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SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

Muga silkworms are often prone to many diseases since, these are non-domesticated and are reared outdoors. Microsporidia, an obligate intracellular pathogen with spore as its active form, causes pebrine disease in these silkworms. The study has attempted to categorise the transcript data of the Nosema obtained from the infected muga silkworm using gene ontology and KEGG pathway studies. A total of 2850 unigene sets were identified out of which 2739 unigenes were placed under biological, cellular as well as molecular function categories based on the gene ontology (GO) terms. 1620 out of these unigenes sets found their orthologous partner in the corresponding Nosema bombycis transcriptome. The unigenes were found to be enriched under organic substance metabolic process, organic cyclic compound binding and intracellular anatomical structure for biological process, molecular function and cellular components respectively. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated majority of the enzymes were found to be mapped under purine and thiamine metabolic pathways, indicating an increase in the energy metabolism required to establish the infection in the silkworms. The putative virulence genes identified in this study are PTP2, PTP3, SWP12 and SWP26 which were found to be expressed in other Nosema species indigenous to India, indicating a probable conservation of these genes, which are primarily involved in establishing host pathogen interactions. The expression of these genes was in detectable levels in the infected silkworm samples. These genes may be validated further through bioassay in order understand their roles in establishing the infection and propagation of the spores. The identified virulence genes may be further targeted to develop diagnostic tools for identification of the pathogen at early stages of infection.

Pebrine is one of the devastating diseases mostly caused by notorious Nosema - a microsporidian infecting silkworms. Identification of novel genes associated with the pathogen plays a key role in developing a reliable diagnostic tool for the detection of disease. Targeting potential biomarkers will help in developing strategies for fast and efficient control measures, which can prevent the spread of infection. This study was to identify genes present commonly in Nosema such as Nosema bombycis, Nosema mylitta, Nosema assamensis and Nosema ricini infecting Indian silkworms in order to find potential gene markers for early pebrine disease diagnosis. Real time PCR was used to validate the genes active early during the infection cycle, confirming the expression of genes and their order of expression. 16S rRNA and β-tubulin were found to be expressed early in infection followed by PTP1 and PTP2, PTP3, SWP5 and MetAP2 genes. These identified molecular markers can be used in addition to conventional gene primers which are traditionally used for the detection of pebrine.

"Pe ' brine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the beta-tubulin gene in the pe ' brine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 x 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV)