二尖梅奇酵母的致病性研究及敏感药物筛选

Pathogenicity Analysis of Yeast Metschnikowia bicuspidata and Sensitive Drug Screening

为探明中华绒螯蟹“牛奶病”病原——二尖梅奇酵母对中华绒螯蟹的致病性,并筛选病原敏感药物用于指导生产,笔者采用自发病蟹处分离的二尖梅奇酵母,通过人工回感健康中华绒螯蟹[平均体质量(6.8±0.7)g]确定其半致死密度,研究温度对其感染的影响,探索二尖梅奇酵母的致病力,并采用牛津杯法测试二尖梅奇酵母对多种药物的敏感性及敏感药物的最小抑菌质量浓度和最小杀菌质量浓度。结果显示:二尖梅奇酵母在水温15℃、感染14 d后,对蟹种的半致死密度为1.05×10^(5)cfu/mL;在10~30℃,中华绒螯蟹发病死亡率与温度呈正相关;在实验室养殖条件下(22℃),注射密度为2.1×10^(3)cfu/mL的病原酵母21 d后可致90%的中华绒螯蟹死亡。葡萄糖酸氯已定醇溶液、制霉菌素、苯扎溴铵、大蒜粗提物等对二尖梅奇酵母有一定的杀灭作用;葡萄糖酸氯已定醇溶液、制霉菌素、苯扎溴铵对二尖梅奇酵母的最小抑菌质量浓度分别为10、10、5 mg/L,最小杀菌质量浓度分别为20、100、10 mg/L;大蒜粗提物对二尖梅奇酵母也具有较好的抑制及杀灭效果,稀释32倍仍可杀灭病原酵母。本研究结果可为中华绒螯蟹“牛奶病”的科学防控提供参考和依据。

In order to explore the pathogenicity and environmental impact factors of yeast Metschnikowia bicuspidate(JMB-1),and to screen pathogenic sensitive drugs for control of“milky disease”in Chinese mitten crab Eriocheir sinensis,the median lethal concentration(LC 50)of JMB-1 isolated from the diseased crab was determined in healthy Chinese mitten crab with body weight of(6.8±0.7)g by challenge experiments,the effect of temperature on infection was studied,and the pathogenicity of JMB-1 was explored.The sensitivity of JMB-1 to drugs was tested by Oxford cup plate assay,and the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of preferred drugs were tested.The results showed that the median lethal concentration(LC 50)of JMB-1 was 1.05×10^(5)cfu/mL in 14 days of infection at water temperature of 15℃,with positive correlation between the mortality of Chinese mitten crab and temperature in the range of 10—30℃.Challenge test revealed that JMB-1 concentration of 2.1×10^(3)cfu/mL led to 90%mortality of Chinese mitten crab in 21 days under laboratory culture conditions(22℃).Chlorhexidine gluconate solution,nystatin and benzalkonium had a better inhibitory effect on JMB-1,with an MIC of 10,10,5 mg/L and an MBC of 20,100,10 mg/L,respectively.The crude extract of garlic also had a good inhibitory effect on JMB-1,and a dilution of 32 times killed JMB-1.The finding provides reference with the scientific prevention and control of"milk disease"in Chinese mitten crab.