Polymerase chain reaction for detection of patent infections of Echinococcus granulosus ("sheep strain”) in naturally infected dogs

Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus ("sheep strain”) was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a Echinococcus multilocularis -specific PCR revealing one mixed infection. Egg-containing faeces from two dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental sample

Inhibition of Haemonchus contortus larval development by fungal lectins

BACKGROUND Lectins are carbohydrate-binding proteins that are involved in fundamental intra- and extracellular biological processes. They occur ubiquitously in nature and are especially abundant in plants and fungi. It has been well established that certain higher fungi produce lectins in their fruiting bodies and/or sclerotia as a part of their natural resistance against free-living fungivorous nematodes and other pests. Despite relatively high diversity of the glycan structures in nature, many of the glycans targeted by fungal lectins are conserved among organisms of the same taxon and sometimes even among different taxa. Such conservation of glycans between free-living and parasitic nematodes is providing us with a useful tool for discovery of novel chemotherapeutic and vaccine targets. In our study, a subset of fungal lectins emanating from toxicity screens on Caenorhabditis elegans was tested for their potential to inhibit larval development of Haemonchus contortus. METHODS The effect of Coprinopsis cinerea lectins - CCL2, CGL2, CGL3; Aleuria aurantia lectin - AAL; Marasmius oreades agglutinin - MOA; and Laccaria bicolor lectin - Lb-Tec2, on cultivated Haemonchus contortus larval stages was investigated using a larval development test (LDT). To validate the results of the toxicity assay and determine lectin binding capacity to the nematode digestive tract, biotinylated versions of lectins were fed to pre-infective larval stages of H. contortus and visualized by fluorescent microscopy. Lectin histochemistry on fixed adult worms was performed to investigate the presence and localisation of lectin binding sites in the disease-relevant developmental stage. RESULTS Using an improved larval development test we found that four of the six tested lectins: AAL, CCL2, MOA and CGL2, exhibited a dose-dependent toxicity in LDT, as measured by the number of larvae developing to the L3 stage. In the case of AAL, CGL2 and MOA lectin, doses as low as 5 μg/ml caused >95 % inhibition of larval development while 40 μg/ml were needed to achieve the same inhibition by CCL2 lectin. MOA was the only lectin tested that caused larval death while other toxic lectins had larvistatic effect manifesting as L1 growth arrest. Using lectin histochemistry we demonstrate that of all lectins tested, only the four toxic ones displayed binding to the larvae's gut and likewise were found to interact with glycans localized to the gastrodermal tissue of adults. CONCLUSION The results of our study suggest a correlation between the presence of target glycans of lectins in the digestive tract and the lectin-mediated toxicity in Haemonchus contortus. We demonstrate that binding to the structurally conserved glycan structures found in H. contortus gastrodermal tissue by the set of fungal lectins has detrimental effect on larval development. Some of these glycan structures might represent antigens which are not exposed to the host immune system (hidden antigens) and thus have a potential for vaccine or drug development. Nematotoxic fungal lectins prove to be a useful tool to identify such targets in parasitic nematodes

Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis

Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation

RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening

RNA interference (RNAi) is a technique to selectively suppress mRNA of individual genes and, consequently, their cognate proteins. RNAi using double-stranded (ds) RNA has been used to interrogate the function of mainly single genes in the flatworm, Schistosoma mansoni, one of a number of schistosome species causing schistosomiasis. In consideration of large-scale screens to identify candidate drug targets, we examined the selectivity and sensitivity (the degree of suppression) of RNAi for 11 genes produced in different tissues of the parasite: the gut, tegument (surface) and otherwise. We used the schistosomulum stage prepared from infective cercariae larvae which are accessible in large numbers and adaptable to automated screening platforms. We found that RNAi suppresses transcripts selectively, however, the sensitivity of suppression varies (40%–>75%). No obvious changes in the parasite occurred post-RNAi, including after targeting the mRNA of genes that had been computationally predicted to be essential for survival. Additionally, we defined operational parameters to facilitate large-scale RNAi, including choice of culture medium, transfection strategy to deliver dsRNA, dose- and time-dependency, and dosing limits. Finally, using fluorescent probes, we show that the developing gut allows rapid entrance of dsRNA into the parasite to initiate RNAi

Polymerase chain reaction for detection of patent infections of Echinococcus granulosus ("sheep strain") in naturally infected dogs

Die Polymerasen-Ketten-Reaktion zur Identifikation von Eiern des Bandwurms Echinokokkus granulosus („Schafstamm“) wurde mit Primern, welche von mitochondrialen Sequenzen abgeleitet wurden, evaluiert. Die Spezifität dieser Primer wurde durch die Untersuchung von DNS anderer E. granulosus Stämme und 14 Helminthen Spezies, welche den Intestinaltrakt des Hundes besiedeln, bestätigt. Dieser PCR-Test wurde zur Untersuchung von 131 Kotproben von kasachischen Hunden (erhalten durch Purgation der Hunde) verwendet. 18 Hunde beherbergten Echinokokkus Würmer und 10 Tiere hatten gemischte Infektionen mit Taenia spp.. Koproantigen Detektion war in 15 Fällen positiv und bei 13 dieser Proben konnten Taeniden-Eier gefunden werden. Acht der Eier- enthaltenden Proben waren in der PCR positiv für Echinokokkus granulosus und vier waren in einem E. multilocularis-spezifischen PCR positiv, ein Zeichen für gemischte Infektionen. Zwei Eier enthaltende Kotproben von zwei Hunden mit Taenia spp. und Echinokokkus waren in beiden PCR negativ. Die Kombination von Eiisolation und PCR wird v.a. auch bei der Untersuchung von Umgebungsproben in epidemiologischen Studien von grossem Wert sein. Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus ("sheep strain") was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a E. multilocularis-specific PCR revealing one mixed infection. Egg-containing faeces from 2 dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental samples

A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia

Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasiteinduced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 M. The inhibition of parasite replication was irreversible at 10 MPPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents

Xenosurveillance proof-of-principle: Detection of Toxoplasma gondii and SARS-CoV-2 antibodies in mosquito blood meals by (pan)-specific ELISAs

Blood-fed insects can be used to analyse the host blood for circulating vertebrate pathogens or antibodies directed against them. We tested whether naturally acquired antibodies in different host species can be detected by host-specific and pan-specific ELISAs in mosquito blood meals. Cat- and alpaca-specific ELISAs could detect antibodies against Toxoplasma gondii or SARS-CoV-2 in blood meals of Aedes japonicus for 48 and at least 24 h, respectively. In the pan-specific ELISA, a conjugated protein A/G and anti-IgY were used to detect antibodies of mammalian and bird hosts. Thus, Toxoplasma antibodies could be detected in mosquitoes fed on blood from humans, chicken, pig, and sheep up to 72 h after the blood meal. The results, however, demonstrated differences in sensitivities between different host species, and the assay requires further evaluation. Xenosurveillance with antibody detection in mosquito blood meals can be an additional surveillance tool that would especially be helpful when it is difficult to sample the potential animal reservoirs

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of ;naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments

Polymerase chain reaction for detection of patent infections of Echinococcus granulosus (“sheep strain”) in naturally infected dogs

Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus ("sheep strain”) was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a Echinococcus multilocularis -specific PCR revealing one mixed infection. Egg-containing faeces from two dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental sample