Anthelmintic resistance (AR) in gastrointestinal nematodes (GIN) is a worldwide problem in all livestock production systems. Several prevalence studies performed internationally demonstrate that AR in cattle parasites is a growing problem; however, there are no published studies on prevalence in the United States (USA). In this study, we performed fecal egg count reduction tests (FECRT) on twelve cow-calf farms in Georgia, USA. On all farms the efficacy of eprinomectin pour-on (Eprinex®) and doramectin injectable (Dectomax®) was tested individually, as well as a combination treatment of doramectin injectable with oral fenbendazole (Safe-guard®). Fenbendazole was also tested individually on 7 farms, and long-acting eprinomectin (LongRange®) on one farm. FECRTs were performed, analyzed and interpreted following the 2023 WAAVP guidelines. Coprocultures were conducted to assess genus-specific efficacy. Overall, 100 % (12/12) and 75 % (9/12) of farms had resistance to at least one of the avermectin drugs tested in one or more GIN species, respectively. FECR ranged from 14.0 % to 96.8 % (mean=61.81 %) and 8.9-97.9 % (mean=66.87 %) for eprinomectin and doramectin, respectively; this difference was not significant (P = 0.924). In contrast, FECR for fenbendazole was high on all 7 farms tested, with just one farm demonstrating a low level of resistance. The combination treatment of doramectin plus fenbendazole was effective on all 12 farms (mean FECR=98.75 %), being significantly higher than for eprinomectin or doramectin (P≤0.001). Where coproculture data were sufficient to evaluate genus-specific reduction, resistance to Cooperia spp., Haemonchus spp. and Ostertagia spp. was present on 11/12 (92 %), 6/8 (75 %), and 4/10 farms (40 %), respectively. These data demonstrate that resistance to avermectin drugs is highly prevalent in Cooperia, Haemonchus, and Ostertagia on beef farms in Georgia. Though these data represent just one southern state, given the high level of movement of cattle in the USA, AR in GIN of cattle is likely to be highly prevalent throughout not only the southern USA, but across much of the country.

Subtherapeutic treatment or 'underdosing' is considered a common problem in the control of parasitic helminths of animals and people and can hasten the emergence of anthelmintic resistance. Increasing reliance on the long-acting macrocyclic lactone, moxidectin, in both veterinary and medical settings may increase exposure of incoming helminth populations to subtherapeutic drug concentrations due to its extended half-life. However, we lack genetic markers to monitor emerging resistance as the mechanism(s) underlying resistance to the macrocyclic lactones are unresolved in parasitic helminths. Furthermore, the impact of prior ivermectin exposure on the evolution of moxidectin resistance is unclear. To test the impact of subtherapeutic selection on the emergence of macrocyclic lactone resistance, we exposed a fully drug susceptible isolate of an economically important parasitic helminth of livestock, Haemonchus contortus, to low but increasing doses of ivermectin or moxidectin in vivo for phenotypic, genomic, and transcriptomic analyses. After a single subtherapeutic dose of ivermectin or moxidectin, we find evidence of selection at a shared genetic locus on Chromosome V, with the signal of selection increasing with subsequent doses. After only three subtherapeutic treatments, ivermectin-selected lines were resistant to a full standard (label) dose of ivermectin. However, moxidectin selected lines remained susceptible to a half dose of moxidectin. This was despite showing higher resistance to ivermectin in vitro and a stronger signal of selection at the Chromosome V locus than the equivalent ivermectin-selected lines. Our findings highlight the rapid selection for anthelmintic resistance with subtherapeutic treatment and implicate the pre-existence of ivermectin and moxidectin resistance haplotypes in a drug-naïve population. We demonstrate that ivermectin selected lines show emerging moxidectin resistance, underpinned by a shared genetic locus of resistance. Finally, we speculate that key differences in the resistance phenotype between ivermectin and moxidectin selected lines relate to differences in the inheritance of resistance within this shared locus, with ivermectin resistance manifesting as dominant trait while moxidectin resistance appears to be recessive.

