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

Abstract

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L₃ encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

Keywords: Allele specific PCR; Diagnostic; Haemonchus; Levamisole; RFLP; Resistance; S168T; SNP.

Graphical abstract

Fig. 1

Schematic representation of primer design. A: Indel spanning primers (Table 1: Hco-Indel-R and Hco-Indel-F). If the deletion is present, then the product size will differ from the full-length allele, allowing for a size discrimination PCR to determine the genotype of the individual. B: Deletion specific primer (Table 1: Hco-Indel-Ins-R). The deletion specific primer lies wholly within the deleted sequence, thus blocking amplification if the deletion is present. C: Schematic representation of allele-specific primer function. The allele-specific primer binds upstream of the discriminating allele in both sensitive and resistant DNA, however, the ability of the primer to facilitate DNA extension during PCR depends specifically on the nucleotide at the 3′ end of the primer. Additional nucleotide mismatches at the 2nd and 5th position from the 3′ end of the primer (indicated by red X) are identical in each set and enhance the destabilisation of the polymerisation complex in the case of an allele specific mismatch at the 3′ position to discriminate between the two alleles: GGT (168T/resistant) and GCT (168S/susceptible). The 3′ mismatch is allele specific, and as shown by the example above will prevent polymerisation and amplification if the S168T variant is present. In the resistant primer assay, the opposite occurs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Summary of acr-8 intron 2 deletion in MHco18(UGA2004) and MHco3(ISE) L₃. Size discrimination PCR (Fig. 1A) was developed to genotype individual L₃ based on the presence or absence of the deletion allele. A deletion specific confirmation PCR (Fig. 1B) was then developed to validate the results of the size discrimination PCR. A: Example gel of single worm PCR of MHco18(UGA2004) and MHco3(ISE) with indel spanning (∼50 bp up and downstream of deletion locus) primer set (Hco-Indel-F/Hco-Indel-R). Odd numbers: MHco18(UGA2004). Even numbers: MHco3(ISE). The 245 bp band corresponds to full length allele, the 149 bp band corresponds to deletion allele. B: Bar chart showing proportion of deletion (D) allele detected by RFLP in LEV resistant MHco18(UGA2004), LEV susceptible MHco3(ISE), and MHco3/18 genetic cross populations. Y axis: genotype frequency. Genotypes DD: homozygous deletion; DF: heterozygous; FF: homozygous full length. C: Single worm PCR with indel internal (will only anneal when deleted sequence is present) confirmation (Hco-Indel-F/Hco-Indel-Ins-R) primer set: 1,3,5 MHco18(UGA2004); 2,4 MHco3(ISE) single L₃ worms; 6 Positive Control (100 ng) pooled L₃ MHco3(ISE) gDNA. D: Single worm PCR with indel spanning (Hco-Indel-F/Hco-Indel-R) primer set: 1,3,5 MHco18(UGA2004), 2,4; MHco3(ISE) single L₃, 6 NTC. The same individual L₃ lysate was used for PCR shown in C and D.

Fig. 3

Summary of RFLP analysis of MHco18(UGA2004), MHco3(ISE) and MHco3/18 L₃. S168T (GCT-GGT) mutation at codon 168 introduces an AvaII restriction site. Restriction Fragment Length Polymorphism (RFLP) was developed to genotype individual MHco3(ISE), MHco18(UGA2004), and MHco3/18 L₃ for the presence of either the GCT (susceptible - S) or the GGT (resistant - R) allele. A: Bar chart showing proportion of GGT allele detected by RFLP in LEV resistant MHco18(UGA2004), LEV susceptible MHco3(ISE), and MHco3/18. Y axis: genotype frequency. B: Detection of GGT by single L₃AvaII RFLP in LEV resistant MHco18(UGA2004), LEV susceptible MHco3(ISE), and MHco3/18 genetic cross. 1: MHco18(UGA2004); 2: MHco3(ISE); 3: MHco3/18.237 bp bands represents 168S GCT sequence. 128 bp and 109 bp bands represent GGT sequence. Predicted genotypes RR: homozygous GGT; RS: heterozygous; SS: homozygous GCT.

Fig. 4

Flow diagram summarising design, establishment, and validation of the AS-PCR for the detection of the S168T variant in multiple H. contortus isolates: the LEV susceptible MHco3(ISE), MHco4(WRS), and MHco10(CAVR), and the LEV resistant MHco18(UGA2004) and MHco3/18, and one phenotypically LEV resistant, and one phenotypically LEV susceptible field isolate from the USA.

Fig. 5

16 LEV resistant MHco18(UGA2004) and 16 LEV susceptible MHco3(ISE) L₃ were analysed on 2% agarose gel: A: MHco3(ISE): 1: Hco-Exon4-F + Hco-168T-R. 2: Hco-Intron4-F + Hco-168S-R. B: MHco18(UGA2004): 1: Hco-Exon4-F + Hco-168T-R. 2: Hco-Intron4-F + Hco-168S-R. Predicted genotypes RR: homozygous GGT; RS: heterozygous; SS: homozygous GCT. “- -” indicates this individual did not amplify by PCR. Only wells that amplified by PCR are counted in the final percentages (Fig. 6; Supplementary Table 3).

Fig. 6

Bar chart showing the proportion of GGT allele detected by AS-PCR in LEV resistant MHco18(UGA2004), LEV susceptible MHco3(ISE), and pre and post single LEV administration MHco3/18, LEV resistant (Farm 001) and LEV susceptible (Farm 002) field populations from the USA. Genotypes RR: homozygous GGT; RS: heterozygous; SS: homozygous GCT.

Fig. 7

Comparison gel showing difference between unmodified (Hco-168T-R/Hco-168S-R) and deoxyinosine modified (Hco-168T [I]-R/Hco-168S [I]-R) primers on geographically divergent LEV susceptible isolates MHco4(WRS) and MHco10(CAVR): Hco-168T-R/Hco-168S-R primers (panels A and B) 1:8 diluted first round exon 4 PCR products from MHco4(WRS) and MHco10(CAVR). Hco-168T [I]-R/Hco-168S [I]-R primers (C and D) 1:20 diluted first-round exon 4 PCR products from MHco4(WRS) and MHco10(CAVR). Lanes 1–8: individual L₃ worms. The same eight individual L₃ from each isolate were used to compare primer sets.

Supplementary Fig. 1

Example gel showing large sample (n = 94) of MHco3(ISE) using size discrimination Indel PCR using primers Hco-Indel-F and Hco-Indel-R.

Supplementary Fig. 2

Example gel showing amplification of Farm 002 by AS-PCR (1 and 3; Hco-168S-R (sensitive) primer; 148 bp band) and ITS2 speciation PCR (2 and 4; 321 bp band) and 100% concordance between poor amplification by AS-PCR and ITS2 PCR indicating a lack of genetic material is responsible for failure of those wells to amplify