Probably the greatest confounding factor limiting interpretation of the FECRT (and the in vitro bioassays) was the species diversity. The presence of a highly dual-resistant population of T. circumcincta contributing only a minor proportion of eggs pre-treatment, allowed for the calculation of a considerably more
effective FECR percentage than was truly observed (McKenna, 1997). The change in species over time on this farm requires a FECRT with species identification to be performed when the farmer would usually treat the sheep – with the age group requiring treatment. The identity of BZ resistant T. axei would allow a veterinary surgeon to suggest that BZ anthelmintics are avoided during the late autumn/winter months if ‘black scours’ was the clinical diagnosis (Craig et al., 2006). However, ideally the FECRT would be repeated when this species was more prevalent. On this farm therefore, reduced anthelmintic efficacy cannot be correctly interpreted without species data. But species identification carries additional costs, and is not routinely performed in the UK. Nevertheless, where an anthelmintic is highly effective, such as in the case of MOX on this farm, the FECRT is a useful confirmation of success. It could also aid diagnosis for causes of weight loss, poor growth and diarrhoea or anaemia not caused by PGE by ruling out anthelmintic resistance, while simultaneously providing beneficial information to the farmer. In addition, although there are limitations to the interpretation of a FECRT as to the number of adults remaining within the host (Cabaret et al., 1998), it is an excellent indicator of the reduction in pasture contamination.
The use of bioassays such as the EHT and LDT provide an understanding of the anthelmintic resistance phenotype of free-living stages of strongyles. Although these may not necessarily reflect the in vivo mechanisms of resistance (Rufener et al., 2009a; Barrère et al., 2012; Kotze et al., 2012), they are quicker than performing a FECRT, identifying anthelmintic efficacy within just 48 hours to a week. Bioassays only require a single faecal sample collection, and do not contribute to drug selection on farm. In this study, the EHT worked
comparatively well and identified the presence of dual-resistant parasites without the need to treat hosts with two anthelmintics. Over the season, the percentage hatch in the definitive dose well reflected recent BZ anthelmintic treatments, reducing as time since the last treatment increased. Variability between replicate wells of a test increased as the species diversity increased, and the abundance of T. circumcincta reduced. An exception was the test performed at the end of August, when there was high variability between wells, despite a high percentage of T. circumcincta, suggesting that there may have been some variation in the resistance phenotype of the T. circumcincta
population at that time. However, without PCR speciation of eggs and L1 larvae, which is time-consuming and can be technically challenging post EHT, the results are difficult to interpret. For both tests, as for coproculture, it is important to remember that not all species will develop equally well at a set temperature, and over a set time period (Dobson et al., 1992). This was potentially observed in the present study, with differences in the control wells pre- and post-
treatment. This may bias results towards those species more suited to the test set-up, compromising interpretation of the anthelmintic efficacy.
While the EHT appeared to detect BZ resistance reasonably well, and has been ring-tested in Europe using lab isolates of H. contortus (von Samson-
Himmelstjerna et al., 2009a), the LDT did not prove as useful in this study. There was little change in the ED50 post-IVM treatment in September (RR = 1.7), despite a considerable reduction in the proportion of IVM sensitive species in
vivo. Similar RRs were also identified between IVM resistant and susceptible
isolates of H. contortus using an agar based LDT, when IVM was the anthelmintic tested (Gill et al., 1995). However, larger RRs were obtained using more polar avermectins, and using a different avermectin as a proxy measure of IVM
resistance may provide more informative measures of IVM phenotype (Gill et al., 1995). In contrast, there was a marked difference between the pre- and post-BZ treatment populations ED50 in the EHT (RR = 12.8). In addition, although the EHT pre-treatment suggested a generally susceptible population, there was the presence of a more resistant sub-population detectable by hatching percentages at the 0.2 and 0.3 mg/ml concentrations (von Samson-Himmelstjerna et al., 2009a). Collectively these results may suggest a similar genetic basis for BZ resistance both in vivo and in vitro in this field population. In contrast, different
genetic mechanisms may underlie in vitro and in vivo IVM resistance, or the in
vitro tests may be inappropriate (George et al., 2017).
Identifying resistance using molecular methods allows for rapid and robust understanding of the resistance status of a population. It can also be performed using eggs, greatly increasing the speed of diagnosis, provided the method is sensitive to extremely low quantities of DNA. For BZ, the presence of the β- tubulin isotype-1 SNPs at codons 167, 198 and 200 have been closely associated with BZ resistance (Kwa et al., 1994; Silvestre and Cabaret, 2002; Ghisi et al., 2007). While these SNPs can be detected in a laboratory setting, no commercial test has been developed for use on farm. In this study, we used pyrosequencing, a technique which provides genotype and some haplotype data for each
individual sequenced. The results from this farm clearly indicate the importance of testing all three codon positions. Codon 200 was the most commonly mutated, as has been found elsewhere (Redman et al., 2015; Ramünke et al., 2016).
However, in some studies, codon 198 was found to have a higher frequency of mutations than codon 200 within the sample population tested (Redman et al., 2015; Esteban-Ballesteros et al., 2017). Most studies have associated leucine at P198 with BZ resistance in T. circumcincta, as was found here, but some have found alanine (Esteban-Ballesteros et al., 2017), which is more commonly identified in H. contortus (Chaudhry et al., 2015), or valine (Avramenko et al., 2019). Although it may be a sequencing error (n=1), the presence of a stop codon was detected at codon 198 in this study. This diversity increases both the cost and the time taken to obtain a conclusive answer. The need to first select and speciate individuals restricts the use of molecular techniques. Newer
techniques, including MiSeq, are now being used to sequence pools of strongyles to determine BZ resistance haplotypes, which may be extremely useful in the future (Avramenko et al., 2019).
