Error-prone protein synthesis in parasites with the smallest eukaryotic genome.

Microsporidia are parasitic fungi-like organisms that invade the interior of living cells and cause chronic disorders in a broad range of animals, including humans. These pathogens have the tiniest known genomes among eukaryotic species, for which they serve as a model for exploring the phenomenon of genome reduction in obligate intracellular parasites. Here we report a case study to show an apparent effect of overall genome reduction on the primary structure and activity of aminoacyl-tRNA synthetases, indispensable cellular proteins required for protein synthesis. We find that most microsporidian synthetases lack regulatory and eukaryote-specific appended domains and have a high degree of sequence variability in tRNA-binding and catalytic domains. In one synthetase, LeuRS, an apparent sequence degeneration annihilates the editing domain, a catalytic center responsible for the accurate selection of leucine for protein synthesis. Unlike accurate LeuRS synthetases from other eukaryotic species, microsporidian LeuRS is error-prone: apart from leucine, it occasionally uses its near-cognate substrates, such as norvaline, isoleucine, valine, and methionine. Mass spectrometry analysis of the microsporidium Vavraia culicis proteome reveals that nearly 6% of leucine residues are erroneously replaced by other amino acids. This remarkably high frequency of mistranslation is not limited to leucine codons and appears to be a general property of protein synthesis in microsporidian parasites. Taken together, our findings reveal that the microsporidian protein synthesis machinery is editing-deficient, and that the proteome of microsporidian parasites is more diverse than would be anticipated based on their genome sequences.

The first study on opportunistic intestinal microsporidiosis in IBD patients receiving immunosuppressive medications in Iran.

Microsporida are known as opportunistic unicellular organisms and have recently been reclassified as fungi that have been frequently reported from patients with congenital and acquired immunity failure disorders, worldwide. However, use of immunosuppressive medications in inflammatory bowel disease (IBD) patients significantly decreases overall immunity, and increases their susceptibility to opportunistic infections. Totally, 71 stool samples were collected from IBD patients consisted of 69 ulcerative colitis (UC) patients and two Crohn's disease (CD) patients. All patients had taken immunosuppressive and/or immunomodulator drugs for at least 3 weeks. DNA was extracted from all stool samples and Nested PCR was performed using genus-specific primers based on small subunit ribosomal RNA (SSU rRNA) gene. Fisher's Exact Test was applied to evaluate statistical association between microsporidia infection and sex, age and types of IBD. Mean of age ± s.d., women and men percentage of the attended patients were 36·17 ± 11·93, 60·6%, and 39·4%, respectively. A 440-bp fragment of SSU rRNA gene attributed to Enterocytozoon bieneusi was amplified from 12·7% of IBD patients. No Encephalitozoon DNA was detected in the samples. No microsporidia-positive sample was found in CD patients. Fisher's Exact Test showed that there was no statistically significant correlation between intestinal microsporidiosis and age, sex, and IBD types with P values: 0·389, 1·00, and 1·00, respectively. This study has shown IBD patients undergoing immunosuppressive/immunomodulators medications, which may be susceptible to intestinal microsporida infection. E. bieneusi is the commonest intestinal microsporidan reported from IBD patients.

In vitro antimicrosporidial activity of gold nanoparticles against Heterosporis saurida.

BACKGROUND: Worldwide, there is a need to expand the number of drugs available to treat parasitic infections in aquaculture. One of the new materials being tested is metal nanoparticles, which have unique chemical and physical characteristics owing to their extremely small size and high surface area to volume ratio. We examined the effectiveness of gold nanoparticles against the microsporidian parasite Heterosporis saurida, which causes severe economic losses in lizard fish, Saurida undosquamis aquaculture. RESULTS: We synthesized gold nanoparticles by chemical reduction of tetrachloroauric acid as a metal precursor. We assessed the antimicrosporidial efficacy of the nanoparticles against H. saurida using an in vitro screening approach, which we had developed previously using the eel kidney cell line EK-1. The number of H. saurida spores produced in EK-1 cells was reduced in a proportional manner to the dosage of gold nanoparticles administered. A cell metabolic activity test (MTT) indicated that the gold nanoparticles did not appear to be toxic to the host cells. CONCLUSIONS: Gold nanoparticles can act as an effective antimicrosporidial agent and hold promise to reduce disease in lizardfish aquaculture. Metal nanoparticles should be considered as an alternate choice for development of new antimicrosporidial drugs to combat disease problems in aquaculture.

[PARASITES OF THE COMMON ROACH (RUTILUS RUTILUS L.) UNDER THE IMPACT OF INDUSTRIAL POLLUTION OF A LAKE].

