes was relatively high when analyzed by DFM, the numbers are lower when analyzed by PCR, because of the relatively low sensitivity of the single step PCR approach and by the fact that with DFM borreliae cannot be distinguished from other spirochetes. Limitation in the detection of Borrelia infections in ticks has  already been reported (Kahl  et al.,  1998; Cisak  et al.,  2006).

The same reason may have influenced the results of the present study, especially the difficulties in amplifying parts of both gene fragments. The fact that only low numbers of borreliae can be present (34% of positive ticks harbor less than 10  borreliae [Hubalek and Halouzka, 1998]), can therefore lead to false-negative results.

Previously there has to be a minimum of about 130  borreliae in a given sample in order to obtain positive PCR results (Nejedlá et al., 2009). Another problem is the lack of standardization of PCR for the identification of Borrelia spirochetes. This lack makes it impossible to  compare the reports on this subject matter. PCR detection of B. burgdorferi s.l. uses various molecular markers, which show varying results (Wodecka et al., 2010).

These already in the literature mentioned varying results are a matter that explains also,  why the PCR results for both gene fragments vary in this study, as the sensitivity for both gene fragments varies greatly.

In the present study, the distribution of the detected species (B. afzelii, B. bavariensis, B. garinii) is in line with previously published data where B. afzelii has been reported to be the most frequent species in  ticks in the Bonn area (Maetzel et al., 2005), which is situated north-west of  our  sampling area.

In another study, the prevalence of borreliae in ticks in Thuringia has been analyzed where B. garinii was the predominant species (Hildebrandt et al., 2003).

Thuringia is situated between the different sampling sites in the present study and the results of  the actual study show a tendency that the abundance of B. garinii/B. bavariensis increases from southern to  central/northern Germany and vice  versa for B. afzelii, which was previously described for  B. garinii and B. afzelii (Rauter and Hartung, 2005).

However, the distribution pattern of the two species does not reflect data reported for Saxony (Bigl et al., 1999), Bonn  (Kurtenbach et al.,  2001; Maetzel et al.,  2005) and the Rhine-Main area (Bingsohn et al.,  2013). In those studies, B. afzelii was detected more frequently than B. garinii.

The distribution of the various species shows spatial and temporal differences, which depend mainly on the activity of the ticks and the presence of adequate reservoir hosts (Gern, 2009; Rizzoli  et al., 2011). Therefore in a certain area where B. garinii predominates, B. afzelii temporarily become the dominant species (Gern, 2009).

In any case, the distribution and prevalence of  various borrelial genospecies varies on a local  and regional scale (Rizzoli et al., 2011), what influences to some extent the appearance of certain symptoms of Lyme borreliosis in a given region. The regional distribution is also of interest because the different Borrelia species are associated with particular reservoir hosts, e.g. B. garinii with birds while B. afzelii and B. bavariensis tend to be associated with rodents (Rizzoli et al., 2011; Rudenko et al., 2011).

One of these two species is in every sampled area of the present study the predominant species (B. afzelii in the west, B. bavariensis in the east). These localities were mainly of rural character where certain mice species are  common, what can explain the distribution. Nevertheless, it has to be taken into consideration that since B. bavariensis, formerly B. garinii OspA type 4, has only recently been classified as a distinct genospecies (Rudenko et al., 2011), it cannot be excluded that sequences deposited in Genbank, which are attributed to  B. garinii, may actually belong to  B. bavariensis.

Culicidae are not considered as  possible vectors for borreliae. Anyway, we could detect borrelial DNA in laboratory-raised adults, which have never fed on blood and have been collected as larvae.

This finding suggests, although it gives no  information about possible vector abilities, at least that there is a mode how borreliae can persist in Culicidae, even from the larval to the adult stage.

The capacity of ticks to successfully transmit borreliae depends on several factors. The blood-feeding act of ticks takes much longer than the one of mosquitoes. Mosquitoes are vessel feeders and generally cannulate dermal capillaries, drawing blood directly from the vessel lumen and rarely from the interstitial tissue.

