Mosquito community influences West Nile virus seroprevalence in wild birds: implications for the risk of spillover into human populations

Mosquito community composition plays a central role in the transmission of zoonotic vector-borne pathogens. We evaluated how the mosquito community affects the seroprevalence of West Nile virus (WNV) in house sparrows along an urbanisation gradient in an area with the endemic circulation of this virus. We sampled 2544 birds and 340829 mosquitoes in 45 localities, analysed in 15 groups, each containing one urban, one rural and one natural area. WNV seroprevalence was evaluated using an epitope-blocking ELISA kit and a micro virus-neutralization test (VNT). The presence of WNV antibodies was confirmed in 1.96% and 0.67% of birds by ELISA and VNT, respectively. The VNT-seropositive birds were captured in rural and natural areas, but not in urban areas. Human population density was zero in all the localities where VNT-positive birds were captured, which potentially explains the low incidence of human WNV cases in the area. The prevalence of neutralizing antibodies against WNV was positively correlated with the abundance of the ornithophilic Culex perexiguus but negatively associated with the abundance of the mammophilic Ochlerotatus caspius and Anopheles atroparvus. These results suggest that the enzootic circulation of WNV in Spain occurs in areas with larger populations of Cx. perexiguus and low human population densities.


Results
In all, 340829 female mosquitoes belonging to 13 species and five genera were trapped. The commonest species was Culex theileri Theobald (n = 282891), followed in descending order by Ochlerotatus caspius Pallas (n = 21155), Culex pipiens Linnaeus (n = 19268), Culex perexiguus Theobald (n = 5939) and Anopheles atroparvus Van Thiel (n = 5387). In addition, 1237 females of the potential WNV vector Culex modestus Ficalbi were captured. The other species were trapped in relatively low numbers and for this reason-and also because they are not involved in the transmission of WNV-were not considered in any of the analyses (with the exception of the species richness calculation). A positive relationship was found between the overall abundance of mosquitoes and the richness of vector species (est = 2.45, z = 6.05, p < 0.001).
Sera obtained from 2544 house sparrows were analysed to detect WNV antibodies. According to the ELISA tests, 50 birds (1.96%) from 18 different localities tested positively (Table 1), while 113 (4.44%) provided doubtful results. Of these birds, 17 (0.67% of the total individuals sampled) had neutralizing antibodies against WNV as confirmed by VNT (Table 1). These 17 WNV-positive birds were captured in five of the 45 studied localities, all of them in rural and natural areas in Huelva province (Fig. 1). WNV seroprevalence in these five localities ranged from 1.6% to 8.5%. Specific USUV-neutralizing antibodies were detected in a single bird (0.04%) captured in a natural area in Seville province. The human population density tended to be lower (0 in all cases) in areas with VNT-positive birds than in areas with negative cases (mean human population = 77.6, range: 0-1,424) (est = −1.90, z = 1.88, p = 0.06).
The relationships between ELISA and VNT seroprevalence rates and the number of mosquitoes captured and species richness are summarised in Tables 2 and 3, respectively. Only those variables included in the selected models (those with ∆AIC ≤ 2 compared to the best model) are shown. WNV seroprevalences estimated by ELISA were positively related to mosquito richness and the number of Cx. perexiguus captured but negatively related to the number of Oc. caspius and Cx. theileri captured. Similarly, for the case of the model based on the WNV seroprevalence according to the VNT, the prevalence of neutralizing antibodies against WNV was positively related to the number of Cx. perexiguus captured (Fig. 2) but negatively associated with the number of both the Oc. caspius and An. atroparvus.

