| "Milky disease",also called "emulsification disease",is a serious epidemic that occurred in the farm of Eriocheir sinensis in Panjin City in 2019.The mortality rate exceeded 20%and caused enormous economic losses to farmers.In order to clarify its pathogen and prevention methods,this experiment conducted research on the isolation and identification of the pathogen,histopathology and the screening of prevention and treatment drugs for milky disease of E.sinensis.The results showed that the diseased crabs were emaciated,the tissues were severely emulsified,and there was a large amount of milky hemolymph.For milky disease in E.sinensis,the microscopic identification showed that the pathogenic agent is a yeast-like organism,the size of which was 0.98-1.55×1.56-2.95?m,most of them are ovoid,budding and reproductive.The purified yeasts were analyzed by electron microscopy(TEM,SEM),and it was found that the purified liquid contained a large number of clumped thallus,and some of the thallus were budding.The cell structure is clearly visible,and most of the yeast cells wre mononuclear or multinucleated.The yeasts formed pink colonies on red bengal,and white colonies on TCBS medium.After PCR amplification,plasmid extraction,and delivery of each product,the results were subjected to BLAST and identified as Metschnikowia bicuspidata.Inaddtion,the the yeast-challenged experiment also identified that the yeast M.bicuspidata was the pathogen of milky disease of Chinese mitten crab in Panjin City.According to the tissues and hemolymph emulsification symptoms of diseased crabs based on the histopathological study,the disease was divided into three stages: lightly infected,moderately infected and highly infected.With the development of the disease,the feeding rate declined gradually or even stops to feed,the hemolymph and cohesion gradually decreases,the yeast content in hemolymph increased,and the color of hemolymph changed from light blue to milky white.Histological analysis of the diseased crabs showed a marked disorder of fibers,discrete necrotic lesions,and high yeast infiltration in the muscles,heart,gills,and other organs.The hepatopancreas and muscles of sick crabs were observed andanalyzed by electron microscope(TEM,SEM).A large number of yeasts were also found in the hepatopancreas and muscles of sick crabs.The drug immersion and antimicrobial susceptibility test was carried out on M.bicuspidata.Among the 15 selected drugs,7 drugs were tested to have well antibacterial effects on M.bicuspidata.While the killing effective of potassium 5-Fluorocytosine,disinfection powder and permanganate was better than other drugs,and the 24 h Lethal concentration was 1ppm,1ppm and 2ppm,respectively.Two of fourteen kinds of drug sensitive tablets of Nystatin and polymyxin B were selected and verified to have better inhibitory effects and highly sensitive on M.bicuspidata.The diameter of the inhibition zone is 30 mm,14mm,respectively,while the other 12 drug sensitive tablets had no antibacterial effect or the antibacterial effect is not obvious.Then,according to the results of the immersion killing experiment and the drug susceptibility experiment,six drugs were selected for the determination of MIC and MBC.Only 5-fluorocytosine and Amphotericin-B were effective,and 5-fluorocytosine MIC and MBC were 10 ppm,100ppm.Amphotericin-B MIC and MBC are 10 ppm and 200 ppm.Disinfecting powder,chlorine disinfectant,copper sulfate,and potassium permanganate also work,but the dosage is too large. |
PDF Full Text Request