Spore Wall Protein Research Articles

MiR-PC-3p-241582_34 Contributes to the Infection of Athetis lepigone by Regulating the Expression of HSWP4 in Nosema bombycis.

Athetis lepigone is a recurring pest in the maize seedling stage under the wheat-maize no-tillage direct seeding system in China's summer maize region. Our previous research identified a highly pathogenic Nosema bombycis to A. lepigone, which spore wall protein plays an important role in the infection process. However, the regulatory mechanism of this spore wall protein is still unclear. In this study, we explored the regulatory mechanism of miRNAs on spore wall proteins. Transcriptome sequencing results showed that expression of the spore wall protein, HSWP4, significantly increased in the germination group compared to dormancy group. Silencing of HSWP4 reduced the number of microsporidian spores breaking through the midgut wall cells of A. lepigone. Association analysis of small RNA and mRNA revealed that the targeting site of miR-PC-3p-241582_34 on HSWP4 was located in the CDS region, and miR-PC-3p-241582_34 had a significant negative regulatory relationship with HSWP4. The dual luciferase reporter assay demonstrated that miR-PC-3p-241582_34 significantly affected the luciferase activity of the HSWP4-3'UTR expression vector (P < 0.05). Delivery of miRNA mimics decreased the expression of HSWP4 and inhibited the behavior of microsporidian spores breaking through the midgut wall of A. lepigone. On the other hand, delivery of inhibitors produced opposite results, indicating that the miR-HSWP4 pathway plays an important role in microsporidian infection of A. lepigone. This study provides a new theoretical basis for understanding the pathogenic mechanism and gene regulation of microsporidia, as well as for the green control of A. lepigone.

NcPTP2, a polar tube protein, interacts with spore wall protein in the parasitic microsporidian Nosema ceranae.

Microsporidia is an obligate intracellular eukaryote, which is capable of parasitizing vertebrates and invertebrates. Nosema ceranae, which can infect both Apis mellifera and Apis cerana, poses a serious threat and causes heavy losses to the worldwide apiculture. During infection, polar tube, a highly specialized invasion structure, ejected from the spore to deliver the sporoplasm into host cells to cause infection. Although seven different polar tube proteins (PTP1 ~ 7) have been reported from various microsporidia and showed key functions associated with spore invasion and proliferation, no systematic analysis on identification and characterization of polar tube proteins from N. ceranae was found. The polar tube proteins 2 (NcPTP2) was identified from the total polar tube proteins of N. ceranae by LC_MS/MS and the transcriptional profile was performed by RT-PCR. Sequence characterization analysis revealed that NcPTP2 was rich in lysine and had a signal peptide at the N-terminal. It had 3 potential O-glycosylation sites and 6 potential N-glycosylation sites. 25 phosphorylation sites were found on serine, tyrosine and threonine sites. Sequence alignment analysis revealed that NcPTP2 was homologous and had conserved cysteine residues with PTP2 proteins from other microsporidia. Indirect immunofuorescence analysis (IFA) and Immunoelectron Microscopy analysis (IEM) confirmed that NcPTP2 was localized on the polar tube of the germinated spores. The interaction between NcPTP2 and spore wall protein in N. ceranae indicated its potential function in anchoring and coiling of polar tube in spore. NcPTP2 was the first subcellular localized polar tube protein in N. ceranae and this work could provide an important basis for further analyzing the biological functions of polar tube proteins and uncovering the infection mechanism of N. ceranae to the host cells.

Increased levels of anti-Encephalitozoon intestinalis antibodies in patients with colorectal cancer.

