Detection of anti-West Nile virus immunoglobulin M in chicken serum by an enzyme-linked immunosorbent assay

Abstract

The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.

FIG. 1.

IgM response of chickens needle inoculated with WN virus versus days p.i. Symbols: •, virus-infected birds; ▴, uninfected cage mates. A P/N value of >2.0 was considered a positive result.

FIG. 2.

Specificity of the chicken WN IgM-capture ELISA. Specimens 1 to 18 previously tested positive by HAI for anti-SLE antibodies; specimens 19 to 47 tested positive for anti-WN virus antibodies. The bar graph shows the results of both WN and SLE IgM ELISAs with these specimens, and the dashed line represents the positive cutoff P/N value below which results are considered negative.

FIG. 3.

Combined WN and SLE chicken IgM-capture ELISA results. Specimens were tested both by WN virus-only and a combined WN-SLE ELISA in which titrated antigens were coincubated. The dashed line represents the positive cutoff P/N value below which results are considered negative.