Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus

Abstract

While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype.

Effects of third generation P-glycoprotein inhibitors on the sensitivity of drug-resistant and -susceptible isolates of Haemonchus contortus to anthelmintics in vitro

“…This is to be expected as it is well known that the major determinants of resistance to this drug are changes in beta-tubulin genes. The WAL isolate is known to show high frequencies of both the F200Y and E198A SNPs associated with resistance to benzimidazole drugs (Kotze et al, 2012). The contribution of P-gps to TBZ resistance in the WAL isolate may be less significant than has been reported for other TBZ-resistant worm isolates in which MDRIs (particularly verapamil) have been reported to increase the sensitivity to this anthelmintic (Beugnet et al, 1997).…”

Genetic evidence for the spread of a benzimidazole resistance mutation across southern India from a single origin in the parasitic nematode Haemonchus contortus

“…Similarly, although the E198A (GCA) mutation was present in 8/23 populations, it was at a low frequency in all cases (maximum frequency of 18%). It is interesting that the E198A (GCA) mutation was identified in so many populations given that it has only been previously reported in just two H. contortus field-derived isolates (from Australia and South Africa) and was not detected in surveys of benzimidazole-resistant H. contortus from the UK, Canada and USA (Ghisi et al, 2007;Rufener et al, 2009;Barrère et al, 2012Barrère et al, , 2013aKotze et al, 2012;Chaudhry, 2015, PhD Thesis, cited earlier). Hence the E198A (GCA) mutation appears to be much more common in southern India than in the other countries examined to date, even though benzimidazole resistance appears less advanced based on the P200Y (TAC) mutation distribution and frequency overall (Hoglund et al, 2009;Barrère et al, 2012Barrère et al, , 2013aRedman et al, 2015;Chaudhry, 2015, PhD Thesis, cited earlier).…”

“…Hence, the F167Y (TAC) mutation may be of similar importance as the F200Y (TAC) mutation for benzimidazole resistance of H. contortus in the UK. A third SNP at codon E198A (GAA to GCA) resulting in a glutamate to alanine substitution has been detected in just two field-derived laboratory passaged populations of H. contortus to date; one from South Africa and one from Australia (Ghisi et al, 2007;Rufener et al, 2009;Kotze et al, 2012). It appears the E198A (GCA) mutation is the rarest of the three benzimidazole mutations globally, although it has not been the focus of many published studies to date.…”

Koyunlarda Haemonchus contortus’un Moleküler Prevalansı, Filogenetik Karakterizasyonu ve Benzimidazol Dirençliliği

“…A point mutation in codon 200 (TTC to TAC), causing a phenylalanine to tyrosine substitution, is the most common SNP and often at high frequency in several countries [1,26,27,30] . While the SNPs at codons 167 (TTC to TAC) and 198 (GAA to GCA) are less common and have been reported in a number of different countries [1,11,20,27,31] . In the present study sequence analyses of the isolates which had an over 39.0% resistant allele frequency revealed the existence of only the widest SNP at codon 200 in H. contortus populations in the study area which is in agreement with above studies [1,26,27,30] .…”