Live attenuated vaccine for nile tilapia, obtained by the deletion of genes from Francisella noatunensis subsp. orientalis.

Abstract

The final goal of this project is to develop a live attenuated vaccine against Francisella noatunensis subspecies orientalis (FNO) that can be applied to Nile tilapia (Oreochromis niloticus). The specific objectives will be: i.) Produce at list one mutant strain of FNO deleting the wbtA, pilA, mglA, clpB, and tolC genes from a non-mutant Brazilian strain isolated in the IP Fish Institute laboratory (0012018). ii) Evaluate the efficacy of the live attenuated vaccine (mutant strains) by the experimental infection test with wild-type FNO. The FNO previously isolated on IP will be subjected to the deletion of genes "operons" by "Lambda red" technique and verified by PCR and sequencing of the genes. The mutant strains will be used for the elaboration of a live attenuated vaccine. The experimental infection of the fish will be conducted in a completely randomized experimental design with seven treatments and four replications. The experimental groups are divided as follows: Nile tilapia inoculated with saline solution and challenged with wild type FNO (G1); Nile tilapia inoculated with mutant FNO strains, deleted on the operon gene (s) wbtA (G2), pilA (G3), mglA (G4), clpB (G5) and tolC (G6) and challenged with wild-type of FNO. Nile tilapia inoculated with saline and not challenged (G7). After 15 days of intraperitoneal inoculation, the G1-G6 groups will be challenged orally with pathogenic wild-type FNO. Survival, clinical or subclinical manifestation of the disease (symptomatology, macroscopic and microscopic lesions, reisolation) will be evaluated. Survival and relative protection data of the vaccine will be analyzed by ANOVA and compared by Tukey (P <0.05). After completion of the two stages of the project, it is expected to produce at least one live attenuated vaccine against FNO.