Bombycis Spores Research Articles

Treatment of Microsporidium Nosema bombycis Spores with the New Antiseptic M250 Helps to Avoid Bacterial and Fungal Contamination of Infected Cultures without Affecting Parasite Polar Tube Extrusion.

Microsporidia are a group of widespread eukaryotic spore-forming intracellular parasites of great economic and scientific importance. Since microsporidia cannot be cultured outside of a host cell, the search for new antimicrosporidian drugs requires an effective antiseptic to sterilize microsporidian spores to infect cell lines. Here, we show that a new polyhexamethylene guanidine derivative M250, which is active against fungi and bacteria at a concentration of 0.5-1 mg/L, is more than 1000 times less effective against spores of the microsporidium Nosema bombycis, a highly virulent pathogen of the silkworm Bombyx mori (LC50 is 0.173%). Treatment of N. bombycis spores that were isolated non-sterilely from silkworm caterpillars with 0.1% M250 solution does not reduce the rate of spore polar tube extrusion. However, it completely prevents contamination of the Sf-900 III cell culture medium by microorganisms in the presence of antibiotics. The addition of untreated spores to the medium results in contamination, whether antibiotics are present or not. Since 0.1% M250 does not affect spore discharging, this compound may be promising for preventing bacterial and fungal contamination of microsporidia-infected cell cultures.

A monoclonal antibody targeting spore wall protein 1 inhibits the proliferation of Nosema bombycis in Bombyx mori.

Microsporidia are obligate intracellular single-cell eukaryotic parasites that can infect almost all kinds of animals and cause microsporidiosis. However, there are a few achievements of microsporidiosis treatments. Nosema bombycis could cause a microsporidiosis condition, pébrine, in the silkworm. Treatment and prevention of pébrine are still the research hotspots in sericulture. In this study, N. bombycis was treated with K2CO3, the spore walls were removed by centrifugation, and then, the alkali-soluble germination proteins were used as antigens to develop monoclonal antibodies (mAbs). Three mAbs were successfully screened and one mAb named G9 showed the highest titer among these monoclonal antibodies after enzyme-linked-immunosorbent serologic assay (ELISA). Mass spectrometry analysis confirmed that the mAb G9 could recognize the N. bombycis spore wall protein 1. Furthermore, the heavy chain and light chain sequences of the G9 monoclonal antibody were cloned, respectively. The vectors that expressing the intact antibodies and the single-chain variable fragments (scFvs) of G9 were constructed, and then, these vectors were used to develop the transgenic silkworm cell lines or transgenic silkworms. The inhibitory effects against N. bombycis were evaluated by the count of microsporidia and qPCR. The scFvs showed better effect on blocking the proliferation of N. bombycis than the intact antibody, and the scFv without the secretory signal peptide was more effective than that with signal peptide. Our study has provided novel strategies for microsporidiosis control and the essential groundwork for the future development of N. bombycis-resistant transgenic silkworms.IMPORTANCEThere are a few reports on the resistance of microsporidia, including Nosema bombycis. Here, the alkali-soluble germination proteins of N. bombycis were used as immunogens to prepare a monoclonal antibody, and its single-chain variable fragments effectively blocked microsporidia infection. Our study has provided novel strategies for microsporidiosis control and demonstrated a useful method for the potential treatment of other microsporidia diseases.

New insights into the effects of UV light on individual Nosema bombycis spores, as determined using single-cell optical approaches.

Nosema bombycis (Nb) is a pathogen causing pebrine in sericulture. Ultraviolet (UV) light exposure is a common physical disinfection method, but the mechanisms underlying UV-based disinfection have only been studied at the population level. In this study, changes in and germination of UV-irradiated spores were observed using Raman tweezers and phase-contrast imaging to evaluate the effects of UV radiation on Nb spores at the single-cell level. We found that irradiation caused the complete leakage of trehalose from individual spores. We also found that more spores leaked as the UV dose increased. There was no significant loss of intracellular biomacromolecules and no marked changes in the peaks associated with protein secondary structures. Low-dose radiation promoted spore germination and high-dose radiation decreased the germination rate, while the germination time did not undergo significant alterations. These results suggest that UV radiation disrupts the permeability of the inner membrane and alters the spore wall, thereby affecting the ability of the spore to sense and respond to extracellular stimuli, which further triggers germination and reduces or stops spore germination. This study provides new insights into the molecular mechanisms underlying conventional disinfection measures on microsporidian spores.

