title: Novel approaches to the control of cyathostomins in equids
Creators: name: Peachey, LE
Publisher: name: University of Liverpool Repository
Year: 2016

description: Cyathostomins, are clade 5 gastrointestinal (GI) nematodes infecting equids. They are associated with a range of pathologies, the most serious of which is larval cyathostominosis, a protein losing enteropathy with a 50 % mortality rate. Importantly, they are the most abundant GI nematode of equids in the developed world and, as they do not induce protective immunity, equids remain at risk of infection throughout their lives. The effective control of cyathostomins is currently threatened by the development of anthelmintic resistance (AR) to all three major classes of anthelmintic licenced for use in equids. Of primary concern is the emerging resistance to the macrocyclic lactones (MLs), which are the mainstay of cyathostomin control. It is therefore important that novel options for controlling cyathostomins are explored. In this study in vitro tests were used to explore a range of options for developing novel treatments against AR cyathostomins and improving the efficacy of the widely used ML, ivermectin (IVM). First the potential use of ethnoveterinary medicines for treatment of cyathostomins in equids in Ethiopia and the UK was explored (Chapters 3, 4 and 5). A participatory rural appraisal was performed in the Oromia region of Ethiopia to identify plants currently used as anthelmintics in equids and other livestock. A total of 37 species of plant were identified, data on dosing and side effects were also recorded. These data were triangulated with a literature review to identify five plants for in vitro screening for anthelmintic activity against cyathostomins. A literature review was used to identify plants for in vitro screening in the UK. A total of 138 publications, reporting the anthelmintic activity of plant extracts against nematodes, were found. The data collected was used to design a ranking system to provide a shortlist of five plant candidates. Shortlisted plants were collected, dried and chemically extracted to give crude extracts, which were screened against cyathostomins in the egg hatch test (EHT) and larval migration inhibition test (LMIT). A total of 7/9 plants screened in this way showed anthelmintic activity in either the EHT and/or the LMIT with median effective concentrations (EC-50s) in the range 0.18-8.90 mg/ml. For some of the most efficacious extracts there were reports of in vivo efficacy against GI nematodes of other veterinary parasitic nematodes, making these good candidates for future in vivo trials. One of the plants shortlisted for the UK, for which there was extensive evidence of anthelmintic activity in the literature, was the cysteine proteinase (CP) containing Carica papaya. Extract from this species, papaya latex supernatant (PLS), could not be processed and tested using the same protocols as the other plant species, as the active compound is an enzyme. The EHT and LMIT were optimised for use with PLS and its efficacy evaluated in chapter 5. It was found that PLS had a potent anthelmintic effect against cyathostomins in the EHT, which was attributable to the action of CPs, with EC-50 values in repeats ranging between 0.12-0.22 µM. PLS also showed efficacy in the LMIT, although this appeared due to another active compound. To the authors knowledge this is the first report of efficacy of PLS against the free living stages of any parasitic nematode, which may indicate that cyathostomins are particularly susceptible to CPs. In chapter 6 the role of P-glycoproteins (P-gps) in IVM resistance in cyathostomins was investigated, with the more specific aim of assessing whether P-gp inhibitors could potentially be used in combination with IVM to improve its efficacy against AR parasites. The transcription of pgp-9, which is putatively associated with IVM resistance in Teladorsagia circumcincta and Haemonchus contortus, was measured after exposure to IVM in in ML resistant (AR) versus anthelmintic naïve (AN) cyathostomin third stage larvae (L3). Pgp-9 expression was significantly increased after incubation with IVM in the AR, but not the AN, parasite population. Two in vitro tests, the LMIT and the larval development test (LDT), were used to assess the effect of a range of P-gp inhibitors (ketoconazole, pluronic 85 and ivermectin aglycone) on IVM efficacy in ML resistant (AR) versus anthelmintic naïve (AN) cyathostomins. In the LMIT ketoconazole and ivermectin aglycone conferred a significant increase in IVM efficacy in the AR parasites, but had less effect on the AN parasites. Pluronic 85 also had a profound effect on IVM efficacy which depended on IVM concentration, however there was no differential effect dependent on parasite population. In the LDT, ketoconazole and pluronic 85 both significantly increased IVM efficacy in both AR and AN parasites. These results suggest that there is a lifecycle stage-specific association of P-gps with AR cyathostomins, and that P-gp inhibitors could potentially be used to improve IVM efficacy in AR cyathostomin populations. In summary in this study has identified a range of potential novel treatments for cyathostomins. The data also suggest a role for P-gps in the emerging resistance to MLs, which could potentially be exploited to improve ML efficacy in AR cyathostomin populations. Importantly, this work will underpin the financial and ethical justification for future in vivo trials on the extracts/compounds tested here.