Expression Characteristics of Virulence Genes of Streptococcus iniae in Infecting Selenotoca multifasciata and Development of Subunit Vaccines

Streptococcus iniae is an important pathogenic bacterium of fish. To screen suitable antigenic proteins and evaluate its immunogenicity, the RT-qPCR method was used to detect the bacterial load and the in vivo induced expression of 7 virulence-related genes of S. iniae in infecting Selenotoca multifasciata. The α-enolase (rENO) encoded by the eno gene was subsequently recombinantly expressed using a prokaryotic system in E. coli, and the immunoprotective effects of an inactivated S. iniae vaccine and an rENO subunit vaccine were finally compared in S. multifasciata. The bacterial load in liver tissue showed a tendency of increasing and then decreasing. At 48 hours post-challenge, the bacterial load reached its peak. The highest mortality rate of fish occurs 48~72 hours post-challenge, exhibiting characteristics of acute infection. Gene expression detection indicated that the relative expression levels of the virulence genes eno, sag, and pgmA at 72 hours post-infection were significantly higher than those at 48 hours. Additionally, their expression levels under the condition of fish body infection were significantly higher than those under in vitro culture conditions. These results indicated that the eno, sagI and pgmA genes are induced to express during infection, and may play key virulence roles. Among them eno had the highest and stable expression at all time points and conditions. The challenge test results after immunization showed that the relative percentage survival of the inactivated S. iniae vaccine and the rENO subunit vaccine were 94.12% and 70.59%, respectively. This indicated that rENO has the potential for development as a subunit vaccine against S. iniae. In summary, we screened three important virulence genes induced by S. iniae after infection of S. multifasciata, and successfully carried out the recombinant expression of the eno gene in a prokaryotic system of E. coli, and evaluated the immune effect of subunit vaccine. The results will provide references for the prevention and control of S. iniae and the development of vaccine.