Microsporidian Infection Research Articles

Silver nanoparticles are effective in controlling microsporidia

Many approaches and technologies have been developed as treatments for microsporidian, infections but effective, broad-spectrum, and sustainable therapeutic approaches have not been found. Silver nanoparticles (AgNPs) have antimicrobial activity and are widely used against many different pathogens. AgNPs provide an opportunity to develop formulations that will control microsporidia. In this study, we synthesized AgNPs via a chemical reduction method and evaluated their formation, morphology, and stability using transmission electron microscopy (TEM) and ultraviolet spectroscopy analysis. We verified that AgNPs could disrupt the spore cell membrane and spore germination of microsporidia Nosema bombycis. This resulted in the release of microsporidia nucleic acids, proteins, and respiratory chain enzymes. The anti-microsporidia activity of AgNPs was studied by measuring the silkworm larvae survival rate and spore genome replication after microsporidia infection. AgNPs have anti-microsporidian activity and could be effective components of formulations for treating or preventing microsporidia infection.

Microsporidian and fungal infections in Lepidoptera and Orthoptera in Bulgaria

Fourteen hundred and sixty three larvae of 10 lepidopteran species collected from trees and bushes in the spring and summer of 2017, 2018 and 2019 from 5 localities in Northwest and South Bulgaria were investigated for presence of microsporidian and fungal pathogens. Also, 77 grasshopper individuals of Poecilimon thoracicus (Orthoptera, Tettiigoniidae) collected from various shrubs and perennial plants in the spring and summer of 2017 were examined. Conducted microscopic analyses showed the presence of microsporidian infection caused by Endoreticulatis poecilimonae in P. thoracicus and fungal infection in the larvae of mottled umber, Erannis defoliaria caused by Entomophaga auliciae. The studies showed that the average infection rate with the microsporidium E. poecilimonae was 57.1%. The prevalence of the fungus Entomophaga auliciae was 100% during the observed epizootic. E. auliciae is an efficient fungal pathogen which causes strong epizootics and can be used as classical or augmentation biological agent.

Microsporidial Stromal Keratitis: Epidemiological Features, Slit-Lamp Biomicroscopic Characteristics, and Therapy.

Microsporidial stromal keratitis is a rare form of infectious keratitis, with only 7 cases reported in the United States to date. This study was performed to evaluate risk factors, clinical features, and response to therapy. A retrospective review of the medical records of all patients diagnosed with microsporidial stromal keratitis seen in the practices of the authors between 1999 and 2020 was performed. Diagnosis was determined by cytology or histopathology in corneal specimens. Risk factors, presence or absence of distinctive clinical features, and response to medical and surgical therapies were recorded. Nine patients-7M:2F, aged 7 to 99 years-with microsporidial stromal keratitis were identified. Exposures to recreational water and hymenopteran insect bites, both epidemiologically linked risk factors for systemic microsporidial infection, were identified in our patients. Presence of stromal edema with features of disciform keratitis and a distinctive granular keratitis were observed in 6 of 9 and 5 of 9 patients, respectively. Poor response to medical therapy was noted. Penetrating keratoplasty was effective in curing the infection. Final visual acuity was 20/40 or better in 6 of 9 patients. In patients with slowly progressive keratitis, history of exposure to recreational water or hymenopteran insects should be sought. In patients with corneal edema consistent with disciform keratitis, with evolution to a granular keratitis, microsporidia should be considered in the differential diagnosis. In cases of established microsporidial stromal keratitis, penetrating keratoplasty should be considered if prompt response to medical therapy is not noted.