The consequences of man-induced transformation of Lake Kostomukshskoe (tailings dump) related to increased mineralization and entry of highly dispersed dredge material not typical for northern lakes were studied using roach parasites as the example. It was found that the roach parasite fauna has been losing rare and scant parasite species of different taxonomic groups, mainly helminthes with an indirect life cycle. Common and dominant species of myxosporidians, monogeneans, trematodes of genera Diplostomum and Tylodelphys have been preserved. The chances of survival are higher for those parasites with direct life cycle. Also the parasite species whose larvae can actively penetrate the host have been survived. The species Ligula intestinalis, Philometra rishta, Pseudocapillaria tomentosa had reported very rare. Fish are infected with these parasites by feeding of Copepoda and oligochaetes.

Caenorhabditis elegans as a model for intracellular pathogen infection.

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole-animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal-related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.

Zinc PVA versus potassium dichromate for preservation of microsporidian spores of human origin.

Microsporidia are emerging opportunistic parasites. Preservation of the biological properties of microsporidian spores is often required in research work. The present study compared two preservatives; zinc polyvinyl alcohol (zinc PVA) and potassium dichromate solutions for preservation of microsporidian spores separated from human faecal samples. After 0, 1, 2 and 4 months of storage, morphological features and staining characters of the spores were assessed by light microscopy in modified trichrome-stained smears and their viability percentages were calculated using acridine orange/ethidium bromide mixture. Also, spore infectivity was evaluated by faecal spore shedding and intestinal spore load in mice orally inoculated with the preserved spores. Results revealed that morphological features, staining characters and viability of the spores were maintained in both solutions throughout the study period. Spore infectivity was completely preserved in zinc PVA solution but showed significant reduction in potassium dichromate solution at the fourth month of the preservation duration.

Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process.

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.

[Staining of microsporidian spores with diamidine phenylindole].

A number of microscopic techniques and dyes are available to diagnose microsporidian infections in invertebrate and vertebrate hosts. Among these, DNA-specific fluorochrome DAPI is widely used to stain DNA in prokaryotic and eukaryotic cells, alone or in combination with other histochemical or fluorescent dyes. Moreover, this dye also binds to membraneous structures and protein complexes. In our studies, DAPI was used to stain spores of microsporidia infecting orthopteran, coleopteran, dipteran and lepidopteran insect hosts. DAPI staining of diplokarya helped to discriminate the Nosema-like microsporidian spores from spore-shaped bodies lacking this characteristic staining. It was found, moreover, that non-DNA staining occurred in many cases and other components of the spores were stained: the exospore, the cytoplasm, the extruded polar filament and the polaroplast. Staining of these structures was feeble as compared to DNA and in most cases did not interfere with nuclear apparatus staining. Feebly stained cytoplasm and exospore clearly indicated unstained zone of endospore, making it easier to diagnose both mono- and diplokaryotic spores. Staining of extruded polar filament allowed to demonstrate viability and to observe some stages of extrusion process of microsporidian spores.

Converging seasonal prevalence dynamics in experimental epidemics.

BACKGROUND: Regular seasonal changes in prevalence of infectious diseases are often observed in nature, but the mechanisms are rarely understood. Empirical tests aiming at a better understanding of seasonal prevalence patterns are not feasible for most diseases and thus are widely lacking. Here, we set out to study experimentally the seasonal prevalence in an aquatic host-parasite system. The microsporidian parasite Hamiltosporidium tvärminnensis exhibits pronounced seasonality in natural rock pool populations of its host, Daphnia magna with a regular increase of prevalence during summer and a decrease during winter. An earlier study was, however, unable to test if different starting conditions (initial prevalence) influence the dynamics of the disease in the long term. Here, we aim at testing how the starting prevalence affects the regular prevalence changes over a 4-year period in experimental populations. RESULTS: In an outdoor experiment, populations were set up to include the extremes of the prevalence spectrum observed in natural populations: 5% initial prevalence mimicking a newly invading parasite, 100% mimicking a rock pool population founded by infected hosts only, and 50% prevalence which is commonly observed in natural populations in spring. The parasite exhibited similar prevalence changes in all treatments, but seasonal patterns in the 100% treatment differed significantly from those in the 5% and 50% treatments. Populations started with 5% and 50% prevalence exhibited strong and regular seasonality already in the first year. In contrast, the amplitude of changes in the 100% treatment was low throughout the experiment demonstrating the long-lasting effect of initial conditions on prevalence dynamics. CONCLUSIONS: Our study shows that the time needed to approach the seasonal changes in prevalence depends strongly on the initial prevalence. Because individual D. magna populations in this rock pool metapopulation are mostly short lived, only few populations might ever reach a point where the initial conditions are not visible anymore.

