| Saprolegniasis is an common disease which is caused by filamentous fungi in aquaculture, it is one of the main factors which can lead to breeding failure. In the adult fish farming, it may also occur Saprolegniasis, if there are no timely measures, it may lead to large-scale disease outbreak and cause huge losses to aquaculture.It has been a long time for us to use "malachite green" to treat the disease in the aquaculture production, and there was a good effect. However, malachite green is highly toxic, high residue, high carcinogenic and high teratogenic. In 2002, the bulletin No. 193 issued by the PRC Ministry of Agriculture and the standard NY 5072 - 2002 "pollution-free food and fisheries guidelines for the use of drugs" clearly ordained the prohibition of use of the malachite green in the fisheries.The prohibition of use of the malachite green made the control of Saprolegniasis lack a safe and effective alternative drug, so there was a very strong demand for the Saprolegniasis drugs in the production of aquaculture. There was not enough study on the aspects of Saprolegniasis drug development in domestic and international research, and there was also no significant progress over the years. After the previous studies in our laboratory, we found a kind of substance of high activity against Saprolegnia in vitro called compound X, in order to further improve its performance, We started the research from two aspects: biological therapy and chemical structure modification, through the processes about the establishment of drug screening models, screening, formulation, safety evaluation, clinical efficacy evaluation and at last we developed an new drug which can alternate "malachite green", it has some innovative and promotional value, it obtained good effect in the practical application, it changed the situation of the Saprolegniasis drug blank in aquaculture and solved the "Malachite green" abuse to a certain extent, it also played an important role in the protection of human health and the quality and safety of aquatic animals.The thesis is divided into four chapters. Chapter I: The comparison of vitro efficacy evaluation of Saprolegnia drugs; Chapter II: Anti-Saprolegnia drugs screening and the subsequent antibacterial effect; Chapter III: Anti-Saprolegnia drugs complex and in vitro drug efficacy evaluation; Chapter IV: anti-Saprolegnia drugs safety evaluation and clinical results. The details are as follows:Chapter I: In order to find a high stability, convenient and efficient vitro drug screening and vitro efficacy evaluation method to facilitate the comparison of the results of drug screening, but also provide a unified and reliable method for the evaluation of drug efficacy in vitro, the common in vitro antifungal susceptibility test methods: liquid dilution and agar dilution methods were used to evaluate the effect of three drugs on anti-Saprolegnia XJ, the strain of which was preserved in our laboratory, the three drugs were gall powder, pentahydrate copper sulfate and malachite green. We evaluated the efficacy in vitro of two different evaluation methods by studying the MIC of spores and mycelium, we drew the following conclusions: The MIC values of drugs on the mycelium were greater than the MIC values on the spores, the MIC values of the hyphae could be used in the drug screening, since the MIC values of liquid dilution were lower than them of agar dilution, the liquid dilution method could be used in the drug screening in order to retain more of the drugs into the next screen and efficacy evaluation in vitro. So we finally confirmed a reliable screening method.Chapter II: From the large number of documents and extensive collection of examples of potential anti-fungal activity of substances (compound or simple substance), we established a drug library to be screened, we started the preliminary screening of drugs into different categories in the drug library, and made the use of liquid dilution method for screening, the drugs which their MIC values of mycelium above the highest concentration should be discarded and we further considered the physical and chemical properties, water solubility, toxicity and the use of cost by looking for relevant information, we got rid of the drugs of high toxicity and high price. At last, the copper sulfate, potassium aluminum sulfate, isothiazolin-ketone, sulfosalicylic acid, ethyl paraben, MBT(dithiocyano-methane) went to the second screening about the drug efficacy evaluation in vitro. Though the comparison of two ways of drug efficacy evaluation in vitro, the results of the efficacy on the spores and mycelium in vitro were obtained. We also studied the subsequent antibacterial effect of two excellent fungicides.Chapter III: In order to investigate the performance of the complex anti-Saprolegnia drug efficacy of the above compounds and the compound of X, we studied the combined antimicrobial effect of copper sulfate, isothiazolin-ketone, sulfosalicylic acid, ethyl paraben and MBT respectively with compound X. We chose one which had a synergy with the compound X, and combined them with different ratios to determine the best mixture ratio between them and found the best mixture ratio was 5:2. We mixed the two drugs with that ratio and added fumed silica together with them and made a new anti-Saprolegnia drug called compound X preparation, the MIC of the spores was 1ppm and the MIC of the mycelium was 5ppm, it was recommended that the concentration of soaking about the compound X preparation in the aquaculture production was 5mg / L in order to get the lowest cost.Chapter IV: In order to verify the application effects of actual production, we studied the safety evaluation and clinical efficacy of the compound X preparation. The safety concentration of compound X preparation to most freshwater fish was 5mg / L or more, it was safe to use 50mg / L or less to eggs of most freshwater fish, 25mg / L or less to eggs of shrimp and crab. Clinical studies had shown that the incidence of Saprolegniasis could be significantly reduced at 5ppm or more (p<0.05), the emergence of crucian carp and micropterus salmoides could not be significantly reduced at 50ppm or less (p>0.05). |
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