Comparison of assays for the detection of West Nile virus antibodies in chicken serum

Abstract

Six tests for the detection of West Nile virus (WNV) antibodies in the serum of experimentally infected chickens were compared. The tests included the hemagglutination-inhibition test (HIT), immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (ELISA) with WNV-infected mouse brain antigen, immunoglobulin G (IgG) indirect ELISA with tickborne encephalitis viral antigen, the microtitre virus neutralization test, the standard plaque reduction neutralization test (PRNT), and the microtitre PRNT (micro-PRNT). Thirty adult chickens, intravenously and intramuscularly inoculated with 10(7) plaque-forming units (PFU) of WNV strain Egypt 101, were bled and given a booster of 10(7) PFU at 7,15, and 21 d postinoculation; the final blood collection was on day 28. Although the micro-PRNT is capable of detecting the highest antibody titres during both early and late infection, because of the technical complexity and time requirements of this test a combination of IgM and IgG ELISAs is recommended for serologic screening. Serum samples that give positive results in the ELISAs can then be tested by the micro-PRNT to determine the specificity of antibodies to WNV.

Figure 1. Distribution of serum titres of antibodies to West Nile virus (WNV) in the microtitre plaque reduction neutralization test (micro-PRNT) in blood collected 7 d after inoculation. Chickens considered as low responders are represented by black columns, high responders by grey columns.

Figure 2. Mean antibody titres and standard errors, determined by the micro-PRNT (A) and the immunoglobulin G enzyme-linked immunosorbent assay (IgG ELISA) (C). Graph B shows IgM ELISA P/N values (see text for explanation) for serum specimens obtained on various days postinoculation (dpi): the group of 13 chickens with high IgM response at 7 d is represented by the solid line; the group of 12 chickens with a lower IgM response at 7 d is represented by the dashed line.

Figure 3. Means and standard errors of serum antibody titres for 25 chickens, determined by the micro-PRNT (circles), the hemagglutination-inhibition test (HIT) (diamonds), the standard PRNT (triangles), and the microtitre virus neutralization test (micro-VNT) (squares).

Figure 4. Rates of detection of WNV antibodies in the serum from all chickens at different times after inoculation with the following tests: IgM ELISA (white bars), micro-VNT (finely cross-hatched bars), IgG ELISA (black bars), HIT (dark grey bars), micro-PRNT (light grey bars), and standard PRNT (thickly cross-hatched bars).