Identification of BmELP as an interaction partner of Bmtutl-519 in the silkworm, Bombyx mori

• The BmELP protein was determined to interact with Bmtutl-519. • BmELP may play an important role in the growth and division of silkworm cells. • N. bombycis infection caused the up-regulation of BmELP expression level, especially in vitro . • BmELP played a role in the process of coping with pathogen invasion. The Turtle protein could act as a signaling receptor or function as a membrane-bound ligand for an unknown receptor. Alternatively, the Turtle protein could function as a co-receptor in a multi-protein receptor complex. Earlier work on the function of Bmtutl-519 suggested that it may act as a cell surface receptor or as a regulatory factor in the infection process of Nosema bombycis . To further investigate the function of Bmtutl-519 in the process of microsporidian infection and the possible regulatory pathways involved, we performed yeast two-hybrid screening on a silkworm midgut cDNA library and identified a Bombyx mori extensin-like protein/BmELP as a binding partner of Bmtutl-519. The interaction between Bmtutl-519 and BmELP was verified by GST pull-down and co-immunoprecipitation assays. Quantitative RT-PCR analysis showed that BmELP was commonly expressed in all of the examined tissues of the silkworm, and the expression was highest in the head. Moreover, BmELP was expressed throughout the entire development period, and the lowest levels of expression were observed at the egg stage. The results of infection experiments showed that N. bombycis infection caused the up-regulation of BmELP expression, especially in vitro . Taken together, our findings may provide new insights into the roles of BmELP and Bmtutl-519 in microsporidian infection.

Human microsporidian pathogen Encephalitozoon intestinalis impinges on enterocyte membrane trafficking and signaling.

Microsporidia are a large phylum of obligate intracellular parasites. Approximately a dozen species of microsporidia infect humans, where they are responsible for a variety of diseases and occasionally death, especially in immunocompromised individuals. To better understand the impact of microsporidia on human cells, we infected human colonic Caco2 cells with Encephalitozoon intestinalis, and showed that these enterocyte cultures can be used to recapitulate the life cycle of the parasite, including the spread of infection with infective spores. Using transmission electron microscopy, we describe this lifecycle and demonstrate nuclear, mitochondrial and microvillar alterations by this pathogen. We also analyzed the transcriptome of infected cells to reveal host cell signaling alterations upon infection. These high-resolution imaging and transcriptional profiling analysis shed light on the impact of the microsporidial infection on its primary human target cell type.This article has an associated First Person interview with the first authors of the paper.

Transcriptome analysis reveals changes in silkworm energy metabolism during Nosema bombycis infection

Energy metabolism is important for the proliferation of microsporidia in infected host cells, but there is limited information on the host response. The energy metabolism response of silkworm (Bombyx mori) to microsporidia may help manage Nosema bombycis infections. We analyzed differentially expressed genes in the B.mori midgut transcriptome at two significant time points of microsporidia infection. A total of 1448 genes were up-regulated, while 315 genes were down-regulated. A high proportion of genes were involved in the phosphatidylinositol signaling system, protein processing in the endoplasmic reticulum, and glycerolipid metabolism at 48 h post infection (h p.i.), and a large number of genes were involved in the TCA cycle and protein processing at 120 h p.i. These results showed that the early stages of microsporidia infection affected the basic metabolism and biosynthesis processes of the silkworm. Knockout of Bm_nscaf2860_46 (Bombyx mori isocitrate dehydrogenase, BmIDH) and Bm_nscaf3027_062 (Bombyx mori hexokinase, BmHXK) reduced the production of ATP and inhibited microsporidia proliferation. Host fatty acid degradation, glycerol metabolism, glycolysis pathway, and TCA cycle response to microsporidia infection were also analyzed, and their importance to microsporidia proliferation was verified. These results increase our understanding of the molecular mechanisms involved in N. bombycis infection and provide new insights for research on microsporidia control. ImportanceNosema bombycis can be vertically transmitted in silkworm eggs. The traditional prevention and control strategies for microsporidia are difficult and time-consuming, and this is a problem in silkworm culture. Research has mainly focused on host gene functions related to microsporidia infection and host immune responses after microsporidia infection. Little is known about the metabolic changes occurring in the host after infection. Understanding the metabolic changes in the silkworm host could aid in the recognition of host genes important for microsporidia infection and growth. We analyzed host metabolic changes and the main participating pathways at two time points after microsporidia infection and screened the microsporidia-dependent host energy metabolism genes BmIDH and BmHXK. The results revealed genes that are important for the proliferation of Nosema bombycis. These results illustrate how microsporidia hijack the host genome for their growth and reproduction.

