M-like Proteins And Antigenicity Of M-like Proteins In Streptococcus Iniae

Streptococcus is one of the most serious bacterium which can induce fish diseases. Many cultural and wild fishes are sensitive to Streptococcus, including limnetic fishes, briny fishes and semi-limnetic fishes. In this study, 3 streptococci isolated from the flounder were proved to be deadly by artificial injection experiments. The strains were identified to be Streptococcus iniae by physiological and biochemical characterization. The rabbit and mice were immunized, and immunofluorescence antibody technology(IFAT) was used to detect the speciality of antiserum. Three different extraction methods (hot acid extraction, alkaline extraction and pepsin digestion) were used for preparing M-like proteins from Streptococcus iniae and Streptococcus dysgalactiae. The M-like proteins of the two streptococci were analyzed by using SDS-PAGE and Western blotting. The Japanese flounder were immunized by M-like protein and NUF693 to investigate the antigenicity of M-like protein. The results are as below.3 strains Streptococci isolated from Japanese flounder were proved to be deadly by artificial injection experiments. NUF849 had strong virulence , and the LD50 of NUF849 was calculated to be 3.7×105CFU/fish. The LD50 of NUF693 and NUF701 were 1.4×107 CFU/fish and 4.6×107 CFU/fish, it indicated that NUF693 and NUF701 had weak virulence.3 strains were indentified by routine characterizations and API ID32E systematic identification, and 3 strains were identified as Streptococcus iniae.A New Zealand rabbit and mice were immunized with Streptococcus iniae NUF849 whole cells, it was found that strain NUF849 could effectively stimulate the rabbit and mice to produce immunity response. The results of immunofluorescence antibody technology (IFAT) showed that the mice antiserum could specially combine to NUF849, and enzyme-linked immunosorbent assay (ELISA) found that the titer of the antiserum were respectively 1600 and 800. IFAT was used to detect the speciality of the antisera. It was found that the antisera could combine to the strain NUF849 specifically, and had cross reaction with S. dysgalactiae and Streptococcus iniae, but had no cross reactions with bacteria belonged to genus Vibrio.Three different extraction methods (hot acid extraction, alkaline extraction and pepsin digestion) were used for preparing M-like proteins from Streptococcus iniae NUF849. The results showed that the different methods can obtain different M-like protein. The hot acid extraction was used to prepare the M-like proteins of the bacteria of Streptococcus iniae and Streptococcus dysgalactiae. The M-like proteins of the two streptococci were analyzed by using SDS-PAGE and Western blotting. By comparing the results of the three methods, the M-like proteins extracted by hot acid had the strongest immunogenicity. The SDS-PAGE diagrams of the M-like proteins of the two streptococci were distinctly different. The M-like proteins extracted from S.iniae contained 7 minor protein bands with molecular weights of 60kDa, 48kDa, 37kDa, 33kDa, 27kDa, 24kDa and 15kDa, the M-like proteins extracted from S.dysgalactiae contained 2 minor protein bands with molecular weights of 68kDa and 42kDa. After SDS-PAGE, in the Western-blot with rabbit antiserum against S.iniae whole cells, only 2 M-like proteins of S. iniae with molecular weights at 60kDa and 37kDa were detected, and only 2 M-like proteins of S. dysgalactiae with molecular weights at 68kDa and 37kDa. It showed that the M-like proteins of the two streptococci had different constitutions and antigenicity. This illuminated that the 37 kDa might be the special antigen of the 7 strains, and was possibly a vaccine candidate against different strains of Streptococcus.In order to prove the antigenicity of the M-like protein and the possibility to be a vaccine candidate, Japanese flounder were immunized by M-like protein and NUF693, and attacked by NUF849. The results showed that the protection ratio of M-like protein and NUF693 were respectively 60% and 30%. This illuminated that the M-like protein had certain protection and certain reference value in vaccine.