Anthelmintic resistance (AR) presents a significant challenge to the treatment of parasites in veterinary medicine, and is emerging as a growing concern in human medicine. Despite the pressing demand for compounds with novel mechanisms of action, the introduction of new anthelmintics to the market has been scarce. In this study, we investigated the in vivo efficacy of compounds previously demonstrated to be inhibitors of phosphoethanolamine methyltransferases (PMTs), enzymes that are highly conserved among parasitic nematodes and represent a novel drug target. Inhibition of PMT enzymes disrupts phosphocholine biosynthesis, which is crucial for parasite viability. This pathway is essential to nematodes and is not present in mammals, making it an attractive and highly selective drug target. Based on previous results obtained from in vitro screenings against both drug-susceptible and multidrug-resistant (MDR) Haemonchus contortus, three candidate compounds with potent in vitro activity were selected for use in a pilot in vivo study. Here, we employed the jird (Meriones unguiculatus) - H. contortus model. The dosing regimens used were selected based on available toxicological data, but since pharmacokinetic-pharmacodynamic (PKPD) profiles of these compounds are not known, dosing regimens were not optimized. As this was a pilot study, small groups of animals were used. Relatively high reductions in worm counts were achieved, ranging from 53.5 to 72.6 %, and for two of the three compounds this reduction was statistically significant (P = 0.008, P = 0.025). These findings further support PMT enzymes as viable drug targets in parasitic nematodes and highlight the need for PKPD studies. Optimizing dosing regimens and exploring synergistic activity could enhance efficacy, advancing the development of novel anthelmintics.

Economic losses and adverse effects on animals' health and productivity due to gastrointestinal parasites constitute a significant challenge for expanding and improving the small ruminant industry in developing countries. This study aimed to determine the diversity and geographic distribution of gastrointestinal parasites infecting small ruminants in Grenada. Seven hundred and thirteen fecal samples from 159 sheep and 554 goats from 64 farms in Grenada, Carriacou, and Petite Martinique were collected for coprological examination. Of 713 sheep and goat samples, 640 were positive for gastrointestinal parasites, yielding an overall prevalence of 90 % (CI (95%), 88 to 92 %). Of the 554 goat samples, 95 % were positive for parasites (525/554; CI (95%), 93 to 97 %), and for the 159 sheep, 72 % were positive (115/159; CI (95%)(,) 65 to 79 %). In goats, the most common parasites observed were Strongyles, 90 % (CI95%, 87 to 92 %); Eimeria spp., 50 % (CI95%, 46 to 54 %); Strongyloides, 7 % (CI95%, 5 to 9 %); Moniezia, 6 % (CI95%, 5 to 9 %); and Trichuris, 5% (CI95%, 4 to 7 %). Sheep showed a similar pattern with the most common parasites identified being Strongyles, 52 % (CI95%, 44 to 59 %); Eimeria spp., 50 % (CI95%, 42 to 57 %); Strongyloides, 6 % (CI95%, 3 to 11 %); Moniezia, 6 % (CI95%, 3 to 10 %); and Trichuris, 3 % (CI95%, 1 to 7 %). The overall prevalence of gastrointestinal nematode (GIN) infections was higher in goats (p < 0.0001), due to the higher prevalence of Strongyles. The proportion of goats with zero parasites was significantly less than the proportion of sheep (p < 0.0001). Larval identification using coproculture analysis performed on pooled fecal samples from each farm to determine the GIN percentages and the overall mean indicated that Haemonchus was the most common genus identified, with an overall mean of 56 %, followed by Trichostrongylus (31 %), Oesophagostomum (13 %) and Cooperia (0.1 %). Nemabiome analysis based on deep amplicon sequencing demonstrated the presence of six nematode species: Haemonchus contortus (42 %), Trichostrongylus colubriformis (38 %), Oesophagostomum columbianum (12 %), Oesophagostomum asperum (7 %), Cooperia punctata (0.4 %) and Cooperia spatulata (0.1 %).

Cases of infection with the canine hookworm Ancylostoma caninum that are resistant to all anthelmintic classes registered in the United States of America (USA) for control of this parasite in dogs were first reported in 2019. Termed Multiple Anthelmintic Drug Resistance (MADR), cases have subsequently been reported in pet dogs across the USA. Recent studies using molecular tests that detect mutations known to cause resistance to benzimidazole anthelmintics have revealed an alarming frequency of this trait in pet dogs throughout the USA, with some reports also from Canada. Since the initial reports in the USA that dog hookworms resistant to benzimidazoles were also resistant to pyrantel and macrocyclic lactones, this genetic evidence suggests that MADR worms are now widely present in the USA. The trait originally evolved on greyhound farms and kennels. However, the problem is no longer limited to greyhounds; recent studies have demonstrated that essentially any breed, age, size and sex of dogs throughout the USA can get infected with drug-resistant and possibly MADR isolates of A. caninum. In this perspective, we review current knowledge of the origin of MADR canine hookworms, tests available to diagnose MADR hookworms, and therapeutic options for treating dogs with such cases. Additionally, we propose research priorities for developing a deeper understanding of the biology of MADR worms, as well as for developing new therapeutic options to address the problem. Adding to the urgency of the situation, A. caninum is a zoonotic parasite, capable of causing disease in humans.