Discussion
Both West Nile virus and USUV antibodies were found in wild house sparrows from southern Spain. The seroprevalence of WNV in house sparrows estimated by VNT was positively related to the abundance of Cx.  17 . It is important to note that WNV has been detected in Spain mainly in Cx. perexiguus and Cx. pipiens pools 14,19 . Moreover, Cx. perexiguus is an abundant ornithophilic mosquito that commonly uses house sparrows as hosts 17,27,28 . Interestingly, we found negative relationships between the abundance of two common mosquito species, An. atroparvus and Oc. caspius, and the prevalence of WNV antibodies in wild house sparrows. Both species have a mammal-biased feeding pattern, even though they can feed on birds 17,27 . Although WNV has been detected in wild collected Oc. caspius 29 , this species is described as an inefficient vector of WNV by the only experimental study of the vector competence of Oc. caspius conducted to date in Europe 13 . At least two factors help explain   the negative association between these two mosquito species and WNV. Firstly, Oc. caspius prefers saltmarshes as larval breeding sites and An. atroparvus is commonest in sand dunes and scrubland, while Cx. perexiguus is frequently found in rice fields 30 . Consequently, Oc. caspius and An. atroparvus are probably more abundant in areas where Cx. perexiguus and/or other potential vector species for WNV such as Cx. pipiens and Cx. modestus are rarer. Secondly, the greater abundance of these mosquito species in the study area, where they feed mainly on mammals that are non-competent hosts for WNV, could lead to a reduction in the overall prevalence of WNV in birds. However, we were not able to identify any mechanisms that might support this hypothesis. Due to its mammal-biased diet and low vector competence, we would expect their abundance to have a low-but not negative-effect on WNV amplification. This is mainly because WNV transmission may be maintained by other vector-competent mosquito species present in the area. In addition, we observed a positive association between mosquito species richness and the seroprevalence detected by ELISA. The same non-significant tendency was found for WNV neutralizing antibodies detected by VNT. ELISA is a less specific technique than VNT and, consequently, individuals with positive sera for ELISA but negative for VNT have probably been exposed to other unidentified flaviviruses antigenically related to WNV. Using a SIR model, Roche et al. 15 concluded that mosquito species richness may increase the transmission success of vector-borne pathogens. However, such an association has never been tested empirically and could be the product of the assumption made in the model that species richness and vector abundance are positively related, a conjecture that, in fact, was supported by our data (see below). Consequently, our results support both the assumption of a positive relationship between vector richness and abundance, and the prediction of a positive relationship between vector richness and pathogen prevalence. Although Cx. perexiguus is the main vector of WNV in the area, other species such as Cx. pipiens and Cx. modestus may contribute significantly to WNV transmission 31,32 . These mosquito species, in addition to the others that co-exist in the area, could play a role in the transmission of certain flaviviruses. A number of flaviviruses have been isolated from mosquitoes (including Cx. pipiens) in Spain 19 , which potentially explains the positive correlation found between ELISA seroprevalence and mosquito species richness.
All positive cases of WNV-specific antibodies by VNT in bird sera were found in Huelva province, where evidence of WNV active circulation has existed since 2003, as demonstrated by the molecular detection of the virus in mosquitoes and the seroprevalence found in birds 33 . In addition, birds with WNV-specific antibodies by VNT were only detected in rural and natural habitats; none of the birds sampled in urban areas (n = 956) were seropositive. Moreover, the negative, marginally significant relationship we found between WNV seroprevalence and human population density may explain why WNV cases in humans are so uncommon in the study area despite the active circulation of the virus between vectors and avian hosts. Our results suggest that WNV, its main vector (Cx. perexiguus) and humans are not all present together in the same places. The seroprevalence of WNV in humans in southern Spain is very low (0.6%) and, mirroring the results for house sparrows in our study, a higher seroprevalence was detected in humans in rural areas than in suburban and urban areas 34 . Moreover, greater numbers of Cx. perexiguus were captured in natural and rural areas than in urban ones; likewise, the abundance of this species decreases as the percentage of land covered by built-up areas increases 35 . Indeed, only Cx. pipiens represents a risk for the transmission of WNV in urban areas 35 .
