Ggermplasm Investigation And Study On Immunology Diagnostic Technique Of Microsporedia Of Honeeybee

Bee nosema disease seriously endangers.beekeeping industry all over the world. In order to effectively treat and prevent the disease, it is essential at first to elucidate the disease's biological features such as the disease's species, infection characteristic and diagnosis. In present study, we establish the method of isolating the microsporidia of honeybee and immunology diagnostic technique. Furthermore, we compared the infection potential to Apis mellifera and Apis cerana in lab. The germplasm identification have been implemented on microsporidia of honeybee using the method which amplified the specific fragment of 16s r RNA gene. The results are as follows.1. Using purification of Nosema bombycis for reference to determine the method of purification that is suitable for microspoiedia of honeybee. This method that combined the differential centrifugation and high speed centrifugation was set up.The favorable conditions for the differential centrifugation were at 500 rpm for 2 min and 1000 rpm for 10 min. The optimum conditions were percoll gradient and centrifugation at 46000g for 90 min. After centrifugation, the microsporidia of honeybee with high purity was isolated.2. Using the primers published for bee nosema disease, an 16s rRNA gene amplification has been done on 16 microsporidia of honeybee samples from Apis mellifera colonies of the mian apiculture areas of China. PCR products were cloned and sequenced. The Homology tree was constructed by the software DNAMAN. According to the results of Blast sequence analysis in NCBI and the comparison with the sequences of Nosema ceranae and Nosema apis published in NCBI, the sequence similarity of them were 99% and 91% of identity. Thus, we concluded that the pathogen of Apis mellifera colonies was Nosema ceranae. The samples we collected did not contain Nosema apis Zender recored in literatures.3. We completed the study on the difference of Nosema cerana to Apis mellifera and Apis cerana. Results showed that the infection rate of Apis mellifera was significantly higher than that of Apis cerana. We found that the infection potential of Nosema ceranae to Apis cerana was stronger than those to Apis mellifera.4. A diagnostic method for the detection of microsporidia of honeybee was developed by the optimization of ELISA protocols. The purified rabbit antibody against Microsporidia of honeybee was used as the first antibody and HRP-sheep anti-rabbit IgG as the second antibody. The developed method can detected the Microsporidia of honeybee at a concentration of 103 spores/mL. The sensitivity of this method is higher than the sensitivity of ordinary optical microscopy.