The prevalence of microsporidiosis in the general population, or within specific groups of individuals/patients, is largely underestimated. The absence of specific seroprevalence tools limits knowledge of the epidemiology of these opportunistic pathogens, although known since the 1980s. Since microsporidia hijack the machinery of its host cell and certain species multiply within intestinal cells, a potential link between the parasite and colorectal cancer (CRC) has been suggested. To explore a potential epidemiological link between microsporidia and CRC, we evaluated the seroprevalence of Encephalitozoon intestinalis among CRC patients and healthy subjects using ELISA assays based on two recombinant proteins, namely rEiPTP1 and rEiSWP1, targeting polar tube and spore wall proteins. ELISA were performed in 141 CRC patients and 135 healthy controls. Patients with CRC had significantly higher anti-rEiPTP1 IgG levels than subjects in the control group. Anti-rEiPTP1 IgG, anti-rEiSWP1 IgG and anti-rEiPTP1 IgA levels were significantly increased among men with CRC compared to healthy men. Women with CRC who had died had higher rEiSWP1 IgG levels than those who were still alive. These higher antibody levels against microsporidia in patients with CRC suggest a relationship between microsporidia and pathophysiology of CRC.

Is Artemia susceptible to the microsporidium Ecytonucleospora hepatopenaei (EHP) infection?

Following the three-stage approach of the WOAH (World Organisation for Animal Health) standard on the confirmation of susceptible species to a specific pathogen, Artemia franciscana juveniles were challenged with immersion exposure (IM) in a homogenate suspension of Penaeus vannamei hepatopancreas infected with Ecytonucleospora hepatopenaei (EHP) and per os administration (PO) with chlorella soaked in EHP-infected hepatopancreatic homogenate suspension. Using TaqMan probe-based quantitative PCR, we detected that loads of EHP in A. franciscana juveniles from both the IM and PO groups were about 101 copies/ng-DNA on the 3rd day post-exposure (3 dpe). However, no more EHP was detectable in the challenged A. franciscana juveniles on 7 dpe. Nested PCR targeting the EHP spore wall protein gene did not produce a target band in A. franciscana juveniles on either 3 dpe or 7 dpe. The confirmative evidence of the absence of sustained infection with EHP in the exposed Artemia was substantiated using in situ DIG-labeling-loop-mediated DNA amplification (ISDL) on A. franciscana juvenile sections, which revealed a few weak grey-blue positive signals appeared on 3 dpe but disappeared on 7 dpe. The results indicated that exposure to EHP resulted in the invasion of Artemia in intestinal epithelial cells, but the infection was unable to persist, confirming Artemia is not susceptible to EHP. Therefore, the research countered the assertion of Artemia as a potential EHP reservoir.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

The Use of the Internal Transcribed Spacer Region for Phylogenetic Analysis of the Microsporidian Parasite Enterocytozoon hepatopenaei Infecting Whiteleg Shrimp (Penaeus vannamei) and for the Development of a Nested PCR as Its Diagnostic Tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

The use of the internal transcribed spacer region for phylogenetic analysis of the microsporidian parasite Enterocytozoon hepatopenaei infecting whiteleg shrimp (Penaeus vannamei) and for the development of a nested PCR as its diagnostic tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2%) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

Revealing the genome of the microsporidian Vairimorpha bombi, a potential driver of bumble bee declines in North America.

