Search

Parasite drug resistance presents a major obstacle to controlling and eliminating vector-borne diseases affecting humans and animals. While vector-borne disease dynamics are affected by factors related to parasite, vertebrate host and vector, research on drug resistance in filarial parasites has primarily focused on the parasite and vertebrate host, rather than the mosquito. However, we expect that the physiological costs associated with drug resistance would reduce the fitness of drug-resistant vs. drug-susceptible parasites in the mosquito wherein parasites are not exposed to drugs. Here we test this hypothesis using four isolates of the dog heartworm (Dirofilaria immitis)—two drug susceptible and two drug resistant—and two vectors—the yellow fever mosquito (Aedes aegypti) and the Asian tiger mosquito (Ae. albopictus)—as our model system. Our data indicated that while vector species had a significant effect on vectorial capacity, there was no significant difference in the vectorial capacity of mosquitoes infected with drug-resistant vs. drug-susceptible parasites. Consequently, contrary to expectations, our data indicate that drug resistance in D. immitis does not appear to reduce the transmission efficiency of these parasites, and thus the spread of drug-resistant parasites in the vertebrate population is unlikely to be mitigated by reduced fitness in the mosquito vector. [ABSTRACT FROM AUTHOR]

In this article the author discusses prescription-only anthelmintic drugs for controlling parasites of U.S. livestock. He argues that their use has gotten out of control in the livestock industry, resulting in multiple-drug resistance in the majority of the most important parasite species of livestock. Topics include the control of nematode parasites, livestock health and productivity, and diagnostic surveillance of parasite infection levels on U.S. farms.

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(TTC>TAC) 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(CAA>CAT). Several benzimidazole resistant A. caninum isolates 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 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(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 summary: Although 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 in A. caninum isolates 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 in A. caninum in pet dogs across the USA. We also identified and showed the functional significance of a novel benzimidazole isotype-1 β-tubulin resistance mutation in A. caninum from 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 many A. caninum populations 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Parascaris equorum and cyathostomins are currently considered the most important parasites of horses and have traditionally been controlled with anthelmintics belonging to three drug classes: benzimidazoles, the tetrahydropyrimidine pyrantel, and macrocyclic lactones. Unfortunately, resistance to benzimidazoles, and to a lesser extent pyrantel, is widespread in cyathostomins around the world. Furthermore, resistance to macrocyclic lactones appears to be in the early stages of development in cyathostomins in multiple locations. In contrast, P. equorum populations have remained susceptible to the three anthelmintic drug classes for a considerably longer period of time. However, over the last 10 years, resistance to macrocyclic lactones has been described in multiple countries. In contrast, resistance to pyrantel has only been described in the USA; resistance to benzimidazoles has yet to be reported. Despite the large number of reports of anthelmintic resistance in both cyathostomins and P. equorum, there are presently no reports that definitively link anthelmintic resistance with clinical problems in horses. However, that generally appears to be due to a publication bias toward well managed horse farms and the lack of appropriate diagnostic methods for rapidly quantifying anthelmintic resistance in these parasites. Management-based, and other, reasons likely responsible for this apparent anomaly are also discussed. Finally, future research priorities in this area, identified from a discussion at the 9th International Conference on Equine Infectious Diseases, are summarized. [Copyright &y& Elsevier]

Abstract: Avermectins and milbemycins (AM) are potent compounds against all major nematode parasites, but their continuous usage has led to the development of widespread resistance in many of the important species of ruminant and equine parasites. The exception to this has been the cyathostomins, where AM resistance was recently first reported only after decades of drug exposure. Data from a Brazilian study suggests that AM resistance has developed in cyathostomins and reports of shortened egg reappearance periods after ivermectin treatment have been published recently from USA and Germany. Thus, AM resistance in cyathostomins is an emerging worldwide concern, but there is only limited amount data on the extent of this problem. To limit the development and spread of AM-resistant cyathostomins the equine industry must implement new strategies for worm control, and the veterinary parasitology community must develop and validate improved protocols for detecting anthelmintic resistance in the field. [Copyright &y& Elsevier]

