title: Candidate anthelmintic resistance-associated gene expression and sequence polymorphisms in a triple-resistant field isolate of Haemonchus contortus.
abstract: An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and ivermectin was isolated from sheep at the University of Georgia, and passaged through experimentally infected goats. We measured the expression of twenty-nine mRNAs encoding drug targets and P-glycoproteins (P-gps), comparing the results to a fully susceptible laboratory passaged isolate. Expression levels of some nicotinic acetylcholine receptor mRNAs were markedly different in UGA/2004. Levels of the Hco-acr-8b mRNA, encoding a truncated subunit, were very high in resistant L3, but undetectable in susceptible larvae, with expression of the full-length Hco-acr-8a mRNA also significant increased. Expression of Hco-unc-63 and Hco-unc-29.3 mRNAs was significantly reduced in the resistant larvae. Expression of the Hco-glc-3 and Hco-glc-5 mRNAs, encoding glutamate-gated chloride channel subunits, were slightly reduced in resistant larvae. We observed significant increases in the expression of the Hco-pgp-2 and Hco-pgp-9 mRNAs in the UGA/2004 larvae, consistent with previous reports; we also saw a decrease in the levels of Hco-pgp-1 mRNA. Treatment of the larvae with ivermectin and moxidectin in vitro produced variable and inconsistent changes in P-gp mRNA levels. The sequences of the β-tubulin isotype 1 mRNAs showed that the resistant larvae had a resistance-associated allele frequency of >95% at codon 200 and ∼40% and codon 167. No changes at codon 198 were present. The presence of the truncated acr-8b mRNA may be a reliable indicator of levamisole resistance, but complex changes in gene expression associated with macrocyclic lactone resistance make the identification of a single genetic marker for this resistance difficult.
authors.affiliation: Dept of Infectious Diseases, University of Georgia, Athens, GA 30602, USA.
authors.forename: Sally M
authors.initials: SM
authors.lastname: Williamson
authors.forename: Bob
authors.initials: B
authors.lastname: Storey
authors.forename: Sue
authors.initials: S
authors.lastname: Howell
authors.forename: Kate M
authors.initials: KM
authors.lastname: Harper
authors.forename: Ray M
authors.initials: RM
authors.lastname: Kaplan
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Wolstenholme
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance; D006041:Goats; D006188:Haemonchiasis / Q000469:parasitology / Q000662:veterinary*; D006189:Haemonchus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D012756:Sheep; D012757:Sheep Diseases / Q000469:parasitology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000871:Anthelmintics; D001562:Benzimidazoles; D018118:Chloride Channels; D015801:Helminth Proteins; C109777:glutamate-gated chloride channels