The Screening Of Antagonistic Bacteria And It’s Antagonism On The Adhesion Of Streptococcus Agalactiae To The Mucus Of Tilapia

Tilapia was a warm water fish widely distributed in tropical and subtropical freshwater and brackish waters. Because of its tender meat, fast growth, high fecundity and strong adaptability, it was promoted by FAO to the whole world in1976as an excellent farming breed. However, streptococcal diseases, especially Streptococcus agalactiae disease have brought great damages to the tilapia farming industry since2008. Up till now, there were no highly effective methods to control the disease. Antagonistic bacteria could compete with the pathogen Streptococcus agalactiae in the initial adherence stage of the pathogen infection, thus blocked the following invasion of the pathogen bacteria and reached the aim of protecting the host. In this paper, the conditions of indirct ELISA method was optimized. Then the characteristics of Streptococcus agalactiae adherence to tilapia mucus at body surface, gill and intestine were studied.59strains of antagonistic bacteria against Streptococcus agalactiae were obtained after screening experiments from different tilapia living environments. The antagonistic effect of these59strains in different mucus by different antagonistic ways were tested and finally two safe and highly angagonistic bacteria were selected for the future Streptococcus agalactiae control in tilapia culture industry. The detailed results were as following:The optimum conditions of the indirect ELISA method are as follows:use10%fetal calf serum and confine for60min; use1:100first antibody at37℃for60min; use1:2000second antibody at37℃for60min, then colorate at37℃for20mins. The minimum effective concentration to Streptococcus agalactiae was1×104CFU/mL. The adherence characteristics of Streptococcus agalactiae to different tilapia mucus were tested by indirect ELISA method. Results showed that top adherence effect were reached when the bacteria concentration was at1×107CFU/mL, pH at7-8, and temperature at35℃-40℃, application time at120min. Within three types of mucus, intestine mucus showed the highest adherence effect, reaching4.052×10CFU/mL; body surface mucus were the second best, at2.825×106CFU/mL; gill mucus were the worst, at6.480×106CFU/mL.In this paper, antagonistic bacteria against tilapia Streptococcus agalactiae were screened using Radial Diffusion Assay (RDA) method. Then these antagonistic bacteria were identified using16S rDNA sequencing technique. A phylogenetic tree was also constructed to analysis the diversity of the antagonistic bacteria. Finally intraperitoneal injection method was used to test if the screened antagonistic bacteria were toxic to tilapia fishes. Results showed that59of the1759strains of bactera isolated from eight freshwater ponds showed stronge antagonistic effect against Streptococcus agalactiae. The diameter of the inhibition zones ranged from10.6mm to30.8mm, with an average value of21.9mm.16S rDNA sequencing and phytogenetic tree analysis indicatedthat these59strains of bacteria belong to5genus,8species, including genus Bacillus (45strains), genus Pseudomonas (5strains), genus Enterococcus (2strains), genus Paenibacillus (2stains) and genus Staphylococcus (5strains). The toxicity tolerance test results showed that22 strains were nontoxic to tilapia fishes. The results of this study will help provide alternative antagonistic bacterial strains for tilapia Streptococcus disease control and prevention in future.The antagonistic effect of these59strains in different mucus by different antagonistic ways were tested, results showed that different antagonistic bacteria had different antagonistic effect in different tilapia mucus through different antagonistic ways. For example, in the same type of mucus, the same antagonistic bacteria showed different levels of antagonistic effect through different antagonistic ways; and in different tilapia mucus, the same antagonistic bacteria showed different levels of antagonistic effect through the same antagonistic way. Combining all test restuls, two (HAINUP40and HAINUP96) safe and highly angagonistic bacteria were selected. These two strains of antagonistic bacteria on tilapia was safe, with0%mortality, Streptococcus agalactiae inhibition zone diameter display these two strains of antagonistic bacteria were respectively28.4±0.2mm and22.2±0.4mm, molecular identification as B. subtilis and B. thuringiensis, thus providing a theoretical basis for the future Streptococcus agalactiae control in tilapia culture industry.