dc.description.abstract
Anthelmintic resistance is an accompaniment to modern livestock industry, which heavily relies on chemotherapy and chemoprophylaxis for maintaining animal health and productivity. While anthelmintic resistance is already established in horses and is a growing issue in cattle, it is, by far, the most severe and most prevalent in the sheep industry. To combat the spread of resistance, researchers are investigating alternative ways of nematode control and formulate recommendations to move towards more sustainable patterns of chemotherapeutic control. A further pillar in the management of nematodes is the understanding of the mechanisms underlying anthelmintic resistance. This knowledge is necessary to implement resistance diagnosis tests and might offer potential new drug targets. Macrocyclic lactones (MLs) and benzimidazoles (BZs) are the most frequently used anthelmintics in the sheep industry and resistance against them has been reported worldwide. Target-site related mechanisms usually are investigated first. For BZs, mutations in the ß-tubulin isotype 1 gene leading to amino acid changes at the drug target site have been found to confer resistance in trichostrongyloid nematodes, which are among the most pathogenic parasites of small ruminants. These mutations have been exploited to set up several tests capable of diagnosing BZ resistance even at very low frequencies of resistance alleles in nematode populations. So far, no target-site related mechanisms have been identified in case of MLs and diagnosis of ML resistance is limited to in vivo and in vitro tests. Drug efflux by P-glycoproteins is a non-target-site related mechanism that has been proposed to play a role in ML resistance particularly. A third possibility that has come to receive more attention in recent years is bioinactivation of MLs and BZs by cytochrome P450 monooxygenases (CYPs). In cancer drug resistance research as well as in the field of insecticide and acaricide resistance CYPs and their contribution to drug resistance are well established concepts.
Reasons to assume that CYPs might also be involved in anthelmintic resistance include, for instance, the metabolism of thiabendazole, a BZ representative, by CYP35D1 in the free-living nematode Caenorhabditis elegans and the detection of BZ metabolites, consistent with CYP activity in the parasitic nematode Haemonchus contortus. Despite these and many other indications, the role of CYPs in anthelmintic resistance is not fully clear.
The present cumulative doctoral thesis elaborates on the likelihood of an involvement of CYPs in ML metabolism in C. elegans and BZ metabolism in H. contortus. The first part was primarily processed by an in vitro approach whereby the larval development of mutant strains was compared to that of the wild-type N2 strain under different ML exposures. In particular, a strain with a temperature-sensitive mutation in the cytochrome reductase gene emb-8 was compared at a non-permissive temperature to the wild-type regarding ivermectin (IVM) and moxidectin (MOX) susceptibility. The second part focused on expression patterns of CYPs in in vitro cultured fourth larvae of H. contortus. Here, constitutive and TBZ-inducible expression was investigated in five different H. contortus isolates with varying levels of phenotypic BZ resistance.
Essentially, the ablation of CYP enzymatic activity by use of an inhibitor and a genetic approach was found to minimally alter the susceptibility to IVM in C. elegans, but not to MOX, both of which are MLs but belong to different groups. CYP14-A5, initially suspected to be involved in ML resistance in C. elegans, is most likely not involved, as its loss of activity was not shown to modulate IVM susceptibility and inducibility of gene expression by IVM or MOX was not observed. Therefore, CYPs can be expected to play no major role in ML resistance in C. elegans. As for BZ resistance in H. contortus, exposure to TBZ did not induce expression of selected CYPs in any of the isolates. However, the highly resistant WR isolate showed a 2.4- 3.7- fold higher constitutive expression of CYP HCOI100383400 in comparison to BZ susceptible isolates. In a previously published study, this CYP has been shown to have elevated transcript levels in the gut – the hypothesized major site of detoxification in nematodes. This is a first hint towards a possible involvement of CYPs in BZ resistance in a parasitic nematode. The WR isolate was also shown to be considerably more resistant than known resistant field isolates while having similar frequencies of the F200Y single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene which along with E198A and F167Y is the primary cause for BZ resistance. The comparison of the frequency of F200Y and phenotypic resistance levels corroborates the long-held assumption of a multi-genic context for BZ resistance. Whether a co-governance of BZ resistance by several mechanisms in general and by the involvement of CYPs in particular is true, cannot be guaranteed with absolute certainty at the present state of research. However, the possibility of such should not be thoughtlessly abandoned, particularly in case of high resistance phenotypes as suggested by the present thesis. There is no question that further experimental investigations are necessary to improve our understanding of metabolism-based detoxification in nematodes.
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