Studies On Detection Of Nosema Bombycis And Pebrine Forecast

Microsporidia are a group of obligate intracellular eukaryotic parasites that are widely distributed in nature, whose host range from invertebrates to vertebrates, including human. Nowadays, about 1,400 species belonging to 160 genera of microsporidia have been reported. Nosema bombycis was firstly discovered from infected Bombyx mori in 1857. This organism can cause pebrine and vertical transmissionvia eggs and bring huge loss to silkworm feeding, so it is defined as the only disease needed for quarantine in sericulture. In Mid-19^th-century, pebrine outbroke in quite several European countries and resulted in heavy loss. Today, more than ten million RMB each year have been lost due to this disease in China. Thus, the identification of silkworm pebrine is very significant in sericulture. To date, the methods to diagnose the silkworm pebrine mainly contain microscopic diagnostics, molecular diagnostics and serological diagnostics, with their advantages and disadvantages. Microscopic examination of female moths have prevalenced 100 years. Serological detection method, based on the specific and sensitive antibody-antigen reaction and the colloidal gold immunization chromatography assay, has broad prospects in the diagnostics of pebrine. In this study, we prepared monoclonal antibodies (MAbs) against N. bombycis and investigated the application of this antibody to rapid detection of silkworm pebrine. The results are as following:1 Preparation and identification of monoclonal antibodies against germinated spores of Nosema bombycis.The N. bombycis spore was purified by differential centrifugation and densithy gradient centrifugation. The antigen was preparated after using the potassium carbonate to trigger the germination of the spore. BALB/c mice were immunized with the dose of 75μg/mouse, and after four times'inoculation, the antibody titer in serum of mice was up to 1:6.4×103 detected by indirect ELISA.The hybridoma cell lines were prepared by fusing the NS₀ myeloma cells and the lymphocytes cells from BALB/c mice spleen using routine technology, with a cell fusion rate up to 97.7% and a positive rate reachs 3.2%. Three hybridoma cell lines to secrete monoclonal antibodies were developed by two subclones, named 1D6-A10,1D₆-E12 and 4D2-D5.The ascites antibody titer of both 1D6-A10 and1D₆-E12 and the cell-culture supernatant antibody titer of 4D₂-D5 were 1:6.4×105,1:1.28×106 and 1:5.12×102, respectively. The specificity of the McAb of 1D₆-A10, 1D6-E12 and 4D2-D5 against N. bombycis was identified by Western-blot (WB), and there were two protein bands with reaction signal, one with 31 kDa while the other with 18 kDa.2. Preparation and identification of monoclonal antibodies against Spore wall protein32(SWP32) of Nosema bombycisl.We purified the N. bombycis spores through differential and density gradient centrifugation. The spore was promoted to germinate by potassium carbonate. Second density gradient centrifugation was used to obtain the empty spore coats. Total spore wall proteins were extracted, and SWP32 was gained as the antigen by SDS-PAGE and electroeluting. Then, BALB/c mice were immunized with dose of 75μg/mouse, and the titer reached to 1:5.12×10⁴ after four times' inoculation. The spleen cells of BALB/c mice and the NSo myeloma cells were fused using PEG-1500. One hybridoma cell line, called C7, was obtained through twice subcloning. Its ascites antibody titer was 1:1.28×10⁶ and cell-culture supernatant antibody titer was 1:2.56×10³.The specificity of the McAb C7 against SWP32 protein of N. bombycis was analyzed through Western-blot (WB) and indirect fluorescent antibody test (IFAT). The WB showed only one band (SWP32) react with antibody but no signal shown in the IFAT, suggesting that the C7-target epitope may be not exhibited on the surface of spore of Nosema bombycis.3.Preliminary application of monoclonal antibody against Nosema bombycis.Monoclonal antibodies were labeled with colloidal gold that was prepared by sodium citrate reduction method. According to immunization chromatography assay,we spotted the sample on the nitrocellulose membrane, and made antibody marked by colloidal gold to flow through this membrane, the labeled antibody and the antigen in sample would bind together and a red spot will form to detect N. bombycis. The results showed this method could detect the purified spores with at least 30 spores per field, the spores in the infected silkworm with more than 60 spores per field, and the spores in the silkworm moth after differential centrifugation can be detected too.