The gut commensal bacterium Enterococcus faecalis LX10 contributes to defending against Nosema bombycis infection in Bombyx mori.

BACKGROUNDMicrosporidia, a group of obligate intracellular fungal‐related parasites, have been used as efficient biocontrol agents for agriculture and forestry pests due to their host specificity and transovarial transmission. They mainly infect insect pests through the intestinal tract, but the interactions between microsporidia and the gut microbiota of the host have not been well demonstrated.RESULTSBased on the microsporidia–Bombyx mori model, we report that the susceptibility of silkworms to exposure to the microsporidium Nosema bombycis was both dose and time dependent. Comparative analyses of the silkworm gut microbiome revealed substantially increased abundance of Enterococcus belonging to Firmicutes after N. bombycis infection. Furthermore, a bacterial strain (LX10) was obtained from the gut of B. mori and identified as Enterococcus faecalis based on 16S rRNA sequence analysis. E. faecalis LX10 reduced the N. bombycis spore germination rate and the infection efficiency in vitro and in vivo, as confirmed by bioassay tests and histopathological analyses. In addition, after simultaneous oral feeding with E. faecalis LX10 and N. bombycis, gene (Akirin, Cecropin A, Mesh, Ssk, DUOX and NOS) expression, hydrogen peroxide and nitric oxide levels, and glutathione S‐transferase (GST) activity showed different degrees of recovery and correction compared with those under N. bombycis infection alone. Finally, the enterococcin LX protein was identified from sterile LX10 fermentation liquid based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis.CONCLUSIONAltogether, the results revealed that E. faecalis LX10 with anti‐N. bombycis activity might play an important role in protecting silkworms from microsporidia. Removal of these specific commensal bacteria with antibiotics and utilization of transgenic symbiotic systems may effectively improve the biocontrol value of microsporidia. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A Field-Friendly Loop-Mediated Isothermal Amplification (FF-LAMP) method for rapid detection of Nosema bombycis in silkworm, Bombyx mori

Pebrine is a destructive disease that exhibits horizontal and vertical transmission and therefore it is the only mandatory quarantine item in sericulture. Here, a field-friendly loop-mediated isothermal amplification (FF-LAMP) method has been developed and validated for the rapid detection of Nosema bombycis, a causative agent of pebrine disease in silkworm, Bombyx mori. FF-LAMP primers were selected and designed for small ribosomal subunit gene and the assay was performed to detect the N. bombycis infection in silkworm. The FF-LAMP reaction was effective at 6 mM MgSO4, 1.4 mM dNTPs at 63 °C. The detection range of LAMP assay was found to be 101 dilutions of N. bombycis spores. Specificity of the primers was tested using DNA isolated from pebrine infected silkworm, pebrine free silkworm and pure N. bombycis by conventional PCR and FF-LAMP assay. Results revealed that the primers were specific to N. bombycis DNA. The FF-LAMP assay was validated in different basic silkworm seed farms with simultaneous microscopic examination of N. bombycis infection. This newly developed method is highly effective, specific, sensitive and rapid in detecting N. bombycis infection, eliminating the DNA purification steps and usage of sophisticated equipment. This method can be used in testing large number of samples making it field friendly method in sericulture industry.

Identification and characterization a novel polar tube protein (NbPTP6) from the microsporidian Nosema bombycis

BackgroundMicrosporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50–500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually.ResultsA novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability.ConclusionsBased on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

The role of Bombyx mori Bmtutl-519 protein in the infection of BmN cells by Nosema bombycis

