The Establishment Of Molecular Detection Techniques And Selection Of Chinese Herbs For Streptococcus Of Tilapia(Oreochromis Niloticus)

Tilapia is the third most consumed fish worldwide,and is of increasing importance in aquaculture due to its high protein content,mild taste and easy preparation.Disease problems,especially in intensive culture mode,constitute the major cause of economic losses in aquaculture.Streptococcicosis is a notorious disease suffered in tilapia,and is mainly caused by infections of Streptococcus agalactiae and S.iniae.Traditionally,a lot of techniques have been adopted for pathogen detection,however,most of which were time-consuming and laborious.Antibiotics have been widely and extensively used to prevent and treat infectious diseases including streptococcicosis in aquaculture.However,the uncontrolled and indiscriminate application of chemotherapeutic agents has given rise to the emergence of numerous antibiotic-resistant bacteria.Therefore,alternatives to traditional detection and controlling measures are urgently required for not only the disease control but also the sustainable development of aquaculture industry.In this study,a set of simple and sensitive molecular early warning detection techniques was established on the basis of PCR technology.The detection kits were applied in a one-year epidemiological survey in Guangdong Province.From 200 kinds of Traditional Chinese medicine,35 kinds of herb that showing inhibitory activity to the growth of S.agalactiae as well as S.iniae were explored the relevance of effective Chinese medicines and screened out in vitro inhibitory compounds.The details are as follows:?1?Screening of primers for specific detection of either S.agalactiae or S.iniae by single PCR.70 pairs of primers were screened according to the seven gene sequences of cfb Genes of Cyclic Adenosine Monophosphate Factor,Sip gene of surface immunogenic protein,hly gene of hemolysin,VaA gene of vaccine antigen A,htra gene of high temperature reguirement A,Fbp gene of fibrinogen binding protein and of 16S rDNA gene of 16S ribosomal RNA in S.agalactiae and S.iniae.Among which,fourteen pairs of specific primers that can specifically detect S.agalactiae or S.iniae were finally selected.These primers were proved to be highly sensitive and the DNA in a concentration as low as 5.6 pg/?l can still be successfully detected.?2?Establishment of triplex PCR assay for S.agalactiae and S.iniae.Three primer pairs of fbpHT,HtrAHT and VaAHT were established for the triplex PCR assay for S.iniae.While three primer pairs of WRVaA,WRsip and WRHtrA of S.agalactiae were established S.iniae detection.The sensitivity of the method for S.iniae was 3.43 pg/?l and 1.20×10³ CFU/mL,and the sensitivity of the method for S.agalactiae was 1.27 pg/?l and 1.48×10³ CFU/mL.?3?A duplex PCR for the simultaneous identification of S.agalactiae and S.iniae.Sip?Surface immunogenic protein?of S.iniae and 16S rDNA?16S ribosomal DNA?of S.agalactiae were assembled and optimized to establish a duplex PCR technique,which could be used to simultaneously detect the two strains.The DNA sensitivities of S.agalactiae and S.iniae were9.84×10^-1 and 9.30×10^-11 pg/?l,respectively.The bacterial cell sensitivities of S.agalactiae and S.iniae were 2.76×10² and 2.51×10² CFU/mL,respectively.?4?Differential PCR detection for S.agalactiae and S.iniae in tilapia with a single pair of primer.A specific primer pair was designed by aligning the whole genome sequence of S.agalactiae and S.iniae.By using the design primers,DNA fragments in sizes of 616 bp and 794bp were amplified from the genomes of S.agalactiae and S.iniae,respectively.That is,S.agalactiae and S.iniae can be simultaneously and differentially detected by using the designed single primer pair in a one-round PCR reaction.The designed PCR assay was discovered with high sensitivity since for both S.agalactiae and S.iniae,the target bands can be successfully amplified even supplied with extremely low concentration of template DNA?1.5 pg/uL?or bacterial cells?5.8×10³?CFU/mL.In order to determine the detection limit of the designed PCR assay on the tissue samples,artificial tilapia specimens were prepared by mixing the tissues with S.agalactiae and S.iniae in various concentrations.A minimum concentration of 10⁵ CFU/g could be successfully amplified for S.agalactiae and S.iniae.?5?Epidemiological study of tilapia Streptococcosis in Guangdong Province.In order to discover the patterns and features of tilapia Streptococcosis outbreak in China,epidemiological investigation that covered a whole year was carried out in Guangdong Province,mainly in the district of Guangzhou,Foshan,Maoming and Huizhou East.Eight strains of pathogenic bacteria were isolated with the method established in this laboratory.The analysis showed that the outbreak of Streptococcosis was closely related to dissolved oxygen,water temperature,pH,nitrite,and total phosphorus.?6?The screening of single Chinese herbs that simultaneously inhibited and killed S.agalactiae and S.iniae.Thirty-five strains were found by screening 200 kinds of Chinese herbal medicines that could effectively inhibit S.agalactiae and S.iniae,ten of which were more effective.?7?The screening of simultaneously killing of Chinese herbal formulas that simultaneously inhibited and killed S.agalactiae and S.iniae.Ten kinds of Chinese herbal medicine with particularly obvious effect in?6?were selected to conduct correlation experiment.Combining experimental results and drug prices,Eleutherococcus senticosus,Siegesbeckia orientalis,Coptis chinensis,and Cyperus rotundus were used to search formula.The results were formula 4 and 5,both of which had good effects not only on Streptococcus agalactiae and Streptococcus iniae,but also on other major pathogenic bacteria of tilapia.Selecting ten kinds of Chinese herbal medicine with particularly obvious effects,doing related experiments,combining experimental results and drug prices,and finally selecting Acanthopanax and Radix Herbs,Rhizoma Coptidis,and Cyperus rotundus were used for compound search to obtain Compounds 4 and 5,both of which had good effects not only on S.agalactiae and S.iniae,but also on other major pathogenic bacteria of tilapia.In summary,a set of molecular detection techniques was established for S.agalactiae and S.iniae in this article and used to conduct epidemiological investigation of the disease in parts of Guangdong Province.Finally,the compound that had a great effect of killing the two bacteria was obtained from Traditional Chinese medicine.