132 research outputs found
Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae
Full text linkTrichuriasis represents a major public health problem in the developing world and is regarded as a neglected disease. Albendazole and mebendazole, the two drugs of choice against trichuriasis display only moderate cure rates, hence alternative drugs are needed. To identify candidate compounds, in vitro drug sensitivity testing currently relies on the adult Trichuris muris motility assay. The objective of the present study was to develop a simple and cost-effective drug sensitivity assay using Trichuris muris first-stage larvae (L1).; Several potential triggers that induce hatching of T. muris were studied, including gastrointestinal enzymes, acidic environment and intestinal microflora. Next, optimal culture conditions for T. muris L1 were determined assessing a wide range of culture media. T. muris L1 were incubated in the presence of mebendazole, ivermectin, nitazoxanide, levamisole or oxantel pamoate at 37°C. The viability of the parasites was evaluated microscopically after 24 hours. The usefulness of fluorescent markers (resazurin, calcein AM, ethidium homodimer-1 or fluorescein-conjugated albumin) in drug sensitivity testing was also assessed.; The established L1 motility assay provided accurate and reproducible drug effect data in vitro. IC50 values for oxantel pamoate, levamisole and nitazoxanide were 0.05, 1.75 and 4.43 μg/mL, respectively. Mebendazole and ivermectin failed to show any trichuricidal effect on L1. No correlation was found between data from the four fluorescent markers and the comparative motility assay.; The motility assay based on L1 was found suitable for drug sensitivity screening. It is rather simple, cost-effective, time-saving and sustains medium-throughput testing. Furthermore, it greatly reduces the need for the animal host and is therefore more ethical. None of the viability markers assessed in this study were found to be satisfactory
Conquering Switzerland: the emergence of Angiostrongylus vasorum in foxes over three decades and its rapid regional increase in prevalence contrast with the stable occurrence of lungworms
No full textAngiostrongylus vasorum, Crenosoma vulpis and Capillaria aerophila are the most common lungworms of domestic and wild canids. We investigated the short- and long-term lungworm prevalence changes in the Swiss fox population with a focus on A. vasorum. Between 2012 and 2017, lungs and hearts of 533 foxes from north-eastern Switzerland were necropsied and blood samples tested for circulating A. vasorum antigen. Angiostrongylus vasorum prevalence increased steadily from 21.5% in 2012 to 81.8% in 2017. In contrast, C. aerophila and C. vulpis prevalences fluctuated between 41.8 and 74.7%, and 3.6 and 14.9%, respectively. Based on 3955 blood samples collected between 1986 and 2017 from three geographic areas and during four time periods, antigen seropositivity increased from 2.4 to 62.0%. In north-eastern Switzerland, seropositivity was initially low (1.9 and 1.7% in the first two time periods) but increased in the following two decades to 22.2 and 62.0%, respectively. Our findings depict the spectacular expansion of A. vasorum in the past three decades. Regionally, the prevalence in foxes increased 4-fold within 6 years in some regions. This underpins the important role of foxes as reservoir hosts, likely explaining the increasing number of cases of canine angiostrongylosis in Switzerland. Our findings are representative of central Europe and may help anticipating future developments in areas where A. vasorum is present but (still) infrequent
Quantitative proteomics analysis of Angiostrongylus vasorum-induced alterations in dog serum sheds light on the pathogenesis of canine angiostrongylosis
No full textBlood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Infections with the blood-dwelling cardiopulmonary nematode Angiostrongylus vasorum in dogs manifest with a broad spectrum of clinical signs including respiratory distress, bleeding diathesis and neurological signs, and are associated with a perturbed blood protein profile in dogs. However, current knowledge does not completely explain the observed pathologies induced by A. vasorum infections, including bleeding disorders. Using sera from experimentally infected dogs, dog serum proteome was analysed by quantitative mass spectrometry methods over several time points before and after inoculation. Following computational analysis, we identified 139 up- and downregulated proteins after infection (log2 ratio cut-off ≥ 1.0; q-value ≤ 0.05). Among upregulated proteins were chitinase 3-like 1 and pulmonary surfactant-associated protein B (log2 fold-changes ≥ 5). Pathway enrichment revealed the complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of downregulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factor XIII-B. These results bring new elements towards understanding the underlying pathomechanisms of bleeding diatheses observed in some A. vasorum-infected dogs