The diagnostics of ruminant parasites remains one of the cornerstones for parasite control best practices. Field veterinarians have several techniques at their disposal (fecal egg count, coproculture, FAMACHA®, plasma pepsinogen, ELISA-Ostertagia, ELISA-Fasciola, Baermann and ELISA-Lungworm) for the identification and/or quantification of gastrointestinal nematodes, lungworms and liver fluke infecting small ruminants and cattle. Each of these diagnostic tools has its own strengths and weaknesses and is more appropriate for a specific production operation and/or age of the animal (young and adults). This review focuses on the usability and interpretation of the results of these diagnostic tools. The most advanced technical information on sampling, storage, advantages and limitations of each tool for different types of production operations and animal categories is provided.

Long-term intensive use of anthelmintics for parasite control of livestock, companion animals, and humans has resulted in widespread anthelmintic resistance, a problem of great socioeconomic significance. But anthelmintic therapy may also select for other biological traits, which could have implications for anthelmintic performance. Here, we highlight recent examples of changing parasite dynamics following anthelmintic administration, which do not fit the definition of anthelmintic resistance. We also consider other possible examples in which anthelmintic resistance has clearly established, but where coselection for other biological traits may have also occurred. We offer suggestions for collecting more information and gaining a better understanding of these phenomena. Finally, we propose research questions that require further investigation and make suggestions to help address these knowledge gaps.

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are infected with A . caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A . caninum in greyhounds was associated with a high frequency of the canonical F167Y(T T C>T A C) isotype-1 β-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A . caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation, Q134H(CA A >CA T ). Several benzimidazole resistant A . caninum isolates from greyhounds with a low frequency of the F167Y(T T C>T A C) mutation had a high frequency of a Q134H(CA A >CA T ) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C . elegans β-tubulin gene ben-1 , by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A . caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(T T C>T A C) and Q134H(CA A >CA T ), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(T T C>T A C) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.

In humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the families Trichostrongylidae, Dictyocaulidae, Chabertiidae, Ancylostomatoidea, and Ascarididae. We characterized these putative PMTs and found that they possess bona fide PMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using an in vitro phosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested against Haemonchus contortus using larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates of H. contortus, with IC50 values (95% confidence interval) of 4.30 μM (2.15-8.28), 4.46 μM (3.22-6.16), 28.7 μM (17.3-49.5), and 0.65 μM (0.21-1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potent in vitro anthelmintic activity.

The faecal egg count reduction test (FECRT) remains the method of choice for establishing the efficacy of anthelmintic compounds in the field, including the diagnosis of anthelmintic resistance. We present a guideline for improving the standardization and performance of the FECRT that has four sections. In the first section, we address the major issues relevant to experimental design, choice of faecal egg count (FEC) method, statistical analysis, and interpretation of the FECRT results. In the second section, we make a series of general recom-mendations that are applicable across all animals addressed in this guideline. In the third section, we provide separate guidance details for cattle, small ruminants (sheep and goats), horses and pigs to address the issues that are specific to the different animal types. Finally, we provide overviews of the specific details required to conduct an FECRT for each of the different host species. To address the issues of statistical power vs. practicality, we also provide two separate options for each animal species; (i) a version designed to detect small changes in efficacy that is intended for use in scientific studies, and (ii) a less resource-intensive version intended for routine use by veterinarians and livestock owners to detect larger changes in efficacy. Compared to the previous FECRT rec-ommendations, four important differences are noted. First, it is now generally recommended to perform the FECRT based on pre-and post-treatment FEC of the same animals (paired study design), rather than on post-treatment FEC of both treated and untreated (control) animals (unpaired study design). Second, instead of requiring a minimum mean FEC (expressed in eggs per gram (EPG)) of the group to be tested, the new requirement is for a minimum total number of eggs to be counted under the microscope (cumulative number of eggs counted before the application of a conversion factor). Third, we provide flexibility in the required size of the treatment group by presenting three separate options that depend on the (expected) number of eggs counted. Finally, these guidelines address all major livestock species, and the thresholds for defining reduced efficacy are adapted and aligned to host species, anthelmintic drug and parasite species. In conclusion, these new guidelines provide improved methodology and standardization of the FECRT for all major livestock species.

