Differentiation of Salmonella-infected and Vaccinated Mice by ELISA-based Detection of Antibodies to Outer Membrane Proteins LptD and PefC

Salmonella infection in livestock is an important problem for both animal and public health. Serological surveillance is widely used to monitor Salmonella infection, but conventional ELISA based on lipopolysaccharide (LPS) cannot distinguish infected animals from those vaccinated with inactivated vaccines. To develop a Differentiating Infected from Vaccinated Animals (DIVA) method, we focused on two Salmonella proteins, LptD and PefC, as alternative antigens for ELISA. Antibodies against both LptD and PefC were clearly detected only in infected mice, while mice inoculated with inactivated vaccines showed no detectable antibody response and were similar to the negative controls. These results indicate that LptD and PefC are recognized specifically during natural infection but not after immunization with an inactivated vaccine. To evaluate the applicability of these ELISAs to a variety of Salmonella serovars, sequence homology analysis was performed, which revealed that lptD was widely distributed among all examined serovars, whereas pefC located on a plasmid was absent in some serovars. This indicated that LptD is a more suitable antigen than PefC for the development of practical diagnostic assays. Based on these findings, ELISA using these antigens has strong potential as a DIVA diagnostic tool for distinguishing infected animals from vaccinated ones. This approach may improve surveillance and control of Salmonella infections in livestock.