[Should the microsporidian spores be treated as dormant stages?].

Spores of bacteria, fungi, microsporidia and other protists are traditionally treated as dormant stages, intended to the long-term survival in the environment and to activation of parasitic forms during the infestation of a new host. However, in the process of examination of insect microsporidia at the molecular cellular levels and also at the level of organisms and populations, we came to a conclusion that spores are very active developmental stages with the entire potential directed to the rapid and successful infestation of new hosts during contact with the later. The work summarizes the original data demonstrating (1) the necessity of the rapid activation of microsporidian spores during host contact, (2) hopelessness of the long retaining of viability by spores of many microsporidia in the environment after leaving host organism; and (3) specific accumulation of metabolic ferments in "dormant" spores, but not in actively proliferating prespore developmental stages. On the basis of these data we conclude that microsporidian spores tend to shorten the period when they stay outside host organism to the maximal degree. The probability of host infestation within the limited time period increases due to diverse modes of transmission of pathogens, accumulation of maximally possible volume of infective spores, and the rapid mobilization of the extrusion apparatus.

[Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis].

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.

[Changes in the behaviour of Moina macrocopa (Crustacea: Cladocera) under the influence of Gurleya sp. (Microsporidia: Gurleyidae)].

It is established, that infestation with Gurleya sp. (Microsporidia, Gurleyidae) changes the behaviour of its host Moina macrocopa (Straus, 1820), a crustacean inhabiting pools. Heavily infested host individuals attach to surface film of water before their death by senescence. As a consequence, their dead bodies filled with spores of the parasite adhere to objects dipped in water that probably facilitates the transfer of spores by birds and cattle to other pools. Weakly infested or uninfested individuals drown after the end of their ontogenesis. Distribution of the microsporidian spores by two different ways (with attached dead bodies of the hosts or with bottom sediments) makes invasion of a new host more probable.

Direct and sensitive detection of a microsporidian parasite of bumblebees using loop-mediated isothermal amplification (LAMP).

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. Although the decline of bee populations affects both wild plants and human food supply, the effects of Nosema spp. infections are not known because it is difficult to obtain infective spores from wild bees due to their low prevalence. Microscopical observation of fecal samples or midgut homogenates and/or PCR are generally used for N. bombi detection. However, the germination rate of microsporidian spore declines if they are kept at 4 °C for a long time or frozen. It is therefore crucial to minimize the diagnosis and isolation time of infective spores from field-collected samples. Therefore, we performed a loop-mediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals.

Behavioural effects of the common brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio).

Research conducted on model organisms may be biased due to undetected pathogen infections. Recently, screening studies discovered high prevalence of the microsporidium Pseudoloma neurophilia in zebrafish (Danio rerio) facilities. This spore-forming unicellular parasite aggregates in brain regions associated with motor function and anxiety, and despite its high occurrence little is known about how sub-clinical infection affects behaviour. Here, we assessed how P. neurophilia infection alters the zebrafish´s response to four commonly used neurobehavioral tests, namely: mirror biting, open field, light/dark preference and social preference, used to quantify aggression, exploration, anxiety, and sociability. Although sociability and aggression remained unaltered, infected hosts exhibited reduced activity, elevated rates of freezing behaviour, and sex-specific effects on exploration. These results indicate that caution is warranted in the interpretation of zebrafish behaviour, particularly since in most cases infection status is unknown. This highlights the importance of comprehensive monitoring procedures to detect sub-clinical infections in laboratory animals.

Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management.

