Development of monovalent recombinant vaccine against streptococcus infection in hybrid red tilapia (Oreochromis spp.)

Tilapia (Oreochromis niloticus) is one of the most cultured species in the world. However, bacterial disease caused by Streptococcus spp. is one of the major problems affecting farmed tilapia worldwide. Hybrid red tilapia is highly susceptible to this disease and mortality may reach up to 70% within a week. Thus, the study was conducted to develop a recombinant vaccine of S. agalactiae, Millud II and S. iniae, TSK_2 previously isolated from infected tilapia. The isolates were successfully characterised as S. agalactiae and S. iniae respectively by species-specific PCR assay. The result confirms the amplification of 300 bp amplicons of 16S rRNA gene and sequence analysis demonstrated 98% similarity with complete genome of S. iniae, TSK_2 in BLASTn study. In addition, PCR amplification with primers specific for S. agalactiae 16S rRNA confirmed the target amplicons of 220 bp from positive control of bacterial DNA for S. agalactiae, Millud II. In order to analyse the antigenicity of S. iniae and S. agalactiae proteins, outer surface proteins (OSPs) of these two bacteria were extracted by sodium lauryl-sarcosinate method and separated by SDS-PAGE. Protein profiling revealed 40 and 52 kDa for S. iniae, while only 52 kDa was observed for S. agalactiae as major proteins. Western immunoblot results demonstrated that rabbit anti-serum of S. iniae could able to detect the antigenic proteins for S. iniae and S. agalactiae in both the homologous and heterologous reactions whereas; the antiserum of S. agalactiae was unable to detect cross-antigenicity. Thus, the immunogenic gene encoding 52 kDa of S. iniae was successfully cloned in pET-32 Ek/LIC plasmid and transformed into E. coli BL21 (DE3). The cloned sequence was identified as 1305 bp nucleotide sequence of sip gene coding for polypeptides of 434 amino acid residues with 99% similarity to other surface immunogenic gene (sip) of S. agalactiae in GenBank. To evaluate efficacy of developed vaccine, 450 fish was vaccinated via intraperitoneal injection with an inoculum containing 250 µl of 1.0 x 10⁷ CFU/ml per fish. Group 1 (90 fish) was vaccinated with formalin killed (FK) recombinant vaccine, group 2 (90 fish) with FK whole cell of S. iniae, group 3 (90 fish) with FK whole cell of S. agalactiae, group 4 (90 fish) with FK E. coli and group 5 (90 fish) was unvaccinated control. Finally all fish were i.p. challenged on week 4 with an inoculum containing 1.32 × 10⁹ CFU/ml live S. agalactiae and S. iniae in different aquariums. Results showed that the recombinant vaccine provides 60% protection against S. agalactiae whereas 100% defence against S. iniae. ELISA assay of serum, mucus and gut lavage fluid of vaccinated and post challenged fish demonstrated significant (p < 0.05) level of systemic and mucosal immunity in recombinant vaccine (group 1). Overall, the developed recombinant vaccine is highly effective to control S. iniae virulence in hybrid red tilapia but have moderate efficacy when challenged with S. agalactiae, suggesting that the vaccine is partially cross protective. Therefore, further improvement may be carried out in the future to induce outstanding protection against streptococcosis