Genome sequence of the cardiopulmonary canid nematode Angiostrongylus vasorum reveals species-specific genes with potential involvement in coagulopathy
No full textAngiostrongylus vasorum is an emerging parasitic nematode of canids and causes respiratory distress, bleeding, and other signs in dogs. Despite its clinical importance, the molecular toolbox allowing the study of the parasite is incomplete. To address this gap, we have sequenced its nuclear genome using Oxford nanopore sequencing, polished with Illumina reads. The size of the final genome is 280 Mb comprising 468 contigs, with an N50 value of 1.68 Mb and a BUSCO score of 93.5%. Ninety-three percent of 13,766 predicted genes were assigned to putative functions. Three folate carriers were found exclusively in A. vasorum, with potential involvement in host coagulopathy. A screen for previously identified vaccine candidates, the aminopeptidase H11 and the somatic protein rHc23, revealed homologs in A. vasorum. The genome sequence will provide a foundation for the development of new tools against canine angiostrongylosis, supporting the identification of potential drug and vaccine targets.Bayer Animal Health GmbH, Leverkusen, GermanySwiss National Science Foundatio
Activity of oxantel pamoate monotherapy and combination chemotherapy against Trichuris muris and hookworms : revival of an old drug
Full text linkBACKGROUND: It is widely recognized that only a handful of drugs are available against soil-transmitted helminthiasis, all of which are characterized by a low efficacy against Trichuris trichiura, when administered as single doses. The re-evaluation of old, forgotten drugs is a promising strategy to identify alternative anthelminthic drug candidates or drug combinations. METHODOLOGY: We studied the activity of the veterinary drug oxantel pamoate against Trichuris muris, Ancylostoma ceylanicum and Necator americanus in vitro and in vivo. In addition, the dose-effect of oxantel pamoate combined with albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin was studied against T. muris in vitro and additive or synergistic combinations were followed up in vivo. PRINCIPAL FINDINGS: We calculated an ED50 of 4.7 mg/kg for oxantel pamoate against T. muris in mice. Combinations of oxantel pamoate with pyrantel pamoate behaved antagonistically in vitro (combination index (CI) = 2.53). Oxantel pamoate combined with levamisole, albendazole or ivermectin using ratios based on their ED50s revealed antagonistic effects in vivo (CI = 1.27, 1.90 and 1.27, respectively). A highly synergistic effect (CI = 0.15) was observed when oxantel pamoate-mebendazole was administered to T. muris-infected mice. Oxantel pamoate (10 mg/kg) lacked activity against Ancylostoma ceylanicum and Necator americanus in vivo. CONCLUSIONSIGNIFICANCE: Our study confirms the excellent trichuricidal properties of oxantel pamoate. Since the drug lacks activity against hookworms it is necessary to combine oxantel pamoate with a partner drug with anti-hookworm properties. Synergistic effects were observed for oxantel pamoate-mebendazole, hence this combination should be studied in more detail. Since, of the standard drugs, albendazole has the highest efficacy against hookworms, additional investigations on the combination effect of oxantel pamoate-albendazole should be launche
The Angiostrongylus vasorum excretory/secretory and surface proteome contains putative modulators of the host coagulation
