Recombinant IpaJ protein for serological detection of Salmonella enterica serovar Pullorum infection in chickens: Production, characterization, and diagnostic evaluation

Background and Aim: Pullorum disease, caused by Salmonella enterica serovar Pullorum, remains an important poultry infection, and reliable serological surveillance is complicated by cross-reactivity of conventional lipopolysaccharide (LPS)-based assays among closely related Salmonella serovars. IpaJ has been proposed as an immunogenic protein antigen with potential utility for serological detection of S. enterica serovar Pullorum infection. This study aimed to produce and characterize recombinant IpaJ (rIpaJ) and preliminarily evaluate its diagnostic performance using field-derived chicken sera from Kazakhstan, compared with a commercial LPS-based enzyme-linked immunosorbent assay (ELISA). Materials and Methods: The ipaJ gene was synthesized, cloned into pET-28, and expressed in Escherichia coli BL21(DE3). The rIpaJ protein was purified under denaturing conditions, refolded, and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blotting, and nano-liquid chromatography-tandem mass spectrometry. An rIpaJ-based indirect ELISA was evaluated using 179 chicken serum samples comprising 15 reference-positive and 164 negative samples. The diagnostic cutoff was calculated from 92 randomly selected negative sera as the mean optical density at 450 nm plus three standard deviations. Diagnostic performance was compared with that of a commercial LPS-based ELISA.