title: Teladorsagia circumcincta: Molecular characterisation of the avr-14B subunit and its relatively minor role in ivermectin resistance.
abstract: Individual mutations (e.g. L256F) and polymorphisms in the avr-14B gene, a glutamate-gated chloride channel subunit, have been associated with ivermectin (IVM) resistance in Caenorhabditis elegans and Cooperia oncophora. The aim of the present study was to determine the full-length coding sequence of the avr-14B subunit homologue in Teladorsagia circumcincta and determine the presence/absence of the putative L256F SNP or any other potential SNPs of interest. Subsequently, we investigated sequence polymorphisms and transcription patterns between four different T. circumcincta isolates: two from Scotland (MTci1 susceptible and MTci5 triple resistant to benzimidazoles, levamisole and IVM) and two from Spain (S-Sp susceptible and R-Sp double resistant to levamisole and IVM). The complete amino acid sequence of the T. circumcincta avr-14B subunit comprises 438 amino acids. Pyrosequencing analysis failed to detect the presence of the L256F mutation in any of the MTci5 or Sp-R samples tested. However, we revealed significant allele frequency changes by means of SSCP analysis of a 106 bp region encompassing the L256F SNP. Allele E showed the greatest change, following IVM exposure in vitro and in vivo, although sequence analysis did not reveal any coding changes. Sequence analysis of the full-length avr-14B coding sequence showed that two SNPs exclusively found in the resistant strain McTi5 (I270F and T305A) are situated in codons involved in the interaction of the receptor with IVM. Moreover, other potentially significant SNPs (K361E and L364M) were identified between transmembrane regions 3 and 4. However, due to the low frequency of all these SNPs, we cannot conclude they confer IVM resistance in T. circumcincta. Moreover, a modest increase in expression of the avr-14B in both resistant isolates has been shown although these differences were not sufficiently great to consider avr-14B to be the sole or even a major determinant of IVM resistance in this species.
authors.affiliation: Instituto de Ganadería de Montana (CSIC-ULE), Finca de Marzanas, 24346 Grulleros, León, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Martínez-Valladares
authors.affiliation: Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Geldhof
authors.affiliation: School of Veterinary Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, 464 Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Jonsson
authors.affiliation: Instituto de Ganadería de Montana (CSIC-ULE), Finca de Marzanas, 24346 Grulleros, León, Spain ; Faculty of Veterinary Medicine, University of León, Campus de Vegazana, 24071 León, Spain.
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Rojo-Vázquez
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh EH26 0PZ, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Skuce
keywords: Anthelmintic resistance; GluCl channels; Ivermectin; Sheep; Teladorsagia circumcincta