【作者】 戎映君；

【导师】 陈集双； 苏松坤；

【作者基本信息】 浙江大学 ， 微生物学， 2006， 硕士

【摘要】 蜜蜂细菌性幼虫病是严重危害蜜蜂正常生长发育的传染性疾病,对养蜂生产和蜂产品质量安全构成重大威胁。至今为止,已报道的蜜蜂细菌性幼虫病包括美洲幼虫腐臭病、欧洲幼虫腐臭病以及蜜蜂粉介病。 本研究论文对2005年浙江省慈溪、富阳、江山等地蜂场发生的一种新的蜜蜂细菌性幼虫病的病原进行了分离和鉴定,并开展了该病原菌的药物敏感性试验研究。实验结果分述如下: 1、在浙江省慈溪、富阳、江山等地采集自然发病的蜜蜂幼虫样品,通过J培养基、EFB培养基、LB培养基上细菌的分离纯化,结果从15份发病幼虫样品中共分离得到了5种细菌纯培养物,取其代表菌株分别标记为L1、L2、L3、L4、L5,其中L2在每份幼虫样品中都分离得到。 2、分离到的5种细菌纯培养物,在实验室通过人工培育的蜜蜂幼虫进行致病性测定。结果接种L2的幼虫,在接种后第2天开始发病,与对照组相比,发病幼虫生长缓慢,虫体失去光泽,有些幼虫腐烂,五天后大部分死亡,3天及5天后平均死亡率分别达60.00%和93.33%,均显著高于对照组;而接种其他四种菌株的幼虫,在观察期间虽有死亡,但是没有出现上述发病症状,其死亡率与对照组相比,无论是3天后还是5天后,均没有显著差异。取接种引起的发病幼虫进行细菌平板分离,结果又得到了与L2菌落形态和菌体形态相同的细菌纯培养物。 3、实验室筛选出的具有致病性的菌株L2,通过蜂群接种实验再次对其致病性进行鉴定。结果从接种完L2菌株后的第二天开始,在实验组的15群蜜蜂中出现了不同程度的发病,发病症状跟自然发病症状基本相同,而对照组的15群蜜蜂中则没有出现异样;分别在接种前和接种后12天进行定群,实验组接种前后蜂群封盖子、群势、幼虫数量的差异极显著,而对照组则没有明显差异。取接种引起的发病幼虫进行细菌平板分离,结果又得到了与L2菌落形态和菌体形态相同的细菌纯培养物。由此得出结论:L2是该蜜蜂幼虫病的一种病原菌。 4、L2通过观察细菌形态特征、生理生化特征以及分析16S rRNA基因序列更多还原

【Abstract】 Bacterial disease of honeybee larvae was contagious. It seriously affected the growth of honeybee, the quality of bee products, as well as the beekeeping industriy. Up to now, three kinds of bacterial diseases of honeybee larvae had been reported, including American foulbrood, European foulbrood and honeybee powdery-scale.In this paper, samples of a new honeybee larvae disease were collected from regions of Zhejiang Province, from 2005 to 2006, and the pathogen was isolated and identified, and its sensitivity to drugs was tested. The results obtained are as the following:1. Five pure cultures of bacteria were separated from fifteen samples on three different culture mediums;we named them as L1, L2, L3, L4 and L5. Among these five pure cultures, only L2 was found in every sample.2. Pathogenicity of these five pure cultures was tested by In-vitro larval rearing. The larvae inoculated with L2 became diseased the day after inoculation. Compared to the healthy larvae, the diseased ones grew slowly, lost their shine, and rot. And most of them were died five days after inoculation. The mortality of the larvae inoculated with L2 was 60.00% three days after inoculation, and 93.33% five days after inoculation, both significantly higher than control group. While the larvae inoculated with other four bacteria have no above-mentioned symptoms, and their mortality was similar to the control group. We could isolate pure culture of a bacterium the same as L2 from the diseased larvae caused by L2.3. Pathogenicity of L2 was confirmed again by the colony trail. The experiment groups became diseased the day after inoculation, while the control groups were healthy. The symptom was similar to the natural-infection. Sealed brood number, colony size and larvae number of experiment groups had significant difference before and twelve days after inoculation, while the control groups didn’t have. We could isolate pure culture of a bacterium the same as L2 from the diseased larvae caused by L2. So we conclude that L2 is a pathogen of this honeybee larvae disease.4. L2 was identified according to the characteristics of morphology , physiological biochemical characteristics and 16S rRNA gene sequence. As a result, we found the morphology and physiological biochemical characteristics of L2 were similar to E. faecium. And its 16S rRNA sequences highly matched to E. faecium, the similarity between them was higher than 99%. In the phylogenetic tree, which was constructed by comparing the 16S rRNA sequences of the L2 with the published 16S rRNA sequences of 41 typical species of Enterococcus, the overall similarity values between strain L2 and the 41 typical species of Enterococcus were 93.9%-99.5%, the top value was between L2 and E. faecium. So L2 was identified as E. faecium.5. Sensitivity of the E. faecium to drugs was tested by inhibition zones trail. The result showed: Among the three royal jelly fragments, only fragment 2 was resisted to E. faecium. Among the eighteen herbal medicine: garlic, indigowoad leaf, bastardtoadflax, hawthorn, gall and liquorice had notable restrain effect;lesser galangal rhizome, largehead atractylodesrhizome, pulsatilla root and goldenthread took the second place;while membranous milkvetch root, honeysuckle flower, rhubarb, indigowoad root, baical skullcap root and amur corktree bark had little restrain effect. According to the above results, we choosed garlic, indigowoad leaf, bastardtoadflax, hawthorn and gall for the compound trail. The result showed that the best prescription was: 410 mg gall, 10 mg bastardtoadflax, 10 mg indigowoad leaf, 10 mg hawthorn and 410 mg garlic in 10 mL water.更多还原