Drug resistance and susceptibility in sheep nematodes: Fitness and the role of anthelmintic combinations in resistance management

Anthelmintic resistance (AR) is a growing challenge within the livestock industry. Current control methods for gastrointestinal parasitism are failing fast and alternative methods are desperately needed. For this to be viable a deeper understanding of parasite biology and behaviour is required. This thesis aims to assess Teladorsagia circumcincta isolates of various AR statuses with in vitro fitness bioassays. This thesis is split into 3 objectives: 1) Investigating differences in the fitness response of isolates of different known AR statuses. 2) Discerning if there are fitness differences between T. circumcincta isolates sourced from different farms. 3) Breaking down an individual isolate into AR sub-populations using drug treatments and assessing if there are intra-isolate fitness differences. The data for studies in this thesis were collected in Ireland (Chapter 4) and New Zealand (Chapters 3, 4, and 5). The cold stress test (CST) measured the number of third stage larvae (L3) developing from eggs stored at 4 °C over time. The larval aging assay (LA) measured the locomotory activity of L3 after exposure to heat stress (30 °C) over time. The exsheathment assay (EX) measured exsheathment percentage of L3. Larval length (LL) used length as a proxy for fecundity. The egg hatch assay (EHA) measured basic egg hatch rate in water at room temperature. Chapter 3 assessed 4 isolates of known AR status (T and W – susceptible; D and M - multi-AR) using the CST, LA, EX, LL, and EHA in vitro fitness bioassays. Farm isolates (26) across Ireland (15) and New Zealand (11) had their anthelmintic efficacy tested and recorded, faecal samples were collected, coprocultured, and assessed by the CST, LA, and EX for Chapter 4. Three isolates (E, K, and W), split by drug treatment into 8 treatment groups were assessed (CST, LA, and EX) and results were compared within isolate for Chapter 5. The Chapter 3 CST found no differences between isolates or for isolate by time. Egg storage at 4 °C for any length of time resulted in significantly decreasing L3 recovered. For the LA, length of time L3 were incubated at 30 °C influenced L3 migration. For the EX, D had significantly higher exsheathment than M and W, and T had a significantly higher exsheathment than W. LL found that M was longer than D, and W was longer than all other isolates. The EHA displayed no significant differences for the isolates. Multivariate multiple regression analysis of Chapter 4 results showed that isolate country of origin significantly impacted fitness for the CST and LA. The efficacy of BZ and LV did not impact fitness. ML efficacy was a weak predictor of fitness across the three assays. Univariate analysis showed fitness differences were driven by the LA. Chapter 5 found several differences in fitness responses. The response of the C group to the CST differed depending on isolate, helping to justify the intra-isolate approach of Chapter 5. Not all results went in the same direction. Treatment groups for K displayed a greater ability to survive cold stress than the C group, while treatment groups for W displayed a reduced ability to survive cold stress than the C group. The LA was non-significant for E and W, but K was nearing significance. Time for L3 to reach 50% migration was non-significant between treatments for all isolates. For the EX, ML group for E was significant, K found all treatment groups were significant, and for W the BZ+LV group was non-significant, with all other treatment groups significantly different to the C group (for each isolate). Findings in this thesis indicate there are detectable and measurable differences in the fitness characteristics between isolates and AR sub-populations within an isolate. These results indicate an exciting new avenue of research. The novel approach of Chapter 5 offers a method not previously used for fitness assessment. Through this it is possible that a greater understanding of T. circumcincta biology.