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.

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.

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.

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.

Clinical Features, Risk Factors, and Treatments of Microsporidial Epithelial Keratitis.

OBJECTIVE: To report the clinical manifestations, risk factors, and treatments of microsporidial epithelial keratitis in Thailand. METHODS: Twenty eyes of 19 patients were diagnosed and the clinical presentations, risk factors, and management were analyzed. RESULTS: Of 19 patients, six patients (32%) had no apparent risk factors. Predisposing factors included soil exposure (6/19, 32%), water contamination (6/19, 32%), and eye liner (1/19, 4%). Twelve cases (63%) were detected in the rainy season. All cases presented with disseminated, punctated, elevated, epithelial keratitis. Corneal scrapings with Gram-chromotrope staining were positive in all patients. Moxifloxacin 0.5% eye drops were given and all 16 patients experienced complete resolution. Three recurrent cases were resolved with only topical moxifloxacin without corneal scraping or swabbing. CONCLUSIONS: Predisposing factors were not found in some patients; thus, corneal scraping with staining should be considered in cases having a high index of suspicion. The incidence is increased during the rainy season; therefore, clinicians should have more awareness during these times. Debridement with topical moxifloxacin eye drops, without any systemic medication, may be an effective treatment. Corneal scraping or swabbing may not be required in recurrences.

Immune signaling pathways activated in response to different pathogenic micro-organisms in Bombyx mori.

The JAK/STAT, Toll, Imd, and RNAi pathways are the major signaling pathways associated with insect innate immunity. To explore the different immune signaling pathways triggered in response to pathogenic micro-organism infections in the silkworm, Bombyx mori, the expression levels of the signal transducer and activator of transcription (BmSTAT), spatzle-1 (Bmspz-1), peptidoglycan-recognition protein LB (BmPGRP-LB), peptidoglycan-recognition protein LE (BmPGRP-LE), argonaute 2 (Bmago2), and dicer-2 (Bmdcr2) genes after challenge with Escherichia coli (E. coli), Serratiamarcescens (Sm), Bacillus bombyseptieus (Bab), Beauveriabassiana (Beb), nucleopolyhedrovirus (BmNPV), cypovirus (BmCPV), bidensovirus (BmBDV), or Nosemabombycis (Nb) were determined using real-time PCR. We found that the JAK/STAT pathway could be activated by challenge with BmNPV and BmBDV, the Toll pathway could be most robustly induced by challenge with Beb, the Imd pathway was mainly activated in response to infection by E. coli and Sm, and the RNAi pathway was not activated by viral infection, but could be triggered by some bacterial infections. These findings yield insights into the immune signaling pathways activated in response to different pathogenic micro-organisms in the silkworm.

[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.

Development of a novel in vitro method for drug development for fish; application to test efficacy of antimicrosporidian compounds.

Few drugs are approved for treating diseases caused by parasites in minor species such as fish. This is due, in part, to the expense of drug development and to the comparatively small market. In vivo effectiveness trials for antiparasitic drugs are costly, time consuming and require ethics approval, therefore an in vitro screening approach is a cost-effective alternative to finding promising drug candidates. We developed an in vitro testing system to test antimicrosporidial compounds against a microsporidian pathogen Heterosporis saurida. Five antiparasitic compounds, albendazole, fumagillin, TNP-70, nitazoxanide and lufenuron, were assayed for antimicrosporidial activity. All compounds reduced the number of H saurida spores in infected cells when applied at a concentration that did not appear to be toxic to the host cells. Albendazole inhibited replication of H saurida by >60 per cent, fumagillin and its analogue TNP-470 inhibited H saurida >80 per cent, nitazoxanide and lufenuron inhibited growth >70 per cent. The data suggest that both fumagillin and its analogous TNP-70 hold the best promise as therapeutic agents against H saurida. The ability to use fish cell cultures to assess drugs against H saurida demonstrates an approach that may be helpful to evaluate other drugs on different microsporidia and host cells.

Unusual fluorescent granulomas and myonecrosis in Danio rerio infected by the microsporidian pathogen Pseudoloma neurophilia.

Abstract Zebrafish are a powerful model organism to study disease. Like other animal models, Danio rerio colonies are at risk of pathogenic infection. Microsporidia, a group of intracellular fungus-like parasites, are one potential threat. Microsporidian spores germinate and spread causing pathological changes in the central nervous system, skeletal muscle, and other anatomic sites. Infection can impair breeding, cause other morbidities, and ultimately be lethal. Previously, detecting microsporidia in zebrafish has required sacrificing animals for histopathologic analysis or microscopic examination of fresh tissues. Here, we show that fish with microsporidial infection often have autofluorescent nodules, and we demonstrate infectious spread from nodule-bearing fish to healthy D. rerio. Histologic analyses revealed that fluorescent nodules are granulomatous lesions composed of spores, degenerating muscle, and inflammatory cells. Additional histologic staining verified that microsporidia were present, specifically, Pseudoloma neurophilia. Polymerase chain reaction (PCR)-based testing confirmed the presence of P. neurophilia. Further PCR testing excluded infection by another common zebrafish microsporidial parasite, Pleistophora hyphessobryconis. Collectively, these studies show that P. neurophilia can induce skeletal muscle granulomas in D. rerio, a previously unknown finding. Moreover, since granulomas autofluoresce, microscopic screening for P. neurophilia infection is feasible in live fish, avoiding the need to sacrifice fish for surveillance for this pathogen.

