Development of indirect enzyme-linked immunosorbent assay for antibody detection against Avibacterium paragallinarum

Abstract

Indirect enzyme-linked immunosorbent assay (I-ELISA) for antibody detection against A. paragallinarum serovars A, B and C was developed in separated plates. One hundred Babcock 308 female layer chickens were randomly divided into five groups of 20 each. Groups 1, 2, 3 and 4 were different positive control groups of immunized chickens with commercial trivalent mineral oil vaccine, and prepared bacterins of Avibacterium paragallinarum serovars A (221), B (0222) and C (Modesto), respectively. The chickens in Group 5 were assigned as a negative control. Positive and negative control sera from the chickens in Groups 1-5 at 2 weeks after the second vaccination were used to calculate sensitivity and specificity of the newly developed I-ELISA. Forty negative control sera (taken before vaccination) were used to evaluate cut-off values of the I-ELISA against each serovar of A. paragallinarum under optimal conditions. The cut-off values of serovars A, B and C, calculated by the mean optical density of all negative sera plus three standard deviations, were 0.334, 0.484 and 0.678, respectively. Efficacy of the developed I-ELISA showed 100% sensitivity for all three serovars of coating antigen, but with low specificity of 30% for all three serovars because of high cross-reactivity among the serovars, while the agreement rate between the I-ELISA and HI assay for serovars A, B and C was around 60%. Nevertheless, the serovar A I-ELISA gave a higher response to serovar A antiserum than to the other two heterologous serovars (p < 0.05). In contrast, the I-ELISA results for B and C did not show any significant differences between the homologous and heterologous serovars. This newly developed I-ELISA could be an alternative method for differentiating between A. paragallinarum-free chickens and those that have received vaccination, but could not clearly differentiate antibodies among the three serovars of A. paragallinarum.