Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Targeting the honey bee gut parasite Nosema ceranae with siRNA positively affects gut bacteria.

BACKGROUND: Gut microbial communities can contribute positively and negatively to host health. So far, eight core bacterial taxonomic clusters have been reported in honey bees. These bacteria are involved in host metabolism and defenses. Nosema ceranae is a gut intracellular parasite of honey bees which destroys epithelial cells and gut tissue integrity. Studies have shown protective impacts of honey bee gut microbiota towards N. ceranae infection. However, the impacts of N. ceranae on the relative abundance of honey bee gut microbiota remains unclear, and has been confounded during prior infection assays which resulted in the co-inoculation of bacteria during Nosema challenges. We used a novel method, the suppression of N. ceranae with specific siRNAs, to measure the impacts of Nosema on the gut microbiome. RESULTS: Suppressing N. ceranae led to significant positive effects on microbial abundance. Nevertheless, 15 bacterial taxa, including three core taxa, were negatively correlated with N. ceranae levels. In particular, one co-regulated group of 7 bacteria was significantly negatively correlated with N. ceranae levels. CONCLUSIONS: N. ceranae are negatively correlated with the abundance of 15 identified bacteria. Our results provide insights into interactions between gut microbes and N. ceranae during infection.

Increased survival of the honey bee Apis mellifera infected with the microsporidian Nosema ceranae by effective gene silencing.

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology.

Bacterium-Expressed dsRNA Downregulates Microsporidia Nosema bombycis Gene Expression.

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain (DE3) to express double-strand RNAs targeting the gene encoding ADP/ATP protein in N. bombycis. The results showed that dsRNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the dsRNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm.

Molecular Characterization of Enterocytozoon bieneusi in Domestic Rabbits (Oryctolagus cuniculus) in Northeastern China.

A study of 426 rabbits from 3 cities in Jilin province (Changchun City and Jilin City) and Liaoning province (Shenyang City) was conducted between May and June 2015. The overall prevalence of E. bieneusi in rabbits was 0.94% (4/426), with 0% (0/116), 1.72% (3/174), and 0.74% (1/136) in Jilin, Changchun, and Shenyang City, respectively. Only 3 farms (farm 1 and farm 3 in Changchun City, farm 8 in Shenyang City) were PCR-positive for E. bieneusi. Moreover, rabbits of more than 6 months (1.72%) had the highest E. bieneusi prevalence, followed by rabbits of 4-6 months (1.26%), 2-3 months (0.58%), and less than 1 month (0%). Analysis of ITS gene of E. bieneusi suggested that all 4 E. bieneusi isolates were genotype D, and were classified as group 1a. The present results first demonstrated the existence of zoonotic E. bieneusi in domestic rabbits in China. Effective control measures should be implemented to prevent E. bieneusi infection in domestic rabbits, other animals, and humans.

[Current Concepts of Human Microsporidiosis].

Microsporidiosis is an ubiquitous opportunistic disease that usually appears in immunocompromised patients: AIDS patients or organ-transplant recipients. The infectious agents of disease are fungi-related obligate intracellular parasites - microsporidia. Alongside with Cryptosporidium and Cytomegalovirus, these parasites are the most common causative agents of diarrhea in HIV-infected patients. Intestinalform of microsporidiosis has been mostfrequently observed, but microsporidia can affect almost any organs of the human body, eyes, lungs, muscles, organs of the nervous system. The present paper overviews the current data on the etiology, pathogenesis, epidemiology, clinical presentation, diagnosis and treatment methods of microsporidiosis.

Fish microsporidia: immune response, immunomodulation and vaccination.

Immune response to fish microsporidia is still unknown and there are current research trying to elucidate the events involved in the immune response to this parasite. There is evidence suggesting the role of innate immune response and it is clear that adaptive immunity plays an essential part for eliminating and then mounting a solid resistance against subsequent microsporidian infections. This review article discusses the main mechanisms of resistance to fish microsporidia, which are considered under four main headings. 1) Innate immunity: the inflammatory tissue reaction associated with fish microsporidiosis has been studied at the ultrastructural level, providing identification of many of the inflammatory cells and molecules that are actively participating in the spore elimination, such as macrophages, neutrophils, eosinophilic granular cells, soluble factors and MHC molecules. 2) Adaptive immunity: the study of the humoral response is relatively new and controversial. In some cases, the antibody response is well established and it has a protective role, while in other situations, the immune response is not protective or it is depressed. Study of the cellular response against fish microsporidia is still in its infancy. Although the nature of the microsporidian infection suggests participation of cellular mechanisms, few studies have focused on the cellular immune response of infected fish. 3) Immunomodulation: glucans are compounds that can modulate the immune system and potentiate resistance to microorganisms. These compounds have been proposed that can interact with receptors on the surface of leukocytes that result in the stimulation on non-specific immune responses. 4) Vaccination: little is known about a biological product that could be used as a vaccine for preventing this infection in fish. In the Loma salmonae experience, one of the arguments that favor the production of a vaccine is the development in fish of resistance, associated to a cellular immune response. A recently proved spore-based vaccine to prevent microsporidial gill disease in salmon has recently shown its efficacy by considerably reducing the incidence of infection. This recent discovery would be first anti-microsporidian vaccine that is effective against this elusive parasite.

