Characterising gastrointestinal nematode epidemiology and benzimidazole resistance in farmed and wild ruminants

The movement of animals between farms and co-grazing with wild ruminants have been associated with increased risk of roundworm infections in livestock and the development and spread of anthelmintic resistance. Gastrointestinal nematode infections (GIN) are ubiquitous in small and large ruminants in both the UK and worldwide, threatening the health and welfare of infected animals and the productivity and economic stability of the sheep and cattle industries, with GIN infections costing an estimated £84 billion per annum (Charlier et al., 2020; Mavrot et al., 2015; Roeber et al., 2013). The data presented in the following chapters has substantially added to the current knowledge on GIN infections in wild and farmed ruminants in the UK, and the current state of benzimidazole resistance in these hosts. This thesis aimed to explore current roundworm epidemiology in UK livestock and wildlife, and to assess the risk posed in the spread of GIN infections and anthelmintic resistance by the introduction of stock onto farm, and the co-grazing with wild ruminants. The most economically important GIN species infecting sheep over the last century are Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus spp. (Burgess et al., 2012; Parnell, 1954; Taylor and Pearson, 1978). In cattle, the main species of economic importance are Ostertagia, Cooperia, Trichostrongylus and Haemonchus (COWS, 2020; Louvandini et al., 2009). GIN infections are most prevalent from mid-summer to autumn and cause parasitic gastroenteritis in effected animals (Burgess et al., 2012; Hamer et al., 2019). In addition, many livestock nematode species have developed resistance to benzimidazoles, one of the original and most widely used anthelmintic classes, first introduced in 1961, with resistance first reported in 1964 (Drudge et al., 1964). Despite the presence of resistance to this compound, benzimidazoles are still commonly used to date. The mechanism of BZ resistance has been well studied over the last 40 years, with the first discovery of an associated SNP in the beta-tubulin isotype-1 gene discovered in the last 1980s, at position 200 (F200Y) (Kwa et al., 1995, 1994). Subsequently, two additional SNPs have been found in numerous trichostrongylid species such as T. circumcincta and T. colubriformis (Silvestre and Humbert, 2002), at positions 198 (E198A, E198L) and 167 (F167Y) to confer benzimidazole resistance. One of the key factors involved in the dissemination of GINs, particularly resistant populations is animal movement and ineffective quarantine practices . Over 10 million sheep and 1 million cattle are sold through ~ 100 livestock auction marts each year in England and Wales (“Livestock Auctioneers’ Association Limited (LAA),” 2023). In addition, previous studies have shown a lack of uptake in quarantine recommendations over the last decade. Morgan et al (2013) found that only 3% of sheep farmers were effectively quarantining. In additional studies by SCOPS (2017) and the UK Government (2019) it was found that only around 50% of sheep and cattle farmers will always quarantine new stock, and effectively (“SCOPS | Sustainable Control of Parasites in Sheep,” 2023). Therefore, animals transported between farms pose a potential threat in the transmission of disease, specifically GINs. Sampling at auction marts provided an insight into the extent of GIN movement in traded animals and allowed us to collect samples originating from a large number of farms and different livestock categories. To explore the role of ‘natural’ animal movement (i.e., wildlife), we also included samples from wild deer in the project. Deer are widespread in the UK, and studies suggest that the range and abundance of deer species in Western Europe is expanding (Ward, 2005), it was therefore important to assess whether they were acting as a reservoir for GIN (particularly anthelmintic resistant populations) and representing an additional risk in the spread of infection through animal movement (Böhm et al., 2007; Ward, 2005). Studies have suggested that both the spread of livestock GINs and potentially resistant strains, could occur through wild deer populations, potentially due to a lack or inability to access effective biosecurity measures (Chintoan-Uta et al., 2014; Lindberg and Vatta, 2006). This thesis utilised applied and molecular parasitological techniques in conjunction with statistical, mathematical and network modelling to investigate roundworm epidemiology, BZ (benzimidazole) resistance and the risk associated with animal movement. Faecal egg counts (FEC) were utilised to determine strongyle prevalence and average count in UK livestock and wildlife (Christie and Jackson, 1982). Next generation sequencing of the Internal Transcribed Spacer 2 (ITS-2) region allowed for species identification of multiple livestock-specific and generalist trichostrongylid species in sheep, cattle and deer populations (Avramenko et al., 2019, 2015; Workentine et al., 2020). Next generation sequencing of the beta-tubulin isotype-1 gene allowed for identification and quantification of SNPs associated with BZ-resistance in livestock trichostrongylid populations (Avramenko, et al. 2019). Statistical modelling, specifically generalised linear models and recursive partitioning was utilised to investigate the effects of multiple host and environmental factors, in relation to strongyle count, prevalence, and species composition. Network modelling was used to