The faecal egg count reduction test (FECRT) is the primary diagnostic tool used for detecting anthelmintic resistance at the farm level. It is therefore extremely important that the experimental design of a FECRT and the susceptibility classification of the result use standardised and statistically rigorous methods. Several different approaches for improving the analysis of FECRT data have been proposed, but little work has been published on how to address the issue of prospective sample size calculations. Here, we provide a complete and detailed overview of the quantitative issues relevant to a FECRT starting from basic statistical principles. We then present a new approach for determining sample size requirements for the FECRT that is built on a solid statistical framework, and provide a rigorous anthelminthic drug efficacy classification system for use with FECRT in livestock. Our approach uses two separate statistical tests, a one-sided inferiority test for resistance and a one-sided non-inferiority test for susceptibility, and determines a classification of resistant, susceptible or inconclusive based on the combined result. Since this approach is based on two independent one-sided tests, we recommend that a 90 % CI be used in place of the historically used 95 % CI. This maintains the desired Type I error rate of 5 %, and simultaneously reduces the required sample size. We demonstrate the use of this framework to provide sample size calculations that are rooted in the well-understood concept of statistical power. Tailoring to specific host/parasite systems is possible using typical values for expected pre-treatment and post-treatment variability in egg counts as well as within-animal correlation in egg counts. We provide estimates for these parameters for ruminants, horses and swine based on a re-examination of datasets that were available to us from a combination of published data and other sources. An illustrative example is provided to demonstrate the use of the framework, and parameter estimates are presented to estimate the required sample size for a hypothetical FECRT using ivermectin in cattle. The sample size calculation method and classification framework presented here underpin the sample size recommendations provided in the upcoming FECRT WAAVP guidelines for detection of anthelmintic resistance in ruminants, horses, and swine, and have also been made freely available as open-source software via our website (https://www.fecrt.com).

OBJECTIVE:To evaluate the efficacy of the 3 major classes of anthelmintics used for the treatment of hookworms in dogs in the US and an extralabel treatment with an FDA-approved product for use in cats in a Labrador kennel with a history of persistent hookworm infections.ANIMALS:22 dogs housed in a single kennel comprised of the following breeds: 19 Labrador Retrievers, 1 English Cocker Spaniel, 1 Chesapeake Bay Retriever, and 1 Boykin Spaniel.PROCEDURES:We performed a fecal egg count (FEC) reduction test using 22 dogs that were allocated randomly to 1 of 5 treatment groups: pyrantel pamoate (Pyrantel pamoate suspension), fenbendazole (Safe-Guard suspension 10%), milbemycin oxime (Interceptor), moxidectin plus imidacloprid (Advantage Multi), and emodepside plus praziquantel (Profender topical solution for cats). FEC was performed on samples collected on days 0 and 11.RESULTS:FEC reductions for the milbemycin oxime, moxidectin plus imidacloprid, and emodepside plus praziquantel groups were 43.9%, 57.4%, and 100%, respectively. The FEC increased following treatment for the pyrantel and fenbendazole groups.CLINICAL RELEVANCE:These data demonstrate that the Ancylostoma caninum infecting the dogs in this kennel are highly resistant to all major anthelmintic classes approved for use in dogs in the US but are susceptible to emodepside. This was the first report of multiple anthelmintic drug-resistant A caninum in a dog kennel that does not involve Greyhounds.

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA 2004) isolates of H. contortus . Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L 3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

AbstractAncylostoma caninumis an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhounds in the USA are infected withA. caninumthat are commonly resistant to multiple anthelmintics. Benzimidazole resistance inA. caninumin greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 β-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread inA. caninumfrom domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistantA. caninumisolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans β-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing onA. caninumeggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, often at high frequency, with prevalences of 49.7% (overall frequency 54.0%) and 31.1% (overall frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.Author SummaryAlthough increasingly common in livestock, no reports of widespread anthelmintic resistance are confirmed in any companion animal or human gastrointestinal nematode parasite to date. The canine hookworm is a common intestinal zoonotic parasite of dogs with severe clinical impacts in young dogs, and for which control is dependent on regular anthelmintic use. We recently reported multiple anthelmintic drug resistance inA. caninumisolates from greyhounds derived from multiple locations in the USA likely caused by long standing intensive treatment regimens in kennels. In this study, we investigated benzimidazole resistance inA. caninumin pet dogs across the USA. We also identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation inA. caninumfrom greyhounds that has not been previously reported in the field for any organism. We then determined that this novel mutation, as well as a previously characterized resistance mutation, were present, often at high frequency, in manyA. caninumpopulations across the USA. This study reports the first evidence of widespread drug resistance for any parasitic nematode of companion animals and illustrates the power of molecular approaches to rapidly assess anthelmintic resistance in a region.

