P-glycoproteins play a role in ivermectin resistance in cyathostomins

Abstract

Anthelmintic resistance is a global problem that threatens sustainable control of the equine gastrointestinal cyathostomins (Phylum Nematoda; Superfamily Strongyloidea). Of the three novel anthelmintic classes that have reached the veterinary market in the last decade, none are currently licenced in horses, hence current control regimens focus on prolonging the useful lifespan of licenced anthelmintics. This approach would be facilitated by knowledge of the resistance mechanisms to the most widely used anthelmintics, the macrocyclic lactones (ML). There are no data regarding resistance mechanisms to MLs in cyathostomins, although in other parasitic nematodes, the ABC transporters, P-glycoproteins (P-gps), have been implicated in playing an important role. Here, we tested the hypothesis that P-gps are, at least in part, responsible for reduced sensitivity to the ML ivermectin (IVM) in cyathostomins; first, by measuring transcript levels of pgp-9 in IVM resistant versus IVM sensitive third stage larvae (L3) pre-and post-IVM exposure in vitro. We then tested the effect of a range of P-gp inhibitors on the effect of IVM against the same populations of L3 using the in vitro larval development test (LDT) and larval migration inhibition test (LMIT). We demonstrated that, not only was pgp-9 transcription significantly increased in IVM resistant compared to IVM sensitive L3 after anthelmintic exposure (p < 0.001), but inhibition of P-gp activity significantly increased sensitivity of the larvae to IVM in vitro, an effect only observed in the IVM resistant larvae in the LMIT. These data strongly implicate a role for P-gps in IVM resistance in cyathostomins. Importantly, this raises the possibility that P-gp inhibitor-IVM combination treatments might be used in vivo to increase the effectiveness of IVM against cyathostomins in Equidae.

Keywords: Anthelmintic resistance; Cyathostomins; Ivermectin; P-glycoproteins.

Graphical abstract

Fig. 1

Maximum likelihood phylogenetic tree demonstrating the relationship between nucleotide sequences for DSW (A1-5 and B1-5) (highlighted red) and published pgp-9 sequences from Cylicocylus elongatus pgp-9 (A.N. KJ701410.1), Haemonchus contortus pgp-9 (A.N. JX430937.1), Teladorsagia circumcincta pgp-9 (A.N. FR691848.1), Caenorhabditis elegans pgp-9 (C47A10.1 wormbase i.d.) and Caenorhabditis briggsae pgp-9 (XM_002638567.1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Bar chart showing transcript levels of pgp-9 mRNA relative to the housekeeping gene, 18S, in third stage larvae in Populations 1 ‘resistant’ and 2 ‘susceptible’, which had been incubated with distilled water (H2O), dimethyl sulfoxide (DMSO) or ivermectin (IVM) (*p = <0.05; **p= <0.001). Within each population, there was a significant effect of the different treatments for Pop 1 (p = 0.005), but not for Pop 2 (p = 0.31).

Fig. 3

Ivermectin (IVM) concentration response curves in Population 1 ‘resistant’ and 2 ‘susceptible’ (Pop 1, Pop 2) in the larval development test (LDT) and larval migration inhibition test (LMIT), with and without the addition of the P-glycoprotein inhibitors ketoconazole (K), ivermectin aglycone (IVM-AG) and pluronic 85 (P85).