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Background: Infection with parasitic nematodes (helminths), particularly those of the order Strongylida (such as Haemonchus contortus), can cause significant and burdensome diseases in humans and animals. Widespread drug (anthelmintic) resistance in livestock parasites, the absence of vaccines against most of these nematodes, and a lack of new and effective chemical entities on the commercial market demands the discovery of new anthelmintics. In the present study, we searched the Global Health Priority Box (Medicines for Malaria Venture) for new candidates for anthelmintic development. Methods: We employed a whole-organism, motility-based phenotypic screening assay to identify compounds from the Global Health Priority Box with activity against larvae of the model parasite H. contortus, and the free-living comparator nematode Caenorhabditis elegans. Hit compounds were further validated via dose–response assays, with lead candidates then assessed for nematocidal activity against H. contortus adult worms, and additionally, for cytotoxic and mitotoxic effects on human hepatoma (HepG2) cells. Results: The primary screen against H. contortus and C. elegans revealed or reidentified 16 hit compounds; further validation established MMV1794206, otherwise known as 'flufenerim', as a significant inhibitor of H. contortus larval motility (half-maximal inhibitory concentration [IC50] = 18 μM) and development (IC50 = 1.2 μM), H. contortus adult female motility (100% after 12 h of incubation) and C. elegans larval motility (IC50 = 0.22 μM). Further testing on a mammalian cell line (human hepatoma HepG2 cells), however, identified flufenerim to be both cytotoxic (half-maximal cytotoxic concentration [CC50] < 0.7 μM) and mitotoxic (half-maximal mitotoxic concentration [MC50] < 0.7 μM). Conclusions: The in vitro efficacy of MMV1794206 against the most pathogenic stages of H. contortus, as well as the free-living C. elegans, suggests the potential for development as a broad-spectrum anthelmintic compound; however, the high toxicity towards mammalian cells presents a significant hindrance. Further work should seek to establish the protein–drug interactions of MMV1794206 in a nematode model, to unravel the mechanism of action, in addition to an advanced structure–activity relationship investigation to optimise anthelmintic activity and eliminate mammalian cell toxicity. [ABSTRACT FROM AUTHOR]

Simple Summary: Parasitic strongyloid nematodes endemic to the gastrointestinal tracts of Australasian marsupials are one of the most diverse groups of mammalian parasites. These nematodes are currently placed in the family Chabertiidae comprising two subfamilies, namely the Cloacininae and Phascolostrongylinae. Their current classification relies primarily on morphological features and has not been validated using molecular data. This study aimed to determine the phylogenetic relationships of the Cloacininae and Phascolostrongylinae within the family Chabertiidae and their relationship with other groups of strongyloid nematodes from non-marsupial hosts, using mitochondrial protein sequence datasets. The findings supported the recognition of the family Cloacinidae, containing the Cloacininae and Phascolostrongylinae, as a monophyletic group within the Strongyloidea. However, the subfamily Phascolostrongylinae was paraphyletic, and the relationships of individual genera corresponded with their host families. Genera of the Cloacininae and Phascolostrongylinae occurring in macropod hosts were more closely related compared to genera of the Phascolostrongylinae occurring in wombats. This study suggests an alternative hypothesis for the origin of marsupial strongyloid nematodes in vombatid hosts that should be explored further using molecular approaches and more widespread sampling. Australasian marsupials harbour a diverse group of gastrointestinal strongyloid nematodes. These nematodes are currently grouped into two subfamilies, namely the Cloacininae and Phascolostrongylinae. Based on morphological criteria, the Cloacininae and Phascolostrongylinae were defined as monophyletic and placed in the family Cloacinidae, but this has not been supported by molecular data and they are currently placed in the Chabertiidae. Although molecular data (internal transcribed spacers of the nuclear ribosomal RNA genes or mitochondrial protein-coding genes) have been used to verify morphological classifications within the Cloacininae and Phascolostrongylinae, the phylogenetic relationships between the subfamilies have not been rigorously tested. This study determined the phylogenetic relationships of the subfamilies Cloacininae and Phascolostrongylinae using amino acid sequences conceptually translated from the twelve concatenated mitochondrial protein-coding genes. The findings demonstrated that the Cloacininae and Phascolostrongylinae formed a well-supported monophyletic assemblage, consistent with their morphological classification as an independent family, Cloacinidae. Unexpectedly, however, the subfamily Phascolostrongylinae was split into two groups comprising the genera from macropodid hosts (kangaroos and wallabies) and those from vombatid hosts (wombats). Genera of the Cloacininae and Phascolostrongylinae occurring in macropodid hosts were more closely related compared to genera of the Phascolostrongylinae occurring in wombats that formed a sister relationship with the remaining genera from macropods. These findings provide molecular evidence supporting the monophyly of the family Cloacinidae and an alternative hypothesis for the origin of marsupial strongyloid nematodes in vombatid hosts that requires further exploration using molecular approaches and additional samples [ABSTRACT FROM AUTHOR]

[Display omitted] Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the ' Pandemic Response Box ' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico- docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially. [ABSTRACT FROM AUTHOR]

