Development of a competitive ELISA based on Brucella neotomae lipopolysaccharide for detecting brucellosis in livestock.

Brucellosis, a global zoonotic threat, requires efficient diagnostic tools for effective surveillance. Commercial competitive enzyme-linked immunosorbent assay (cELISA) predominantly utilizes smooth lipopolysaccharides (S-LPS) extracted from B. abortus and B. melitensis as key antigens for brucellosis serodiagnosis. However, culturing pathogens requires facilities with high biosafety, which is operationally complex and economically demanding. In this study, we developed a cELISA using LPS extracted from B. neotomae, which can be handled more facilely in biosafety level 2 conditions, and analyzed clinical adaptability of the cELISA. The optimized cELISA demonstrated lower detection limits, which was 2-4 times more analytically sensitive than commercial kit by detecting sera against B. melitensis and B. abortus. No cross-reactivity was observed with sera infected with other bacteria, including E. coli, Salmonella, Y. enterocolitica, and M. tuberculosis. The diagnostic sensitivity and specificity of the cELISA were 100 % (40/40) and 100 % (40/40), respectively. The coefficients of variation were less than 10 %. Moreover, compared to the commercial kit, the developed ELISA achieved agreement of 92.51 % across 427 sera from vaccinated livestock, and agreement of 96.98 % across 696 sera from non-vaccinated livestock. In conclusion, the cELISA exhibits excellent sensitivity, specificity and repeatability, indicating its potential for brucellosis diagnosis in livestock.