RESUMEN
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.
Asunto(s)
Bombyx , Nosema , Animales , Esporas Fúngicas/genética , Esporas Fúngicas/química , Esporas Fúngicas/metabolismo , Proteínas Fúngicas/química , Nosema/genética , Nosema/química , Nosema/metabolismo , Bombyx/genética , Clonación MolecularRESUMEN
Microsporidia are intracellular fungal parasites and they are the most common pathogens for sericulture. Microsporidian sp. can cause pebrine, a dreadful disease and lead to destructive disorder in Muga silkworm, Antheraea assamensis Helfer by vertical and horizontal transmission. This disease is the key factor obstructing the developmental progress of Muga culture in India. Nevertheless, molecular identification and characterization of pathogen associated with pebrine disease in A. assamensis is not yet established. Insect bioassay studies revealed that microsporidian infection in Muga silkworm, A. assamensis Helfer significantly reduced (P < 0.005) cocoon weight, pupal weight, shell weight and silk ratios. A new set of PCR primers suitable for amplification of small subunit ribosomal RNA (SSU-rRNA) of microsporidia infecting A. assamensis have been designed. The amplicon was cloned, sequenced and analysed. Microsporidia pathogen of wild silk moth A. assamensis has been identified at genus level as Nosema sp. AA1. Phylogeny of Nosema sp. AA1 was constructed on the basis of SSU-rRNA sequence and it has a close evolutionary relationship with microsporidian pathogens of other wild silkmoths. The arrangement and organization of the rRNA genes inferred that Nosema sp. AA1 belongs to true Nosema group and not to members of the Nosema/Vairimorpha group.
RESUMEN
Microsporidiosis (Pebrine) caused by the microsporidian parasite is one of the important devastating disease which affect the silk production leading to an unprofitable harvest. Till date ribosomal RNA (rRNA) gene was used as a target for detection of microsporidian species. In this study, we describe conventional and SYBR green based real-time PCR techniques alternatively targeting ß-tubulin gene for quantitative detection of microsporidia infecting both the mulberry and non-mulberry silkworms. The modified DNA extraction method followed in our study was found to be easy, economical and could be used for both conventional and real time PCR as template. The real time qPCR revealed the expression of ß-tubulin gene in different infected tissues of the silkworm Bombyx mori. The sensitivity of the SYBR green based real time PCR was found to be 100 times more than the conventional PCR and PCR was found more sensitive than the microscopic examination. The developed method did not produce any false positive results with the other silkworm pathogens and healthy silkworm. The data suggest that both the developed PCR methods targeting ß-tubulin gene could be used effectively in quarantine process at seed centres for early detection of microsporidian infection in silkworms.
RESUMEN
Nosema bombycis is a spore-forming parasite causing microsporidiosis in silkworm Bombyx mori. Methionine aminopeptidase 2 (MetAP2), an essential gene of N. bombycis, is a target for the anti-microsporidian drug Fumagillin, an antibiotic derived from Aspergillus fumigatus. In this study, a 1077 bp full-length cDNA of the MetAP2 gene of N. bombycis was cloned and characterized. Furthermore, the expression study of the MetAP2 gene revealed a ubiquitous expression during all the developmental stages of the silkworm B. mori. The phylogenetic analysis of the MetAP2 gene of N. bombycis revealed the MetAP2 gene sequences to be highly conserved in nature. The present study also includes the validation of the anti-microsporidian drug Fumagillin against the MetAP2 gene of N. bombycis. The findings revealed that Fumagilin-B could also suppress the N. bombycis multiplication in the silkworm B. mori, thereby proving the therapeutic role of Fumagillin against microsporidian infection. This is the first-ever report regarding the characterization of the MetAP2 gene in the Indian isolate of N. bombycis and also towards the usage of Fumagillin in the control of microsporidiosis in B. mori.
RESUMEN
Sugar transporters play an essential role in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. These genes exist as large multigene families within the insect genome. In insects, sugar transporters not only have a role in sugar transport, but may also act as receptors for virus entry. Genome-wide annotation of silkworm Bombyx mori (B. mori) revealed 100 putative sugar transporter (BmST) genes exists as a large multigene family and were classified into 11 sub families, through phylogenetic analysis. Chromosomes 27, 26 and 20 were found to possess the highest number of BmST paralogous genes, harboring 22, 7 and 6 genes, respectively. These genes occurred in clusters exhibiting the phenomenon of tandem gene duplication. The ovary, silk gland, hemocytes, midgut and malphigian tubules were the different tissues/cells enriched with BmST gene expression. The BmST gene BGIBMGA001498 had maximum EST transcripts of 134 and expressed exclusively in the malphigian tubule. The expression of EST transcripts of the BmST clustered genes on chromosome 27 was distributed in various tissues like testis, ovary, silk gland, malphigian tubule, maxillary galea, prothoracic gland, epidermis, fat body and midgut. Three sugar transporter genes (BmST) were constitutively expressed in the susceptible race and were down regulated upon BmNPV infection at 12h post infection (hpi). The expression pattern of these three genes was validated through real-time PCR in the midgut tissues at different time intervals from 0 to 30hpi. In the susceptible B. mori race, expression of sugar transporter genes was constitutively expressed making the host succumb to viral infection.