Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus

Abstract

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr- 8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing “resistant” allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous “resistant” genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the “resistant” allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

“…However, this was not supported by our results; given that the majority of individuals in a LEV susceptible population are homozygous for the deletion, it is unlikely to serve as an effective diagnostic marker of LEV resistance. Our results are more consistent with a recent study using droplet digital PCR comparing the presence of the intron 2 deletion in Swedish field-derived and laboratory populations of H. contortus ; although there was a difference in genotype frequencies between resistant and susceptible isolates, the homozygous deletion was present in a high proportion of susceptible isolates ( Baltrušis et al, 2021 ).…”

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

“…Our results are more consistent with a recent study using droplet digital PCR comparing the presence of the intron 2 deletion in Swedish field-derived and laboratory populations of H. contortus; although there was a difference in genotype frequencies between resistant and susceptible isolates, the homozygous deletion was present in a high proportion of susceptible isolates (Baltrušis et al, 2021).…”

The presence and relative frequency detection of the levamisole-resistance-associated S168T substitution in hco-acr-8 in Haemonchus contortus

“…The samples used in this study have been described in our previous work ( Baltrušis et al, 2021 ). Briefly, in order to establish the presence or absence and frequencies of the mutation S168T in hco-acr-8 (HCON_00151270), genomic DNA (for extraction procedures please refer to Baltrušis et al, 2021 ), belonging to individual, adult H. contortus and mostly H. contortus larvae populations (see Supplementary Table 1 ) was used.…”

“…Up until recently, the prevailing hypothesis regarding the major mechanism responsible for levamisole resistance in H. contortus was based on the observation that some phenotypically resistant isolates appeared to express a truncated transcript of hco-acr-8 (hco-acr-8b) as a result of the (63bp) deletion in the intron 2 ( Fauvin et al, 2010 ; Barrère et al, 2014 ). However, growing interest in hco-acr-8 and its role in resistance has resulted in findings inconsistent with this hypothesis ( Chagas et al, 2016 ; Baltrušis et al, 2021 ; Doyle et al, 2022 ). The most recent data suggests that a non-synonymous mutation (A G C→A C C; protein coding strand) in the exon 4 of this gene, resulting in the S168T substitution, could, instead, be the major determinant of levamisole resistance and serve as a potential molecular marker of its identification in parasite populations ( Doyle et al, 2022 ; Antonopoulos et al, 2022 ).…”

“…In this study, we aimed to analyze the samples at our disposal from the previous study ( Baltrušis et al, 2021 ) with a newly developed allele-specific PCR (AS-PCR) and droplet digital (dd) PCR approaches to investigate the presence or absence of the mutation A G C→A C C at codon position 168 (hereafter referred to as mutation S168T) in hco-acr-8 in six phenotypically characterized and three Swedish, field H. contortus isolates. In addition, the ddPCR assay was also employed to perform relative frequency analyses of the mutation S168T using genomic DNA from locally recovered larvae cultures (where levamisole was shown to be 100% efficacious).…”