Author ORCID Identifier

https://orcid.org/0000-0001-9309-9879

Year of Publication

2019

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Veterinary Science

First Advisor

Dr. Martin K. Nielsen

Abstract

Anthelmintic resistance of parasites infecting livestock animals is a global problem resulting in decreased animal welfare and production losses. Horses are not exempt from this issue as wide-spread anthelmintic resistance exists among the equine cyathostomins and Parascaris spp. Of the three drug classes available for treating equine intestinal helminths anthelmintic resistance, defined as less than 90-95% drug efficacy, exist to all three. New pharmaceutical control regimens and the elucidation of parasite drug response mechanisms are needed.

Two studies were carried out evaluating combination deworming regimens. A population of cyathostomins with known resistance to the benzimidazole (BZ) and pyrimidine drug classes maintained in a herd of Shetland ponies was used. Fecal egg counts were performed every two weeks and used to evaluate drug efficacy. The first study evaluated the combination of a BZ and pyrimidine drug for four consecutive treatments, and compared the individual drug efficacies before and after combination use. The first combination treatment exhibited an additive effect at 76.6%, but the subsequent three combination treatments decreased to approximately 40%. There was no significant difference between the initial and final efficacies of individual drugs (BZ, p=0.4421; pyrimidine, p=0.8361). It appears the combination treatment selected for double-drug resistant adult parasites. The timeframe of this study (1 year) and the one year lifespan of adult cyathostomins prevented observations of combination treatment on subsequent generations, however given the sustainability of resistance in this cyathostomin population, it seems unlikely efficacy would improve over time. The second study examined the combination of a BZ drug with a macrocyclic lactone (ML) drug. This parasite population was 100% naïve to the ML drug class. This study was carried out in a similar manner to the first, except only two combination treatments were given. ML exhibited 100% efficacy when it was used alone, or in combination. The initial and final BZ efficacy did not significantly differ (p=0.9890). In summary, the results described herein do not support the use of combination treatments where resistance is prevalent, but more long term studies are needed to fully understand the long-term effects on subsequent generations.

The in vitro maintenance of Parascaris spp. provides opportunity for various molecular analyses. An objective motility scoring assessment allowed for continuous monitoring of worm viability. In this study, several saline solutions, nutrient supplements, environmental conditions, and Roswell-Park Memorial Institute medium 1640 (RPMI-1640) were evaluated for the longevity and viability of adult Parascaris spp. Overall, RPMI-1640 resulted in better longevity (168 hours) and significantly better viability (pParascaris spp. to in vitro drug exposure. Oxibendazole at 10 µg/mL for 24 hours and ivermectin at 1 µg/mL for three hours were employed, and worms were used for transcriptomic analyses to identify drug response mechanisms. The top four genes which were significantly different between drug treated and control groups were: cyp4504C1, sup-9, frmd4a, and klhdc10. It is hypothesized that cyp4504C1 and klhdc10 are drug detox mechanisms, while sup-9 and frmd4a may be indirect response related to the drug effects. Their expression was further evaluated using quantitative RT-PCR, however there was no significant difference in any gene expression between groups. It should be noted that there are several limitations associated with the qPCR method, and the lack of significance should not rule out the possible involvement of these genes and more research on drug response mechanisms is needed.

In summary, there is very little research regarding combination deworming in horses, and their current use is largely due to some success for ruminant parasites, but the current work summarized herein does not support their use. Finally, until now the lack of in vitro methods for equine helminths has significantly delayed the elucidation of drug response mechanisms. This was the first whole-transcriptome approach for any ascarid parasite and uncovered proteins with possible involvement in drug metabolism or compensate for the toxic effects Overall, the research surrounding anthelmintic resistance in livestock helminths, particularly in horses, is lacking and the resistance crisis demands further investigation.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2019.225

Funding Information

This work was financially supported by Zoetis, the Gluck Equine Research Foundation, and the Burroughs-Wellcome Fund.

Supplementary file, gene list all.txt (892 kB)
Supplemental Data: List of all genes

Supplementary file, gene list drug vs control.txt (4 kB)
Supplemental Data: List of genes between all drug treated and controls

Supplementary file, gene list IVM vs IVMC.txt (2 kB)
Supplemental Data List of genes between IVM and IVM controls

Supplementary file, gene list OBZ vs OBZC.txt (8 kB)
Supplemental Data List of genes between OBZ and OBZ controls

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