RNA Interference-Mediated Knockdown of Genes Encoding Spore Wall Proteins Confers Protection against Nosema ceranae Infection in the European Honey Bee, Apis mellifera.

Nosema ceranae (Opisthosporidia: Microsporidia) is an emergent intracellular parasite of the European honey bee (Apis mellifera) and causes serious Nosema disease which has been associated with worldwide honey bee colony losses. The only registered treatment for Nosema disease is fumagillin-b, and this has raised concerns about resistance and off-target effects. Fumagillin-B is banned from use in honey bee colonies in many countries, particularly in Europe. As a result, there is an urgent need for new and effective therapeutic options to treat Nosema disease in honey bees. An RNA interference (RNAi)-based approach can be a potent strategy for controlling diseases in honey bees. We explored the therapeutic potential of silencing the sequences of two N. ceranae encoded spore wall protein (SWP) genes by means of the RNAi-based methodology. Our study revealed that the oral ingestion of dsRNAs corresponding to SWP8 and SWP12 used separately or in combination could lead to a significant reduction in spore load, improve immunity, and extend the lifespan of N. ceranae-infected bees. The results from the work completed here enhance our understanding of honey bee host responses to microsporidia infection and highlight that RNAi-based therapeutics are a promising treatment for honey bee diseases.

Microscopic and Molecular Description of the Microsporidian Parasite,Heterosporis sp., Infecting Lizardfish, Saurida undosquamis in the Persian Gulf

Background: The lizardfish is an economically important fish in the Persian Gulf with high rates of parasitic infections. Microsporidia species, as opportunistic parasites, cause several disorders, which in turn result in economic problems. Objectives: The main objective was to evaluate Heterosporis sp. infection in Persian Gulf lizardfish using the small subunit ribosomal RNA phylogenetics to describe and classification of the unknown microsporidia species as well as morphological characteristics. Methods: The abdominal cavities of fifty specimens of lizardfish, Saurida undosquamis, were examined using morphological and molecular techniques. Some irregular whitish cyst-like were fixed for histopathological and transmission electron observations. The small subunit ribosomal genomic DNA was studied and a 1,279 bp genomic sequence was amplified and investigated for molecular analysis. Results: Twenty-two (out of fifty) specimens were infected with irregular whitish microsporidian cysts. Light and electron microscopic findings revealed round cysts containing large numbers of monomorphic and ovoid spores with a posterior vacuole. Polar tube coiled between six and eight-times, in one row. The large xenoma (hypertrophied parasitizing host cells) was encapsulated by a host-derived thick connective tissue in pathological samples. The phylogenetic analysis showed that despite some morphological similarity of the Persian Gulf microsporidia sp. to Glugea spp., the most closely related species with minimum genetic distance to Heterosporis anguillarum isolated is Japanese eels (Anguillajaponica). Conclusions: This is the first phylogenetic report of microsporidian infections in mesenteric tissues of lizardfish S. undosquamis in Iran including morphological and molecular markers, to introduce novel species

Wide geographic distribution of overlooked parasites: Rare Microsporidia in Gammarus balcanicus, a species complex with a high rate of endemism.