Plant-based supplement containing B-complex vitamins can improve bee health and increase colony performance.

It is common knowledge that nutritive stress resulting from decreased diversity and quality of food, pollution of food sources and beekeeping errors may lead to increased susceptibility of bees to pathogens and pesticides. The dearth of adequate food is frequently compensated with supplements. Thus, this research was aimed to study the effects of the plant-based supplement B + on colony strength (assessed according to open and sealed brood area, honey and pollen/bee bread reserves, and the number of adult bees). In addition, Nosema ceranae spores and viruses were quantified and the level of infestation with Varroa destructor assessed. The experiment was conducted in late summer and early spring. In colonies which were given B + in feed a significant increase (p < 0.05) in the parameters of colony strength were noticed in comparison to the control (colonies fed on sugar syrup). Moreover, it was proven that the bees from these colonies had significantly lower (p < 0.05) N. ceranae spore counts, and acute bee paralysis, deformed wing and sacbrood virus loads. Our results suggest that the addition of B + supplement to the colonies provide them with nutrients, contribute to their strengthening, might prevent nutritive stress and increase the success of bees in combating pathogens.

Transmission of Pseudoloma neurophilia in Laboratory Zebrafish (Danio rerio) When Using Mass Spawning Chambers and Recommendations for Chamber Disinfection.

Pseudoloma neurophilia, a microsporidium that primarily infects neural tissues, is a common pathogen in laboratory zebrafish. The risk of parasite transmission with different spawning apparatuses and the effectiveness of disinfection are unknown. In this study, we spawned uninfected zebrafish with P. neurophilia-infected zebrafish in either 50 L mass spawning chambers (MSCs) or 1 L standard breeding tanks (BTs). Fish were spawned once or thrice, with and without chamber disinfection between uses, to evaluate risk of vertical and horizontal transmission. Six disinfection protocols were tested to determine which effectively eliminated residual spores. We demonstrated that three consecutive uses of an MSC significantly increased the risk of transmission to other fish when compared to the use of BTs or only one spawning event in an MSC (both p < 0.0001). Vertical transmission was not detected with any method. Disinfection with ∼100 ppm bleach soak (pH ∼7.0), 75 ppm Wescodyne® soak, and 175 ppm Wescodyne Plus spray was 100% effective in eliminating spores from the MSCs. Disinfection of MSCs before spawning did not decrease P. neurophilia transmission when infected fish remained present in the breeding population. Researchers should avoid using endemically infected fish in MSCs to minimize transmission of pathogens within their colonies.

Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine.

Pseudoloma neurophilia (Microsporidia) is the most common pathogen found in zebrafish Danio rerio research facilities. The parasite is associated with marked emaciation. Zebrafish laboratories usually disinfect eggs to prevent transmission of pathogens, typically with chlorine at 25 to 50 ppm for 10 min. The ability of chlorine to kill spores of P. neurophilia and 2 other microsporidia, Glugea anomala and Encephalitozoon cuniculi, was evaluated using 2 viability stains. SYTOX Green was used to visualize dead spores, and live spores were identified by their ability to extrude polar tubes in Fungi-Fluor solution following UV exposure. Results with both stains were similar at various chlorine concentrations for P. neurophilia and G. anomala, but Fungi-Fluor was not useful for E. cuniculi, due to the much smaller spore size. Using the SYTOX stain, we found that 5 ppm chlorine for 10 min causes 100% death in spores of E. cuniculi, which was similar to findings in other studies. In contrast, the spores of P. neurophilia and G. anomala were much more resistant to chlorine, requiring >100 or 1500 ppm chlorine, respectively, to achieve >95% spore death. Repeating chlorine exposures with spores of P. neurophilia using solutions adjusted to pH 7 increased the efficacy of 100 ppm chlorine, achieving >99% spore inactivation. We corroborated our viability staining results with experimental exposures of zebrafish fry, achieving heavy infections in fry at 5 to 7 d post-exposure in fish fed spores treated at 50 ppm (pH 9). Some fish still became infected with spores exposed to 100 ppm chlorine (pH 9.5). This study demonstrates that spores of certain fish microsporidia are highly resistant to chlorine, and indicates that the egg disinfection protocols presently used by most zebrafish research facilities will not prevent transmission of P. neurophilia to progeny.