In conclusion, this study provides evidence of the central role of Cx. perexiguus in the enzootic circulation of WNV in southern Spain. The fact that WNV seropositive birds were found in both natural and rural areas, and tended to be present in areas with lower human densities, may explain the low incidence of WNV in humans in the area despite the local circulation of this virus between mosquitoes and wild birds.

Materials and Methods
Study area. This study was conducted in Andalusia, southern Spain (Fig. 1). This area is characterized by a Mediterranean climate with most precipitations concentrated during winter, while summer represents a long dry season. The study was conducted in 2013 at 45 different sites in Cadiz, Huelva and Seville provinces (southern Spain). The sampling sites (15 in each province) were situated in geographically close groups of three, each with one locality in a natural habitat, one in a rural habitat and one in an urban habitat (Fig. 1). The mean distance between localities within the same triplet was 5,740 m. Selection of the three habitat categories was performed after visual inspection of the areas based on the following criteria: urban habitats contained more densely human-populated areas than the other two habitat types; rural habitats had higher density of livestock than urban and natural areas; and natural habitats were selected on the basis of both lower human and livestock densities than in the other two habitat types, and a generally better conserved landscape.

Mosquito and bird sampling and identification. Mosquitoes were captured at the 45 sampling sites in
April-December, the period with maximum mosquito activity in southern Spain 30,36 . We used BG-sentinel traps baited with BG-lure and dry ice as a source of CO 2 , which is considered an effective method for mosquito diversity and abundance characterization 35 . At each site, once every 45 days, three traps were operated for 24 hours in each of the three localities of the same triplet. Overall, 135 traps (3 traps x 45 localities), with a mean distance between traps of 119 m (range 20-636 m), were employed during each mosquito trapping session for a total trapping effort of 810 trap nights. Mosquito sampling was conducted during days with favourable weather conditions (e.g. clear nights without rain). This procedure was repeated during 5-6 trapping sessions throughout the study period. Female mosquitoes were identified to species level following the morphological keys in Schaffner et al. 37 and Becker et al. 38 . Mosquitoes belonging to the univittatus complex were identified as Culex perexiguus based on male genitalia (see Harbach 39 ). For the case of samples compromising several thousands of mosquitoes captured per trap per night, we visually identified 500 individuals. These 500 mosquitoes were separated in five groups of 100 individuals, which were weighted to the nearest 0.001 g. This approach was used to estimate the proportion of individuals of each species for the rest of the sample based on the weight of the total number of mosquitoes captured 35 . Mosquito species richness, which ranged from 2 to 10, was calculated as the number of different species captured at each locality during the sampling period 35 . For each locality, the mean number of captures of the five commonest mosquito species in the study area (Anopheles atroparvus, Ochlerotatus caspius, Culex theileri, Culex pipiens and Cx. perexiguus) and of Cx. modestus, a potential WNV vector in the area 14,17,31 , were calculated.
House sparrows were sampled using mist-nests at the same localities during capture sessions in July-October, i.e. immediately after the breeding season to maximize the capture of juvenile birds and to better reflect virus circulation during the season from hatching until capture. Each bird was individually marked with a metal ring, sexed and aged 40 . A blood sample was taken from the jugular vein of each bird using a sterile syringe and preserved in a cool-box during the fieldwork session. In the laboratory, blood was allowed to clot at 4 °C overnight and was then centrifuged for 10 minutes at 4,000 rpm to separate the serum from the cellular fractions. Serum samples were frozen at −80 °C until further analysis. WNV antibodies detection. Serum samples from birds were analysed with the epitope-blocking ELISA Kit Ingezim West Nile Compac (INGENASA, Madrid, Spain) to determine the presence of WNV antibodies 41 . Positive results from ELISA may reflect past infections by WNV or even other unidentified flaviviruses circulating in the area. The cut-off value of this commercial ELISA test is set at 30% percentage of inhibition, while samples showing a percentage of inhibition between 30% and 40% are considered doubtful samples as established by the manufacturer 41 . All samples producing ELISA positive and/or doubtful results were subsequently analysed by a comparative micro virus-neutralization test (VNT) using WNV (strain Eg-101) and Usutu virus (USUV; strain SAAR1776) since the circulation of these flaviviruses has been demonstrated in the study area 42 . This confirmatory test allow to differentiate the specific antibodies against WNV from those elicited by other related flaviviruses. Neutralization titres were assigned based on the highest dilution of each serum capable of neutralizing the infection in vitro. Separate VNT were performed using serial (two-fold) dilutions (1:10-1:1280) of each serum sample using a micro-VNT method 21 . For a given sample, WNV-specific antibody responses were assigned when the observed VNT titres against WNV were at least four times higher than those observed against USUV 43 .