Spirochetemia caused by B. burgdorferi s.l. is a transient, process of short duration whereas spirochetes can persist in the skin for a very long time over years as shown in patients with Acrodermatitis chronica atrophicans. Thus, the destruction of skin tissue by tick  feeding facilitates the uptake of borreliae to a much greater degree than does the cannulation of capillaries by mosquitoes.

For these reasons, it has been previously suggested that mosquitoes do not play a role as competent vectors (Matuschka and Richter, 2002).

These works did not take into account that also various biotic and abiotic factors, such as climatic conditions, vegetation type and human management of the area (by direct influence on the behavior/activity of the vector), as well as host behavior, abundance and susceptibility influence the transmission (Gern, 2009).

While it has been clearly established, that ticks are the primary vectors of Lyme disease spirochetes, it may be possible that mosquitoes play a role as an occasional mechanical vector.

Borreliae have already been found in  overwintering female mosquitoes (Halouzka et al., 1998; Sanogo et al.,  2000) and larvae (Zˇ ákovska et al., 2004) supporting the results of the present study in which laboratory-reared adults were tested positive for borreliae, although they lacked any opportunity to get infected via a blood meal. This may also indicate a transstadial and/or transovarial transmission within mosquito populations.

In summary, here we  show that different mosquito species and even genera harbor borrelial DNA from B. afzelli,  B. garinii, and B. bavariensis. The questions whether mosquitoes can serve as a secondary mechanical vector remains unclear.

Thus further studies are required to confirm natural transmission of spirochetes via mosquitoes to a competent host.

Our results also raise the questions, if mosquitoes have functional-related proteins enabling at least the attachment and survival of borreliae. With the detection of borreliae in laboratory hatched specimens, we could show for the first time that borreliae endure the metamorphosis from larvae to pupae and finally to  adults in mosquitoes, as the adult specimens were analyzed without having a prior blood meal. Nevertheless, the question of how mosquito larvae acquire borreliae remains unclear and is a focus of ongoing studies.

Table 2

Distribution of the identified Borrelia species in the respective mosquitoes. The table summarizes the sampling point, the accession numbers as well as the strain with the highest similarity found in GenBank and their respective accession number. Borrelia species have been identified by  nested-PCR targeting the flaB and ospA genes.


Gene Borrelia species Mosquito species Number of pooled individuals Sampling point Length of sequence Accession number comparison Strain/isolate Accession number Similarity
FL B. afzelii Ae. cinereus 2 Wielenbach 151 KJ810661.1 Not stated LN650604 100
Oc. communis 10 Wielenbach 191 KJ810661.1 Not stated LN650605 99
Oc. sticticus 1 Wielenbach 162 CP009212.1 Tom3107 LN650606 99
Oc. sp 2 Rhön 201 CP009212.1 Tom3107 LN650607 99
Oc. sp 1 Nussloch 168 KJ810661.1 Not stated LN650608 100
Oc. sp 1 Nussloch 136 KJ810661.1 Not stated LN650609 99
B. garinii Ae. cinereus 10 Görlitz 3 149 KF894055.1 iso: 66-S-12 LN650612 100
Ae. vexans



Ae. vexans





Neißewiesen Hagen- werder Neißewiesen 215








iso: 66-S-12



iso: 66-S-12











Ae. vexans




Hagen- werder Berlin  








iso: 66-S-12







Ae. vexans 10 Berlin 165 KF894055.1 iso: 66-S-12 LN650616 100
Ae./Oc. sp



Ae./Oc. sp





Neißewiesen Hagen- werder










iso: 66-S-12



iso: 66-S-12









Cs. subochrea 1 Darmstadt 124 KF894055.1 iso: 66-S-12 LN650611 100
Cx. pipi- ens/torrentium Oc. communis 1












Not stated


iso: 66-S-12









Oc. communis




Hagen- werder Neißewiesen  








iso: 66-S-12







Hagen- werder
OspA B. afzelii Ae. cinereus 2 Wielenbach 276 GU826936.1 iso: 991 LN650621 99
Oc. caspius 10 Berlin 134 FJ546608.1 Strain IPT179 LN650624 100
Oc. sticticus 10 Haarsee 184 FJ546608.1 Strain IPT179 LN650622 99
B. bavariensis Ae. vexans


Oc. cataphylla













Strain mutant


Strain mutant








Oc. communis










Strain mutant






Oc. communis



(Wiese) Ullersdorf  





Strain mutant






Oc. dorsalis










Strain mutant






Oc. geniculatus










Strain mutant






Oc. sticticus




Görlitz 3






Strain mutant






B. garinii


Oc. sticticus
















Conflict of interest

No one of the authors has  any  conflicts of interest.