Pollinators are vital for food security and the maintenance of terrestrial ecosystems. Bumblebees are important pollinators across northern temperate, arctic, and alpine ecosystems, yet are in decline across the globe. Vairimorpha bombi is a parasite belonging to the fungal class Microsporidia that has been implicated in the rapid decline of bumblebees in North America, where it may be an emerging infectious disease. To investigate the evolutionary basis of pathogenicity of V. bombi, we sequenced and assembled its genome using Oxford Nanopore and Illumina technologies and performed phylogenetic and genomic evolutionary analyses. The genome assembly for V. bombi is 4.73 Mb, from which we predicted 1,870 protein-coding genes and 179 tRNA genes. The genome assembly has low repetitive content and low GC content. V. bombi's genome assembly is the smallest of the Vairimorpha and closely related Nosema genera, but larger than those found in the Encephalitozoon and Ordospora sister clades. Orthology and phylogenetic analysis revealed 18 core conserved single-copy microsporidian genes including the histone acetyltransferase (HAT) GCN5. Surprisingly, V. bombi was unique to the microsporidia in not encoding the second predicted HAT ESA1. The V. bombi genome assembly annotation included 265 unique genes (i.e. not predicted in other microsporidia genome assemblies), 20% of which encode a secretion signal, which is a significant enrichment. Intriguingly, of the 36 microsporidian genomes we analyzed, 26 also had a significant enrichment of secreted signals encoded by unique genes, ranging from 6 to 71% of those predicted genes. These results suggest that microsporidia are under selection to generate and purge diverse and unique genes encoding secreted proteins, potentially contributing to or facilitating infection of their diverse hosts. Furthermore, V. bombi has 5/7 conserved spore wall proteins (SWPs) with its closest relative V. ceranae (that primarily infects honeybees), while also uniquely encoding four additional SWPs. This gene class is thought to be essential for infection, providing both environmental protection and recognition and uptake into the host cell. Together, our results show that SWPs and unique genes encoding a secretion signal are rapidly evolving in the microsporidia, suggesting that they underpin key pathobiological traits including host specificity and pathogenicity.

Microsporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission.

Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission.

Identification of Enterocytozoon hepatopenaei in Mud Crabs (Scylla serrata) Co-infected with White Spot Syndrome Virus in Taiwan

Background: The high mortality rate and stunted growth of marine shrimp in the farm have raised concerns about the outbreak of co-infection involving Enterocytozoon hepatopenaei (EHP) and white spot syndrome virus (WSSV), which could lead to significant economic loss. In a mixed-culture farm consisting of Scylla serrata and Peneaus vannamei located in Yunlin Country, Taiwan, were reported mortality cases on culture animals. Methods: Based on clinical signs, histology and molecular analyses, the disease was diagnosed in this study. In the histopathology examination, infected organs, such as the carapace and gills, were stained with hematoxylin and eosin (H and E), then observed under microscope. For the molecular analysis, SalI DNA fragment (WSSV) and spore wall protein (SWP) gene (EHP) were targeted to detected the pathogen in animal host. Result: The cumulative mortality data for this co-infection indicates that S. serrata (1.5% mortality) exhibits higher susceptibility compared to P. vannamei ( greater than 99% mortality). Phylogenetic tree analysis reveals that the spore wall protein (SWP) gene of EHP and the SalI DNA fragment of WSSV strain extracted from S. serrata are genetically similar to those extracted from Peneaus spp., indicating the potential for cross-species infection. Histopathological examination of lesion tissue observed the presence of WSSV basophilic intranuclear inclusion bodies in the carapace tissue and gill fragments of S. serrata. The source of the disease outbreak is highly suspected to be the feed used, which consisted of raw shrimp and fish. This study provides the first documented case of natural EHP infection with co-infection of WSSV in S. serrata.

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.

A study on the natural host range of Enterocytozoon hepatopenaei in different species of shrimp and co-habiting aquatic fauna

Enterocytozoon hepatopenaei (EHP) is a serious pathogen impacting farmed shrimpproduction in Asian countries. Though many aquatic organisms were reported to besusceptible to EHP, the full range of susceptible hosts, which have the potential to transmitthe pathogen to shrimp has not been identified. In this study, a range of farmed and wildinvertebrates and other cohabiting fauna from different ecological niches located in thecoastal and inland saline areas were subjected to PCR-based detection employing primerstargeting three different genes coding for small subunit (ssu) rRNA, spore wall protein(SWP) and β-tubulin. The PCR analysis with ssu-rRNA primers showed positive amplificationin Penaeus vannamei, Penaeus monodon, Penaeus indicus, marine shrimps, aquatic insect,mud crabs, freshwater crab and violet clam. PCR using SWP primers detected EHP inP. vannamei, P. monodon, aquatic insect and marine crabs, while the β-tubulin primersresulted in positive amplification only in mud crabs and P. vannamei. The PCR ampliconsrevealed 99-100% identity with the sequences of EHP. The present study forms the firstattempt to screen a wide range of aquatic fauna for EHP employing three different PCR testsand the findings are significant as these organisms have the potential to be carriers of EHPand are likely to transmit the parasite to shrimp culture systems. Keywords:Co-habiting aquafauna, Enterocytozoonhepatopenaei, EHP, Host range, Shrimp culture

