蜜蜂螺原体的分离鉴定及致病性研究

Identification and Pathogenicity of a Spiroplasma Isolated from Honeybee

从患"爬蜂病"的蜜蜂体内分离到一株螺原体M10,具有典型的螺原体形态和运动性,能透过0.22μm孔径的滤膜,在含青霉素浓度为2000U/mL的R-2培养基中生长良好。该菌株生长需要血清,能利用葡萄糖、精氨酸、不能利用尿素,其16S rDNA序列与Spiroplasma melliferum BC-3(=ATCC33219)同源性为99.86%。通过饲喂菌液的方式,发现供试蜜蜂4d开始出现"爬蜂病"病症,15d内71%的蜜蜂死亡,说明M10对蜜蜂具有较强的致病性,且感染致死的蜜蜂体内螺原体的分离率为100%,利用螺原体特异性16S rDNA引物在感染致死的蜜蜂的不同部位(头、胸、腹、足)均能扩增出螺原体16S rDNA,反映了螺原体对蜜蜂的系统性侵染。

M10, a spiroplasma isolated from honeybee showed a helical morphology and a contractile movement in R-2 medium. The isolate M10 could pass through a 220nm membrane and resist to penicillin （2000U/ml）. It must grow in medium with serum. Glucose could be used as its source of carbon instead of sucrose. Arginine could be metabolized and urea could not. The 16S rDNA of M10 shared 99.86% similarity with the typical strain Spiroplasma meUiferum BC-3 （ = ATCC33219）. M10 can cause serious disease to honeybees through feeding cultures, the honeybees showed symptoms of spiroplasmosis around 4 days after feeding and the death rate reached to 71% around 15 days. The isolation rate of spiroplasmas from dead honeybees was as high as 100%. Using primers of Spiroplasma-specific 16S rDNA, 16S rDNA of spiroplasma was amplified in the various parts of honeybees, such as heart, chest, abdomen and appendage of the dead honeybees. The results indicated that M10, which caused spiroplasmosis, could infect honeybees systemically.