Development and potential use of Pseudomonas plecoglossicida mutant ΔOmpRΔrpoS as a live attenuated vaccine against visceral white nodules disease in large yellow croaker (Larimichthys crocea)

Abstract

Pseudomonas plecoglossicida has become a major pathogen adversely affecting the farming of the large yellow croaker (Larimichthys crocea) in which it causes visceral white nodules disease (VWND), a highly devastating disease that leads to severe economic losses. The development of a live attenuated vaccine (LAV) will be an effective and beneficial strategy to control the disease in farmed L. crocea. In this study, we constructed an attenuated P. plecoglossicida mutant by knocking out the OmpR and rpoS genes using the allelic exchange method and analyzed the growth kinetics in vitro. Then, the use of the mutant ΔOmpRΔrpoS strain as a potential LAV was evaluated. We found no significant difference in the in vitro growth kinetics between the mutant and wild-type (WT) strains. The ΔOmpRΔrpoS strain showed a 1.17 × 107-fold decrease in virulence in L. crocea compared with the parental WT strain. Fish vaccinated twice via intraperitoneal (IP) injection with the ΔOmpRΔrpoS strain had a relative percent survival (RPS) of 72.25% and 64.90%, when challenged at 4 and 6 weeks post-initial vaccination (wpiv), respectively. Vaccinated fish developed a much higher P. plecoglossicida-specific antibody response than the unvaccinated fish. In ΔOmpRΔrpoS-vaccinated fish, the immune-related genes were remarkably upregulated after P. plecoglossicida challenge, and a lower bacterial load was observed in the spleen of these fish, unlike the control fish, which had high bacterial loads. Moreover, the ΔOmpRΔrpoS strain showed no adverse effects on the growth rate of large yellow croaker. Taken together, these results suggest that the ΔOmpRΔrpoS mutant is a promising LAV candidate against P. plecoglossicida infection.