Parasites and other symbionts deeply influence host organisms, and no living organism can be considered to have evolved independent of its symbionts. The first step towards understanding symbiotic influences upon host organisms is a strong supporting knowledge of parasite/symbiont diversity. Parasites of freshwater amphipods are diverse, with Microsporidia being a major group. These intracellular parasites impact gammarid fitness in different ways, ranging from reduced fitness to increased fecundity. Many Microsporidia have been recorded using molecular data, with multiple taxa pending formal taxonomic description. While some parasites are common, others are known only through sporadic records of single infections. In this study, we focus on rare/sporadic microsporidian infections within Gammarus balcanicus, a host species complex with a high level of endemism. In addition to enriching our knowledge on Microsporidia parasite diversity in amphipod hosts, we test whether these symbionts are specific to G. balcanicus or if they are the same taxa infecting other gammarid species. Of 2231 hosts from 87 sites, we catalogued 29 sequences of “rare” Microsporidia clustering into 19 haplogroups. These haplogroups cluster into 11 lineages: four pre-described taxa (Cucumispora roeselum, C. ornata, C. dikerogammari and Enterocytospora artemiae) and seven ‘Molecular Operational Taxonomic Units’, which are known from previously published studies to infect other European amphipod species. Our study significantly widens the geographic range of these Microsporidia and expands the known spectrum of hosts infected. Our results suggest that these parasites are ancient infections of European gammarids. For some host-parasite systems, we hypothesize that the common parasite ancestors that infected the hosts' common ancestors, diversified alongside host diversification. For others, we observe Microsporidia taxa with wide host ranges that do not follow host phylogeny.

Genetic bioengineering of overexpressed guanylate binding protein family BmAtlastin-n enhances silkworm resistance to Nosema bombycis

Microsporidia are obligate single-celled eukaryote parasites. Microsporidian infection can cause large economic losses to beneficial insects such as silkworms and honey bees. Identification of resistance biomacromolecules and breeding of transgenic lines resistant to the microsporidian Nosema bombycis are important for disease management. We previously used transcriptome analysis to identify a guanylate binding protein family BmAtlastin-n gene that was significantly upregulated after Nosema bombycis infection, and we determined that the molecule was highly expressed in resistance-related tissues such as the midgut, fat body and the epidermis. The transgenic silkworm line overexpressing BmAtlastin-n biomolecules had economic characters similar to those of non-transgenic lines. The transgenic OE-BmAtlastin-n lines had significantly improved survival after microspore infection. We used RT-PCR and H&E staining to show that the number of spores in the transgenic lines was significantly lower than in the control lines. In this study, we identified a BmAtlastin-n macromolecule with resistance to N. bombycis and developed a transgenic line. The results improved understanding of the GBP protein family and provided biomacromolecule material for the treatment and prevention of microsporidia.

Distribution and Occurrence of Vairimorpha plodiae (Opisthokonta: Microspora) in the Indian Meal Moth, Plodia interpunctella (Lepidoptera: Pyralidae) Populations: An Extensive Field Study

The Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae) is one of the most important stored product pests. Fumigation plays a significant role in the management of insect pests in stored-products. However, the use of fumigants is problematic because of their effects on the environment and high costs. Entomopathogenic organisms are environmentally friendly control agents and suppress pest populations under natural conditions. In this study, distribution and occurrence of a microsporidian pathogen, Vairimorpha plodiae (Opisthokonta: Microspora) in the populations of P. interpunctella from 12 localities representing Turkey between 2019 and 2020 are presented for the first time by confirming its effectiveness on natural populations. The presence of the microsporidian pathogen was found in 11 of 12 (91.7%) populations. In total, 863 of 3,044 samples were infected by the pathogen. Infection mean was 28.4% for all populations. Our results showed that V. plodiae infection reached to a considerably high prevalence (88.77%) in P. interpunctella populations and varied from 5.1 to 88.7% between the populations. In addition, microsporidia infections have been identified throughout Turkey. We found that V. plodiae can infect all life stages of P. interpunctella. Totally, 623 (28.5%) of 2187 larvae, 14 (37.8%) of 37 pupae, 226 (27%) of 820 adults were found to be infected by the pathogen. There were considerable differences between the dead and living larvae. The microsporidian infection was found in 26 (11.6%) of 225 living larvae, whereas it was found in 595 (30.5%) of 1,952 dead larvae. These results confirm that the microsporidia pathogen has a high spreading potential in P. interpunctella populations and can be a natural biological suppression factor on pest populations.

Prevalence of Microsporidiosis in Different Hosts in Turkey: A Meta-analysis.

