Development and evaluation of recombinant protective antigen (rPA)-based indirect-ELISA for sero-monitoring of anthrax-specific antibodies in sheep.

Protective antigen (PA), a key exotoxin of Bacillus anthracis, is involved in the pathogenesis of anthrax, leading to 'sudden death' of affected herbivores. PA is also known to induce protective neutralizing antibodies following vaccination. Although an effective anthrax live spore vaccine is used to immunize susceptible animals, pre- and post-vaccination antibody titres are not regularly monitored, which is of immunological significance in endemic areas, and it is important to evaluate the vaccine efficacy at the field level. Hence, this study describes a standardized indirect-ELISA, a simple and rapid immuno-assay based on recombinant protective antigen (603 aa, ∼67 kDa) to detect anti-PA-specific IgG antibodies in sheep serum samples to assess serological evidence of exposure and/or vaccination to anthrax. Further, rPA-iELISA was used to assess prevailing antibody titers in randomly collected sheep (n = 1289) serum samples from three different states representing anthrax-endemic (Telangana) and non-endemic (Punjab and Haryana) geographical regions of India. A standardized rPA antigen (200 ng/well) based indirect-ELISA was found to possess an area under the curve (AUC) of 0.980 and 95 % CI of 0.932-0.992 with sensitivity of 93.7 %, and specificity of 94.9 % (p < 0.001) against an optimal cut-off criterion of >25.3 PP (>0.46 OD). A higher degree of sero-positivity in Telangana (>66.98 %), and a lower positivity was noted in Punjab and Haryana (6.83 % and 15.95 %, respectively) among sheep. The study indicated the potential utility of rPA-iELISA for sero-monitoring of anthrax / anti-PA-specific IgG antibodies in sheep.