Isolation And Identification Of Streptococus Agalactiae From Tilapia And Immunogenicity Of Protein His-Sip Recombinant Protein

Tilapia originated in the Middle East and Africa and became one of the main species of aquaculture in the world. Tilapia is an extremely suitable fish for farming due to the fact that they are fast growing, fast propagation and very tolerant in regards to water conditions and are generally speaking very hardy fish. Tilapia is also an important freshwater cultured fish in China, with an annual production of 1.2 million tons. Guangdong province alone produces over 500,000 tons of tilapia each year. Strong disease resistance is considered one of the most important characteristics of tilapia, which helped it to become a worldwide culture species. However, in recent years Streptococcus infection of tilapia has occurred in various countries, overturning the myth that tilapia is hardy and disease-resistant. In fact, Steptococcus disease may become a major threat to the future of tilapia industry.In the summer of 2009 and 2010, a disease breakout spread tilapia farms in major cultivation areas of Southern China, especially in Guangdong and Hainan provinces. The diseased fish showed typical clinical signs of streptococcal-infection. Compared to earlier reported cases, the disease was more widespread and detrimental. High infectivity (>50%) and mortality rate (>95%) were observed in some farms. This paper presented our research works on it, including isolation and identification of pathogens, establishment of rapid identification of Streptococus agalactiae and Streptococcus iniae with duplex PCR assay, molecular serotyping, and preparing of recombinant Surface immunogenic protein Sip and analysis of its immunogenicities.1 Isolation and identification of pathogen, and antibiotic sensitivity assays Over twenty bacteria strains were isolated from diseased tilapia cultured in several farms of Guangdong and Hainan provinces. Artificial infection experiments showed that these isolates possessed strong virulence. 7 bacteria strains with strong virulence were chosen for antibiotics sensitivity test and identifications. Antibiotic sensitivity assays showed that among 29 antibiotics tested 13 were sensitive, 7 were resist, while 9 items showed various reactions among strains. The isolates were identified as Streptococcus agalactiae by biochemistry assays of ID 32 STREP test, Lancefield test and other assays. Molecular analysis of 16S rRNA and cfb genes (GBS-specific gene cfb, CAMP factor) were used for further identification. BLAST showed that 16S rRNA and cfb genes of all the strains possessed high similarities with their counterparts registered in GenBank, and with each other. Taken together, these experiment results showed that pathogen caused high mortality of tilapia in both provinces in 2009 was S. agalactiae. The result was helpful for tilapia disease control and prevention, and for development of streptococcus vaccine for tilapia.2 Rapid identification of Streptococus agalactiae and S. iniae with duplex PCR assayStreptococus agalactiae and S.iniae are two common pathogens of fish Streptococcus diseases. Symptoms of disease caused by both Streptococcus pathogens were quite similar, and it was difficult to distinguish the two pathogens from each other directly from the symptoms. Therefore it is necessary to develop a rapid identification method in order to control the disease effectively and rapidly. A rapid duplex PCR assay for identification of S. agalactiae and S. iniae was established, according to the cfb gene sequence of S. agalactiae and 16S rRNA gene sequence of S. iniae, A 474bp fragment special for S. agalactiae and a 296bp fragment for S. iniae were produced. The method is quite sensitive, and can detect a template concentration as low as 3.2×10-3 ng/μL for S. agalactiae and 3.0×10-2 ng/μL for S. iniae, respectively. The double PCR method provides a sensitive and accurate method for rapid identification of S. agalactiae and S. iniae. 3 Molecular typing of the isolated strains of Streptococcus agalactiaeMultiplex PCR assay of the alpha C protein (ACP) gene and cps gene was employed to identify their molecular serotype (MS). Amplification of the ACP gene produced a 400-bp bca fragment, suggesting that these isolates belong to MS Ia, Ib or II; amplification of cps produced a 790-bp amplicon, indicating that they belong to MS Ia/III-3. An additional PCR based on nucleotide difference in the cps H-I region of MS Ia and III further suggested that the isolates belong to serotype MS Ia. Moreover, multi-locus sequence typing (MLST) indicated that these strains were of sequence type 7 (ST-7). These results showed that isolates from different regions of China shared the same MS and ST. However, none of the isolated ST-7 GBS corresponded to the capsular serotype, suggesting that these fish GBS possessed specific molecular characteristics not present in human or other animals. Data from this study will facilitate the understanding of epidemiology and nosogenesis of tilapia GBS and the establishment of effective disease prevention methods.4 Cloning, expression and immunogenicity analysis of Surface immunogenic protein(Sip)Surface immunogenic protein Sip is a kind of immune related protein that exists in group B Streptococcus. In this present study, Sip gene was amplified from the genomic DNA of Streptococcus agalactiae Guangdong strain isolated from pond-cultured tilapia in Guangdong Province. Prokaryotic expression vector pColdII-Sip was constructed and transferred into E.coli BL21 (DE3) strain. The recombinant protein was induced to express and SDS-Page analysis showed that the target protein was solubly expressed. The purification of the protein was up to 98% after purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the recombinant protein, tilapia (Oreochromis niloticus, GIFT strain) was immunized by intraperitoneal injection of the recombinant protein. Recorded relative survival percent (RPS) of the vaccinated groups ranged from 70% to 87%. Enzyme-linked immunosorbent assay (ELISA) showed that the reciprocal serum titers of the immunized fish was 1: 128000. The result showed that the recombinant protein possessed strong immunogenicity, and Sip gene could be a candidated gene for genetic engineering vaccine.