Establishment Of Rapid Method For Detection Of Streptococcus Agalactiae And Researches On Immunoprophylaxis Of Streptococcusis In Tilapia

Tilapia is an important commercial freshwater fish in South China. However, with the development of its culture, diseases become more and more serious. Diseases induced by bacterial pathogens including Pseudomonas fluorescens, Edwardsiellatarda, Aeromonas hydrophila, Streptococcus iniae and Streptococcus agalactiae are very common and produce great damage to its culture. Although antibiotics have effect on the prevention and treatment of tilapia disease, they have lots of disadvantage such as drug resistance, drug residue, environmental pollution. In order to promote the sustainable and healthy development for tilapia farming, it is very urgent to explore the safe and efficient disease control methods.In order to detect tilapia bacterial pathogens quickly and accurately, we use molecular biology methods to establish PCR methods for rapid identification of Streptococcus agalactiae. To explore the safe and efficient control measures on tilapia streptococcal disease, this paper separated Hainan local virulent strain of Streptococcus agalactiae and used a spectrophotometer to establish a method for rapid determination of the concentration of the vaccine. What is more, this paper studied the effection of immunzed by different methods,inclouding injection,immersion and oral, and established of a suitable processes for hypertonic immune method on tilapia. Contents and results are as follows:(1) Established PCR method for rapid detection of Streptococcus agalactiae. In this paper, specific gene primers are designed to establish a PCR method for rapid detection of S. agalactiae. Results showed that when PCR reaction was:95℃for5min,94℃for1min,56℃for2min,72℃for1min and72℃for10min could simultaneously detect the bacteria. It also showed detection threshold of S. agalactiae was102cell/mL.We collected17S. agalactiae from Guangdong and Hainan by using this method, while identification by16S rRNA further proved the accuracy of this rapid detection method.(2) Typing of Streptococcus agalactiae. With analysis the16S rRNA of S. agalactiae from Guangdong and Haina, the results showed that Streptococcus agalactiae genotype hand non-differences, and both had a strong pathogenicity, thus broadin spectrum preparation of the vaccine(3) Preparation and concentration determination of inactivated vaccine Streptococcus agalactiae.Streptococcus agalactiae-was excitated andenlage cultivated by BHI from:-80℃and inactivated in4℃by0.4%formaldehyde for24h, then accessed the safe and efficient safety and efficacy testing. For rapid and accurate determination of the concentration of the vaccine, we established a rapid determination of vaccine concentratio by spectrophotometer. The results showed that the detection wavelength is600nm, the inactivated vaccine absorbance value (X) and the concentration (Y) was positively correlated (r=0.9983, df=6, P<0.01), which curve is:y=11.277x-2.0229.(4) Protective efficacy of different methods forimmune of tilapia.50day-old tilapia were immunedby injection, immersion and oral respectively, the results showed that after injection or immersion, tilapia got significantly improvement on resistance of Streptococcus agalactiae, in which RPS of tilapia by injection for1and2times was66.67%and78.33%, RPS of hypertonic immunization for1and2times were21.67%and28.33%, RPS ofisotonic immersion method1and2times were18.33%and3.33%and1.67%for oral.A variety of specific and non-specific indicator test results also showed that to antibody titers, serum protein, lysozyme activity, and alkaline phosphatase activity were significantly higher (P <0.05) after injection or immersion, which showed that an injection or immersion method can be used for tilapia immunization.(5) Immersion immune for different day-old tilapia.This paper used isotonic and hypertonic immunization for10day-old,20day-old and50day-oldtilapia, results showed that:After isotonic and hypertonic immunization for1times,RPS of10day-old was6.67%and0; RPS of20day-old tilapia were15%and10%, respectively; RPS of50day-old tilapia were13.33%and15%; And afterimmunizationfor2times, RPS of10day-old wass were3.33%and1.67%;2RPS of20day-old tilapia were38.33%to26.67%, respectively RPS of50-day-old tilapia were26.67%and16.67%.Growth affect after different hypertonic immunization showed that10day-old tilapiawere significantly decreased while20day-old and50day-oldhad no significant effect on growth rate. Comprehensive immune protection and growth rate impact, hypertonic immersion for2has the best protective immunity.