Development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera

Abstract

Serological tests, such as agglutination and indirect hemagglutination assay (IHA), have been used to identify antibodies against Pasteurella multocida in poultry sera, but none are highly sensitive. An enzyme-linked immunosorbent assays (ELISA) has been used with varying degrees of success in attempts to monitor seroconversion in vaccinated poultry, but are not suitable for diagnosis. Commercial ELISA kits are available for chickens and turkeys, but not for ducks. The present study reports development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between the two diagnostic tests. An in-house indirect ELISA was developed using a heat extract antigen of P. multocida strain X-73 as a coating antigen and horseradish peroxidase conjugated goat anti-duck IgG antibody (dIgG-HRP). The checkerboard titration method was done using sera from ducks immunized with P. multocida bacterin as positive sera and 1day old duckling sera as negative sera. The heat extract antigen at 1μg/ml, sample serum at a dilution of 1:100, and dIgG-HRP 1:2000 were optimal concentrations for the assay. The cut-off value was 0.200. Of the duck sera, 89.05% (244/274) were considered seropositive by ELISA. Estimates for sensitivity and specificity of ELISA were higher than prior values with medians of 94.7% [95% posterior probability interval (PPI)=89.6–98.2%] and 87.2% (PPI=68.2–98.3%). Estimates for sensitivity of IHA were lower than prior values (median=97.6, PPI=93.2–99.7%) while the specificity was close to the prior value (median=76.5, PPI=65.8–85.4%). This finding suggests that an in-house indirect ELISA can be used to detect duck antibody to fowl cholera.