The Attenuation Mechanism and Live Vaccine Potential of a Low-Virulence Strain Obtained by Rifampicin Passaging Culture.

The rifampicin-resistant strain E9-302 of strain 669 (WT) was generated by continuous passage on BHI agar plates containing increasing concentrations of rifampicin. E9-302 was attenuated significantly by 119 times to zebrafish compared to WT in terms of the 50% lethal dose (LD). Zebrafish vaccinated with E9-302 via intraperitoneal (IP) injection at a dose of 1 × 10 CFU/fish had relative percentage survival (RPS) rates of 85.7% when challenged with wild-type via IP 14 days post-vaccination (dpv). After 14 days of primary vaccination with E9-302 via immersion (IM) at a dose of 4 × 10 CFU/ml, a booster IM vaccination with E9-302 at a dose of 2 × 10 CFU/ml exhibited 65.2% RPS against challenge with wild-type via IP 7 days later. These results indicated that the rifampicin-resistant attenuated strain E9-302 had potential as a live vaccine against infection. A previously unreported amino acid site change at position 142 of the RNA polymerase (RNAP) β subunit encoded by the gene associated with rifampicin resistance was identified. Analysis of the whole-genome sequencing results revealed multiple missense mutations in the virulence-related genes and in E9-302 compared with WT, and a 189 bp mismatch in one gene, whose coding product was highly homologous to glycosyltransferase family 39 protein. This study preliminarily explored the molecular mechanism underlying the virulence attenuation of rifampicin-resistant strain E9-302 and provided a new target for the subsequent study of the pathogenic mechanism of .