This  research was funded by  the ERA-Net  BiodivERsA, with the  national  funders German Research Foundation  (DFG   KL 2087/6-1), Austrian Sciend Fund (FWF  I-1437) and the French National  Research  Agency (ANR-13-EBID-0007-01), part of the 2013  BiodivERsA call for research proposals, by the research funding programme “LOEWE–Landes-Offensive zur  Entwicklung Wissenschaftlich-ökonomischer  Exzellenz” of Hesse’s Ministry of Higher Education, Research, and the Arts, and by the Senate Competition  Committee  grant  (SAW-2014-SGN-3) of the Leibniz Association. Also we want to thank Arno Koenigs (Institute of Medical  Microbiology and Infection Control, Frankfurt am  Main) for proof reading of the manuscript.


Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.  25,  3389–3402.

Becker, N., Petric, D., Zgomba, M., Boase, C., Dahl, C., Lane, J., Kaiser, A., 2010. Mosquitoes and Their Control. Kluwer Academic/Plenum, New York, NY.

Bigl, S., Müller, L., Pönitz, G., Mickel, C., Klapper, B.M., 1999. Untersuchungen zur Epidemiologie der Borreliose im Freistaat Sachsen 1997 Verbreitung von Borrelia burgdorferi in Zecken. Bundesgesundheitsbln Gesundh. Gesundh. 42, 219–225.

Bingsohn, L., Beckert, A., Zehner, R., Kuch, U., Oehme, R., Kraiczy, P., Amendt, J.,

  1. Prevalences of tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in Ixodes ricinus populations of the Rhine-Main region, Germany. Ticks Tick Borne Dis. 4, 207–213,

Casjens, S.R., Fraser-Liggett, C.M., Mongodin, E.F., Qiu,  W.G.,  Dunn, J.J., Luft,  B.J., Schutzer, S.E., 2011. Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate. J. Bacteriol. 193, 1489–1490.

Cisak, E., Wójcik-Fatla, A., Stojek, N.M.,  Chmielewska-Badora, J., Zwolin´ ski,  J., Buczek, A., Dutkiewicz, J., 2006. Prevalence of Borrelia burgdorferi genospecies in Ixodes ricinus ticks from Lublin region (Eastern Poland). Ann. Agric. Environ. Med. 13,  301–306.

Doby, J.M., Anderson, J.F., Coutarmanach, A., Magnarelli, L.A., Martin, A., 1986 Lyme disease in Canada with possible transmission by  an insect. Zbl. Bakt. Hyg. A 263, 488–490.

Gern, L., 2009. Life cycle of Borrelia burgdorferi sensu lato and transmission to humans. In: Lipsker, D., Jaulhac, B. (Eds.), Lyme Borreliosis. Curr. Probl. Dermatol. 37,  18–30.

Grimm, D., Tilly,  K., Byram, R., Stewart, P.E., Krum, J.G., Bueschel, D.M.,  Schwan, T.G., Policastro, P.F., Elias, A.F., Rosa, P.A., 2004. Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for  infection of mammals. Proc. Natl. Acad. Sci. U. S. A. 101, 3142–3147, 0306845101.

Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for  Windows 95/98/NT. Nucleic Acids Symp. Ser.  41,  95–98.

Halouzka, J., 1998. Borreliae in Aedes  vexans and hibernating Culex  pipiens molestus mosquitoes. Biologia (Bratisl.) 48,  123–124.

Halouzka, J., Postic, D., Hubálek, Z., 1998. Isolation of the spirochaete Borrelia afzelii from the mosquito Aedes  vexans in the Czech Republic. Med. Vet.  Entomol. 12, 103–105.

Halouzka, J., Wilske, B., Stünzner, D., Sanoge, Y.O., Hubálek, Z., 1999. Isolation of Borrelia afzelii from overwintering Culex  pipiens biotype molestus mosquitoes. Infection 27,  275–277.