Surveillance of Vittaforma corneae in hot springs by a small-volume procedure.

Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence supporting the need for proper control and regulations at hot spring recreational waters in order to avoid health risks from this pathogen. Finally, we have developed a small volume procedure for detecting V. corneae in water samples and this has proved to be very useful.

Molecular detection and species identification of Enterocytozoon bieneusi isolated from immunocompetent Orang Asli in Malaysia.

Most studies of opportunistic infections focus on immunocompromised patients. However, there is a lack of information on microsporidiosis in healthy people (immunocompetent) worldwide. This study aimed to detect and identify microsporidia species in immunocompetent Orang Asli living in Pahang, Malaysia. Orang Asli is a collective term for a group of indigenous people that usually reside in the interior regions of Peninsular Malaysia. They comprise about 0.7% of the total population in Malaysia and 76% of them lived below the poverty line i.e., poor housing conditions with the lack of access to safe drinking water and adequate sanitation, contaminated environment, high illiteracy rate and unhygienic practices by these people. Stool samples were collected from 209 Orang Asli and analyzed for detecting the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis by polymerase chain reaction assay targeting small subunit ribosomal RNA gene. E. bieneusi was detected in 8 individuals (3.83%). This infection was commonly found in males than females (5.2% vs. 2.7%). All infected Orang Asli were adults, with a mean age of 44years. Diarrhea and other gastrointestinal symptoms were reported in one case (12.5%) among individuals infected with this species. These findings clearly show that exposure to E. bieneusi may actually be common than reported. The accurate detection and identification of microsporidian species by molecular technique will improve therapy, clinical manifestations and prognosis of this infection, as no antiparasitic therapy has been approved for E. bieneusi. It is hoped that these findings will allow the formulation of better health management and disease prevention advisories, and improvement in the standards of health in similar communities.

Implementation of a Zebrafish Health Program in a Research Facility: A 4-Year Retrospective Study.

In the past two decades, zebrafish (Danio rerio)-based research has contributed to significant scientific advances. Still, husbandry and health programs did not evolve at the same pace, as evidenced by the absence of general guidelines. Health monitoring is essential to animal welfare, to permit animal exchanges across facilities, to contribute to robust experimental results, and for data reproducibility. In this study, we report a health program implemented in a zebrafish research facility to prevent, monitor, and control pathogen, and disease dissemination. This program includes quarantine, routine health screening of sentinels, and nonroutine screenings of retired animals and sick/moribund individuals. An extensive list of clinical signs, lesions, and pathogens was monitored based on: daily observation of fish, necropsy, histology, and bacterial culture. The results indicate that the combined analysis of sentinels with the evaluation of sick/moribund animals enables a comprehensive description not only of pathogen prevalence but also of clinical and histopathologic lesions of resident animals. The establishment of a quarantine program revealed to be effective in the reduction of Pseudoloma neurophilia frequency in the main aquaria room. Finally, characterization of the colony health status based on this multiapproach program shows a low prevalence of lesions and pathogens in the facility.

Facility Design and Health Management Program at the Sinnhuber Aquatic Research Laboratory.

The number of researchers and institutions moving to the utilization of zebrafish for biomedical research continues to increase because of the recognized advantages of this model. Numerous factors should be considered before building a new or retooling an existing facility. Design decisions will directly impact the management and maintenance costs. We and others have advocated for more rigorous approaches to zebrafish health management to support and protect an increasingly diverse portfolio of important research. The Sinnhuber Aquatic Research Laboratory (SARL) is located ∼3 miles from the main Oregon State University campus in Corvallis, Oregon. This facility supports several research programs that depend heavily on the use of adult, larval, and embryonic zebrafish. The new zebrafish facility of the SARL began operation in 2007 with a commitment to build and manage an efficient facility that diligently protects human and fish health. An important goal was to ensure that the facility was free of Pseudoloma neurophilia (Microsporidia), which is very common in zebrafish research facilities. We recognize that there are certain limitations in space, resources, and financial support that are institution dependent, but in this article, we describe the steps taken to build and manage an efficient specific pathogen-free facility.

Controlling Endemic Pathogens-Challenges and Opportunities.

