Buckley, C. 3 neighboring countries (Morocco, Portugal, and France) (13). 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 blood 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 (excess weight 800 g) might be involved in this pattern. Birds were captured in Doana (376N, 69W) in a walk-in trap in October 2003 (3 capture INHA antibody sessions) and from September through February in 20042005 (12 sessions) and 20052006 (14 sessions). Overall, 853 captures of 515 different birds were conducted (17 captures/bird). Blood was obtained from the tarsal vein and allowed to clot, and serum was stored at 20C. 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 explained (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 computer virus were included for every sample at a 1:10 dilution. Cytotoxic samples were excluded from your analysis. Seroconversion was defined as a bird that was seronegative when first captured and became seropositive at recapture with an antibody titer that experienced increased 4-fold (8). Seroreversion was defined as a seropositive bird whose antibody titer decreased below the cut-off value of Tartaric acid 20 Tartaric acid at recapture. The interassay coefficient of variance of titers, expressed as log10(calculated using an internal control repeated in 5 different assays, mean 2.56, standard deviation 0.35) was Tartaric acid 13.67%. This variance is similar to that observed in individual samples and repeated in different assays. In a series of 27 samples tested twice, the imply fluctuation observed was 0.29 log10units (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 computer 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 computer virus; 93.6% were>4 higher (Table 1). These results suggested that this neutralizing antibody response was generated by WNV or an antigenically related WNV-like computer virus. == Table 1. Antibody titers against West Nile computer virus (WNV) and Usutu computer virus in 47 serum samples from common coots, Doana, Spain. == Comparisons between years were restricted to data from October, the only month sampled in all 3 years. For analysis of variance 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 Tartaric acid of WNV-neutralizing antibodies was highest in October 2003, intermediate in October 2004, and least expensive in October 2005 (222.80, df 2, p<0.0001, p<0.05 for all those pairwise comparisons) (Determine 1). Juvenile (<1 12 months of age) birds experienced lower antibody prevalences than adults in October (27.14, df 1, p = 0.008). Antibody prevalence increased throughout the 20042005 season (28.45, df 2, p = 0.02), but not during the 20052006 season (21.10, df 2, p = 0.58) (Figure 1). == Physique 1. == Prevalence of common coots with neutralizing antibodies against West Nile computer virus (WNV), Doana, Spain, 20032006. Figures above bars indicate sample size for each period. Error bars show 95% confidence.