Abstract
Pullorum disease remains an epidemic in the poultry industry in China. The causing pathogen is a host-restricted Salmonella enterica serovar Pullorum, which can spread through both horizontal and vertical transmissions. To eradicate the pullorum disease from poultry farms, it is necessary to specifically monitor the prevalence of the bacterial infection in adult chicks. In this study, we constructed a new competitive ELISA method based on the development of monoclonal antibodies (MAbs) against a specific immunogen of S. Pullorum, IpaJ protein. In total, eight MAbs against IpaJ were prepared using the purified recombinant His-IpaJ protein as the immunogen. Characterization of the eight MAbs demonstrated that 4G5 can be used as the competitive antibody in ELISA. A competitive ELISA was subsequently developed using purified MBP-IpaJ as the capture (0.5 μg/ml) and the HRP-labeled 4G5 (0.14 μg/ml) as the competitive antibody, respectively. A specificity test demonstrated that the ELISA assay can differentiate antisera of S. Pullorum-infected chickens from that of S. Gallinarum and S. Enteritidis. Furthermore, 4 out of 200 clinical antisera collected from a poultry farm were detected to be S. Pulloram positive using this method. The plate agglutination test (PAT) and the previously established indirect ELISA confirmed that these positive antisera reacted specifically with S. Pullorum. We propose that the established competitive ELISA assay based on MAb against IpaJ protein, is a novel and quick method that can detect S. Pullroum infection in the poultry industry.
Highlights
Avian salmonellosis remains a big threat to the poultry industry and the public health system
The recombinant bacteria BL21(DE3)-pColdI-ipaJ was induced by 0.5 mM IPTG, and the SDS-PAGE results confirmed that His-IpaJ was successfully expressed in the host cells (Figure 1B)
The His-IpaJ protein was purified from polyacrylamide gels (Figure 1C)
Summary
Avian salmonellosis remains a big threat to the poultry industry and the public health system. The pullorum disease has mostly been eradicated in many developed countries in Europe, America, and Asia, apart from sporadic incidences in free-ranging backyards It remains endemic in some developing countries like China [1]. The traditional method to diagnose the disease is to identify the pathogen in infected chickens through biochemical tests or molecular detection methods including PCR analysis [2,3,4]. All of these methods need the bacteria to be extracted from the chicken samples, which is time-consuming. New methods based on the detection of antibodies can improve the efficiency of pathogen detection, as opposed to the traditional methods [5]
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