An elicitation exercise was conducted to collect and identify pressing questions concerning the study of helminths in livestock, to help guide research priorities. Questions were invited from the research community in an inclusive way. Of 385 questions submitted, 100 were chosen by online vote, with priority given to open questions in important areas that are specific enough to permit investigation within a focused project or programme of research. The final list of questions was divided into ten themes. We present the questions and set them briefly in the context of the current state of knowledge. Although subjective, the results provide a snapshot of current concerns and perceived priorities in the field of livestock helminthology, and we hope that they will stimulate ongoing or new research efforts. Highlights Important questions on helminths of livestock were elicited across the research community and prioritised by online vote. The approach contrasts with traditional review formats and seeks to identify questions agreed to be both important and feasibly addressed by focused research projects. A shortlist of 100 questions is presented with supporting text to provide perspective. The article is intended to stimulate new and ongoing research on helminth infections, in support of sustainable global livestock health and productivity. [ABSTRACT FROM AUTHOR]

The article presents the discussion on regional and global impacts. Topics include research also focusing on the sustainability of regional and international aquaculture with local implications for food security and climate change adaptation; and animals during the COVID-19 pandemic, pet ownership, and academic performance and stress management among SGU veterinary students.

The article discusses how the Saint George's University School of Veterinary Medicine (SGU-SVM) in Grenada, West Indies particularly focuses on one-to-one medicine, evidence-based primary care, and diversity support, it provides high-quality veterinary education in a multicultural tropical setting. It states that before graduating from an accredited school, students spend their first three years conducting preclinical research in Grenada.

Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes have been controlled using broad-spectrum anthelmintics; however, cyathostomin resistance to each anthelmintic class has been reported and populations insensitive to more than one class are relatively commonplace. The faecal egg count reduction test (FECRT) is considered the most suitable method for screening anthelmintic sensitivity in horses, but is subject to variation and is relatively time-consuming to perform. Here, we describe a larval migration inhibition test (LMIT) to assess ivermectin (IVM) sensitivity in cyathostomin populations. This test measures the paralysing effect of IVM on the ability of third stage larvae (L3) to migrate through a pore mesh. When L3 from a single faecal sample were examined on multiple occasions, variation in migration was observed: this was associated with the length of time that the L3 had been stored before testing but the association was not significant. Half maximal effective concentration (EC50) values were then obtained for cyathostomin L3 from six populations of horses or donkeys that showed varying sensitivity to IVM in previous FECRTs. Larvae from populations indicated as IVM resistant by FECRT displayed significantly higher EC50 values in the LMIT than L3 from populations classified as IVM sensitive or L3 from populations that had not been previously exposed to IVM or had limited prior exposure. The analysis also showed that EC50 values obtained using L3 from animals in which IVM faecal egg count reduction (FECR) levels had been recorded as <95% were significantly higher than EC50 values obtained using L3 from animals for which FECR was measured as >95%. For one of the populations, time that had elapsed since IVM administration had an effect on the EC50 value obtained, with a longer time since treatment associated with lower EC50 values. These results indicate that the LMIT has value in discriminating IVM sensitivity amongst cyathostomin populations, but several factors were identified that need to be taken into account when executing the test and interpreting the derived data. [ABSTRACT FROM AUTHOR]

Objective--To compare larvicidal regimens of fenbendazole and moxidectin for reduction and suppression of cyathostomin fecal egg counts (FEC) in a transient herd of embryo transfer-recipient mares. Design--Randomized, complete block, clinical trial. Animals--120 mares from 21 states, residing on 1 farm. Procedures--An initial fecal sample was collected from each mare; mares with an FEC > 200 eggs/g were assigned to treatment groups. Eighty-two horses received fenbendazole (10.0 mg/kg [4.5 mg/lb], PO, q 24 h for 5 days) or moxidectin (0.4 mg/kg [0.18 mg/lb], PO, once); FEC data were analyzed 14, 45, and 90 days after treatment. Results--Mean FEC reduction was 99.9% for moxidectin-treated mares and 41.9% for fenbendazole- treated mares 14 days after treatment. By 45 days, mean FEC of fenbendazoletreated mares exceeded pretreatment counts; however, FECs of moxidectin-treated mares remained suppressed below pretreatment values for the duration of the 90-day study. Fecal egg counts were significantly different between groups at 14,45, and 90 days after treatment. Conclusions and Clinical Relevance--Failure of the 5-day regimen of fenbendazole to adequately reduce or suppress FEC suggested inadequate adulticidal and larvicidal effects. In contrast, a single dose of moxidectin effectively reduced and suppressed FEC for an extended period. Given the diverse geographic origins of study mares, these results are likely representative of cyathostomin-infected mares in much of the United States, confirming previous findings indicating that fenbendazole resistance in cyathostomins is widespread and that moxidectin remains an effective treatment for control of these important parasites. [ABSTRACT FROM AUTHOR]