OBJECTIVE: To investigate the incidence and epidemiologic factors involved in the development of microsporidial keratitis. The association of host immune status and clinical pattern, clinical features, and the role of fluoroquinolone monotherapy in treatment are also examined. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: All cases (124 patients, 134 eyes) of microsporidial keratitis confirmed with modified trichrome stain positive of corneal scrape over a 4-year period. METHODS: Epidemiologic factors were observed. Host immune status with human immunodeficiency virus (HIV) serology and CD4/CD8 analysis was performed when consent was obtained. Visual acuity (VA) and slit-lamp examination throughout the course of keratitis was recorded. Treatment used included topical fluoroquinolones (ciprofloxacin 0.3%, moxifloxacin 0.5%, gatifloxacin 0.5%, levofloxacin 0.5%, or norfloxacin 0.3%) as monotherapy or in combination with topical fumagillin and/or systemic albendazole. Where corneal edema developed, ultrasound corneal pachymetry was recorded. MAIN OUTCOME MEASURES: Demographic features and epidemiologic factors, including host immune status. Clinical features and disease course, including the response to different therapeutic regimes. RESULTS: Patients ranged in age from 11 to 68 years (mean, 31.9; median, 30) with a male:female ratio of 8:1 (females n = 17 [13.7%]). We performed HIV serology and CD4/CD8 in 45.9% of cases (n = 57); all the cases tested were negative with normal T-cell indices. Epidemiologic factors included soil exposure (50%), contact lens wear (21.1%), and topical steroid treatment (17.1%). The VA on presentation ranged from 20/20 to 20/100 (median, 20/30) with no loss in lines of VA on resolution. Common features were follicular papillary conjunctivitis and coarse punctate epithelial lesions in 3 patterns--diffuse, peripheral, and paracentral--evolving into nummular keratitis before resolution. Resolution occurred in 99% of cases on topical fluoroquinolone monotherapy. Four patients had recurrent disease that resolved with repeat fluoroquinolone or fluoroquinolone/oral albendazole combination. Two new clinical features were identified--diffuse endotheliitis (19.4%) with corneal edema and limbitis. CONCLUSIONS: This study identifies an increasing incidence of microsporidial keratitis in Singapore with a strong correlation with prior soil exposure. Diffuse endotheliitis and limbitis have not been described and resolves with topical steroid therapy. Topical fluoroquinolone monotherapy is a valid treatment option.

Infection of Drosophila melanogaster by Tubulinosema kingi: stage-specific susceptibility and within-host proliferation.

Despite its importance as a model organism very little is known about the interaction between Drosophila and its microsporidian pathogens. Here we report on the relative susceptibility of Drosophila melanogaster life history stages to infection by Tubulinosema kingi, and on patterns of pathogen proliferation. We find that only larvae can be infected, and that this susceptibility decreases with larval age. Following infection, the pathogen shows little subsequent proliferation in larvae, a limited amount in pupae while it replicates greatly in adults. We present evidence that the host launches a cellular immune response after infection with the pathogen, although its effectiveness remains to be demonstrated.

Examination of naturally exposed bottlenose dolphins (Tursiops truncatus) for Microsporidia, Cryptosporidium, and Giardia.

Bottlenose dolphins (Tursiops truncatus) captured in the estuarine waters off the coasts of South Carolina and Florida were examined for the presence of Microsporidia, Cryptosporidium sp., and Giardia sp. DNA extracted from feces or rectal swabs was amplified by polymerase chain reaction using parasite-specific small subunit ribosomal RNA gene primers. All positive specimens were subjected to gene sequence analysis. Of 83 dolphins, 17 were positive for Microsporidia. None was positive for Cryptosporidium or Giardia. Gene sequence data for each of the positive specimens were compared with data in GenBank. Fourteen specimens were found similar to, but not identical to, the microsporidian species Kabatana takedai, Tetramicra brevifilum, and Microgemma tinca, reported from fish, and possibly represent parasites of fish eaten by dolphins. Gene sequence data from 3 other specimens had approximately 87% similarity to Enterocytozoon bieneusi, a species known primarily to infect humans and a variety of terrestrial mammals, including livestock, companion animals, and wildlife. It is not clear if these specimens represent a species from a terrestrial source or a closely related species unique to dolphins. There were neither clinical signs nor age- or gender-related patterns apparent with the presence of these organisms.

Late-Onset Microsporidial Keratitis in Femtosecond Astigmatic Keratotomy After Laser-Assisted Phacoemulsification.

PURPOSE: To present a case of microsporidial keratitis in a femtosecond laser-created astigmatic keratotomy (AK) incision. METHODS: Case report. RESULTS: A 65-year-old Middle Eastern man presented 2 months after uncomplicated femtosecond laser-assisted cataract surgery (FLACS) and AK with mildly decreased vision and corneal edema in the operative eye. Shortly after treatment with topical corticosteroids, a fulminant corneal infiltrate manifested along the temporal arcuate incision. Multiple corneal scrapings sent for laboratory analysis were inconclusive. Two weeks after the initial presentation, a deep tissue sample was obtained using a 27-gauge cannula passed within the arcuate incision. The gram stain was directly observed, revealing intracellular microsporidial spores. The patient was treated with oral albendazole 400 mg once daily over 2 weeks and topical voriconazole 1% and fumagillin 3 mg/mL eye drops over 10 weeks. During this course, visual function steadily recovered as the infiltrate coalesced and ocular inflammation subsided. CONCLUSIONS: This is the first reported case of microsporidial keratitis presenting as a late-onset infection after femtosecond laser-assisted AK.

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria.

Microsporidia are small (1-20 micron) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2,3], and are reported to lack mitochondria [2,4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2,4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5-9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.