Molecular characterization of turtle-like protein in whiteleg shrimp (Litopenaeus vannamei) and its role in Enterocytozoon hepatopenaei infection.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects shrimp hepatopancreas, causing growth retardation and disease susceptibility. Knowledge of the host-pathogen molecular mechanisms is essential to understanding the microsporidian pathogenesis. Turtle-like protein (TLP) is part of the immunoglobulin superfamily of proteins, which is widely distributed in the animal kingdom. TLP has multiple functions, such as cell surface receptors and cell adhesion molecules. The spore wall proteins (SWPs) of microsporidia are involved in the infection mechanisms. Some SWPs are responsible for spore adherence, which is part of the activation and host cell invasion processes. Previous studies showed that TLP from silkworms (Bombyx mori) interacted with SWP26, contributing to the infectivity of Nosema bombycis to its host. In this study, we identified and characterized for the first time, the Litopenaeus vannamei TLP gene (LvTLP), which encodes an 827-aa protein (92.4 kDa) composed of five immunoglobulin domains, two fibronectin type III domains, and a transmembrane region. The LvTLP transcript was expressed in all tested tissues and upregulated in the hepatopancreas at 1 and 7 days post-cohabitation (dpc) and at 9 dpc in hemocytes. To identify the LvTLP binding counterpart, recombinant (r)LvTLP and recombinant spore wall protein 1 of (rEhSWP1) were produced in Escherichia coli. Coimmunoprecipitation and enzyme-linked immunosorbent assays demonstrated that rLvTLP interacted with rEhSWP with high affinity (KD = 1.20 × 10-7 M). In EHP-infected hepatopancreases, LvTLP was clustered and co-localized with some of the developing EHP plasmodia. Furthermore, LvTLP gene silencing reduced the EHP copy numbers compared with those of the control group, suggesting the critical role of LvTLP in EHP infection. These results provide insight into the molecular mechanisms of the host-pathogen interactions during EHP infection.

Non-coding RNAs identification and regulatory networks in pathogen-host interaction in the microsporidia congenital infection

BackgroundThe interaction networks between coding and non-coding RNAs (ncRNAs) including long non-coding RNA (lncRNA), covalently closed circular RNA (circRNA) and miRNA are significant to elucidate molecular processes of biological activities and interactions between host and pathogen. Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-feeding industry. However, little is known about ncRNAs that take place in the microsporidia congenital infection. Here we conducted whole-transcriptome RNA-Seq analyses to identify ncRNAs and regulatory networks for both N. bombycis and host including silkworm embryos and larvae during the microsporidia congenital infection.ResultsA total of 4,171 mRNAs, 403 lncRNA, 62 circRNAs, and 284 miRNAs encoded by N. bombycis were identified, among which some differentially expressed genes formed cross-talk and are involved in N. bombycis proliferation and infection. For instance, a lncRNA/circRNA competing endogenous RNA (ceRNA) network including 18 lncRNAs, one circRNA, and 20 miRNAs was constructed to describe 14 key parasites genes regulation, such as polar tube protein 3 (PTP3), ricin-B-lectin, spore wall protein 4 (SWP4), and heat shock protein 90 (HSP90). Regarding host silkworm upon N. bombycis congenital infection, a total of 14,889 mRNAs, 3,038 lncRNAs, 19,039 circRNAs, and 3,413 miRNAs were predicted based on silkworm genome with many differentially expressed coding and non-coding genes during distinct developmental stages. Different species of RNAs form interacting network to modulate silkworm biological processes, such as growth, metamorphosis and immune responses. Furthermore, a lncRNA/circRNA ceRNA network consisting of 140 lncRNAs, five circRNA, and seven miRNAs are constructed hypothetically to describe eight key host genes regulation, such as Toll-6, Serpin-6, inducible nitric oxide synthase (iNOS) and Caspase-8. Notably, cross-species analyses indicate that parasite and host miRNAs play a vital role in pathogen-host interaction in the microsporidia congenital infection.ConclusionThis is the first comprehensive pan-transcriptome study inclusive of both N. bombycis and its host silkworm with a specific focus on the microsporidia congenital infection, and show that ncRNA-mediated regulation plays a vital role in the microsporidia congenital infection, which provides a new insight into understanding the basic biology of microsporidia and pathogen-host interaction.