By most measures, the University of Utah Centralized Zebrafish Animal Resource is a successful zebrafish core facility: we house ∼4000-5000 tanks for over 16 research groups; provide services and equipment for ∼150 users; are currently undergoing an expansion by 3000 tanks; and have been praised by institutional and national regulatory agencies for the cleanliness and efficiency of our facility. In recent years, we have implemented new programs to improve the overall health of our colony and believe we have seen a reduction in apparently sick fish. However, there are still deficiencies in our monitoring and pathogen control programs. Our histopathology sample sizes have been insufficient to estimate prevalence, but our sentinel tank program reveals the presence of Pseudoloma neurophilia and myxozoan, presumably Myxidium streisinger, in our facility. As we develop protocols to further reduce the burden of disease, we are focused on defining our baseline, establishing goals, and implementing methods to monitor our progress. The data generated by this approach will allow us to evaluate and implement the most cost-effective protocols to improve fish health.

[Highly Active AntiRetroviral Therapy and opportunistic protozoan infections]. / La Highly Active AntiRetroviral Therapy e le infezioni protozoarie opportuniste.

Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern has been observed in the HAART era, characterised by a high rate of relapses, which could be explained by the increased survival rate resulting from the effective antiretroviral therapy. A 64.8% decrease of the visceral leishmaniasis incidence was detected after HAART began to be used extensively in Spain. In a large cohort study carried out in ten European countries and in Australia, the relative risk to contract cryptosporidiosis as the first AIDS defining disease was reduced by 96% in the HAART era. In Italy, the relative risk of death for cryptosporidiosis reduced of 74% in the period 1997-98, when HIV-positive persons received HAART. In a large study carried out in Italy, isosporiasis was included in the group of opportunistic infections, of which the relative hazards showed a reduction of 95% in the HAART era. Since 1997, there was the evidence that the use of HAART in persons with advanced HIV infection can improve chronic diarrhoea and lead to disappearance of Enterocytozoon bieneusi from the stools. Although the reconstitution of the cellular immunity seems to be the main factor influencing the reduction of OPIs in persons with AIDS who undergo HAART, there are clinical and microbiological evidences, as well as in vitro and in vivo results, which indicate direct effects of HIV-PIs on the proteases of opportunistic parasites. These findings stress the existence of non-conventional unexpected benefits of PIs in HAART against protozoa. In addition, this benefit of PIs has been demonstrated also for Candida albicans secreted aspartyl proteases and for P. carinii acid proteases. In spite of these important results, HIV PIs are still very toxic for humans, specially in cases of very long treatment, and no clinical trial has been carried out for persons at risk, such as children and pregnant women, because the priority was to reduce the severity of HIV and not the evaluation of possible side effects of the therapy. It follows that further researches are needed to establish the non-conventional use of HIV PIs. Furthermore, the study of PIs against specific aspartyl proteases of those opportunistic protozoa that cause severe and intractable diseases, could be considered an alternative way towards the development of new drugs that may prove effective against these infections.

[Parasitoses in immune deficiences]. / Parazytozy w stanach niedoborów immunologicznych.

The congenital or acquired cause the state of immune deficiency. To acquired factors belong immunosuppressive therapy after grafting and in systemic diseases as infections with HIV. There is a number of parasitic organisms, mainly protozoa, which preferentially settle in immunocompromised persons. The opportunistic parasites are present in the nearest environment. Some of them were newly recognized as human invaders. The state of immune deficiency may reactivate latent infections, that occurs with Toxoplasma gondii infection. Some parasitic infections which are benign and self- resolving, when affecting immunocompetent hosts, become fulminant or disseminated and very often life - threatening in immunosuppressed individuals.

Screening alternative therapies to control Nosemosis type C in honey bee (Apis mellifera iberiensis) colonies.

Nosemosis type C caused by the microsporidium Nosema ceranae is one of the most widespread of the adult honey bee diseases, and due to its detrimental effects on both strength and productivity of honey bee colonies, an appropriate control of this disease is advisable. Fumagillin is the only veterinary medicament recommended by the World Organization for Animal Health (OIE) to suppress infections by Nosema, but the use of this antibiotic is prohibited in the European Union and few alternatives are available at present to control the disease. In the present study three therapeutic agents (Nosestat®, Phenyl salicylate and Vitafeed Gold®) have been tested to control N. ceranae infection in honey bee colonies, and have been compared to the use of fumagillin. None of the products tested was effective against Nosema under our experimental conditions. Low consumption of the different doses of treatments may have had a strong influence on the results obtained, highlighting the importance of this issue and emphasizing that this should be evaluated in studies to test therapeutic treatments of honey bee colonies.

Demonstrated efficacy of a pilot heterologous whole-spore vaccine against Microsporidial gill disease in rainbow trout.

Intraperitoneal vaccines using whole viable spores of the microsporidian Glugea anomala or Glugea hertwigi reduced the numbers of branchial xenomas by 80% and 91%, respectively, after a standard experimental infection of juvenile rainbow trout with the microsporidian Loma salmonae. Similar significant results were obtained when killed-spore preparations were used.