PGP expression in Cooperia oncophora before and after ivermectin selection

Abstract

The aim of this study was to investigate genetic selection and P-glycoprotein (PGP) expression in three different isolates of Cooperia oncophora before treatment and after ivermectin (IVM) injection. Adult parasites were recovered from nine calves experimentally infected with the isolates represented by one IVM susceptible laboratory isolate, and two field isolates showing signs of phenotypic macrocyclic lactone resilience according to the faecal egg count reduction test. Five males and five females per isolate were examined both pre- and post-IVM treatment giving a total of 60 worms. A sequence from C. oncophora (Con-pgp) was identified, showing 83 % similarity to Pgp-9 of Caenorhabditis elegans. Primers specific to putative Con-pgp-9 mRNA were designed, generating a 153-bp PCR product. Total RNA was prepared from all worms, and Con-pgp-9 expression was measured by quantitative real-time reverse transcription PCR. Our results showed that mean PGP concentrations were four to five times higher in female as compared to male worms. No significant differences in gene expression between experimental groups pre- and post-IVM selection were detected. However, PGP gene expression tended to be increased by IVM treatment in male worms (p = 0.091), with 70 % higher mean expression in treated than in untreated male worms. Amplified fragment length polymorphism analysis did not demonstrate any bottleneck effect within the different isolates post-treatment. The total mean gene diversity values were 0.158 and 0.153 before and after treatment, respectively. Inbreeding coefficient in subpopulations compared to total population FST was 0.0112, suggesting no genetic differentiation between or within the investigated isolates in relation to treatment. In conclusion, comparison of Con-pgp-9 expression showed no significant difference before and after treatment, but some tendency towards increasing expression in male worms.

The role of drug efflux systems in anthelmintic resistance in parasitic nematodes

“…However, there were no significant differences observed between the expression patterns of P-gp genes before and after IVM treatment in C. oncophora and H. contortus (Williamson and Wolstenholme, 2012;Areskog et al, 2013). Therefore, the role of these trans-membrane proteins as a drug efflux mechanism in nematodes is still clouded and needs further investigation.…”

“…The expression patterns of P-gps in nematodes seem to be quite variable, with some reports linking them to AR (Dicker et al, 2011;Williamson et al, 2011) and other studies finding no association (Williamson and Wolstenholme, 2012;Areskog et al, 2013). The increased transcription of several P-gp genes in the drug-resistant WAL larvae suggests that these particular P-gps may play a role in the resistance phenotype shown by this isolate.…”

“…These proteins are proposed to be important players in the development of resistance to IVM because there have been reports describing an over-expression of multiple P-gps in different nematodes resistant to this class of anthelmintic (Sangster and Gill, 1999;Le Jambre et al, 2000;Dicker et al, 2011). The up-regulation of helminth P-gps gene expression enhances the parasite's ability to survive IVM exposure (Areskog et al, 2013) and also supports its role in the development of resistance to this drug. These efflux proteins are also called multi-drug resistance transporters and have been identified as a mechanism of resistance both in mammals and nematodes.…”

“…Therefore, ABC transporters might be involved in active efflux of anthelmintic drugs away from their target sites, resulting in decreased drug concentration and increased parasite survival. It has been suggested that commonly used anthelmintics such as ivermectin (IVM), levamisole (LEV) and thiabendazole are substrates of ABC transporters (Kerboeuf and Guegnard, 2011;Falasca and Linton, 2012), and there is inconsistent evidence that exposure to anthelmintics modulates the expression patterns of different ABC transporters in nematodes Areskog et al, 2013;. Modulation of the abundance or activity of these transport proteins (particularly Pglycoprotein) has been suggested as a possible mechanism of AR in nematodes (Blackhall et al, 1998a).…”

“…contortus and Cooperia oncophora worms collected from animals treated with IVM compared to the worms collected from untreated control animals (Areskog et al, 2013;Alvarez et al, 2015).…”