Hård, S., 1966. Erythema chronicum migrans (Afzelii) associated with mosquito bite. Acta Derm. Venerol. 46,  473–476.

Hildebrandt, A., Schmidt, K.H., Wilske, B., Dorn, W., Straube, E., Fingerle, V., 2003. Prevalence of four species of Borrelia burgdorferi sensu lato and coinfection with Anaplasma phagocytophila in Ixodes ricinus ticks in central Germany. Eur. J. Microbiol. Infect. Dis.  22,  364–367.

Hubalek, Z., Halouzka, J., 1998. Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe. Parasitol. Res.  84,  167–172.

Ivanova, L.B., Tomova, A., González-Acun˜ a, D., Murúa, R., Moreno, C.X., Hernández, C., Cabello, J., Cabello, C., Daniels, T.J., Godfrey, H.P., Cabello, F.C., 2014. Borrelia chilensis, a new member of the Borrelia burgdorferi sensu lato complex that extends the range of this genospecies in the southern hemisphere. Environ. Microbiol. 16,  1069–1080,

Ivanova, N.V., deWaard, J., Hebert, P.D.N.,  2006. An inexpensive, automation-friendly protocol for  recovering high quality DNA. Mol. Ecol.  Notes 6, 998–1002.

Jaffé,  J., 1907. Spirochaeta culicis nov. spec. Arch. F. Protist. 9, 100–107.

Kahl, O., Gern, L., Gray, J.S., Guy, E.C., Jongejan, F., Kirstein, F., Kurtenbach, K., Rijpkema, S.G.T., Stanek, G., 1998. Detection of Borrelia burgdorferi sensu lato in ticks: immunofluorescence assay versus polymerase chain reaction. Zentralbl. Bacteriol. Microbiol. Hyg. 287, 205–210.

Kosik-Bogacka, D., Bukowska, K., Kuz´ na-Grygiel, W., 2002. Detection of Borrelia burgdorferi sensu lato in mosquitoes (Culicidae) in recreation areas of the city of Szczecin. Ann. Agric. Environ. Med. 9, 55–57.

Kosik-Bogacka, D., Kuz´ na-Grygiel, W., Bukowska, K., 2004. The prevalence of spirochaete Borrelia burgdorferi sensu lato in ticks Ixodes ricinus and mosquitoes Aëdes  spp. within a selected recreational area in the city of Szczecin. Ann. Agric. Environ. Med. 11,  105–108.

Kosik-Bogacka, D., Kuz´ na-Grygiel, W., Górnik, K., 2006. Borrelia burgdorferi sensu lato infection in mosquitoes from Szczecin area. Folia Biol.  54,  55–59.

Kosik-Bogacka, D., Kuz´ na-Grygiel, W., Jamborowska, M., 2007. Ticks and mosquitoes as vectors of Borrelia burgdorferi s.l. in the forested areas of Szczecin. Folia Biol.  55,  143–146.

Kurtenbach, K., De Michelis, S., Sewell, H.S., Etti, S., Schäfer, S.M., Hails, R., Collares-Pereira, M., Santos-Reis, M., Haninc¸ ová, K., Labuda, M., Bormane, A., Donaghy, M., 2001. Distinct combinations of Borrelia burgdorferi sensu lato genospecies found in individual questing ticks from Europe. Appl. Environ. Microbiol. 67,  4926–4929.

Li, X., Neelakanta, G., Liu, X., Beck, D.S., Kantor, F.S., Fish, D., Anderson, J.F., Fikrig, E.,

  1. Role of outer surface protein D in the Borrelia burgdorferi life cycle. Infect. Immun. 75, 4237–4244,

Luger, S.W.,  1990. Lyme disease transmitted by  a biting fly.  N. Engl. J. Med. 322, 1752.

Lundström, J.O., Andersson, A.C., Bäckman, S., Schäfer, M.L., Forsman, M., Thelaus, J., 2011. Transstadial transmission of Francisella tularensis holarctica in mosquitoes, Sweden. Emerg. Infect. Dis.  17,  794–799, 3201/eid1705.100426.