Bmtutl-519 is an isoform of the Bombyx Turtle protein and a member of the immunoglobulin superfamily (IgSF). The relative expression level of Bmtutl-519 was significantly upregulated when BmN cells were infected by Nosema bombycis. The subcellular localization of Bmtutl-519 was studied using an indirect immunoinfluscent assay (IFA), Co-localization assay, Western blotting, and enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bmtutl-519 expression plasmid. The results indicate that Bmtutl-519 is distributed in both the cytoplasm and the cell membrane of BmN cells. Bmtutl-519 may be involved in the infection process of N. bombycis as a cell surface receptor or regulatory factor. Interaction analysis of Bmtutl-519 with NbSWP26, a spore wall protein of N. bombycis involved in host cell adherence and infection, showed that the C-terminal heparin-binding motif (HBM) of NbSWP26 mediates the interaction between these two proteins. Mutation of the NbSWP26 HBM at K208G, K209G, K210G, and K213G led to a loss of the ability to bind the Bmtutl-519 protein. Spore adherence and infection assays showed that Bmtutl-519 enhances the binding ability of N. bombycis to the host cell surface, but this did not enhance host cell infection by N. bombycis. In contrast, the sustained high expression of Bmtutl-519 in BmN cells inhibited the proliferation of N. bombycis spores.

Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells.

Nosema bombycis is a destructive, obligate intracellular parasite of the Bombyx mori. In this study, a single-chain variable fragment (scFv) dependent technology is developed for the purpose of inhibiting parasite proliferation in insect cells. The scFv-G4, which we prepared from a mouse G4 monoclonal antibody, can target the N. bombycis spore wall protein 12 (NbSWP12). Indirect immunofluorescence assays showed that NbSWP12 located mainly on the outside of the N. bombycis cytoskeleton, although some of it co-localized with β-tubulin in the meront-stage of parasites. When meront division began, NbSWP12 became concentrated at both ends of each meront. Western blotting showed that scFv-G4 could express in Sf9-III cells and recognized native NbSWP12. The transgenic Sf9-III cell line showed better resistance than the controls when challenged with N. bombycis, indicating that NbSWP12 is a promising target in this parasite and this scFv dependent strategy could be a solution for construction of N. bombycis-resistant Bombyx mori.

Functional characterization of an aquaporin from a microsporidium, Nosema bombycis.

Microsporidia are a diverse group of eukaryotic organisms, capable of causing parasitic infections in both vertebrates and invertebrates. During the germination process, there is an increase in the osmotic pressure of microsporidian spores. As part of this study, we cloned a homologous aquaporin gene in Nosema bombycis, and named it Nosema bombycis aquaporin (NbAQP). Sequence analysis revealed that the NbAQP contains an open reading frame with a length of 750 bp and encodes a polypeptide of 249 amino acids. Amino acid sequence homology was greater than 50% that of five aquaporins from other microsporidian species. Indirect immunofluorescence (IFA) and immunogold electron microscopy showed NbAQP to be located predominantly in the spore wall of N. bombycis spores. The results of qRT-PCR analysis revealed that NbAQP expression remained high 0 h after inoculation and decreased sharply to 24 h, increased gradually from 2 days and peaked at 6 days. After expression of NbAQP in Xenopus laevis oocytes, it was observed that NbAQP can promote rapid penetration of water into oocytes. The associated permeation rate was 2–3 times that of the water-injected and uninjected oocytes. Antibody blocking experiments showed that the inhibition rate of spore germination was approximately 28% after antibody blocking. The difference in germination rate between the control group and the NbAQP group was significant (P < 0.05). This study shows for the first time that N. bombycis contains functional water channel proteins and provides a platform suitable for further research into the mechanisms underlying the regulation of NbAQP protein expression. Further study of NbAQP and their inhibitors may have significance for prevention of microsporidiosis.

Ricin-B-lectin enhances microsporidia Nosema bombycis infection in BmN cells from silkworm Bombyx mori.

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion.

Quantitative Proteomic Analysis of Germination of Nosema bombycis Spores under Extremely Alkaline Conditions.

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH > 10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for microsporidia. Here we show, using an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated, respectively, by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analyses in vitro and in vivo. Furthermore, the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine, and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape of N. bombycis spores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general.

Exogenous gene can be integrated into Nosema bombycis genome by mediating with a non-transposon vector.