Results from this PhD and a recent study in Ireland (Keegan et al., 2017a), indicate that BZ resistance in T. circumcincta may be more complicated than just the presence of SNPs at these three codons, and that the presence of resistant individuals may be missed by molecular methods focusing solely on these. In this study, the presence of homozygous ‘susceptible’ offspring
Ireland, Keegan et al. (2017a) identified the presence of adults surviving oxfendazole treatment, which were genotyped as heterozygous or homozygous ‘susceptible’ at each of the BZ SNPs. Similar ideas of alternative BZ resistance mechanisms have been suggested previously. Originally, selection at both β- tubulin isotype-1 and β-tubulin isotype-2 was noted in BZ resistant isolates
compared to susceptible isolates (Beech et al., 1994; Lubega et al., 1994). It was suggested that BZ resistance could occur following mutation in β-tubulin isotype- 1 but that high level phenotypic resistance was possible with deletion of β- tubulin isotype-2 in H. contortus (Kwa et al., 1993). In Caenorrhabditis elegans, multiple ben-1 gene variants have been associated with BZ resistant phenotypes, and the N2 Bristol strain, commonly used in research, was unusual in having the codon 200 SNP (Driscoll et al., 1989; Hahnel et al., 2018). Similar to H.
contortus, highly BZ resistant C. elegans strains had a deletion of ben-1 (Driscoll
et al., 1989). Resistance phenotype may also vary by codon (Silvestre and Cabaret, 2002) and lifecycle stage (Barrère et al., 2012; Kotze et al., 2012). In addition to the target gene(s), studies have reported other mechanisms which may increase BZ resistance or tolerance, including the multidrug transporters, the P-glycoproteins (Blackhall et al., 2008). It is clear from EHTs that there are a range of resistance phenotypes even within a single isolate population, with only a subset of worms able to survive at higher concentrations. This would suggest a range of genotypes underlying this trend, which may be more relevant in some field populations than others.
Although molecular methods can be extremely useful and reliable, despite the potential for multiple mechanisms of resistance, they are comparatively
expensive. For example, taking the Animal and Plant Health Agency (APHA, a standard lab for diagnostic testing in the UK) as a guide, a FECRT would cost a farmer £25.20 for a McMaster FEC for a single sheep (pre- and post-treatment), providing five or more animals were included. A parasitological examination of a single faecal sample would be £34.30. Morphological identification of L3 was priced at £172.30 (price data accessed July 2019). No EHT or LDT was included in the price list. In this study, the approximate cost of speciating 96 individual strongyles to identify four species by a multiplex PCR was £37.25, not including staff costs. The pyrosequencing assay, not including speciation prior to
tubulin isotype-1 SNPs. Although these costs would be offset by the size of the flock, and therefore the cost-benefit spread over many individual sheep, for many farmers the cost would be likely prohibitive, especially in the face of known resistance. As such, any molecular tests used in research settings need first to be adapted for commercial use, so that either by scale of use, or a cost- reduction in materials, such tests become cost-effective for farmers. For BZ resistance in T. circumcincta, which is widespread, the uptake of molecular testing is unlikely. However, if resistance was not yet identified, or there was potential to monitor development of resistance with different management practices, the uptake of such tests might be more likely, for example for emerging resistance of N. battus (Morrison et al., 2014) or cattle parasites (Winterrowd et al., 2003), or for IVM resistance.
As both the pyrosequencing and microsatellite results showed, the T.
circumcincta population on this farm was highly diverse and did not appear to
segregate into sub-populations at different times of year, in different age groups of sheep or following either BZ or IVM treatment in September. On this farm the presence of multiple genetic mutations associated with BZ resistance, and multiple haplotypes carrying these alleles, suggests that a soft sweep has occurred as observed elsewhere in the UK (Redman et al., 2015). This may be related to multiple origins of resistance within the farm, for example several resistant haplotypes may have been present since the establishment of the farm. Alternatively, the habit of replacing stock each year may have sequentially introduced new resistance alleles into the T. circumcincta population (Redman et al., 2015). In the UK, the stratified flock system, with many farms buying and selling stock at various times of year, may help to maintain species diversity but also increase the chance of resistance being introduced onto a farm. In France, where many farms are established and remain ‘closed’ with no new stock bought in, resistance was found to be linked to the number of farms of origin at
establishment (Silvestre et al., 2000). Fewer β-tubulin isotype-1 haplotypes were identified in French T. circumcincta populations compared with UK populations (Skuce et al., 2010). Due to the practice of quarantine drenching (a necessity to reduce the spread of disease and resistant parasites), the likelihood of
introducing new sensitive haplotypes, with potentially higher fitness than resistant parasites (if such fitness costs exist), is unlikely. In the presence of
such diversity, studies investigating single genes can also increase the chance of false positive association of a SNP with a resistance phenotype (Laing et al., 2016; Rezansoff et al., 2016), and this may be why there is so much
contradictory evidence regarding the mechanism of IVM resistance.