Study On The Major Pathogenic Saprolegnia Species In Freshwater Aquaculture Of China And Saprolegniasis Early Outbreak Detection

Saprolegniasis is one of the most common diseases among the fresh water fishspecies in China. This disease causes huge economic losses to the aquaculture industryworldwide. The disease will attack an existing injury on the fish and can spread quicklyto healthy tissue. Determining the concentration of Saprolegnia spores is crucial forunderstanding the spreading potential of this disease. Having a rapid, highly specific,accurate method for the diagnosis of Saprolegnia spores is essential for ensuring that theappropriate treatments are administered and for reducing economic losses. Tools fordirectly monitoring Saprolegnia spores in aquatic environments are needed.As a result,60isolates of suspected pathogenic Saprolegnia strains collected from China wereidentificated by characteristics of morphology and molecular biolog. We establishedand evaluated two measures—Fluorescence quantitative PCR and LAMP technologyusing different primers according to the principles of each method.We also used theSaprolegnia spores in an infective challenge assay and Our purpose was to investigatethe relationship between the concentration of Saprolegnia spore and outbreak ofSaprolegniasis by monitoring the water using specific qpcr. The main results are asfollows:1. Identification of major pathogenic Saprolegnia species in freshwater aquacuture ofChinaThis study used Restriction Fragment Length Polymorphism (RFLP) and InternalTranscribed Spacer (ITS) PCR sequencing technology to identify60isolates ofsuspected pathogenic Saprolegnia strains collected from7provinces including ShangHai, Jiang Su, Jiang Xi, Si Chuan, He Bei, Zhe Jiang and Hu Bei. Taxonomical analysiswas also performed with strains of Saprolegnia reported in recent years. Comparativestudies on physiological and molecular characteristics of pathogenic strains isolatedfrom both infected fishes and eggs were carried out. The results showed that the60isolates could be classified into10special genetypes and all the isolates belonged to theclade of S. ferax, S. australis and S. parasitia by ITS-RFLP. Based on the phylogenetictree, S. ferax and S. parasitia were the main Chinese pathogenic Saprolegnia strains. Inaddition, no significant difference existed in morphology of both asexual and sexual organs between the two S.ferax isolates from infected fishes and eggs. The same isolatecould infect amphibian eggs and fishes.2.Establishment Real-time PCR and LAMP assays for Saprolegnia spores detectionIn this study, two detection methods, real-time PCR and loop-mediated isothermalamplification (LAMP), were developed for the detection of Saprolegnia spores bytargeting parts of the internal transcribed spacer (ITS) region and the5.8S rRNA geneof the nuclear ribosomal DNA cistron. We compared the sensitivity of both assays withthat of a conventional PCR assay. Results indicated that both assays specifically detectSaprolegnia, and no cross-reaction was found with other samples. The sensitivity of theLAMP method is equivalent to that of real-time PCR, i.e.,100-times higher than that ofconventional PCR.3.The relationship between the concentration of Saprolegnia spores and outbreak ofSaprolegniasisWe use Real-time PCR detection assays for monitoring the relationship betweenSaprolegnia spores concentrations in nature waters.The results demonstrated that grasscarp (Ctenopharyngodon idellu) can be infected by exposure to104(spore/ml)zoospores of S. ferax, while grass carp can be dead by exposure to106(spore/ml)spores.In addition,this study which used LAMP technology for the first time forquantitative detection of Saprolegnia spores, will further facilitate studies ofSaprolegnia spore dynamics during saprolegniasis outbreaks, which will broaden ourknowledge of the biology of this pathogen. The LAMP assays does not require specialequipmentand is more suitable for Saprolegniasis outbreak alert.