Human density quantification. We estimated the density of human population in the studied areas as the number of people living in a grid of 250 × 250 m. This information was obtained from the Andalusian Institute of Statistics and Cartography based on the number of residents registered in the local population census on 1 January 2013 (Base de Datos Longitudinal de Población de Andalucía). This variable was log-transformed to normalize its distribution.

Statistical analyses.
To estimate WNV seroprevalence we controlled for variables that operate at individual level (i.e. age, sex and date of capture) and others that operate at locality level (i.e. mosquito species richness and abundance of the different mosquito species). For this reason two-step analyses were performed. First, we fitted a generalized linear model to the seroprevalence of WNV using binomial distributed errors and including bird sex (fixed factor: male or female), age (fixed factor: juvenile or adult), month (continuous variable) and locality (fixed factor) as independent variables. Two different models were fitted using the results of ELISA and VNT as the dependent variable, respectively. Second, least square means (lsmeans) were calculated by retaining bird age and sampling locality, the only two significant factors explaining variance between individuals in WNV seroprevalence according to the previous models. This procedure allowed us to calculate both the ELISA and VNT seroprevalences for each of the 45 localities while controlling for the potential confounding effect of bird age. Third, two Linear Mixed-effects Models (LMM) were fitted using the lsmeans for ELISA and VNT seroprevalences as dependent variables. 'Province' and 'triplets' were included as random factors to account for the geographical stratification of the sampling design, and models were fitted using maximum likelihood and normal distributed errors. The independent variables included in these models were the three habitat categories (fixed factor: urban, rural or natural), the number of captures of each of the six main mosquito species found and species richness (continuous variables). Variance Inflation Factors (VIF) were checked to exclude collinearity between independent variables 44 and Akaike's Information Criterion (AIC) was used to select the best final models for each ELISA and VNT LMM model. Parameters were estimated by model averaging of all models with ∆AIC ≤ 2 45 , which were considered to similarly support the data. To normalize their distribution, the numbers of each mosquito species captured were log-transformed and the distribution of all predictors and model residuals were checked using qq plots in R software. We calculated the respective marginal coefficient of determination (R 2 ) for the fixed and random effects of the models according to Nakagawa & Schielzeth 46 .
Finally, two additional LMMs were fitted. One to test the model assumption of Roche et al. 15 of a positive correlation between mosquito richness and total abundance, and the other to compare the density of human population, as measured in Ferraguti et al. 35 , at sampling sites with and without VNT positive birds. All the statistical analyses were conducted in R (v. 2.14.2; R Development Core Team 2005) using the packages vegan, lme4, car, arm, Matrix, Rcpp, MASS, MuMIn and lsmeans.
Ethics statement. Bird sampling and mosquito trapping were performed with the necessary permits issued by the regional Department of the Environment (Consejería de Medio Ambiente, Junta de Andalucía) and in accordance with relevant guidelines and regulations. Procedures were approved by the Ethical Committee of CSIC and complied with current Spanish laws. Surveys and sampling on private land and in private residential areas were conducted with all the necessary permits and consent, and in the presence of owners. This study did not affect any endangered species.