Monitoring drug efficacies in equid nematodes

Abstract

Advances in technology are an important weapon in the fight against anthelmintic resistance. Improved diagnostics are therefore crucial to enable targeted selective treatments and avoid unnecessary anthelmintic use. In common with other grazing animals, equines are at risk of infection with intestinal parasites, and it is crucial that effective control measures are maintained. Evidence suggests the re-emergence of large Strongyles such as Strongylus vulgaris given reduced treatment of cyathostomins. Thus, minimising cyathostomin anthelmintic exposure, and controlling the pathogenic large strongyles is a key balancing act. Faecal egg counts (FECs) are the standard method of diagnosing the level of parasitic infection in horses and other grazing animals. Testing before treatment is an important factor in slowing the appearance of anthelmintic resistance in nematode parasites of horses. The FECPAKG2 allows farmers to perform FECs on their own sheep and cattle, without the need for any specialist parasitology knowledge. The current work has optimised the FECPAKG2 (G2) method for horses, and validated this against the original FECPAK (G1), using faecal samples from 114 horses in Wales and New Zealand. No significant difference was observed between the FECs obtained using the two methods (rmANOVA: F1,37 = 0.052, p = 0.821, ŋ2p = 0.001) with no effect noted linked to the country of origin. In addition, the accuracy of the G2 method was not affected by FEC level (r = -0.251 (CI: 0.030, -0.472) p= 0.124 n = 39). Further improvements to the G2 method such as a lower detection limit are possible with planned improvements in the imaging process. Following validation, the FECPAKG2 approach was used to identify anthelmintic resistance to allow the assessment of altering equine nemabiomes pre and post anthelmintic treatment. DNA from twelve paired faecal samples, pre- and posttreatment with Fenbendazole were sequenced using the MinION (Oxford Nanopore Technologies Ltd.). Six major cyathostomin species were identified across all equine nemabiomes. The most numerous species was Cyathostomum catinatum (39% of pre-treatment sequences) with this species displaying consistent resistance to Fenbendazole with 96% still remaining after treatment. It was noted that BZ resistance is potentially linked to predilection site within the GI tract. These findings are consistent with the idea that anthelmintic resistance develops more quickly when sub-lethal doses are administered, and suggests that there is insufficient Fenbendazole remaining in the gut to effectively control helminths towards the end of the gastro-intestinal tract. FECPAKG2 and MinION nemabiome sequencing were also trialled and found to be effective for helminth monitoring in zebra. The work also demonstrated that the cyathostomin nemabiome sequencing method was effective even at low FEC levels. The zebra in this study harboured the same species of cyathostomin as had been found in the equine samples. However, the zebra nemabiomes suggests that the history of repeated BZ administration may have influenced the host nemabiomes towards those species least responsive to BZ treatment. Given the work conducted the FECPAKG2 represents an acceptable method for equine FECs. It is hoped that the user-friendliness of the method will increase the uptake of FECs amongst horse owners, either by direct use of the technology or through their veterinary practice, hence slowing the development of anthelmintic resistance. It was also concluded that MinION sequencing offers a useful method of determining which species of strongyle are present, facilitating a differential diagnosis between more or less pathogenic species and enabling informed treatment decisions to be made. Interestingly, the entire process of FEC and nemabiome sequencing are portable for use in the field, and thus could be utilised to monitor strongyle infections of exotic equids in wild populations.

Date of Award	2020
Original language	English
Awarding Institution	Aberystwyth University
Sponsors	Knowledge Economy Skills Scholarships
Supervisor	Russ Morphew (Supervisor) & Peter Brophy (Supervisor)