The efficacy of corneal debridement in the treatment of microsporidial keratoconjunctivitis: a prospective randomized clinical trial.

PURPOSE: To evaluate the efficacy of corneal debridement in the treatment of clinically diagnosed cases of microsporidial keratoconjunctivitis. DESIGN: Prospective, double-masked randomized clinical trial. METHODS: Patients with clinical features such as multifocal, coarse, raised, punctate, round to oval epithelial lesions in the cornea in slit-lamp examination with mild to moderate conjunctival congestion, suggestive of microsporidial superficial keratoconjunctivitis, were included in the prospective study. All patients were randomized into 2 groups. Group 1 patients underwent debridement with the help of a sterile #15 blade on a Bard-Parker handle, whereas only conjunctival swabs were taken from Group 2 patients. All patients were treated with ocular lubricants. RESULTS: One hundred and twenty patients with clinical features suggestive of microsporidial superficial keratoconjunctivitis were included in the study. The mean age was 34.3 ± 13.6 years (Group 1) and 35.8 ± 16.2 years (Group 2) (P = .59). The mean duration of symptoms was 6.8 ± 3.9 days (Group 1) and 7.2 ± 4.6 days (Group 2) (P = .61). Baseline characteristics showed no difference between the 2 groups. The primary outcome was the time from the presentation to complete resolution (ie, absence of corneal lesions) of the clinical signs and symptoms. The secondary outcomes were final visual acuity and residual corneal side effects and/or scarring, if any. The mean resolution time of the corneal lesions was 5.7 ± 4.0 days (Group 1) and 5.9 ± 3.9 days (Group 2) (P = .83). There was no significant difference in final visual outcome in the 2 groups. No serious side effects were observed. CONCLUSION: Debridement does not have any significant advantage in terms of resolution of the corneal lesions and final visual outcome in cases of microsporidial keratoconjunctivitis.

Parasites of freshwater fishes in North America: why so neglected?

Fish parasitology has a long tradition in North America and numerous parasitologists have contributed considerably to the current knowledge of the diversity and biology of protistan and metazoan parasites of freshwater fishes. The Journal of Parasitology has been essential in disseminating this knowledge and remains a significant contributor to our understanding of fish parasites in North America as well as more broadly at the international level. However, with a few exceptions, the importance of fish parasites has decreased during the last decades, which is reflected in the considerable decline of funding and corresponding decrease of attention paid to these parasites in Canada and the United States of America. After the 'golden age' in the second half of the 20th Century, fish parasitology in Canada and the United States went in a new direction, driven by technology and a shift in priorities. In contrast, fish parasitology in Mexico has undergone rapid development since the early 1990s, partly due to extensive international collaboration and governmental funding. A critical review of the current data on the parasites of freshwater fishes in North America has revealed considerable gaps in the knowledge of their species composition, host specificity, life cycles, evolution, phylogeography, and relationships with their fish hosts. As to the key question, "Why so neglected?" this is probably because: (1) fish parasites are not in the forefront due to their lesser economic importance; (2) there is little funding for this kind of research, especially if a practical application is not immediately apparent; and (3) of shifting interests and a shortage of key personalities to train a new generation (they switched to marine habitats or other fields). Some of the opportunities for future research are outlined, such as climate change and cryptic species diversity. A significant problem challenging future research seems to be the loss of trained and experienced fish parasitologists. This has 2 major ramifications: the loss of expertise in identifying organisms that other biologists (e.g., ecologists, molecular biologists, evolutionists) work with, and an incomplete comprehension of ecosystem structure and function in the face of climate change, emerging diseases, and loss of biodiversity.

Are there genetic trade-offs between immune and reproductive investments in Tribolium castaneum?

Parasites impose strong selection on hosts to defend themselves, which is expected to result in trade-offs with other fitness traits such as reproduction. Here we test for genetic trade-offs between reproductive traits and immunity using Tribolium castaneum lines that were subject to experimental evolution. The lines have been exposed to contrasting sexual selection intensities via different sex ratios (female-biased, equal and male-biased). After 56 generations, the lines have significantly diverged and those experiencing high sexual selection have evolved males who are superior competitors for reproductive success, and females who are more resistant to multiple mating. All selected lines were assessed for both an immune measurement (phenoloxidase (PO) activity) and host resistance to the microsporidian Nosema whitei after two generations of relaxed selection. In contrast to our expectations we did not find any evidence for a genetic trade-off between investment in reproduction and immunity. Both PO and Nosema resistance did not differ between lines, despite their divergences in reproductive investment due to variation in sexual selection and conflict. Nevertheless, overall we found that females had higher PO activities and in the Nosema free control treatment survived longer than males, suggesting that females generally invest more in PO and survival under control conditions than males. This result fits the Bateman's principle, which states that females gain fitness through increased immunity and longevity, while males gain fitness through increased mating success.

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.

Microsporidia and coccidia as causes of persistence diarrhea among liver transplant children: incidence rate and species/genotypes.

We determined species/genotype(s) of enteric microsporidia and coccidia causing diarrhea among 44 liver transplant children in Shiraz Nemazee hospital using acid-fast-trichrome staining and polymerase chain reaction-sequencing techniques. Enterocytozoon bieneusi (genotype D), Cryptosporidium (parvum and meleagridis) were detected in 6.81% and 11.36% of the children, respectively.

Evolutionary conservation of the ribosomal biogenesis factor Rbm19/Mrd1: implications for function.

Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-ß4-strand in RBD6 is essential for function.

[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.

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.