No full textAngiostrongylus vasorum is a cardiopulmonary nematode of canids and is, among others, associated with bleeding disorders in dogs. The pathogenesis of such coagulopathies remains unclear. A deep proteomic characterization of sex specific A. vasorum excretory/secretory proteins (ESP) and of cuticular surface proteins was performed, and the effect of ESP on host coagulation and fibrinolysis was evaluated in vitro. Proteins were quantified by liquid chromatography coupled to mass spectrometry and functionally characterized through gene ontology and pathway enrichment analysis. In total, 1069 ESP (944 from female and 959 from male specimens) and 1195 surface proteins (705 and 1135, respectively) were identified. Among these were putative modulators of host coagulation, e.g., von Willebrand factor type D domain protein orthologues as well as several proteases, including serine type proteases, protease inhibitors and proteasome subunits. The effect of ESP on dog coagulation and fibrinolysis was evaluated on canine endothelial cells and by rotational thromboelastometry (ROTEM). After stimulation with ESP, tissue factor and serpin E1 transcript expression increased. ROTEM revealed minimal interaction of ESP with dog blood and ESP did not influence the onset of fibrinolysis, leading to the conclusion that Angiostrongylus vasorum ESP and surface proteins are not solely responsible for bleeding in dogs and that the interaction with the host’s vascular hemostasis is limited. It is likely that coagulopathies in A. vasorum infected dogs are the result of a multifactorial response of the host to this parasitic infection.University of Zurich, SwitzerlandBusiness Unit Animal Health, German
Investigations on potential drug candidates and metabonomics-based diagnostic biomarker discovery for human soil-transmitted helminthiases
Full text linkSoil-transmitted helminthiases (STHs) are diseases caused by nematode worms. The most common species affecting humans are Ascaris lumbricoides, Necator americanus Ancylostoma duodenale, Trichuris trichiura and Strongyloides stercoralis. More than 1 billion people are infected globally. Most at risk are the 3 billion poorest people of the world, particularly children. Heavy infections cause iron-deficiency anemia to growth stunting and intellectual retardation. STHs occur often concomitantly with other infections such as malaria. Currently, STHs morbidity control relies on only five drugs (albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin). These present limited efficacies, especially when administered in single dose against T. trichiura and hookworm infections. Although anthelmintic drug resistance has not yet appeared as a major public health problem, emergence of drug resistance may be inevitable. The situation is precarious and new drugs are urgently needed. In addition, existing tools for in vitro drug sensitivity testing are based on a viability assessment assay which lacks convenience and hinders high-throughput screening rates. Also, the lack of accurate diagnostic tools to detect STHs and malaria infections hampers an optimal management of these diseases.
This work aimed first to set up nematode-rodent models at the Swiss Tropical and Public Health Institute (Swiss TPH), improve drug screening assays and evaluate potential new treatments for human STHs. Prior to this thesis, monepantel (AAD 1566), tribendimidine, nitazoxanide and oxantel pamoate had been identified as potential drug candidates for STHs. Secondly, we aimed to strengthen our understanding of the impact of a murine malaria and hookworm co-infection on the host’s metabolism and explore the potential of metabolic profiling as multiplexing diagnostic tool.
Once the animal models corresponding to human helminthiases have been established, (Ancylostoma ceylanicum, N. americanus and Trichuris muris), the Alamar Blue, the MTT and the acid phosphatase assays, as well as the xCELLigence System, isothermal microcalorimetry, and the feeding-inhibition assay (A. ceylanicum only) were tested and compared to the current assay of choice, the motility assay. For T. muris, the Alamar Blue assay compared most favorably to the motility assay since it is precise and cost-effective. For A. ceylanicum, no alternative assay was found better than the motility assay for testing on L3, whereas the xCELLigence System was found accurate and convenient for adult worms.
The potential of monepantel was assessed against A. ceylanicum, N. americanus, T. muris, Ascaris suum and Strongyloides ratti. In vivo, the veterinary drug showed good and moderate activities respectively, against A. ceylanicum (10 mg/kg: 100% worm burden reduction) and N. americanus (10 mg/kg: 58.3% worm burden reduction), but failed to show sufficient anthelmintic properties in the other three models.
