Identification of zoonotic genotypes of Giardia duodenalis

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.This study was partially funded by the European MED-VET-NET project contract FOOD-CT-2004-506122, and by the Dutch Food and Consumer Product Safety Authority (VWA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Human Islet Amyloid Polypeptide Transgenic Mice: In Vivo and Ex Vivo Models for the Role of hIAPP in Type 2 Diabetes Mellitus

Human islet amyloid polypeptide (hIAPP), a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2). To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process

Deliverable 6.1 - Demonstration prototype of the EuroMix model toolbox

This document describes in short the new features in a demonstration prototype of the EuroMix toolbox, developed as MCRA 8.2. An important aim of the EuroMix project is to develop and implement a web-based platform (the EuroMix toolbox) including data and models accessible to all key-actors in risk assessment and risk management. In the EuroMix project the development of a mixture selection module based on exposure was prioritised, because the choice of chemicals for the experiments depended on this. A mixture selection module was therefore developed, based on a method called sparse non-negative matrix under-approximation (SNMU). The mixture selection module was then applied to French and Dutch data, leading to a list of suggested chemicals for each adverse outcome pathway in the project

Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia

Background: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. Results: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. Conclusions: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.Fil: Kazimírová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Hamšíková, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Spitalská, Eva. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Minichová, Lenka. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Mahríková, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Caban, Radoslav. Široká ; EslovaquiaFil: Sprong, Hein. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Fonville, Manoj. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kocianová, Elena. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaqui

Self-reported symptoms and health complaints associated with exposure to Ixodes ricinus-borne pathogens

Background: The impact of infections with tick-borne pathogens (TBPs) other than Borrelia burgdorferi (s.l.) and tick-borne encephalitis virus (TBEV) on public health in Europe remains unclear. Our goal is to evaluate whether the presence of these TBPs in ticks can be associated with self-reported health complaints. Methods: We enrolled individuals who were bitten by I. ricinus between 2012 and 2015 and collected their relevant demographic and clinical information using a self-administered online questionnaire. A total of 4163 I. ricinus ticks sent by the participants were subject to molecular analyses for detection of specific TBPs. Associations between the presence of TBPs in ticks and self-reported complaints and symptoms were evaluated by means of a stepwise approach using a generalized linear model (GLM). Results: Of 17 self-reported complaints and symptoms significant in the univariate analyses, 3 had a highly significant association (P < 0.01) with at least one TBP in the multivariate analysis. Self-reported Lyme borreliosis was significantly associated (P < 0.001) with B. burgdorferi (s.l.) infection. Facial paralysis was associated (P < 0.01) with infection with B. miyamotoi, N. mikurensis and R. helvetica. Finally, a significant association (P < 0.001) was found between nocturnal sweating and A. phagocytophilum. Conclusions: We found associations between the presence of TBPs in ticks feeding on humans and self-reported symptoms. Due to the subjective nature of such reports and the fact that infection was determined in the ticks and not in the patient samples, further prospective studies utilizing diagnostic modalities should be performed before any clinical outcome can be causally linked to infection with TBPs. Graphical Abstract: [Figure not available: see fulltext.]

Aberrant Receptor-Mediated Endocytosis of Schistosoma mansoni Glycoproteins on Host Lipoproteins

BACKGROUND: Bilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification. METHODS AND FINDINGS: We studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites. CONCLUSIONS: Transfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen

Vaccination against Bm86 Homologues in Rabbits Does Not Impair Ixodes ricinus Feeding or Oviposition

