Due to major problems with drug resistance in parasitic nematodes of animals, there is a substantial need and excellent opportunities to develop new anthelmintics via genomic-guided and/or repurposing approaches. In the present study, we established a practical and cost-effective whole-organism assay for the in vitro-screening of compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). The assay is based on the use of exsheathed L3 (xL3) and L4 stages of H. contortus of small ruminants (sheep and goats). Using this assay, we screened a panel of 522 well-curated kinase inhibitors (GlaxoSmithKline, USA; code: PKIS2) for activity against H. contortus by measuring the inhibition of larval motility using an automated image analysis system. We identified two chemicals within the compound classes biphenyl amides and pyrazolo[1,5-α]pyridines, which reproducibly inhibit both xL3 and L4 motility and development, with IC50s of 14-47 μM. Given that these inhibitors were designed as anti-inflammatory drugs for use in humans and fit the Lipinski rule-of-five (including bioavailability), they show promise for hit-to-lead optimisation and repurposing for use against parasitic nematodes. The screening assay established here has significant advantages over conventional methods, particularly in terms of ease of use, throughput, time and cost. Although not yet fully automated, the current assay is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation. The current assay is highly adaptable to many parasites of socioeconomic importance, including those causing neglected tropical diseases. This aspect is of major relevance, given the urgent need to deliver the goals of the London Declaration (http://unitingtocombatntds.org/resource/london-declaration) through the rapid and efficient repurposing of compounds in public-private partnerships.

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Background-The porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.Methods-Different enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.Results-Whereas the L3 N-glycome was simpler and more dominated by phosphorylcholinemodified structures, the male and female worms express a wide range of core fucosylated Nglycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered 'male', while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc 2-4 PC 2-4 motifs not observed in our previous studies on other nematodes. Conclusion-The interspecies tendency of class V nematodes to share most, but not all, Nglycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.General significance-Unusual methylated, core fucosylated and phosphorylcholinecontaining N-glycans vary between stages and genders in a parasitic nematode.

BackgroundCystoisospora suis is the causative agent of porcine neonatal coccidiosis, a diarrheal disease which affects suckling piglets in the first weeks of life. Detection of oocysts in the faeces of infected animals is frequently hampered by the short individual excretion period and the high fat content of faecal samples. We analysed oocyst excretion patterns of infected piglets, evaluated different detection methods for their detection limit and reproducibility, and propose a sampling scheme to improve the diagnosis of C. suis in faecal samples from the field using a protocol for reliable parasite detection.ResultsBased on a hypothesized model of the course of infection on a farm, three samplings (days of life 7–14-21 or 10–15-20) should be conducted including individual samples of piglets from each sampled litter. Samples can be examined by a modified McMaster method (lower detection limit: 333 oocysts per gram of faeces, OpG), by examining faecal smears under autofluorescence (lower detection limit: 10 OpG) or after carbol-fuchsin staining (lower detection limit: 100 OpG). Reproducibility and inter-test correlations were high with (R2 > 0.8). A correlation of oocyst excretion with diarrhoea could not be established so samples with different faecal consistencies should be taken. Pooled samples (by litter) should be comprised of several individual samples from different animals.ConclusionsSince oocyst excretion by C. suis-infected piglets is usually short the right timing and a sufficiently sensitive detection method are important for correct diagnosis. Oocyst detection in faecal smears of samples taken repeatedly is the method of choice to determine extent and intensity of infection on a farm, and autofluorescence microscopy provides by far the lowest detection limit. Other methods for oocyst detection in faeces are less sensitive and/or more labour- and cost intensive and their usefulness is restricted to specific applications.

Cystoisospora suis is a coccidian species that typically affects suckling piglets. Infections occur by oral uptake of oocysts and are characterized by non-hemorrhagic transient diarrhea, resulting in poor weight gain. Apparently, primary immune responses to C. suis cannot readily be mounted by neonates, which contributes to the establishment and rapid development of the parasite, while in older pigs age-resistance prevents disease development. However, the presence of extraintestinal stages, although not unequivocally demonstrated, is suspected to enable parasite persistence together with the induction and maintenance of immune response in older pigs, which in turn may facilitate the transfer of C. suis-specific factors from sow to offspring. It is assumed that neonates are particularly prone to clinical disease because infections with C. suis interfere with the establishment of the gut microbiome. Clostridia have been especially inferred to profit from the altered intestinal environment during parasite infection. New tools, particularly in the area of genomics, might illustrate the interactions between C. suis and its host and pave the way for the development of new control methods not only for porcine cystoisosporosis but also for other mammalian Cystoisospora infections. The first reference genome for C. suis is under way and will be a fertile ground to discover new drugs and vaccines. At the same time, the establishment and refinement of an in vivo model and an in vitro culture system, supporting the complete life cycle of C. suis, will underpin the functional characterization of the parasite and shed light on its biology and control.