Identification and Evaluation of Novel Antigen Candidates against Salmonella Pullorum Infection Using Reverse Vaccinology

Abstract

Pullorum disease, caused by the Salmonella enterica serovar Gallinarum biovar Pullorum, is a highly contagious disease in the poultry industry, leading to significant economic losses in many developing countries. Due to the emergence of multidrug-resistant (MDR) strains, immediate attention is required to prevent their endemics and global spreading. To mitigate the prevalence of MDR Salmonella Pullorum infections in poultry farms, it is urgent to develop effective vaccines. Reverse vaccinology (RV) is a promising approach using expressed genomic sequences to find new vaccine targets. The present study used the RV approach to identify new antigen candidates against Pullorum disease. Initial epidemiological investigation and virulent assays were conducted to select strain R51 for presentative and general importance. An additional complete genome sequence (4.7 Mb) for R51 was resolved using the Pacbio RS II platform. The proteome of Salmonella Pullorum was analyzed to predict outer membrane and extracellular proteins, and was further selected for evaluating transmembrane domains, protein prevalence, antigenicity, and solubility. Twenty-two high-scored proteins were identified among 4713 proteins, with 18 recombinant proteins successfully expressed and purified. The chick embryo model was used to assess protection efficacy, in which vaccine candidates were injected into 18-day-old chick embryos for in vivo immunogenicity and protective effects. The results showed that the PstS, SinH, LpfB, and SthB vaccine candidates were able to elicit a significant immune response. Particularly, PstS confers a significant protective effect, with a 75% survival rate compared to 31.25% for the PBS control group, confirming that identified antigens can be promising targets against Salmonella Pullorum infection. Thus, we offer RV to discover novel effective antigens in an important veterinary infectious agent with high priority.

Keywords: PstS; S. Pullorum; chick infection model; immunogenicity; reverse vaccinology.

Figure 1

Virulence assays of S. Pullorum R51 and additional seven S. Pullorum clinical isolates in 16-day-old chick embryos and 1-day-old chicks. (A) Chick Embryos, (B) Chicks.

Figure 2

(A) Complete genome sequence of S. Pullorum R51; (B) Overview of the reverse vaccinology steps used in this study to predict potential vaccine candidates against the S. Pullorum R51 strain.

Figure 3

SDS-PAGE analysis of 18 recombinant target proteins expressed in E. coli Rosetta after purification. The arrows indicate the protein band for further studies.

Figure 4

ELISA analysis of sera collected on day 14 post-immunization (day 11 post-hatching). The antisera from chicks mock-vaccinated with PBS were set as a negative control (NC). Statistical analysis was compared to the NC group. p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****).

Figure 5

Bacterial loads in the spleen and liver were determined on day 7 after infection with S. Pullorum R51. Statistical analysis compared to NC group. p < 0.05(*), p < 0.01(**). (A) Liver, (B) Spleen.

Figure 6

Survival percentage of PBS, SinH, SthB, PstS, and LpfB immunizing chicks infected with S. Pullorum R51. Survival of chicks was monitored for 21 days.