Isolation And Identification Of The Pathogen Of Streptococcicosis And Screening Of Its Controlling Chinese Herbal Medicines For Tilapia Cultured In Hainan

In Recent years, tilapia cultured in southern china such as Hainan, Guangdong and Guangxi provinces outbreaks serious streptococcicosis and its pathogen was proved mainly Streptococcus agalactiae(SA). The incidence rate of this disease is about20%-50%and its mortality is more than50%. The disease have become one of the main factors which impede the sustainable development of tilapia culture. Moreover, because SA can escape from being killed by antibiotics by the macrophages in its host, the effect of antibiotics on controlling this disease is not so good. Chinese herbal medicine(CHM) is a natural medicine and possesses many advantages such as having multiple targets, without antimicrobial resistance and drug residues. It was used more and more extensive for disease control in aquaculture and is regarded as a good selection for streptococcicosis control. Therefore, the pathogen was isolated and identified from the diseased tilapia with typical symptoms of streptococcicosis in Hainan. CHMs with good anti-SA activities were rapidly screened and the optimal conditions for extracting the active substances from the screened CHMs were studied.Two strains of dominant bacteria, named as QH-726and WC-806, were isolated from tilapia with the typical symptoms of streptococcus disease cultured in Qionghai and Wenchang, The results of artificial challeng by intraperitoneal injection showed that both islolated trains had strong pathogenicity against tilapia and its symptoms were similar to those of natural infection. Both of the islated stains were identified as Streptococcus agalactiae (SA) by their morphological characteristics, physiological and biochemical characteristics and16S rRNA test.The extraction of CHM against SA was preliminary screened from75kinds of the CHMs by using ultrasonic and boiling water bath methods and102kinds of the CHMs by using water bath extraction at50℃and water bath at room temperature in a micro plate. The CHMs with inhibitation rate above5%were screened again with agar diffusion method. The minimum inhibitation concentration and minimum bactericidal concentration were checked for the screened medicines by two times dilution agar diffusion method. The results showed that, the inhibitation rate of ten CHMs by ultrasonic extraction method, eight CHMs by boiling water bath extraction method, fourteen CHMs by water bath extraction at50℃and thirty CHMs by water bath extraction at room temperature were above5%. The results of secondly screening using agar diffusion method showed that,8CHMs by ultrasonic extraction method,8CHMs by boiling water bath extraction method,14CHMs by water bath extraction at50℃and21CHMs by water bath extraction at room temperature had good anti-SA effects. The accuracy of screening were82.3%.The infection model was established for challenging tilapia against SA by intraperitoneal injection and the treatment effect of the CHM with good anti-SA activity was studied by using artificial feed pellete containing10%of CHM to feed tilapia after intraperitoneally injected with SA. The results showed that, the extraction of1CHMs by ultrasonic method,4CHMs by water bath at50℃and3CHMs by water bath at room temperature had good effects on curing the streptococcicosis. The protective rates of8CHMs were100%,77.8%,55.6%,33.3%,75.0%,62.5%,25.0%and12.5%. Median effective doses (ED50) and the minimum effective doses against SA of3screened CHMs with protective rates above75%were determined. The ED50were4.934%,4.483%and3.908%, and the minimum effective doses were0.052%,0.013%and0.001%, respectively.The effective ingredients in the CHM are complex and their contents are relatively low, so it is difficult to isolate them. In order to improve the aquired rates of effective ingredients from CHMs, the optimal conditions for extracting the active substances from TCH15were studied with single factor method and response surface methodology. The results showed that the optimal conditions for extracting anti-SA ingredients from TCH were as follows:the ratio of liquid and material37.80:1.0(mL:g), temperature86.7℃, infiltration time104min and extraction time182min. Under these optimal extracting conditions, the predicted extreme value of aquire rate for active ingredient against SA was25.95%and its actual extraction rate was (25.87±0.04) after four parallel tests. It is2.59times higher than the data befroe optimization.In brief, a method for rapid screening the effective CHMs against SA was established in this paper, the effect of the selected CHMs on curing the tilapia artificially infected with SA and the optimal extraction conditions of the CHM against SA were also studied. The results of this study will be helpful for aquiring safe and effective CHMs for controlling streptococcicosis in tilapia and have good effects on improving the survival rates for tilapia culture and the quality of tilapia product.