Abstract: The generally warm, moist environmental conditions in the southern United States (U.S.) are ideal for survival and growth of the egg and larval stages of Haemonchus contortus and other gastrointestinal nematodes (GIN) of sheep and goats. Consequently, infection with GIN is the greatest threat to economic small ruminant production in this region. With anthelmintic resistance now reaching epidemic proportions in small ruminants in the U.S., non-chemical control alternatives are critically needed. The Southern Consortium for Small Ruminant Parasite Control (SCSRPC) was formed in response to this crisis and over the last decade has successfully validated the use of several novel control technologies, including FAMACHA© for the implementation of targeted selective treatments (TST), copper oxide wire particles (COWP), nematode-trapping fungi, and grazing or feeding hay of the high-tannin perennial legume sericea lespedeza [Lespedeza cuneata (Dum.-Cours. G. Don)]. Producer attitudes toward GIN control in the U.S. have been shifting away from exclusive dependence upon anthelmintics toward more sustainable, integrated systems of parasite control. Some novel control technologies have been readily adopted by producers in combination with appropriate diagnostic tools, such as FAMACHA©. Others techniques are still being developed, and will be available for producer use as they are validated. Although new drugs will likely be available to U.S. goat and sheep producers in the future, these will also be subject to development of anthelmintic resistance. Therefore, the adoption and implementation of sustainable GIN control principles will remain important. With emerging markets for grass-fed or organic livestock, there will continue to be a critical need for research and outreach on development and on-farm application of integrated GIN control systems for small ruminants in the U.S. for the foreseeable future. [Copyright &y& Elsevier]

Background: The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum. Methods: Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene. Results: Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases. Conclusions: These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 IC 50 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., Competing Interests: The authors declare no conflict of interest.

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 recommendations 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 recommendations, 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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)., Competing Interests: Conflict of interest The authors declare that they have no direct conflict of interest with this work., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Published by Elsevier B.V.)

Ancylostoma caninum is the most prevalent nematode parasite of dogs. We confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the USA. Cases of MDR hookworms appear to be highly overrepresented in greyhounds. The aims of this study were to evaluate the drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from greyhounds of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite treatment with anthelmintics, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA), respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 54 animals. Mean and median IC 50 and IC 95 values for the EHA were 5.3 μM, 3.6 μM, and 24.5 μM, 23.4 μM, respectively. For the LDA, the median IC 50 value was >1000 nM. These values ranged 62-81 times higher than our susceptible laboratory isolate. Only post-treatment samples were available. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools. Deep sequencing of the isotype 1 β-tubulin gene only revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples. These clinical, in vitro, and genetic data provide strong evidence that greyhound dogs in the USA are infected with MDR A. caninum at very high levels in prevalence and infection intensity., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A population of Haemonchus contortus that was highly resistant to benzimidazoles and avermectin/milbemycins with a subpopulation that was resistant to levamisole, was replaced with a susceptible laboratory isolate of H. contortus in a flock of sheep. The anthelmintic susceptibility and population genetics of the newly established population were evaluated for 3.5 years using in vivo, in vitro, and molecular methods. Successful replacement of the resistant population with a susceptible population was confirmed using phenotypic and genotypic measurements; larval development assay indicated full anthelmintic susceptibility; albendazole treatment yielded 98.7% fecal egg count reduction; pyrosequence genotyping of single nucleotide polymorphisms in positions 167 and 200 of the isotype-1 beta tubulin gene were present at 0.0 and 1.7%, respectively; microsatellite genotyping indicated the background haplotype was similar to the susceptible isolate; and haplotypes of the isotype-1 beta tubulin gene were similar to the susceptible isolate. To sustain the susceptibility of the new population, targeted selective treatment was implemented using albendazole. Surprisingly, within 1.5 years post-replacement, the population reverted to a resistant phenotype. Resistance to albendazole, ivermectin, and moxidectin was confirmed via fecal egg count reduction test, larval development assay, and pyrosequencing-based genotyping. Targeted selective treatment was then carried out using levamisole. However, within one year, resistance was detected to levamisole. Population genetics demonstrated a gradual change in the genetic structure of the population until the final population was similar to the initial resistant population. Genetic analyses showed a lack of diversity in the susceptible isolate, suggesting the susceptible isolate had reduced environmental fitness compared to the resistant population, providing a possible explanation for the rapid reversion to resistance. This work demonstrates the power of combining molecular, in vitro, and in vivo assays to study phenotypic and genotypic changes in a field population of nematodes, enabling improved insights into the epidemiology of anthelmintic resistance., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)