Anthelmintic resistance is an increasing problem in many gastrointestinal parasites of grazing animals. Among these, the equine roundworm, Parascaris spp., has developed wide-spread resistance to macrocyclic lactones over the past decades. Additionally, there are recent observations of emerging treatment failure of both tetrahydropyrimidine and fenbendazole. Therefore, the aims of this study were to further investigate the occurrence of fenbendazole resistance on breeding farms and to explore potential management-related risk factors associated with resistance in Parascaris spp. in Sweden. Eleven farms with 92 foals positive for Parascaris spp. were included in a faecal egg count reduction test during the years 2021-2023. According to the clinical protocol of the guidelines of the World Association for the Advancement of Veterinary Parasitology, fenbendazole resistance was present on four farms with efficacies varying from 45 % to 96 %. Having previously reported reduced efficacy on one of these farms, we can now confirm that fenbendazole resistance in Parascaris spp. has established. Farms with more than 40 yearly born foals had a significantly higher probability of having resistant Parascaris spp. Populations compared with smaller farms, (generalized linear model (GLM), t = 70.39, p < 0.001). In addition, there was a correlation between the number of foals on the farm and the frequency of yearly treatments showing that farms with < 20 foals were notably inclined to administer treatments twice during the first year (GLM, t=2.76, p < 0.05) in contrast to larger farms with > 40 foals that were using more frequent treatment intervals. In conclusion, this study confirms the establishment of fenbendazole resistance in Parascaris spp. populations on Swedish stud farms with the number of foals on the farm identified as a risk factor for development of anthelmintic resistance.

Over the preceding decades, the widespread dependence on anthelmintic drugs for managing nematodes in grazing equids has given rise to resistance against commonly used anthelmintics in various countries. This study explores the prevalence of anthelmintic resistance across 44 horse farms in Ireland. Anthelmintic efficacy was evaluated through fecal egg count reduction (FECR) tests employing the mini-FLOTAC technique. Resistance to benzimidazoles was identified in 12 out of 14 farms (FECR range: 0.00% to 86.2%). Ivermectin resistance was observed on two farms, one with an FECR of 80.70% and another with an FECR of 96.10% (lower 95% high probability density interval (HPD) <90%, 11.70%). On the remaining six farms, the reduction with ivermectin still exceeded 95%. The reduced efficacy of moxidectin was noted on two farms (FECR = 86.90% and 93.50%) and on a third farm with an FECR of 99.50 and a lower HPD interval < 90% at 24.00%. In summary, these findings emphasize the urgent need for alternative strategies in equine strongyle control that reduce reliance on anthelmintics and prioritize effective management practices on Irish equine farms to hinder the impending development of drug-resistant parasite populations.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examined the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) in the BZ response as well as to determine if another beta-tubulin acts redundantly with ben-1. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1. We found that the loss of ben-1 conferred the maximum level of resistance following exposure to a single concentration of albendazole, and the loss of a second beta-tubulin gene did not alter the level of resistance. However, additional traits other than larval development could be affected by the loss of additional beta-tubulins, and the roles of other beta-tubulin genes might be revealed at different albendazole concentrations. Therefore, further work is needed to fully define the possible roles of other beta-tubulin genes in the BZ response.

The population of South American camelids (SAC) has been steadily growing in Europe, where they are confronted with the regional endoparasite population of ruminants. As there are no anthelmintic drugs registered for use against nematode infections in SACs, anthelmintics (AH) available for ruminants or horses are usually applied. Reports indicating potential failures in administered AH are increasing. However, the generally low egg counts in SACs complicate the application of resistance tests in the field. The present study reports a follow-up study on SAC farms where anthelmintic resistance (AR) was suspected. The aims were (i) to repeat faecal egg count reduction tests (FECRTs) on potentially affected farms identified in a previous study with larger sample sizes, (ii) to verify suspected AR of Haemonchus contortus against benzimidazoles (BZ) by performing a single-nucleotide polymorphism (SNP) analysis using digital polymerase chain reaction (dPCR), and (iii) to apply the mini-FLOTAC technique for more reliable results at low egg counts in line with current recommendations. Seven farms (9-46 animals each) were examined by coproscopy, larval differentiation and SNP analysis. A FECRT was performed on six of these farms with moxidectin (three farms), monepantel (two farms) and ivermectin (one farm). The FEC was calculated according to the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines with the clinical protocol (a newly introduced variant of FECRT which can be used for smaller sample sizes and lower egg counts on the cost of sensitivity) and an expected efficacy of 99%. A high level (> 90%) of BZ-resistance-associated SNPs on codon 200 of H. contortus was observed on all farms. With the FECRT, resistance was demonstrated for ivermectin (74% FECR), while it remained inconclusive for one farm for moxidectin treatment. Sustained efficacy was demonstrated for the remaining treatments. This study showed an advanced level of BZ resistance in H. contortus of SACs and the development of AR against macrocyclic lactones on some farms. Thus, constant monitoring of AH treatment and sustainable worm control methods both need to be applied.

