Identification Of Pathogenic Bacterium Causing A Novel Honeybee Larvae Disease And Drug Selection

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.