Nosema bombycis, a microsporidium, is a pathogen of pebrine disease of silkworms, and its genomic DNA sequences had been determined. Thus far, the research of gene functions of microsporidium including N. bombycis cannot be performed with gain/loss of function. In the present study, we targeted to construct transgenic N. bombycis. Therefore, hemocytes of the infected silkworm were transfected with a non-transposon vector pIZT/V5-His vector in vivo, and the blood, in which the hemocyte with green fluorescence could be observed, was added to the cultured BmN cells. Furthermore, normal BmN cells were infected with germinated N. bombycis, and the infected cells were transfected with pIZT/V5-His. Continuous fluorescence observations exposed that there were N. bombycis with green fluorescence in some N. bombycis-infected cells, and the extracted genome from the purified N. bombycis spore was used as templates. PCR amplification was carried out with a pair of primers for specifically amplifying the green fluorescence protein (GFP) gene; a specific product representing the gfp gene could be amplified. Expression of the GFP protein through Western blotting also demonstrated that the gfp gene was perfectly inserted into the genome of N. bombysis. These results illustrated that exogenous gene can be integrated into N. bombycis genome by mediating with a non-transposon vector. Our research not only offers a strategy for research on gene function of N. bombycis but also provides an important reference for constructing genetically modified microsporidium utilized for biocontrol of pests.

Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae

Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis–silkworm interactions due to its quantitative function.

Phagocytic Uptake of Nosema bombycis (Microsporidia) Spores by Insect Cell Lines

Microsporidia are highly specialized obligate intracellular parasites that can infect a wide variety of animals ranging from protists to mammals. The classical concept of the parasite invasion into a host cell involves its polar tube acting as a needle-syringe system. However, recent studies show microsporidian spores can also gain access to host cells by phagocytosis. The present study investigated the phagocytic uptake process of causative agent of the pebrine disease, Nosema bombycis, in several insect cell lines. We observed KOH-treated spores and cold-storaged spores can be easily uptaken by all the studied cell types 4 h post inoculation. In contrast, large numbers of freshly recovered spores remained in the culture medium. To further investigate the intracellular fates of KOH-treated spores and cold-storaged spores, electron and fluorescence microscopy were performed. No intracellular germination or subsequent parasite development were observed. Intracellular spores can be detected in host cells by polyclonal antibody 7 d post inoculation, suggesting phagocytized N. bombycis could not be digested by these non-professional phagocytes. Our results suggest that, phagocytic uptake of N. bombycis spores might represent a defense mechanism of the host cells and the intact spore wall barrier enable freshly recovered spores to keep resistance to this mechanism.

Reduced infectivity and environmental resistance of microsporidian Nosema bombycis after spore wall proteins-removal treatment

Nosema bombycis is a highly virulent causative agent of the pebrine disease in sericulture. The only infective stage of the parasite is the environmentally resistant spore, on which the protein-rich spore wall supposedly contributes to their infection and resistance. In this study, we explore the potential roles of spore wall proteins (SWPs) of mature N. bombycis spores by evaluation of their performances on staining, infectivity, and resistance, following SWPs-removal treatment. Although no significant structural change is discerned by ultrastructural micrographs, the treated spores showed enhanced eosin B staining with strong shades of reddish purple or violet. Moreover, their infectivity to silkworms was significantly affected, resulting in 38.89% compared to 61.11% for the untreated control. Further infectivity bioassays show clearly that they are more susceptible to sodium chloride and heating pretreatment. In all, the enhanced permeability, decreased infectivity and environmental resistance of the SWPs-removal spores indicated thatSWPs play multiple roles in the infection and resistance for N. bombycis. These results have significant implication for controlling the pebrine disease in sericultrue. Key words: Nosema bombycis, microsporidia, spore wall proteins, infectivity, resistance.

Inactivation and mechanisms of chlorine dioxide on Nosema bombycis

Biological tests demonstrated that the inactivation of Nosema bombycis ( N. bombycis) spores by chlorine dioxide (ClO 2) occurs very fast and is highly sensitive. The lowest effective inactivation dosage and time was 15 mg/mL for 30 min. The inactivation of spores was additionally verified by using double color fluorescence stain and spore germination testing. A series of biological changes, including a large number of substrates that were leaked out from the spores included proteins, DNA, polysaccharide, K +, and Ca 2+, occurred a short time after N. bombycis spores were treated with ClO 2. In addition, the lipid of spores was disrupted and ATPase activity was inhibited, which resulted in the destruction of the inner structure of the spores.