Anthelmintic resistance in small ruminants is a serious worldwide problem. To reduce their spread, it is essential to know the prevalence of helminths on farms and the control practices adopted. As these studies in the Calabria region of southern Italy are fragmentary and outdated, a study on the prevalence of helminths in small ruminant holdings in this area has been conducted. The measures implemented to control helminths were also evaluated through questionnaires administered to farmers. In particular, on 90 farms (45 sheep and 45 goats), 1800 faecal samples from 900 sheep and 900 goats were collected in the spring. Using the FLOTAC dual technique, parasitological examinations demonstrated the presence of gastrointestinal nematodes in 100% of sheep and goat farms, followed by Nematodirus spp. (84.44% sheep and 48.89% goats), Moniezia spp. (73.33% sheep and 35.56% goats), Trichuris ovis (48.89% sheep and 42.22% goats), lungworms (28.89% sheep and 42.22% goats), Strongyloides papillosus (40% sheep and 26.67% goats), Dicrocoelium dendriticum (13.33% sheep and 26.67% goats), Calicophoron daubneyi (6.67% sheep and 31.11% goats), Fasciola hepatica (6.67% sheep and 4.44% goats), and Skrjabinema ovis (4.44% sheep and goats). The questionnaires showed that 82% and 85% of the farmers had applied pasture rotation, and that 93.3% and 86.6% had used anthelmintics in the previous year for sheep and goats, respectively. Only 24.4% of sheep farmers and 11.3% of goat farmers had carried out parasitological tests prior to treatments. The most used classes of anthelmintics were macrocyclic lactones and benzimidazoles, and only in 21.6% and 15.6%, for sheep and goats, respectively, was drug rotation carried out. These results denote that helminths represent a health problem for small ruminants and highlight a lack of knowledge of parasite control strategies among farmers. In these conditions, anthelmintic resistance phenomena could develop over time. Therefore, it is necessary to implement all possible strategies for the control of helminths, and to prevent the spread of anthelmintic resistance phenomena on farms in southern Italy.

Soil-transmitted helminth (STH) infections, commonly treated with benzimidazoles, are linked to resistance through single nucleotide polymorphisms (SNPs) at position 167, 198, or 200 in the β-tubulin isotype 1 gene. The aim of this study was to establish a novel genotyping assay characterized by its rapidity and specificity. This assay was designed to detect the presence of SNPs within the partial β-tubulin gene of Trichuris trichiura. This was achieved through the biallelic discrimination at codons 167, 198, and 200 by employing the competitive binding of two allele-specific forward primers. The specificity and reliability of this assay were subsequently confirmed using Trichuris samples isolated from captive primates. Furthermore, a molecular study was conducted to substantiate the utility of the β-tubulin gene as a molecular marker. The assays showed high sensitivity and specificity when applied to field samples. Nevertheless, none of the SNPs within the β-tubulin gene were detected in any of the adult worms or eggs from the analyzed populations. All specimens consistently displayed an SS genotype. The examination of the β-tubulin gene further validated the established close relationships between the T. trichiura clade and Trichuris suis clade. This reaffirms its utility as a marker for phylogenetic analysis.

Ancylostoma caninum is a widely prevalent parasitic nematode in dogs across the world. There has been a notable increase in reports of anthelmintic resistance in A. caninum within the United States of America in recent years, which has led us to investigate the potential of this scenario in Canada. The study objectives were to assess the prevalence of A. caninum in two different groups, including a colony of rescued dogs in Canada and three imported Greyhound dogs from USA, and to evaluate the efficacy of two benzimidazole (BZ) anthelmintics against A. caninum, complemented with a molecular genetic analysis adapted to low prevalence. Fecal samples were collected at pre- and post-treatment with fenbendazole for the native shelters-origin group, and a combination of anthelmintic formulations, including the pro-BZ febantel for the USA-origin group. The coprology analyses found several genera of internal parasites. Canine ancylostomiasis was the most prevalent parasitosis with 30.77% in the native group and 100% in the USA group, but with overall low average of A. caninum eggs per gram. Through the fecal egg count reduction test (FECRT), applying a cut-off at 90% as baseline of egg reduction for successful efficacy, BZ showed variable efficacy. Furthermore, molecular analysis confirmed the presence of A. caninum in both groups of dogs and found differences in the genetics linked to BZ resistance on the A. caninum β-tubulin isotype 1 gene. In the isolate from the native group, both codons 167 and 200 were homozygous without the presence of single nucleotide polymorphism (SNP). In contrast, the selected isolate from the USA group, showed a homozygous allele at position 200 and a heterozygous SNP at position 167. The latter was congruent with the low efficacy in FECRT and agrees with the recent findings of USA A. caninum isolate resistant phenotype to the BZ anthelmintics. The limitations of the study include an overall low eggs-per-gram in both canine groups, and the shortage of additional fecal samples from the USA group, restraining the molecular analysis only to one out of the three Greyhounds. This study provided some insights on the efficacy of BZs against A. caninum and revealed the presence of BZ resistant isolates in imported dogs in Quebec, Canada. All this information should be considered, for choosing the best strategy in the control of A. caninum using anthelmintic drugs.