Microsporidia are opportunistic obligate intracellular pathogens which infect many vertebrate and invertebrate hosts. This study aimed at investigating all evidence about microsporidia infection in human and other vertebrate hosts in Turkey. This study covered all prevalence studies, related to microsporidiosis in Turkey until April 2020, that were found in Web of Science, PubMed, Scopus, and ULAKBIM databases were considered in this meta-analysis. A total of 168 studies were identified in the systematic literature research. After the initial assessment, only 15 articles (12 humans and three other vertebrates) were included for meta-analysis. Data analysis was carried out using the Revman 5.3 (Review Manage 5.3) software. With the evaluation of these studies, it was found that the prevalence of microsporidia in humans (n=6.707) and other vertebrate hosts (n=506) was 13.4% and 15.2%, respectively. The risk ratio in the patient groups was 2.87 compared to the control group [95% confidence interval (CI): 1.20-6.87, I2=87%, p<0.00001]. There was no difference between genders and parasite prevalence (95% CI: 1.00-1.39, I2=18%, p=0.29). The prevalence of microsporidia was also found to be high in patients with diarrhea (95% CI: 1.09-1.58, I2=86%, p=0.0001) and in immunosuppressed individuals (95% CI: 1.86-3.70, I2=16%, p=0.31). Although there are few studies on the prevalence of these parasites, the results of this meta-analysis provides extensive information about the current situation in Turkey.

Characterization of a Murine Model for Encephalitozoon hellem Infection after Dexamethasone Immunosuppression.

Background: Encephalitozoon hellem (E. hellem) belongs to a group of opportunistic pathogens called microsporidia. Microsporidia infection symptoms vary and include diarrhea, ocular disorders and systemic inflammations. Traditionally, immunodeficient animals were used to study microsporidia infection. To overcome the difficulties in maintenance and operation using immunodeficient mice, and to better mimic natural occurring microsporidia infection, this study aims to develop a pharmacologically immunosuppressed murine model of E. hellem infection. Methods: Wild-type C57BL/6 mice were immunosuppressed with dexamethasone (Dex) and then E. hellem spores were inoculated into the mice intraperitoneally. Control groups were the Dex-immunosuppressed but noninoculated mice, and the Dex-immunosuppressed then lipopolysaccharide (LPS)-treated mice. Mice body weights were monitored and all animals were sacrificed at the 15th day after inoculation. Tissue fragments and immune cells were collected and processed. Results: Histopathological analysis demonstrated that E. hellem inoculation resulted in a disseminated nonlethal infection. Interestingly, E. hellem infection desensitized the innate immunity of the host, as shown by cytokine expressions and dendritic cell maturation. We also found that E. hellem infection greatly altered the composition of host gut microbiota. Conclusions: Dex-immunosuppressed mice provide a useful tool for study microsporidiosis and the interactions between microsporidia and host immunity.

Prevalence and Genetic Characterization of Cryptosporidium and Microsporidia Infecting Hematological Malignancy Patients.

To evaluate the prevalence of Cryptosporidium and Microsporidia, associated risk factors and species identification in patients with haematological malignancies (HM). A total of 148 consecutive patients with HM and 101 healthy subjects were evaluated for Cryptosporidium and Microsporidia using modified Kinyoun and modifiedTrichrome staining. Clinical, demographic and laboratory parameters were studied. The species of Cryptosporidium and Microsporidia were studied using PCR-RFLP. Of 148 HM patients initially screened, 47 were excluded from the final analysis due to inadequate clinical records. Patients with HM [n = 101, 63 (62.4%) male] more often had Cryptosporidium than healthy subjects [n = 101, 65 (74.4%) male] [3/101 (3%) vs. 0/101 (0%), p = 0.02]. Two of 101 (2%) HM patients and none of the healthy subjects had Microsporidia (p = 0.155). Diarrhea was more prevalent in HM patients with Cryptosporidium than those without [3, 100% vs. 39/96, 40.62%; p = 0.04). Both patients infected with Microsporidia presented with persistent diarrhea and fever. Cryptosporidium hominis was identified in all the three HM patients. Enterocytozoon bieneusi was identified in one HM patient infected with Microsporidia, which was classified as genotype Ind2. Cryptosporidium and Microsporidia may infect HM patients leading to overwhelming diarrhea. The commonest species of Cryptosporidium and Microsporidia found to infect HM patients are C. hominis and E. bieneusi.

