• Animals
• Drug Resistance
• Biotransformation
• Nematoda/drug effects
• Nematoda/metabolism
• Nematoda/enzymology
• Anthelmintics/pharmacology
• Anthelmintics/metabolism
• Anthelmintics/pharmacokinetics
• Humans

• Haemonchus contortus
• Automated motility assay
• Detoxification pathways
• Xenobiotics

• Haemonchus/drug effects
• Haemonchus/metabolism
• Animals
• Anthelmintics/pharmacology
• Xenobiotics/metabolism
• Inactivation, Metabolic/drug effects
• Levamisole/pharmacology
• Larva/drug effects
• Ivermectin/pharmacology
• Albendazole/pharmacology
• Albendazole/analogs & derivatives
• Aminoacetonitrile/analogs & derivatives
• Aminoacetonitrile/pharmacology
• Inhibitory Concentration 50

• BDMX_2023_1#49499052 CSIC - Udelar for the Posgraduate scholarship
• Research project 366 Comisión Sectorial de Investigación Científica de la Universidad de la República
• Programa de Desarrollo de Ciencias Básicas (PEDECIBA, Uruguay)
• CSIC – Udelar for the Posgraduate scholarship

• Gene
• Haemonchus
• Ivermectin
• P-glycoprotein
• Resistance

• Animals
• Male
• Anthelmintics/pharmacology
• ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
• Drug Resistance
• Gene Expression
• Haemonchiasis/veterinary
• Haemonchiasis/parasitology
• Haemonchus/drug effects
• Haemonchus/genetics
• Ivermectin/pharmacology
• Mexico
• Phenotype
• Sheep
• Sheep Diseases/parasitology

• Allele specific PCR
• Diagnostic
• Haemonchus
• Levamisole
• RFLP
• Resistance
• S168T
• SNP

• Animals
• Haemonchus
• Levamisole/pharmacology
• Drug Resistance/genetics
• Anthelmintics/pharmacology
• Anthelmintics/therapeutic use
• Receptors, Cholinergic/genetics
• Haemonchiasis/drug therapy
• Haemonchiasis/veterinary
• Haemonchiasis/parasitology

• Wellcome Trust
• MR/T020733/1 Medical Research Council

• Anthelmintic screening
• Automated motility assay
• Benzimidazole derivatives
• Development assay
• Haemonchus contortus
• New anthelmintics

• anthelmintic
• development
• helminths
• mechanism
• resistance

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr-8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing “resistant” allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous “resistant” genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the “resistant” allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.

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Acr8b – levamisole resistance marker investigated in single worms and larval cultures.

• AR
• Anthelmintic resistance
• Haemonchus contortus
• Hco-acr-8b
• Levamisole
• Resistance
• ddPCR

• Animals
• Haemonchiasis/drug therapy
• Haemonchiasis/veterinary
• Haemonchus/genetics
• Levamisole/pharmacology
• Polymerase Chain Reaction
• Sheep
• Sheep Diseases

• Haemonchus
• abomasum
• anthelmintic resistance
• diagnosis
• goat
• health management
• prevention
• sheep
• treatment

• Anthelmintic resistance, sheep, Argentina
• Drug combination
• Levamisole
• Moxidectin

• C. elegans
• anthelmintics
• drug resistance
• natural diversity

For more than four decades, the free-living nematode Caenorhabditis elegans has been extensively used in anthelmintic research. Classic genetic screens and heterologous expression in the C. elegans model enormously contributed to the identification and characterization of molecular targets of all major anthelmintic drug classes. Although these findings provided substantial insights into common anthelmintic mechanisms, a breakthrough in the treatment and control of parasitic nematodes is still not in sight. Instead, we are facing increasing evidence that the enormous diversity within the phylum Nematoda cannot be recapitulated by any single free-living or parasitic species and the development of novel broad-spectrum anthelmintics is not be a simple goal. In the present review, we summarize certain milestones and challenges of the C. elegans model with focus on drug target identification, anthelmintic drug discovery and identification of resistance mechanisms. Furthermore, we present new perspectives and strategies on how current progress in C. elegans research will support future anthelmintic research.

• Anthelmintic drug
• Anthelmintic resistance
• Caenorhabditis elegans
• Mode of action
• Parasitic nematode

• Animals
• Anthelmintics/therapeutic use
• Caenorhabditis elegans
• Drug Discovery
• Nematoda

• R01 AI153088 NIAID NIH HHS
• R21 AI121836 NIAID NIH HHS
• T32 GM008449 NIGMS NIH HHS

• Anthelmintics
• Drug discovery
• Haemonchus contortus
• Novel chemical entities
• Phenotypic screening
• Repurposing

• Anthelmintic
• Haemonchus contortus
• Larval development assay
• Levamisole
• Resistance
• Trichostrongylus colubriformis

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Teladorsagia circumcincta is an economically significant nematode (roundworm) pathogen affecting sheep and goats in temperate regions of the world. The widespread use of prophylactic treatment has resulted in rapid selection for anthelmintic (anti-worm drug) resistance in this and other species of livestock parasites. The mechanism of resistance is not well understood because most studies have focused on the role of candidate genes using simplistic models of single gene selection, despite evidence that the evolution of resistance is more complex. Here, we report on a comprehensive whole-genome analysis that elucidated resistance-associated genes, which was facilitated by developing a pair of T. circumcincta strains sharing a largely common genetic background but differing markedly in their susceptibility to anthelmintic drugs. The results show that multiple genetic factors contribute to anthelmintic resistance in a variety of ways, including possible reduction/modulation in target site sensitivity, reduced target site expression, and increased drug efflux, to name a few. This suggests that drug resistance in these parasites is a multifactorial quantitative trait rather than a simple discrete Mendelian character. With this study, we established a genomics-based experimental paradigm for investigating anthelmintic resistance, at a time when its medical importance is rapidly increasing.

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchuscontortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite.

•

Identification of ABC-B full and half transporter genes in Dirofilaria immitis.

• Dirofilaria immitis
• Genetic identification
• P-glycoprotein

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L₃ to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L₃ were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

•

We studied interaction of anthelmintics with ABC transporters in Haemonchus contortus.

• ABC transporters
• Anthelmintics
• Haemonchus contortus
• P-glycoprotein
• Resistance

• Amino-acetonitrile derivates
• Anthelmintic resistance
• Benzimidazoles
• Haemonchus contortus
• Macrocyclic lactones
• Resistance mechanisms

• Anthelmintic drugs
• Anthelmintic resistance
• Anthelmintic targets
• Molecular markers
• Receptors

• R01 AI047194 NIAID NIH HHS