A proteomic‐based approach for the characterization of some major structural proteins involved in host–parasite relationships from the silkworm parasite Nosema bombycis (Microsporidia)

Nosema bombycis is the causative agent of the silkworm Bombyx mori pebrine disease which inflicts severe worldwide economical losses in sericulture. Little is known about host-parasite interactions at the molecular level for this spore-forming obligate intracellular parasite which belongs to the fungi-related Microsporidia phylum. Major microsporidian structural proteins from the spore wall (SW) and the polar tube (PT) are known to be involved in host invasion. We developed a proteomic-based approach to identify few N. bombycis proteins belonging to these cell structures. Protein extraction protocols were optimized and four N. bombycis spore protein extracts were compared by SDS-PAGE and 2-DE to establish complementary proteomic profiles. Three proteins were shown to be located at the parasite SW. Moreover, 17 polyclonal antibodies were raised against major N. bombycis proteins from all extracts, and three spots were shown to correspond to polar tube proteins (PTPs) by immunofluorescent assay and transmission electron microscopy immunocytochemistry on cryosections. Specific patterns for each PTP were obtained by MALDI-TOF-MS and MS/MS. Peptide sequence tags were deduced by de novo sequencing using Peaks Online and DeNovoX, then evaluated by MASCOT and SEQUEST searches. Identification parameters were higher than false-positive hits, strengthening our strategy that could be enlarged to a nongenomic context.

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

Enterococcus sp. was isolated from the midgut of silkworm against the germination ofNosema bombycis spores. Identification was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes ofEnterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose.Enterococcus sp. was clustered together withEnterococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes ofEnterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe ofE. mundtii. So, we can make conclusion the 16S rDNA segment ofEnterococcus sp. can hybridise with species-specific probe ofE. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm.

Biochemical characterization of the microsporidian Nosema bombycis spore proteins

Spore proteins of the microsporidian Nosema bombycis, from the silkworm Bombyx mori, were analysed by SDS–polyacrylamide gel electrophoresis. The protein profile of partially solubilized spores showed three major peptide bands of molecular weight 68, 94 and 100 kDa. On complete solubilization, it showed peptide bands ranging from 17 to 68 kDa. Attempts to purify the 17 kDa infection-specific protein showed aggregation of this protein to higher molecular size proteins. Partial peptide analysis of the different peptides exhibited similar patterns suggesting the probablity of processing during the infective cycle. Reconstitution assay showed the reversible nature of this processing. N-terminal sequencing showed homology to heat shock proteins. The low molecular weight 17 kDa protein also showed very high protease activity.

Production of Monoclonal Antibodies Against Nosema bombycis and their Utility for Detection of Pebrine Infection in Bombyx mori L.

Latex agglutination assay based on monoclonal antibodies (MCAs) described in this communication may be useful for detection of Pebrine infection in silkworm. Four murine MCAs were produced against Nosema bombycis spore. In ELISA all 4 MCAs (IgM isotype) reacted with alkali treated Nosema spores and to variable extent with acetone precipitated surface protein. However, MA-310 and MA-542 showed a low degree of cross reactivity with BmNPV. In contrast, MA-503 and MA-515 were devoid of reactivity with BmNPV, B. thuringiensis, S. marcescens, Azotobactor, Rhizobium and normal hemolymph protein in ELISA. Latex beads sensitized with a combination of MA-503 and MA-515 (50 μg each per ml of 0.4% latex beads) could detect 1×105 Nosema spores per test. Sensitization of the latex beads with the cocktail of these two MCAs through protein-A bridge further led to a 10-fold increase in the sensitivity (1×104 spores/test) of the assay. No agglutination was observed in presence of BmNPV, Rhizobium, Azotobactor, E. coli, B. thuringiensis, S. marcescens and normal hemolymph protein indicating the specificity of the test. The results obtained by latex agglutination assay on hemolymph samples of infected as well as normal larvae collected from field, II instar larvae infected in the laboratory and from infected mother moth revealed 100% correlation with results by microscopic examination.