Detection of Intestinal Parasites and Genetic Characterization of Enterocytozoon bieneusi in Hedgehogs from China.

Microsporidia are a group of unicellular and opportunistic intestinal parasites in which Enterocytozoon bieneusi is a frequent species causing microsporidial infections in humans. Many domesticated and wild animals have been shown to be hosts of E. bieneusi and other microsporidia. The role of hedgehogs in the ecology of microsporidia is unclear; therefore, we investigated the prevalence and genetic diversity of E. bieneusi, Cryptosporidium, and Blastocystis spp. in hedgehogs (Erinaceus amurensis) collected from Hubei Province in Central China. PCR amplification of the internal transcribed spacer region of the ribosomal DNA indicated that 9.8% (4/41) hedgehogs were positive to E. bieneusi, but none (0/41) was positive to Cryptosporidium and Blastocystis spp. Phylogenetic analysis showed the strains detected from the hedgehogs belong to four novel genotypes (EA1-EA4), which were most closely related to type IV of group 1c. This study demonstrated that hedgehogs are hosts of E. bieneusi and may play a role in the transmission of E. bieneusi to humans in the process of being caught and slaughtered.

Intestinal Microsporidia Infection in Leukemic Children: Microscopic and Molecular Detection.

Microsporidia infection was originally described as an immunocompromised associated pathogen. Limitations to correct microscopic diagnosis of microsporidia include size of the organism presenting a challenge even to a highly competent laboratory expert. The present study aimed to detect microsporidia infection among leukemic children. The performance of modified trichrome stain and PCR in the diagnosis of microsporidia was evaluated with further speciation. Stool samples of 100 leukemic children on chemotherapy were examined microscopically for microsporidia. DNA was extracted from all samples. Amplification was performed by conventional and nested PCR. Sequencing of amplified products was performed on unspeciated samples. Microsporidia were detected in 23% of the children by MTS and 29% by PCR. The 29 positive samples were subjected to PCR for speciation. Enterocytozoon bieneusi was found to predominate in 20 cases, Encephalitozoon intestinalis in three cases, two cases had co-infection, and the remaining four samples were not amplified with either E. bieneusi or E. intestinalis specific primers. By DNA sequencing of the unspeciated samples, three samples shared high homology with Encephalitozoon hellem and one sample with Encephalitozoon cuniculi. Referring to PCR as a gold standard, MTS exhibited 72.4% sensitivity and 97.2% specificity with 90% accuracy. Among a number of studied variables, diarrhea and colic were significantly associated with microsporidia infection when diagnosed by either technique. The use of sensitive and discriminative molecular tools will contribute to determining the true prevalence of microsporidiosis and possibly their potential transmission source depending on species identification.

DEVELOPMENT OF MICROSPORIDIOLOGY IN RUSSIA

The study of microsporidia and microsporidioses of wild animals in Russia has been initiated in the 60-s of the past century. In the European part of country, microsporidia, infecting agricultural insect pests (All-Russian Institute of Plant Protection), freshwater arthropods and fishes (State Research Institute of Lake and River Fisheries) and the blood-sucking insects such as horseflies (Biology Institute, Karelian Scientific Center) were studied. In the Western Siberia, microsporidia of blood-sucking mosquitoes were studied (Tomsk University). As a result, by 2000, as many as 118 species and 47 genera of microsporidia were found, including 20 taxa new to science, from 100 animal species. Currently, descriptions of new taxa and taxonomic revision of the previously described taxa are performed using the molecular phylogenetic analysis. The novel data on speciation of microsporidia have been obtained, and the coevolution of parasites and their host insects have been confirmed for microsporidia of blood-sucking mosquitoes. During the study of the structure and physiology of microsporidia, it has been demonstrated, that the secretory proteins of microsporidia migrate into the nucleus of the host cell; the factors of parasites suppressing host cell apoptosis and the presence of specific organelles related to the energy metabolism have been revealed; the mitosomes have been found in the spores and not the prespore stages of microsporidia. The role of the Golgi complex in the formation of the extrusion apparatus, as well as the absence of the vesicular secretory transport in microsporidia, has been shown for the first time. For the first time in Russia, cases of microsporidia infection in HIV-infected patients have been identified. Currently, attention is paid to the development of a new universal taxonomic system of microsporidia combining molecular characteristics with a description of the structural and developmental features of each taxon of the parasites. Microsporidia possess many remarkable structural and functional differences from any other organisms thus substantiating an independent field of biological research: “microsporidiology”.