Maetzel, D., Maier, W.A.,  Kampen, H., 2005. Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks (Acari: Ixodidae) in urban and suburban Bonn, western Germany. Parasitol. Res.  95,  5–12.


Magnarelli, L.A., Anderson, J.F., Barbour, A.G., 1986. The etiologic agent of Lyme disease in deer flies, horse flies and mosqitoes. J. Infect. Dis.  154, 355–358.

Magnarelli, L.A., Freier, J.E., Anderson, J.F., 1987. Experimental infections of mosquitoes with Borrelia burgdorferi, the etiologic agent of Lyme disease. J. Infect. Dis.  156, 694–695.

Magnarelli, L.A., Anderson, J.F., 1988. Ticks and biting insects infected with the etiologic agent of Lyme disease, Borrelia burgdorferi. J. Clin.  Microbiol. 26, 1482–1486.

Matuschka, F.J., Richter, D., 2002. Mosquitoes and soft ticks cannot transmit Lyme disease spirochetes. Parasitol. Res.  88,  283–284, s00436-001-0584-1.

Margos, G., Wilske, B., Sing, A., Hizo-Teufel, C., Cao,  W.C., Chu, C., Scholz, H., Straubinger, R.K., Fingerle, V., 2013. Borrelia bavariensis sp.  nov. is widely distributed in Europe and Asia.  Int. J. Syst. Evol.  Microbiol. 63,  4284–4288,

Mohrig, W., 1969. Die  Culiciden Deutschlands. Parasitologische Schriftenreihe, vol.

  1. Gustav Fischer Verlag, Jena.

Morens, D.M.,  Folkers, G.K., Fauci, A.S., 2004. The challenge of emerging and re-emerging infectious diseases. Nature 430, 242–249.

Nejedlá, P., Norek, A., Vostal, K., Zˇ ákovska, A., 2009. What is the percentage of pathogenic Borrelia in spirochaetal findings of mosquito larvae. Ann. Agric. Environ. Med. 16,  273–276.

Pal,  U., Yang, X., Chen, M., Bockenstedt, L.K., Anderson, J.F., Flavell, R.A., Norgard, M.V., Fikrig, E., 2004a. OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J. Clin.  Invest. 113, 220–230,

1172/JCI19894, PMC 311436. PMID  14722614.

Pal,  U., Li, X., Wang, T., Montgomery, R.R., Ramamoorthi, N., Desilva, A.M., Bao,  F., Yang, X., Pypaert, M., Pradhan, D., Kantor, F.S., Telford, S., Anderson, J.F., Fikrig, E., 2004b. TROSPA, an Ixodes scapularis receptor for  Borrelia burgdorferi. Cell 119, 457–468,

Picken, R.N., 1992. Polymerase chain reaction primers and probes derived from flagellin gene sequences for  specific detection of the agents of lyme disease and North American relapsing fever. J. Clin.  Microbiol. 30,  99–114.

Picken, M.M., Picken, R.N., Han, D., Cheng, Y., Strle, F., 1996. Single-tube nested polymerase chain reaction assay based on flagellin gene sequences for detection of Borrelia burgdorferi sensu lato. Eur. J. Clin.  Microbiol. Infect. Dis. 15, 489–498.

Rauter, C., Hartung, T., 2005. Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis. Appl. Environ. Microbiol. 71,  7203–7216.

Rijpkema, S.G., Tazelaar, T., Molkenboer, M., Noordhoek, G., Plantinga, G., Schouls, L.M., Schellekens, J.E., 1997. Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by  PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clin. Microbiol. Infect. 3, 109–116.

Rizzoli, A., Hauffe, H.C., Carpi, G., Vourc’h, G.I., Neteler, M., Rosà, R., 2011. Lyme borreliosis in Europe. Eur. Surveill. 16 (27), pii=19906.

Rudenko, N., Golovchenko, M., Grubhoffer, L., Oliver Jr., J.H., 2011. Updates on Borrelia burgdorferi sensu lato complex with respect to public health. Ticks Tick  Borne Dis.  2, 123–128, 002.

Sanogo, Y.O., Halouzka, J., Hubálek, Z., Neˇ mec, M., 2000. Detection of spirochetes in,  and isolation from, culicine mosquitoes. Folia Parasitol. 47,  79–80.