This study reports the spatial and temporal distribution of ascarid and strongylid nematodes in Thoroughbred horses by age category across different climatic zones in Australia over an 18-month period. Faecal samples (n = 2046) from individual horses were analysed using the modified McMaster technique for faecal egg counts (FECs). Strongylids were identified using PCR-directed next-generation sequencing of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA. Yearlings had the highest prevalence (82%) of strongyle eggs followed by weanlings (79%), foals (58%), wet mares (49%) and dry mares (46%). For Parascaris spp., foals had the highest prevalence (35%) followed by weanlings (21%) and yearlings (10%). The highest mean FECs for Parascaris spp. were observed in foals (525 eggs per gram [EPG] of faeces) while those for strongyles were in yearlings (962 EPG). Among horses that were classified as adults at the time of sampling, 77% (860 of 1119) of mares were low (i.e., <250 EPG) strongyle egg-shedders. Mean strongyle FEC counts were highest in the Mediterranean (818 EPG) followed by summer (599 EPG), winter (442 EPG), and non-seasonal (413 EPG) rainfall zones. Twenty-six nematode species were detected, with Cylicostephanus longibursatus (26.5%), Cylicocyclus nassatus (23.7%) and Coronocyclus coronatus (20.5%) being the most frequently detected species. Their richness and relative abundance varied with horse age, season and climatic zone. In addition, Strongylus equinus and Triodontophorus spp. (T. brevicauda and T. serratus) were also detected. This comprehensive study elucidates spatial (climatic zone) and temporal (i.e., seasonal) trends in prevalence and burdens of intestinal nematodes in Australian horses using non-invasive conventional and molecular methods. The information presented in this study is crucial for developing integrated management strategies to control horse parasites in farmed horses. [Display omitted] • A longitudinal approach was used to study intestinal nematodes of horses using the McMaster technique and nemabiome approach. • The farm-level prevalence of ascarid and strongylid nematodes ranged from 0–29% and 46–95%, respectively. • Only up to 17% of the adult horses were classified as high egg shedders (>500 eggs per gram). • The climate (Mediterranean type), season (autumn) and horse age (younger horses) had significant effect on egg shedding. • Nemabiome results showed higher abundance of selected species, affected by horse age, season and climate. [ABSTRACT FROM AUTHOR]

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10–14%) and 72% (70–74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251–500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses. [Display omitted] • A cross-sectional study of 62 Thoroughbred farms revealed a high prevalence of ascarid and strongylid nematodes. • Majority of the adult horses were low strongylid egg shedders (i.e., < 250 eggs per gram). • Horse age and diverse climatic conditions impacted the parasite diversity and egg shedding patterns. • Cylicostephanus longibursatus, Coronocyclus coronatus and Cylicocyclus nassatus were the most abundant species. • Study provided insights for tailoring parasite control strategies in horses. [ABSTRACT FROM AUTHOR]

This review is aimed to (i) appraise the literature on the use of molecular techniques for the detection, quantification and differentiation of gastrointestinal helminths (GIH) of equids, (ii) identify the knowledge gaps and, (iii) discuss diagnostic prospects in equine parasitology. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we retrieved 54 studies (horses: 50/54; donkeys and zebras: 4/54) from four databases. Polymerase chain reaction (PCR) was employed in all of the studies whereas PCR amplicons were sequenced in only 18 of them. Other techniques used (including modifications of PCR) were reverse line blot, quantitative (q)PCR, restriction fragment length polymorphism, nested-PCR, PCR-directed next-generation sequencing, Southern blotting, single strand conformation polymorphism, PCR-enzyme linked immunosorbent assay, matrix-assisted laser desorption/ionisation-time of flight and random amplification of polymorphic DNA. Most of the studies (53/54) used nuclear ribosomal RNA (including the internal transcribed spacers, intergenic spacer, 5.8 S, 18 S, 28 S and 12 S) as target loci while cytochrome c oxidase subunit 1 and random genomic regions were targeted in only three and one studies, respectively. Overall, to date, the majority of molecular studies have focused on the diagnosis and identification of GIHs of equids (i.e. species of Anoplocephala , Craterostomum , cyathostomins, Oesophagodontus , Parascaris , Strongylus , Strongyloides and Triodontophorus), with a recent shift towards investigations on anthelmintic resistance and the use of high-throughput nemabiome metabarcoding. With the increasing reports of anthelmintic resistance in equid GIHs, it is crucial to develop and apply techniques such as advanced metabarcoding for surveillance of parasite populations in order to gain detailed insights into their diversity and sustainable control. To the best of our knowledge, this is the first systematic review that evaluates molecular investigations published on the diagnosis and quantification of equid GIHs and provides useful insights into important knowledge gaps and future research directions in equid molecular parasitology. • The second internal transcribed spacer is the main marker used in molecular diagnostics. • Whole genome sequencing of equid gastrointestinal helminths would support molecular diagnostics. • Use of molecular tools in investigating anthelmintic resistance is increasing. • Metabarcoding techniques should be used to monitor parasite populations to achieve sustainable control. [ABSTRACT FROM AUTHOR]