This is the first study evaluating for the presence of resistance alleles in ovine trichostrongyloids in Austria, focusing on sheep flocks within a transhumance management system. No FECRT could be performed because after several reports of treatment failure with BZs from attending veterinarians, farmers were not willing to use BZs to deworm their sheep. Consequently, all study animals were treated with moxidectin. The combination of FECRT for ML and molecular tests for BZs allowed estimation of resistance status regarding both drug classes in parallel. While it may be argued that the SNP-analysis is only an indirect diagnosis of resistance, there is convincing evidence available that the percentage of resistance alleles containing the exchanges F167Y, E198A or F200Y strongly correlates with the results of the FECRT and/or the EHT [24, 35, 37, 42, 43] while susceptible populations have very low levels of these resistance alleles. We also want to highlight that FECRT and EHT can even lead to inconclusive results in field populations with mixed infections . Thus, by analysing specifically a single species, molecular analyses can overcome the limitations of in vivo and in vitro tests with mixed field samples.
Although all three SNPs have been reported to be associated with AR to the benzimidazoles, codon 200 remains the most prevalent and arguably the most important .
The F200Y exchange was found to be highly prevalent in H. contortus and T. colubriformis on the tested farms. T. circumcincta populations generally had lower F200Y allele frequencies, greater than 40% in five of the nine populations tested. A recent study identified the F200Y exchange at frequencies up to 13% in benzimidazole-susceptible strains of T. circumcincta, while resistant strains had allele frequencies above 60% . This suggests that phenotypic benzimidazole resistance could be possible in some of the T. circumcincta populations tested within this study. The interaction between the F200Y and F167Y polymorphisms is not fully understood however, it has been suggested for T. circumcincta that F200Y mutation may predominate initially with the frequency of F167Y increasing with phenotypic resistance . From the results presented, it could be hypothesised that BZ-resistance may be at an advanced stage in T. colubriformis in the region tested. Unfortunately, due to limited sample material, we were not able to perfom the SNP analysis on codon 167 (F200Y) of Haemonchus contortus although this SNP remains an important polymorphism in other regions of the world [24, 25]. However, the nearly complete manifestation of F200Y in the Haemonchus-samples suggests a high prevalence of clinically relevant BZ resistance.
Thus, it can be concluded that a high percentage of BZ resistant H. contortus and T.colubriformis are present in sheep in this region of Austria, while the frequency of resistant T. circumcincta remains moderate. This also supports anecdotal reports of poor efficacy of BZs in sheep in the investigated area. When compared to recent data , the frequency of resistance alleles of H. contortus in Austria is amongst the highest in Europe. This may be due to a long-standing “dose and move” practice on many Austrian farms practicing transhumance. Mutations leading to AR can arise due to the very large worm population sizes and be rapidly enriched due to strong selection by frequent strategic treatments in local nematode populations .
Similar high levels of BZ resistance were observed in goats in the alpine region of Italy [1, 46] supporting the assumption that resistance alleles associated with AR were spread through the introduction of resistant strains via animal movement and communal high altitude grazing. Dose and move strategies and animal movement are also common in Styria and Salzburg  and might be an important driving factor for the spread of AR on all farms investigated. In addition, strategies to maintain a refugium of anthelmintic-susceptible parasites, known to reduce the rate of development of anthelmintic resistance in GIN , may be especially difficult to implement when animals from different farms and management strategies are herded together on communal pastures. The deworming frequency in the study populations was low (1-2 treatments per year), but the deworming of all animals in spring before they are turned out to the mountain pastures clearly reduces the refugium for susceptible worms.
Haemonchus sp. are sensitive to cold weather  , and few Haemonchus larvae will survive the harsh conditions of the alpine winter. Consequently, Haemonchus overwinters largely in hypobiosis within the host, which accounts for the high prevalence observed in some regions with northern boreal/continental climate [25, 35, 47, 48]. When all animals are treated before winter turn-in, Haemonchus encounters a significant bottleneck for survival  , which increases the selective advantage for resistant worms that overwinter in their host and survive spring treatment, quickly increasing the proportion of resistant individuals on summer pastures.
Considering the present results in combination with the high fecundity of Haemonchus , there is considerable concern that the population of BZ resistant Haemonchus in Austria may expand further in size and geographic range e.g. through treatment errors, animal movement and/or the spread by wildlife hosts . Indeed, Haemonchus increased in relation to other trichostrongyloids in faecal cultures examined during the course of the grazing season . This trend was independent of BZ treatment as the sheep involved in the study were only treated with moxidectin during the investigated time period. It should, however, be mentioned that also macrocyclic lactone treatment might select for the variants F200Y and F167Y of Haemonchus .
One of the most popular strategies to slow the rate of development of AR is targeted selective treatment . This leaves selected animals untreated. Untreated animals will then shed eggs of worms that were not exposed to anthelmintics, so presumably susceptible to the AH. These eggs of untreated animals will dilute eggs produced by any resistant worms which survived treatment within treated animals, and thus ameliorate the strong effects of drug selection by maintaining susceptible parasites in refugia [11, 20, 47]. However, this strategy requires that a sufficient proportion of alleles associated with a susceptible phenotype are present within the population. The results of the present study suggest a lack of susceptibility-associated alleles at codon 200 in the H. contortus populations. Therefore, leaving some sheep untreated would not be effective in reducing resistance allele frequencies and thus BZ resistance, unless a susceptible population of H. contortus was introduced . Ideally, susceptibility of this introduced worm population should be tested at the genetic level. It has also to be considered that adding only H. contortus larvae into the population would increase the relative prevalence of this highly pathogenic species in the overall trichostrongyloid population potentially resulting in greater production losses, so until more evidence of this strategy is provided we would not recommend this approach. Nevertheless, selective treatment is still considered to be an appropriate strategy for the region under investigation in this study, especially for slowing down selection of AR against other anthelmintic classes.
The fact that the prevalence of BZ resistance genotypes was very high in three flocks where ML resistance was also observed shows that multi-drug-resistant trichostrongyloid nematodes of sheep can also occur in extensive management systems such as transhumance in alpine Austria. If no alternative strategies are implemented in the near future, it must be expected that AR will be widespread and irreversible . Information and instruction to farmers by veterinarians is strongly recommended, especially since a considerable part of the Austrian sheep population is kept by farmers with sideline enterprises or hobby farmers who may not have sufficent knowledge and experience with novel antiparasitic management.
In the longer term, economical and sustainable management of grazing livestock will likely depend on three main strategies: (1) rational use of licensed anthelmintic drugs to slow down the selection of resistant populations using approaches such as targeted selective treatment , (2) the development of new effective anthelmintic drug classes and (3) the improvement of strategies that do not depend on the administration of anthelmintics . For the latter there are a number of approaches such as vaccination, breeding and grazing management. Vaccination against haemonchosis is a promising development for farms heavily afflicted by this worm [55–57]. The first commercial vaccine, Barbevax®, has been evaluated in several countries and is available in Australia since 2014 . However, the high frequency of vaccination required in the first two years would be very difficult to implement under transhumance farming management. Animals kept in transhumance are not easely accessible and this will intensify the work load for farmers tremendously. Breeding sheep that are resilient or resistant to worm infections is another feasible strategy  and appropriate grazing management is still the cornerstone of non-chemical strategies [47, 60].