Schuijt, T.J., Hovius, J.W., van Burgel, N.D., Ramamoorthi, N., Fikrig, E., van Dam, A.P., 2008. The tick salivary protein Salp15 inhibits the killing of serum-sensitive Borrelia burgdorferi sensu lato isolates. Infect. Immun. 76, 2888–2894,

Schwan, T.G., Piesman, J., 2000. Temporal changes in outer surface proteins A and C of the Lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J. Clin.  Microbiol. 38,  382–388.

Sinton, J.A., Shute, P.G., 1939. Spirochaetal infections of mosquitoes. J. Trop. Med. Hyg. 42,  125–126.

Skotarczak, B., 2014. Why are there several species of Borrelia burgdorferi sensu lato detected in dogs and humans? Infect. Genet. Evol.  23,  182–188, http://dx.

Skuballa, J., Petney, T., Pfäffle, M., Oehme, R., Hartelt, K., Fingerle, V., Kimmig, P., Taraschewski, H., 2012. Occurrence of different Borrelia burgdorferi sensu lato genospecies including B. afzelii, B. bavariensis, and B. spielmanii in hedgehogs (Erinaceus spp.) in Europe. Ticks Tick  Borne Dis.  3, 8–13, 1016/j.ttbdis.2011.09.008.

Thelaus, J., Andersson, A., Broman, T., Bäckman, S., Granberg, M., Karlsson, L., Kuoppa, K., Larsson, E., Lundmark, E., Lundström, J.O., Mathisen, P., Näslund, J., Schäfer, M., Wahab, T., Forsman, M., 2014. Francisella tularensis subspecies holarctica occurs in Swedish mosquitoes, persists through the developmental stages of laboratory-infected mosquitoes and is transmissible during blood feeding. Microb. Ecol.  67,  96–107,

Tilly,  K., Krum, J.G., Bestor, A., Jewett, M.W., Grimm, D., Bueschel, D., Byram, R., Dorward, D., Vanraden, M.J., Stewart, P., Rosa, P., 2006. Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect. Immun. 74,  3554–3564,

Vennestrøm, J., Egholm, H., Jensen, P.M., 2008. Occurrence of multiple infections with different Borrelia burgdorferi genospecies in Danish Ixodes ricinus nymphs. Parasitol. Int. 57,  32–37.

Wodecka, B., Leon´ ska, A., Skotarczak, B., 2010. A comparative analysis of molecular markers for  the detection and identification of Borrelia spirochetes in Ixodes ricinus. J. Med. Microbiol. 59,  309–314, 08-0.

Zˇ ákovska, A., Jörkova, M., Sˇ ery´ , O., Dendis, M., 2004. Spirochetes in Culex  (C.) pipiens s.l. larvae. Biologia (Bratisl.) 59,  283–287.

Zˇ ákovska, A., Cˇ apková, L., Sˇ ery´ , O., Halouzka, J., Dendis, M., 2006. Isolation of Borrelia afzelii from overwintering Culex  pipiens biotype molestus mosquitoes. Ann. Agric. Environ. Med. 13,  345–348.

Zeman, P., 1998. Borrelia infection rates in ticks and vectors accompanying human risk acquiring Lyme borreliosis in a highly endemic region in Central Europe. Folia Parasitol. 45,  319–325.

Christian Melaun a , Sina  Zotzmann a , Vanesa Garcia Santaella a , Antje Werblow a , Helga Zumkowski-Xylander b , Peter Kraiczy c , Sven  Klimpel a,∗

a) Goethe-University, Institute for Ecology, Evolution and Diversity, Senckenberg Biodiversity and Climate Research Centre, Senckenberg Gesellschaft für Naturforschung, Frankfurt am Main, Germany

b) Senckenberg Museum of Natural History Görlitz, Görlitz, Germany

c) University Hospital of Frankfurt, Institute of Medical Microbiology and Infection Control, Frankfurt am Main, Germany

Please cite this article in press as: Melaun, C., et al., Occurrence of Borrelia burgdorferi s.l. in different genera of mosquitoes (Culicidae) in Central Europe. Ticks Tick-borne Dis. (2015),

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