ABSTRACT

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.

Results: The purified rIpaJ had an approximate molecular mass of 32 kDa and retained antibody-binding activity after refolding. Mass spectrometry identified IpaJ with a Mascot score of 1845 and 54% sequence coverage. The diagnostic cutoff was 0.515. The rIpaJ-based ELISA correctly identified 14/15 positive and 157/164 negative sera, yielding 93.3% sensitivity, 95.7% specificity, 66.7% positive predictive value, 99.4% negative predictive value, and 95.5% accuracy. The commercial LPS-based ELISA showed 100% sensitivity and 83.5% specificity, with 27 false positive results compared with seven for the rIpaJ assay. The specificity difference was significant (p < 0.001), whereas sensitivity did not differ significantly (p = 1.000).

Conclusion: The rIpaJ-based ELISA showed strong preliminary diagnostic performance and substantially fewer false-positive reactions than the commercial LPS-based assay. These findings support rIpaJ as a promising antigen for serological detection of pullorum disease. Larger independent cohorts and heterologous Salmonella serum panels are required before serovar-specific diagnostic performance and routine applicability can be established.

Keywords: avian salmonellosis, diagnostic accuracy, enzyme-linked immunosorbent assay, IpaJ, pullorum disease, recombinant antigen, Salmonella enterica serovar Pullorum, serological diagnosis.