ivermectin

P-glycoproteins play a role in ivermectin resistance in cyathostomins

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.

Volume
7
Issue
3
Publication date
Country

Development of the larval migration inhibition test for comparative analysis of ivermectin sensitivity in cyathostomin populations

Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes have been controlled using broad-spectrum anthelmintics; however, cyathostomin resistance to each anthelmintic class has been reported and populations insensitive to more than one class are relatively commonplace. The faecal egg count reduction test (FECRT) is considered the most suitable method for screening anthelmintic sensitivity in horses, but is subject to variation and is relatively time-consuming to perform. Here, we describe a larval migration inhibition test (LMIT) to assess ivermectin (IVM) sensitivity in cyathostomin populations. This test measures the paralysing effect of IVM on the ability of third stage larvae (L3) to migrate through a pore mesh. When L3 from a single faecal sample were examined on multiple occasions, variation in migration was observed: this was associated with the length of time that the L3 had been stored before testing but the association was not significant. Half maximal effective concentration (EC50) values were then obtained for cyathostomin L3 from six populations of horses or donkeys that showed varying sensitivity to IVM in previous FECRTs. Larvae from populations indicated as IVM resistant by FECRT displayed significantly higher EC50 values in the LMIT than L3 from populations classified as IVM sensitive or L3 from populations that had not been previously exposed to IVM or had limited prior exposure. The analysis also showed that EC50 values obtained using L3 from animals in which IVM faecal egg count reduction (FECR) levels had been recorded as <95% were significantly higher than EC50 values obtained using L3 from animals for which FECR was measured as >95%. For one of the populations, time that had elapsed since IVM administration had an effect on the EC50 value obtained, with a longer time since treatment associated with lower EC50 values. These results indicate that the LMIT has value in discriminating IVM sensitivity amongst cyathostomin populations, but several factors were identified that need to be taken into account when executing the test and interpreting the derived data.

Publication date
Country

The p-glycoprotein inhibitor ketoconazole causes a reversion to sensitivity in ivermectin resistant cyathostomins in vitro

Laura Peachey
Jacqui. B. Matthews
Gina L. Pinchbeck
Faith A. Burden
Jane E. Hodgkinson
Presentation date

Anthelmintic resistance is a growing problem in both the developed and developing world; of most concern is the level of resistance detected against the potent macrocylic lactone (ML) anthelmintics. To identify and target a common mechanism of resistance to anthelmintics would allow potential modification of existing drugs, and may even enable the prediction and prevention of the development of resistance to novel drugs. There is a growing body of evidence that P-glycoproteins (P-gps) are involved in resistance to the MLs in many parasitic nematodes of humans and veterinary species (Ardelli et al, 2011). P-gps belong to class two of the ATP binding cassette (ABC) transporter protein superfamily; they are responsible for the active removal of xenobiotic compounds from cells. The cyathostomins are gastrointestinal nematodes of equids that cause significant pathology. Recently resistance to MLs has been described in cyathostomins (Molento et al, 2008), its mechanisms have not yet been elucidated.

Country
Not published as conference proceedings

The p-glycoprotein inhibitor ketoconazole causes a reversion to ivermectin sensitivity in cyathostomins in vitro

Laura Peachey
Jacqui. B. Matthews
Gina L. Pinchbeck
Faith A. Burden
Nikki Stradling
Jane E. Hodgkinson
Presentation date

Anthelmintic resistance is a major veterinary and public health issue globally, of most concern is the level of resistance to the macrocyclic lactones. Recent studies have identified a role in resistance for the ATP binding cassette (ABC) drug transporters, P-glycoproteins (P-gps). This study demonstrates the effect of the P-gp inhibitor ketoconazole on the efficacy of ivermectin (IVM) against equid cyathostomin larvae using the larval migration inhibition test (LMIT). Third stage cyathostomin larvae (L3) were cultured from two populations; 1) with recent history of IVM resistance in vivo and 2) naive to anthelmintic exposure. The sensitivity to IVM in each group (n=8) was characterised using the LMIT. The IVM LMIT was repeated for each sample with and without the addition of 10µM ketoconazole. Probit analysis was performed on grouped data from each population to give LC-50 values. The LC-50 value for IVM in Populations 1 and 2 was 4.9 and 2.4µg/ml respectively indicating that Population 1 has a resistant phenotype in comparison to Population 2. Addition of 10µM ketoconazole to IVM in Population 1 caused a drop in LC-50 value from 5.8 to 1.6µg/ml. In Population 2 the effect of the addition of ketoconazole was negligible (1.1 to 0.9µg/ml). This study demonstrates that the P-gp inhibitor ketoconazole causes reversion to a sensitive phenotype in IVM-resistant cyathostomins, inferring that P-gps play a role in their resistance to IVM. This work will be corroborated by investigation into P-gp genes and their expression in cyathostomins.

Country
Not published as conference proceedings

Efficacy of two endectocides (moxidectin and ivermectin) against strongylus spp. parasites in UK donkeys

Elena Barrio
F. J. Vasquez
Presentation date

The Donkey Sanctuary is a UK based charity in Sidmouth, Devon. Its mission is to transform the quality of life for donkeys, mules and people worldwide through greater understanding, collaboration and support, and by promoting lasting, mutually life-enhancing relationships. The Donkey Sanctuary has a total of seven farms in the UK including a reception farm for new arrivals (quarantine) where the study is based. The relinquished and rescued donkeys by can arrive to the quarantine farm from different origins, for that reason, it is important to follow a health control program on arrival which includes a full coprological exam; this will determine the need of using an specific worming protocol. One of the most prevalent parasites is Strongylus spp. and it is very common to find animals with a moderate egg count who are asymtomathyc. The general recommendation is to treat donkeys with an egg count of 300 to 400 eggs per gram (The two antihelmintics used during the isolation period were two endectocides: moxidectin (Equest, 0,4 mg/Kg) and ivermectin (Eqvalan, 0,2 mg/Kg).

The worming protocol used for those animals that have been considered healthy on arrival and that they did not have received a recent antihelmintic treatment was to recieve an initial treatment with Moxidectin with a quarantine period of 48 hour without access to pasture. Those animals that have recieved a recent worming treatment( within the last 6 weeks) with ivermectin or moxidectin, they were only treated if the faecal egg count was higher than 50 epg or a different type of parasite was found (for example pulmonary nematodes). No other actions were taken in those animals with Strongylus spp. after the first faecal egg count results if they had been treated with moxidectin, otherwise the previous described protocol had been followed. 

Country
Not published as conference proceedings
Subscribe to ivermectin