better understand farmer’s transportation trends, and to conduct a risk analysis of potential movement trends. These results in conjunction with the species composition data and the BZ-resistance allele frequencies in sheep and cattle, highlighted the potential risk of spread of infection through traded livestock and the importance of effective biosecurity. Utilising mathematical modelling, specifically the GLOWORM-FL model (Rose et al., 2015) allowed outputs to be created to better inform stakeholders of the need for effective quarantine treatment of purchased stock. Of the sheep sampled (n = 491), 443 had a positive count with a mean strongyle eggs per gram (epg) of 217±23 epg (range 0-11268). T. circumcincta was the most prevalent species, identified via next generation sequencing of the ITS-2 gene, found in 100% of samples tested (n = 262), comprising 57% (range 1-99) of the total composition. In addition, species including T. vitrinus, T. colubriformis and H. contortus were found. SNPs were found at positions 167, 198 and 200 in four trichostrongylid species: T. circumcincta, T. axei, T. colubriformis and H. contortus, with T. circumcincta having the highest SNP frequency overall, with F200Y mutation identified in 61% of samples with a frequency of 69±6% (12-100%). Of the cattle sampled (n=311), 210 had a positive strongyle count, with a mean count of 20±3 epg (range 1-747). Both multiplex-tandem PCR (MT-PCR) and next generation sequencing found the most prevalent species to be O. ostertagi and C. oncophora. Very low levels of BZ-resistant SNPS were identified in GINS of cattle, with resistant alleles found in C. oncophora, O. ostertagi and T. axei, also found at low frequency, with two populations of O. ostertagi identified as having the F200Y mutation, at 2-5% frequency. Samples collected from farmed deer (n=341) had a strongyle count of 25±2 epg (0-324) with 89% of those analysed having a positive strongyle count. Only three strongyle species were found in farmed deer samples, O. venulosum, O. leptospicularis and low levels of C. oncophora. Wild deer faecal samples collected from 645 deer, had a mean count of 32±4 epg (0-2376) and positive counts were observed in 77% of samples. The most prevalent species found by ITS-2 sequencing were O. venulosum found in 76% of populations at a mean composition of 42% and O. leptospicularis found in 80% of populations at a mean composition of 42%. In addition, other livestock specific species such as O. ostertagi and T. circumcincta were found. BZ-resistant SNPs were only identified in five wild deer populations and two farmed deer populations, with the most prevalent SNP being F200Y in T. circumcincta, found at high frequencies in four of the samples. In addition to looking at the roundworm epidemiology and BZ-resistant SNP allele frequency in multiple ruminant species, farmers transportation trends were analysed using spatial and network models. Traded sheep (n=407) were transported on average 61±4 km (range 0-512) to market and 163±7 km (1-550) to farm following sale. From a subset of 153 farms, only 42% of sheep farmers attended their nearest livestock market for sale. Traded cattle (n=340) travelled an average of 59±5 km (2- 800) to market and 80 ± 5 km (2-509) to farm post sale, and only 30% attended their nearest livestock sale. Finally, the GLOWORM-FL mathematical model (Rose, et al. 2015) was used to quantify potential pasture contamination and therefore proposed infection risk post introduction of new stock on farm. The models were run for two main trichostrongylid species: T. circumcincta, and O. ostertagi. Simulations were designed to explore the difference in potential pasture contamination resulting from farmers implementing (i) best practice recommended quarantine strategies, (ii) administering an ineffective anthelmintic as a quarantine treatment and (iii) no quarantine. The models also looked at regional and time-based differences in potential pasture contamination following the introduction of animals onto farm, with the goal to use these model outputs to create resources to educate stakeholders in the importance of effective quarantine and promote uptake of industry recommendations. The findings highlight the importance of quarantine and effective treatments for traded ruminants in the UK. In addition, the data shows potential risk factors that may increase likelihood of spread of infection, and the need to better disseminate information to stakeholders on the prevalence of anthelmintic resistance in roundworms. With such a large number of animals being transported through auction markets every day in the UK, and the potential risks involved in the introduction of stock onto farm, this thesis acts as a starting point for additional research into animal movement as a risk factor in the spread of anthelmintic resistance and how this could be minimised in future. In addition, this thesis explored the use of mathematical modelling as a resource for knowledge exchange, further development of the prototype resources presented here could be adapted into a web resource for stakeholders. By exploring roundworm prevalence and anthelmintic resistance in wild deer populations, this thesis also acknowledged other biosecurity risks, such as cograzing with livestock, to help provide an updated understanding of risk to UK farmers. This thesis has provided an insight into the risk associated with animal movement (both trade and wildlife) which could inform the development of effective biosecurity recommendations for the UK livestock industry.