Application of PCR-based diagnostic tools that target Enterocytozoon hepatopenaei for the molecular detection of a Vittaforma-like microsporidium that infects Penaeus vannamei from Costa Rica

Several PCR methodologies are available for the detection of Enterocytozoon hepatopenaei (EHP) that target the SSU rRNA gene. However, these methodologies are reported as unsuitable for the detection of EHP due to specificity issues. Here, we report the applicability of two commonly used SSU rRNA methodologies for the detection of additional microsporidia from the genus Vittaforma that is present in cultured Penaeus vannamei from Costa Rica. The molecular detection of DNA of the novel microsporidia can only be achieved using SSU rRNA targeting methodologies and does not cross-react with the highly specific spore wall protein gene PCR detection method.

Detection of Enterocytozoon hepatopenaei (EHP) (microsporidia) in several species of potential macrofauna-carriers from shrimp (Penaeus vannamei) ponds in Malaysia

Infection by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) has become a significant problem in the shrimp cultivation industry in Asian countries like Thailand, China, India, Vietnam, Indonesia, and Malaysia. The outbreak of this microsporidian parasite is predominantly related to the existence of macrofauna-carriers of EHP. However, information about potential macrofauna-carriers of EHP in rearing ponds is still limited. In this study, the screening of EHP in potential macrofauna-carriers was conducted in farming ponds of Penaeus vannamei in three states in Malaysia, namely Penang, Kedah, and Johor. A total of 82 macrofauna specimens (phyla: Arthropoda, Mollusca, and Chordata) were amplified through a polymerase chain reaction (PCR) assay targeting genes encoding spore wall proteins (SWP) of EHP. The PCR results showed an average prevalence of EHP (82.93%) from three phyla (Arthropoda, Mollusca and Chordata). The phylogenetic tree generated from the macrofauna sequences was revealed to be identical to the EHP-infected shrimp specimens from Malaysia (MW000458, MW000459, and MW000460), as well as those from India (KY674537), Thailand (MG015710), Vietnam (KY593132), and Indonesia (KY593133). These findings suggest that certain macrofauna species in shrimp ponds of P. vannamei are carriers of EHP spores and could be potential transmission vectors. This study provides preliminary information for the prevention of EHP infections that can be initiated at the pond stage by eradicating macrofauna species identified as potential vectors.

Towards characterizing of Enterocytozoon hepatopenaei (EHP) spore wall proteins with feature identification and analogy modeling

Enterocytozoon hepatopenaei (EHP) is a parasite that mostly affects shrimp; it is linked to spore-forming and obligatory intracellular microsporidia and causes growth retardation and body length fluctuation, which results in financial consequences for the shrimp industry in Asian nations. However, the introduction of technologies to detect EHP has been complicated by the absence of a thorough knowledge of the properties of spore wall proteins (SWPs) linked to host infection. Thus, characterization, feature identification, 3D modeling of protein sequence, prediction of transmembrane regions, motif analysis, as well as model validation of SWPs can help improve drug delivery in the future. Conservation analysis was performed using ClustalW. InterPro, MEMEsuite, and TMHMM Server v2.0 were used to perform motif analysis, family domain prognosis, and transmembrane region analysis, respectively. Finally, the Alphafold2 server was used for analogy modeling and 3D layout. Different model validation servers were used to validate the protein structural model. Physicochemical features for SWPs were analyzed and found molecular weight around 16.701 KDa-38.314 KDa, theoretical pI 5.25–9.05, instability index 32.04–48.89, aliphatic index 76.49–99.84, grand average of hydropathicity (GRAVY) – 0.059 to −0.892. Each SWP was discovered to be hydrophilic. There was no significant region detected in the conserved region analysis. Both a lower and higher E value were found by the motif analysis. For most of the proteins, there were no transmembrane regions. Finally, analogous 3D models for each protein were created and verified using various bioinformatics tools and software. This investigation could aid in the further research of diseases and the development of drug delivery systems.