Human tick-borne diseases that are transmitted by Ixodes ricinus, such as Lyme borreliosis and tick borne encephalitis, are on the rise in Europe. Diminishing I. ricinus populations in nature can reduce tick exposure to humans, and one way to do so is by developing an anti-vector vaccine against tick antigens. Currently, there is only one anti-vector vaccine available against ticks, which is a veterinary vaccine based on the tick antigen Bm86 in the gut of Rhipicephalus microplus. Bm86 vaccine formulations cause a reduction in the number of Rhipicephalus microplus ticks that successfully feed, i.e. lower engorgement weights and a decrease in the number of oviposited eggs. Furthermore, Bm86 vaccines reduce transmission of bovine Babesia spp. Previously two conserved Bm86 homologues in I. ricinus ticks, designated as Ir86-1 and Ir86-2, were described. Here we investigated the effect of a vaccine against recombinant Ir86-1, Ir86-2 or a combination of both on Ixodes ricinus feeding. Recombinant Ixodes ricinus Bm86 homologues were expressed in a Drosophila expression system and rabbits were immunized with rIr86-1, rIr86-2, a combination of both or ovalbumin as a control. Each animal was infested with 50 female adults and 50 male adults Ixodes ricinus and tick mortality, engorgement weights and egg mass were analyzed. Although serum IgG titers against rIr86 proteins were elicited, no effect was found on tick feeding between the rIr86 vaccinated animals and ovalbumin vaccinated animals. We conclude that vaccination against Bm86 homologues in Ixodes ricinus is not an effective approach to control Ixodes ricinus populations, despite the clear effects of Bm86 vaccination against Rhipicephalus microplus

The MCRA toolbox of models and data to support chemical mixture risk assessment

A model and data toolbox is presented to assess risks from combined exposure to multiple chemicals using probabilistic methods. The Monte Carlo Risk Assessment (MCRA) toolbox, also known as the EuroMix toolbox, has more than 40 modules addressing all areas of risk assessment, and includes a data repository with data collected in the EuroMix project. This paper gives an introduction to the toolbox and illustrates its use with examples from the EuroMix project. The toolbox can be used for hazard identification, hazard characterisation, exposure assessment and risk characterisation. Examples for hazard identification are selection of substances relevant for a specific adverse outcome based on adverse outcome pathways and QSAR models. Examples for hazard characterisation are calculation of benchmark doses and relative potency factors with uncertainty from dose response data, and use of kinetic models to perform in vitro to in vivo extrapolation. Examples for exposure assessment are assessing cumulative exposure at external or internal level, where the latter option is needed when dietary and non-dietary routes have to be aggregated. Finally, risk characterisation is illustrated by calculation and display of the margin of exposure for single substances and for the cumulation, including uncertainties derived from exposure and hazard characterisation estimates.</p

Tekenradar.nl, een webplatform over tekenbeten en de ziekte van Lyme

Tekenradar.nl is een webplatform voor onderzoek naar teken en tekenoverdraagbare aandoeningen zoals de ziekte van Lyme. Het RIVM en Wageningen University hebben de website in 2012 gezamenlijk opgericht. Tekenradar.nl geeft informatie over tekenbeten en de ziekte van Lyme en een actuele voorspelling van de tekenactiviteit. Mensen kunnen hun tekenbeet of erythema migrans en de vermoedelijke geografische locatie waar de beet is opgelopen melden op de site. Tot voorjaar 2013 konden deze melders van een tekenbeet worden uitgenodigd de teek op te sturen voor onderzoek, om gedurende anderhalf jaar periodiek vragenlijsten in te vullen waarin wordt gevraagd naar hun gezondheid. In één van de nog lopende onderzoeken wordt onderzocht in hoeverre een eenmalige preventieve dosis antibioticum na een tekenbeet helpt om het ontstaan van de ziekte van Lyme te voorkomen. In dit artikel geven wij een overzicht van het bereik van Tekenradar.nl onder het Nederlands publiek, en een samenvatting van lopende onderzoeken en eerste resultaten

Cell-Sized confinement in microspheres accelerates the reaction of gene expression

Cell-sized water-in-oil droplet covered by a lipid layer was used to understand how lipid membranes affect biochemical systems in living cells. Here, we report a remarkable acceleration of gene expression in a cell-sized water-in-oil droplet entrapping a cell-free translation system to synthesize GFP (green fluorescent protein). The production rate of GFP (VGFP) in each droplet remained almost constant at least for on the order of a day, which implies 0th-order reaction kinetics. Interestingly, VGFP was inversely proportional to radius of droplets (R) when R is under 50 μm, and VGFP in droplets with R ∼ 10 μm was more than 10 times higher than that in the bulk. The acceleration rates of GFP production in cell-sized droplets strongly depended on the lipid types. These results demonstrate that the membrane surface has the significant effect to facilitate protein production, especially when the scale of confinement is on the order of cell-size