1Taylor I. Heckman, 1Khalid Shahin, 1Esteban Soto
1Aquatic Animal Health Laboratory, Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, California, 95616, USAStreptococcus iniae is a Gram-positive, opportunistically zoonotic bacterium infective to a variety of farmed and wild fish species worldwide. Current methods of controlling outbreaks rely heavily on antibiotic administration, but variable efficacies and concerns with human health and antibiotic resistance make this an unsustainable long-term strategy. Vaccination efforts have also been met with limited success, due to high intraspecific variability. There are few commercial vaccines available for S. iniae and the current vaccines for used in aquaculture are restricted to killed whole-cell preparations. Live attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they robustly induce both arms of immunity and may provide longer lasting protection through less stressful routes of administration. One of the few LAV in the US aquaculture industry is AQUAVAC-ESC, derived from a strain of Edwardsiella ictaluri attenuated by multiple passages in ascending concentrations of rifampin. We hypothesized that accumulation of mutations in S. iniae is achievable by this method and can lead to the generation of immunogenic LAV that confer protection to vaccinated fish, when challenged with wild-type (WT) strains. Previous multilocus sequence analysis determined the presence of distinct clades of S. iniae affecting cultured fish in the North American continent. Three representative strains from different clades and host types were selected for attenuation, and three lineages of rifamycin resistant strains were generated from each WT parental strain (n=9). Strains were passaged in increasing increments Rifamycin SV sodium salt, from the minimal inhibitory concentration of 0.312 μg/mL to a final concentration of 250 μg/mL. The 25 μg/mL and the 250 μg/mL passages were selected for comparison to the WT parents by in vitro assays evaluating growth in liquid media, extent of capsulation, survival in whole blood from Nile tilapia (O. niloticus) and cytotoxicity in the endothelial O. mossambicus bulbus arteriosus cell line. At least one passaged isolate from each clade demonstrated significant reduction (p < 0.05) in whole blood survival and/or cytotoxicity in endothelial cells. Potential LAV candidates (n=3 for each clade) were selected based on the in vitro assays for assessment of cross-reactivity and protectivity. Ten adult Nile tilapia (O. niloticus) were immunized by injection and blood collected 30 days post-immunization. Anti-S. iniae IgM produced by immunized tilapia was quantified by ELISA.