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.

Enterocytozoon bieneusi in raw milk of cattle, sheep and water buffalo in Turkey: Genotype distributions and zoonotic concerns

Raw milk is a continued threat to public health due to possible contamination with zoonotic pathogens. Enterocytozoon bieneusi is one of the most prevalent pathogenic fungi in a wide range of vertebrate hosts, causing diarrheal disease. Although there has been some evidence, the role and potential risk of raw milk of dairy animals in the transmission dynamics of E. bieneusi is not clear. Therefore, we aimed to determine the occurrence and genotypes of E. bieneusi in raw milk of dairy animals in several farms of the Central Anatolia Region. We also investigated if there is a relation between the presence of E. bieneusi and mastitis. Genomic DNAs from a total of 450 raw milk including 200, 200 and 50 samples from cattle, sheep and water buffalo respectively were analyzed using nested PCR, targeting the internal transcribed spacer of E. bieneusi. Totally milk samples of 9 (4.5%) dairy cattle, 36 (18.0%) sheep, and 1 (2.0%) water buffalo were PCR-positive. A significant relationship was determined between mastitis and the presence of E. bieneusi. Sequence analysis revealed the presence of eight genotypes: two known (ERUSS1, BEB6) and six novel genotypes (named as TREb1 to TREb6). The genotype ERUSS1 and BEB6 were the most common genotypes, found in all cattle and sheep farms. Phylogenetic analysis clustered all the identified genotypes in Group 2. This study provides novel findings that contribute to the transmission dynamics and molecular epidemiology of E. bieneusi. Our study also highlighted the potential risk of raw milk for public health with respect to microsporidia infections.

Nitric oxide plays a crucial role in midgut immunity under microsporidian infection in Antheraea pernyi

The insect gut participates in initial local immune responses by producing reactive oxygen and nitrogen species as well as anti-microbial peptides to resist pathogenic invasions. Nitric oxide (NO), a signaling and an immune effector molecule synthesized by the enzyme NO synthase (NOS), mediates an early step of the signal transduction pathway. In this study, we evaluated NO levels after Nosema pernyi infection in Antheraea pernyi gut. NOS activity was higher in the microsporidia-infected gut of A. pernyi than in that of control. Three NOS-related genes were cloned, and their spatio-temporal expression patterns were evaluated. ApNOS2 was expressed quickly in the midgut after N. pernyi infection. Sodium nitroprusside, dihydrate (SNP), or Nω-L-nitro-arginine methyl ester, hydrochloride (L-NAME), altered the NO content in A. pernyi midgut. Anti-microbial peptides (AMPs) in the groups exposed to N. pernyi plus SNP and N. pernyi plus L-NAME exhibited higher and lower expression, respectively, relative to the control. These results indicate that microsporidia infection triggers short-term activation of NO and NOS genes in the A. pernyi gut that is downregulated after 24 h. Notably, infection rates can be influenced by a NOS inhibitor. Furthermore, NO can be induced by pathogens. Similarly, NO content in the A. pernyi gut also influences AMPs in humoral immunity and some immune-related genes. Our results suggest that nitric oxide plays a vital role in A. pernyi gut immunity.