Development of ELISA-based diagnostic methods for the detection of haemorrhagic septicaemia in animals

Haemorrhagic septicaemia (HS) is an acute infection of cattle and buffaloes caused by the B:2 serotype of Pasteurella multocida. This disease is highly endemic in South Asia. In some peracute cases, there is 100% mortality in infected animals within a few hours of infection. Therefore, timely diagnosis of infection may contribute to its treatment and control to minimize economic losses. The current work reported the development of ELISA-based assays for the detection of anti-P. multocida antibodies and pathogen i.e. P. multocida. Owing to high immunogenicity, membrane proteins (MPs) extracted from local isolates of P. multocida serotype B:2 (PM1, PM2, and PM3) were employed as a potential diagnostic antigen for the development of indirect ELISA (i-ELISA) to detect HS antibodies in animals. MPs extracted from PM1, PM2 and PM3 isolates showed very low heterogeneity; hence MPs from the PM3 isolate were selected for the development of i-ELISA. The concentration of MPs (as coating antigen) of 3.13 μg/well and test sera dilution 1:100 was found to be optimal to perform i-ELISA. The developed method was validated through the detection of anti-P. multocida antibodies in sera of mice, immunized with MPs and formalin killed cells from the three local isolates (PM1, PM2 and PM3) of P. multocida. The significantly higher antibody titer in immunized mice was determined compared to unimmunized mice with the cut off value of 0.139. To detect P. multocida directly from the blood of infected animals, whole cell-based ELISA (cb-ELISA) assay was developed. A better detection signal was observed in the assay where bacterial cells were directly adsorbed on plate wells as compared to poly L-lysine (PLL) assisted attachment at a cell concentration of 10⁶ CFU and 10⁷ CFU respectively. The developed assays can be scaled up and potentially be used for the rapid detection of HS antibodies to gauge the immune status of the animal as well as vaccination efficacy and pathogen detection.