Identification of immunogenic proteins and evaluation of recombinant PDHA1 and GAPDH as potential vaccine candidates against Streptococcus iniae infection in flounder (Paralichthys olivaceus)

Streptococcus iniae is a major Gram-positive pathogen that causes invasive disease in fish worldwide. In this study, in order to identify immunogenic proteins for developing highly effective vaccine against S. iniae, whole-cell lysate proteins of S. iniae were analyzed by western blotting using flounder anti-S. iniae antibodies, and two positive protein bands of molecular weight 37 kDa and 40 kDa were screened, which were identified as pyruvate dehydrogenase E1 subunit alpha (PDHA1), BMP family ABC transporter substrate-binding protein (BMP) and L-lactate dehydrogenase (LDH), as well as ornithine carbamoyltransferase (OCT), lactate oxidas (LOx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by mass spectrometry. Subsequently, the six recombinant proteins were produced and used to immunize healthy flounder, and the relative percent survival (RPS) value was 72.73%, 27.27%, 36.36%, 9.09%, 36.36% and 63.64% respectively after intraperitoneal challenge with live S. iniae, revealing that rPDHA1 and rGAPDH produced higher relative percent survival than formalin-killed S. iniae (36.36%). To further investigate the protective efficacy of rPDHA1 and rGAPDH, the proliferation of surface membrane immunoglobulin-positive (sIg(+)) lymphocytes in peripheral blood leucocytes, the total serum IgM, specific IgM against S. iniae and RPS were detected. The results showed that rPDHA1, rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg(+) lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher RPS (62.5% and 75%, respectively) again. These results demonstrated that rPDHA1 and rGAPDH are promising vaccine candidates against S. iniae infection in flounder.