Tribendimidine, a Chinese anthelmintic, and its metabolites dADT and AdADT were tested using the hookworm models A. ceylanicum and Heligmosomoides bakeri. In A. ceylanicum-infected hamsters, a single oral dose of 10 mg/kg resulted in 74.8% worm burden reduction. In the H. bakeri model, a single oral dose of 2 mg/kg achieved a worm burden reduction of 100%. The metabolite AdADT showed moderate activity against both parasites. The combination tribendimidine-levamisole displayed an additive to synergistic behavior in the A. ceylanicum model in vivo.
Nitazoxanide, an anti-protozoal drug was evaluated against A. ceylanicum and T. muris. In vitro, it had a marked effect on A. ceylanicum adult worms (IC50 = 0.74 µg/ml) and on T. muris L3 and adult worms (IC50s = 0.27 and 12.87 µg/ml, respectively). However, the drug lacked efficacy in both models in vivo.
The “old” anthelmintic oxantel pamoate was studied in the A. ceylanicum, N. americanus and T. muris models. The drug lacked anti-hookworm activity in vivo (10 mg/kg), but showed promising trichuricidal properties in vitro and in vivo (ED50 = 4.7 mg/kg). Moreover, the combination oxantel pamoate-mebendazole revealed highly synergistic properties.
Murine H. bakeri and Plasmodium berghei single and co-infection (delayed and simultaneous) models were established for metabolic analysis. Urine and plasma samples were subjected to 1H nuclear magnetic resonance (NMR) spectroscopy and subsequent multivariate analysis in order to identify infection-discriminating metabolic fingerprints. Characteristic metabolic fingerprints have been found for each of the infection scenarios. We detected two unknown metabolites and confirmed the accumulation of urinary pipecolic acid in P. berghei-infected mice. Pipecolic acid may therefore represent a candidate for human malaria diagnostics. 1H NMR spectroscopy was found powerful for detecting metabolic changes in the co-infection model, but still presents some drawbacks as diagnostic tool in its actual form
Fox serum proteomics analysis suggests host-specific responses to angiostrongylus vasorum infection in canids
No full textDogs infected with the cardiopulmonary nematode Angiostrongylus vasorum may suffer from respiratory distress and/or bleeding disorders. Descriptions of clinical signs in foxes are rare, despite high prevalence. To evaluate the impact of infection on coagulation and immune response, serum proteins from eight experimentally infected foxes before and after inoculation (day 0, 35, 84, 154) were subjected to differential proteomic analyses based on quantitative data and compared to available data from dogs. The number of proteins with differential abundance compared to the uninfected baseline increased with chronicity of infection. Bone marrow proteoglycan, chitinase 3-like protein 1 and pulmonary surfactant-associated protein B were among the most prominently increased proteins. The abundance of several proteins involved in coagulation was decreased. Enriched pathways obtained from both increased and decreased proteins included, among others, “platelet degranulation” and “haemostasis”, and indicated both activation and suppression of coagulation. Qualitative comparison to dog data suggests some parallel serum proteomic alterations. The comparison, however, also indicates that foxes have a more adequate immunopathological response to A. vasorum infection compared to dogs, facilitating persistent infections in foxes. Our findings imply that foxes may be more tolerant to A. vasorum infection, as compared to dogs, reflecting a longer evolutionary host–parasite adaptation in foxes, which constitute a key wildlife reservoir.‘Forschungskredit’ from the University of Zurich, SwitzerlandBayer Vital GmbH, Business Unit Animal Health, German
Helminth extracellular vesicles in host–parasite interactions
No full textExtracellular vesicles (EVs) have been characterized from many species of parasitic helminths, and recent experimental evidence supports important functions for their cargo in host–parasite relationships as immunomodulatory mediators. Here we summarize available data on the effects of parasite-derived EVs, including their protein and/or small RNA contents, on their hosts
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