Parasites are highly prevalent in poultry; thus, the management of parasites is a key component in the profitable production of poultry. The most common nematode parasite of poultry, Heterakis gallinarum, typically causes no direct pathology but is the vector of Histomonas meleagridis, a highly pathogenic protozoan parasite that causes blackhead disease. There are no approved treatments for H. meleagridis, making control reliant on controlling the helminth vector. In the United States, the benzimidazole anthelmintic fenbendazole (FBZ) is the only approved treatment for H. gallinarum. We were contacted by an industry veterinarian regarding clinical problems with histomoniasis despite frequent anthelmintic treatments. Given that we had recently diagnosed FBZ resistance in the closely related parasite Ascaridia dissimilis, we were interested to determine if H. gallinarum had also evolved resistance. An initial on-farm pilot study using 20 birds suggested that FBZ was poorly effective, therefore a larger controlled study was initiated. Heterakis gallinarum eggs were isolated from litter at the farm and used to infect 118 chicks. Treatment groups included a non-treated control, a label-, and a 2×-label dose of FBZ, with 36 birds per group divided into two replicates of 18 birds. Three weeks post-hatch, birds were infected with 150 embryonated eggs. Two weeks post-infection treated birds were administered either a label- or 2× label-dose of FBZ in water for five days (SafeGuard® Aquasol, 1 mg/kg BW). To increase the likelihood that all birds consumed the full intended dose, the dosage was calculated using 1.25 times the average body weight. One-week post-treatment, birds were euthanized, and parasites enumerated. There were no significant differences in worm numbers recovered from any of the three groups (p-value = 0.3426), indicating that both dosages of FBZ failed to provide the expected levels of efficacy. These data provide strong evidence that H. gallinarum has developed resistance to FBZ on this farm. Consequently, on this farm, or any farm with FBZ-resistant H. gallinarum, H. meleagridis will continue to cycle in an unrestricted manner despite administration of anthelmintic treatments. Given recent evidence of increasing problems with histomoniasis, and the fact that resistance was documented on the first farm we investigated, further investigations are needed to determine the prevalence of resistance in H. gallinarum on poultry farms. These data, when viewed together with our recent findings of FBZ resistance in A. dissimilis on multiple farms, suggest that drug resistance in ascarid nematodes may be an emerging problem in the US poultry industry.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths.

ABSTRACTDue to their ubiquity, management of parasites is a common and important factor for profitable production of poultry.Heterakis gallinarum, the cecal nematode, is the most common nematode parasite of poultry. While typically causing no pathology on its own,H. gallinarumis the vector ofHistomonas meleagridis, a protozoan parasite that causes blackhead disease.Histomonas meleagridisis highly pathogenic in turkeys, potentially causing high mortality. In contrast, disease caused byH. meleagridisis much less severe in chickens, where it primarily reduces productivity without manifestations of clinical disease. There are no approved treatments forH. meleagridis, making control reliant on control of the helminth vector through the use of fenbendazole (FBZ) the only drug labeled for treatment ofH. gallinarumin the United States We were contacted by an industry veterinarian regarding health-related concerns in a broiler-breeder house due to histomoniasis, despite frequent anthelmintic treatments. Since we had recently diagnosed resistance to FBZ inAscaridia dissimilis, a closely related nematode of turkeys, we were interested to determine ifH. gallinarumhad also evolved resistance to FBZ.Heterakis gallinarumeggs were isolated from litter collected from the house and used to infect 108 chickens. Treatment groups included a non-treated control, a label-dose and a 2X-label dose of FBZ, with 36 birds per group divided into two replicate pens of 18 birds each. Birds were placed at 1-day post hatch, and at 3 weeks of age were infected with 150 embryonated eggs via oral gavage. Two weeks post infection, treated birds were administered a minimum of either a label- or 2X label-dose of FBZ in water for 5 days (SafeGuard®Aquasol, 1mg/kg BW). To ensure that all birds consumed the full intended dose at a minimum, the dosage was calculated using 1.25 times the average body weight. One-week post treatment, birds were euthanized, ceca removed, and parasites enumerated. Efficacy was calculated by comparing the total numbers of worms recovered from each treatment group to the numbers recovered in the non-treated control group. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.81). There also was no efficacy benefit to treatment with a 2X dose;H. gallinarumworm counts were reduced by 42.7% and 41.4%, for the label and 2X dosages, respectively. These data provide strong evidence thatH. gallinarumhas developed resistance to FBZ. Consequently, in houses infected with FBZ-resistantH. gallinarum,H. meleagridiswill be able to cycle through the birds in an unrestricted manner. Further investigation is needed to determine the prevalence of resistance inH. gallinarum onchicken farms, but it is clear this has the potential to have a large-scale economic impact on the poultry industry. These data when viewed together with our recent findings of FBZ resistance inA. dissimilis, suggest that drug resistance in ascarid nematodes may be an important emerging problem on poultry operations.