一种新的蜜蜂细菌性幼虫病病原的分离鉴定

Isolation and identification of a new bacterial pathogen infecting larvae of honeybee (Apis mellifera) Perish

2005年早春,在浙江部分地区出现了一种严重的蜜蜂细菌性幼虫病,该病导致蜜蜂幼虫颜色发黄,失去光泽;严重时幼虫死亡腐烂。从10批发病死亡幼虫样品中,分离得到并保存了5类纯培养物。通过蜂群接种试验和实验室人工培养的幼虫接种,确定L2菌株能引起与自然发病相似的症状,且能从接种发病的幼虫上再次分离到相同菌株,证明L2菌株是该蜜蜂细菌性幼虫病的致病菌。进一步对分离到的该致病菌从发病特征、病原形态学、生理生化特性、16SrRNA序列等方面进行了分析鉴定,结果显示:该菌株属于肠球菌属的屎肠球菌(Enterococcusfaecium),不是目前报道的任何一种已知蜜蜂细菌性幼虫病的病原。

A bacteroidal disease of honeybee （Apis mellifera ） larvae was found in some regions of Zhejiang Province, China , in early spring 2005. The diseased larvae lost its shine, became yellow and rotted when serious. This symptom was different to any bacteroidal disease of honeybee larval been reported. So, it is considered to be a new bacteroidal disease of honeybee larval. Five pure cultures of bacteria were separated from ten collections of diseased honeybee larvae, named as L1, L2, L3, L4 and L5. Among these five pure cultures, only L2 could make the healthy honeybee larvae become diseased in both field and lab test. The symptom caused by L2 was similar to the natural-infection. From the diseased larvae caused by L2 could isolate bacteria the same as L2. Thus L2 was determined as the causing agent of this bacteroidal disease of honeybee larval. L2 was identified according to the characteristics of morphology, physiological biochemical characteristics and 16S rRNA gene sequence. As a result, the morphology and physiological biochemical characteristics of L2 were similar to E. faecium. And its 16S rRNA sequences highly matched to E. faecium, the similarities between them were higher than 99%. The overall similarity values between L2 and the published 16S rRNA sequences of 41 typical species of Enterococcus were 93.9% - 99.5%, the top value was between L2 and E. faecium. In the phylogenetic tree, L2 and E. faecium were assembled in the same ramification. So L2 was identified as E. faecium. Although Enterococcus faecium was known as pathogen to many post, account for 12% of all nosocomial infections, only second to E. coli, but there is no report about this bacteria infects honeybee up to now. So it is a new pathogen to honeybee. The isolation and identification of pathogen of this new bacteroidal larvae disease, afford a good feasibility for available prevention and cure to this new disease.