Survey on achieving recombinant DNA vaccine (live delivery) against streptococcosis for Rainbow trout (Oncorhynchus mykiss) immunization

The main purpose of this study was survey on achieving recombinant DNA vaccine (live delivery) against streptococcusis for Rainbow trout (Onchorhynchus mykiss) Immunization. Initially, a total of 515 samples were collected from the head kidney of diseased fish (weigh 50_ 200g) in 72 farms of 8 provinces. Approximately, 40% (206 samples) of specimens were infected to Streptococcus species. Then were isolated 172 DNA samples and consequently, five pathogenic species have been identified, including S. iniae, S. faecium, S. agalactiae, S. dysgalatiae, and S. uberis. The enzyme phosphoglucomutase (PGM) has recently been discovered to play an important role in polysaccharide capsule production and virulence in S. iniae. Therefore, was initially isolated S. iniae and cloning phosphoglucomutase gene. Then, the PGM gene was amplified successfully and cloned in pTZ57R cloning vector. The recombinant plasmid was sub cloned into pETD uet-l expression vector by restriction enzymes and confirmed by PCR. Meanwhile, for amplifying simA and cpsD genes were used universal primers pNZ8148 and special for simA and cpsD genes.The recombinant bacteria Lactococcus lactis (NZ9000) was used for transformation the plasmid into Lactococcus lactis. Vaccination was performed by oral, bath and injection (peritoneal) methods. The efficiency of g2 was better than g1in these three methods and all of groups. The determination of IgM level, or detection of anti S. iniae antibody was carried out by using ELISA. The results revealed that there was a significant (p<0.05) difference between the level of IgM in all three methods and experiment groups compare to control group. The results of challenge of vaccinated fish with S. iniae showed that fish RPS in all of groups except group 3 (42.63 percent) were more than 50 percent while, in control group was 21.43 percent. The highest fish RPS was belong to group 11(61.25 percent) and statistical analyses revealed that significant (p<0.05) difference between fish vaccinated RPS, compare to control group.