Anthelmintic resistance in livestock animals has been spreading across the world in prevalence and severity. As a result, researchers are exploring alternative strategies to combat this issue, and one promising avenue is the utilization of medicinal plants. This study aims to investigate the anthelmintic efficacy of the crude ethanolic extract (CEE) derived from the leaves of Juglans regia against one of the most detrimental nematode parasites affecting poultry, namely Ascaridia galli (A. galli). For the in vitro studies, adult A. galli worms were collected from the naturally infected chickens and the efficacy of CEE was measured at the concentration of 25, 50, and 100 mg/ml using adult worm motility inhibition (WMI) assay. In addition, levamisole (0.55 mg/ml) was used as the positive control. Likewise, Phosphate buffered saline (PBS) was used as the negative control. For the in vivo studies, CEE of J.regia at the doses of 500, 1000, and 2000 mg/kg were evaluated in chickens experimentally infected with A. galli. The anthelmintic efficacy was monitored using faecal egg count reduction (FECR) and worm count reduction (WCR) assays. In vitro studies revealed significant (P < 0.001) anthelmintic effects of CEE of J.regia on the motility of A. galli worms at different hours post-exposure. At the concentration of 100 mg/ml, CEE resulted in 96.5% inhibition of worm motility at 24 h post-exposure. While the synthetic anthelmintic drug, levamisole caused the highest inhibition of worm motility (100%) at the same time period. The in vivo anthelmintic activity of CEE of J. regia demonstrated a maximum effect on day 14 post-treatment by inducing 67.28% FECR and 65.03% WCR. We observed no significant difference (P > 0.05) in worm counts between the negative control group and the chickens treated with CEE at the dosage of 500 mg/kg. Together, the results of the present study suggest that CEE of J. regia leaves possess anthelmintic properties and could be a potential source of novel anthelmintic compounds for controlling helminth parasites.

Helminth infections in grazing ruminants are a major issue for livestock farming globally, but are unavoidable in outdoor grazing systems and must be effectively managed to avoid deleterious effects to animal health, and productivity. Next-generation sequencing (NGS) technologies are transforming our understanding of the genetic basis of anthelmintic resistance (AR) and epidemiological studies of ruminant gastrointestinal parasites. They also have the potential to not only help develop and validate molecular diagnostic tests but to be directly used in routine diagnostics integrating species-specific identification and AR into a single test. Here, we review how these developments have opened the pathway for the development of multi-AR and multispecies identification in a single test, with widespread implications for sustainable livestock farming for the future.

Gastrointestinal nematodes (GINs) are a significant threat to the sustainability of global sheep production. Periparturient ewes play a key role in GIN epidemiology, with increased GIN faecal egg counts (FECs) in these ewes resulting in heavy pasture contamination that facilitates parasitic gastroenteritis in immunologically naïve lambs later during the grazing period. Traditionally, blanket anthelmintic treatment would suppress GIN egg outputs in these ewes and subsequent pasture contamination. However, farmers are now advised to implement targeted selective treatment (TST) to reduce anthelmintic use and subsequent anthelmintic resistance development, yet, there is currently limited evidence to determine optimal TST strategies in ewes. In this study, the characteristics of 226 ewes on seven Welsh farms were assessed postlambing to identify factors associated with their individual strongyle FECs using negative binomial mixed model analysis. Nemabiome analysis was conducted on 34 ewes across two study farms using the Oxford Nanopore MinIon platform with an aim of identifying factors associated with variations in ewe nemabiome composition within flocks. The best-fitted model of ewe FEC incorporated ewe body condition score, dag score, breed, and an interaction effect between ewe age and litter size as fixed factors. The addition of a mean FEC value for ewes of a specific litter size on each farm further improved model fit and reduced between-farm variance in the model. Nemabiome analysis revealed significant variation in within flock nemabiome diversity on individual farms, with significantly reduced nemabiome diversity recorded in ewes exhibiting dags and in twin-bearing ewes on respective farms, whilst T. circumcincta was present as a significantly higher proportion of the nemabiome in Suffolk ewes and twin bearing ewes (P < 0.05) in respective flocks. Our data demonstrate that commonly recorded ewe characteristics can be exploited to predict individual periparturient ewe FEC and subsequently may be used as a guide for TST strategies on sheep farms once specific TST thresholds are identified to deliver the optimal balance between minimal pasture contamination and maximal GIN refugia. This study is the first to utilise Oxford Nanopore MinIon sequencing to evaluate the nemabiome of sheep, and to molecularly assess the nemabiome of individual ruminants within a flock/herd, with results indicating that significant within flock variations in nemabiome composition which may have implications for TST and flock management strategies.