In Silico-Based Vaccine Design Against Hepatopancreatic Microsporidiosis in Shrimp

Enterocytozoon hepatopenaei (EHP) is a spore-forming intracellular parasite that produces an economically devastating disease in farmed shrimp called hepatopancreatic microsporidiosis (HPM), known to slow the growth of hosts in aquaculture. Spore wall proteins are implicated in the recognition of host cells in microsporidia. Many parasites have been demonstrated to detect heparin, a glycosaminoglycan (GAG) molecule present on cell surfaces, as a host. The sequencing and characterization of several EHP strains started to reveal information regarding pathogen biology, pathogenicity, and spore wall proteins must bind to the shrimp cell surface to infect it, and failure to do so results in ineffective infection. Despite these devastating complications, there is still no cure or vaccine for the parasites. In this regard, an immunoinformatics method was used to generate an epitope-based vaccine against this pathogen. The immunodominant T-cell and B-cell epitopes were identified using the spore wall proteins of EHP. The final constructed vaccine sequence was developed to be immunogenic, non-allergenic as well as have better solubility. Molecular dynamics simulation revealed significant binding stability and structural compactness. Finally, using Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher CAI value, which was then included in the cloning vector pET2+ (a). Altogether, our outcomes imply that the proposed peptide vaccine may be a good option for HPM prophylaxis. HIGHLIGHTS Hepatopancreatic microsporidiosis (HPM) is known to slow the growth of shrimp There are approved drugs or vaccines to control this disease In-silico approach and immunoinformatics has some advantage previously in fishes The design vaccine candidate would have some advantages to combat the disease for sustainable shrimp production GRAPHICAL ABSTRACT

Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

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.

Human Microsporidiosis: A Narrative Review of Serodiagnostic Tests

Context: Microsporidiosis is an emerging disease in both immunocompetent and immunocompromised patients. Diagnosis of human microsporidiosis is challenging. Parasitological tests using staining methods have limitations for detecting spores in stool samples. The performance of molecular methods for diagnosing human microsporidiosis is satisfactory, but these methods are expensive and time-consuming. Serodiagnosis tests for the detection of antibodies have insufficient specificity. Detecting microsporidia antigens seems to be an appropriate method for diagnosing human microsporidiosis. Therefore, the present study reviewed serological approaches to assess the current status of these methods for diagnosing human microsporidiosis. Evidence Acquisition: All related published articles were searched. The search terms were “microsporidiosis”, “microsporidiasis”, “human microsporidiosis”, and “human microsporidiasis”, combined with the search terms “diagnosis”, “serodiagnosis”, “immunodiagnosis”, “antigen detection”, and “antibody detection”. Data were extracted from the articles that met our eligibility criteria. Immunodiagnosis studies in experimentally infected animals were excluded. Results: Few studies have evaluated the performance of serological tests for diagnosing human microsporidiosis. The IgG response against spore wall protein or polar tube antigens remains at least three years after infection and shows more cross-reactivity with other species. Therefore, the western blot should be conducted as a confirmatory method. Serodiagnosis tests using monoclonal antibodies against microsporidia showed less cross-reactivity and more efficacy compared to polyclonal antibodies. Conclusions: Indirect fluorescent antibody test based on the monoclonal antibody is highly effective in diagnosing microsporidia species in clinical samples.