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A serosurvey for neutralizing antibodies against West Nile virus (WNV) in common coots (Fulica atra) was conducted in Donana, Spain. Antibody prevalence was highest in 2003, intermediate in 2004, and lowest in 2005. Some birds seroreverted <1 year after first capture. Seroconversion of birds suggests local circulation of the virus.

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In western Mediterranean countries, the frequency of outbreaks of West Nile virus (WNV) infection has increased in recent decades. Evidence for WNV circulation in Spain has remained elusive, although WNV foci have recently been identified in 3 neighboring countries (Morocco, Portugal, and France) (1-3). Recent WNV activity in Spain has been shown by serologic screening in humans, with detection of WNV-specific immunoglobulin M (4) and identification of the first clinical case in 2004 (5). In avian hosts, WNV-neutralizing antibodies have been found in chicks of wild migratory birds in southern Spain (6). However, interpretation of serologic data is not straightforward because antibodies in chicks may be the result of maternal transmission through eggs (7). To ascertain local circulation of WNV in Spain, we designed a capture-recapture study in which serum samples from wild birds were obtained at different times.

The Study

We focused on the partially migratory common coot (Fulica atra) because of its high seroprevalence for WNV detected during a preliminary screening of 72 bird species (J. Figuerola et al., unpub, data). Reasons for this high seroprevalence remain unclear, although preference of this bird for mosquito-rich habitats and its relative size (weight [approximately equal to] 800 g) might be involved in this pattern. Birds were captured in Donana (37[degrees]6′N, 6[degrees]9′W) in a walk-in trap in October 2003 (3 capture sessions) and from September through February in 2004-2005 (12 sessions) and 2005-2006 (14 sessions). Overall, 853 captures of 515 different birds were conducted (1-7 captures/bird).

Blood was obtained from the tarsal vein and allowed to clot, and serum was stored at -20[degrees]C. All birds were marked with numbered metal rings. Age was determined by plumage characteristics before the birds were released. Neutralizing antibody titers for WNV (strain Eg101) were determined by using a micro-virus neutralization test as described (6). Only birds that showed neutralization (absence of a cytopathic effect) at dilutions >1:20 were considered seropositive. Controls for cytotoxicity in the absence of virus were included for every sample at a 1:10 dilution. Cytotoxic samples were excluded from the analysis.

Seroconversion was defined as a bird that was seronegative when first captured and became seropositive at recapture with an antibody titer that had increased 4-fold (8). Seroreversion was defined as a seropositive bird whose antibody titer decreased below the cut-off value of 20 at recapture. The interassay coefficient of variation of titers, expressed as [log.sub.10] (calculated using an internal control repeated in 5 different assays, mean 2.56, standard deviation 0.35) was 13.67%. This variation is similar to that observed in individual samples and repeated in different assays. In a series of 27 samples tested twice, the mean fluctuation observed was 0.29 [log.sub.10] units ([approximately equal to] 2-fold). To obtain accurate measurements of titers, particularly when assessing seroconversion/seroreversion, we analyzed samples at least twice, and when results differed, they were assayed again until a consistent result was obtained. Specificity of the test was assessed by parallel neutralization against Usutu virus (strain SAAR 1776), a flavivirus found in wild birds that belongs to the same serogroup as WNV, with a panel of sera positive for WNV by micro-virus neutralization test. All titers were higher for WNV than for Usutu virus; 93.6% were [greater than or equal to] 4x higher (Table 1). These results suggested that the neutralizing antibody response was generated by WNV or an antigenically related WNV-like virus.

Comparisons between years were restricted to data from October, the only month sampled in all 3 years. For analysis of variation in antibody prevalence within seasons, data were grouped into 2-month intervals. Prevalence was analyzed by generalized linear models with binomial distributed error, logit link, and randomly choosing 1 observation per bird.

Prevalence of WNV-neutralizing antibodies was highest in October 2003, intermediate in October 2004, and lowest in October 2005 ([chi square] 22.80, df 2, p<0.0001, p<0.05 for all pairwise comparisons) (Figure 1). Juvenile (<1 year of age) birds had lower antibody prevalences than adults in October ([chi square] 7.14, df 1, p = 0.008). Antibody prevalence increased throughout the 2004-2005 season ([chi square] 8.45, df 2, p = 0.02), but not during the 2005-2006 season ([chi square] 1.10, df 2, p = 0.58) (Figure 1).

Of 95 birds captured in 2 consecutive years, 59% had no detectable antibodies in either year, 21% seroreverted, 6.3% seroconverted, and 13.7% had antibodies in both years. Seroconversion confirms that WNV circulation is present in the study area, and seroreversion indicates that antibody titers decreased. Antibodies persisted for > 1 year in some birds, although whether this was caused by reinfection, which would stimulate the antibody response, is uncertain.

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