New codon 198 β-tubulin polymorphisms in highly benzimidazole resistant Haemonchus contortus from goats in three different states in Sudan

Abstract

Background: Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. Methods: Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro-and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 β-tubulin in DNA extracted from pooled thirdstage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. Results: Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC 50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. Conclusions: To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.

Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta-tubulin alleles

“…Recent results indicate that BZ resistance is heterogeneous (Crook et al, 2016;Howell et al, 2008). Population-wide sequencing of beta-tubulin alleles have found different alleles in the same population (Avramenko et al, 2019;Hinney et al, 2020;Mohammedsalih et al, 2020;Redman et al, 2015;von Samson-Himmelstjerna et al, 2009). The frequency of each allele likely is determined both by its level of resistance and its effect on growth.…”

“…One untested hypothesis of a possible interplay between resistance and fitness consequences was observed at the 198 position. The E198V allele has been detected at low frequencies in populations that also harbor the E198L allele (Avramenko et al, 2019;Mohammedsalih et al, 2020), suggesting that the E198V allele (Supplemental Fig. 1) confers lower levels of resistance and/or has a fitness detriment.…”

“…Other beta-tubulin alleles beyond the canonical alleles have been identified using population sequencing approaches (Avramenko et al, 2019;Mohammedsalih et al, 2020;Redman et al, 2015). These alleles are not present in all parasites in a population, so they might confer different levels of BZ resistance and/or effects on growth.…”

“…After exposure to BZ compounds, the frequencies of certain beta-tubulin alleles increase in parasitic nematode populations, including F167Y, E198A, E198I, E198K, E198L, E198V, E198Stop, and F200Y (Avramenko et al, 2019;Ghisi et al, 2007;Kwa et al, 1994;Mohammedsalih et al, 2020;Redman et al, 2015;von Samson-Himmelstjerna et al, 2009;Silvestre and Cabaret, 2002 (Kwa et al, 1995). Since the introduction of the CRISPR-Cas9 system in C. elegans, experiments on gene function at the single amino acid level have become possible.…”

“…These three putative resistance alleles are considered canonical because they are commonly found in resistant populations (Avramenko et al, 2019;Hinney et al, 2020;Redman et al, 2015). Recently, improved nematode genotyping techniques have enabled the detection of additional mutations at the 198 position: E198V, E198L, E198K, E198I, and E198Stop (Avramenko et al, 2019;Mohammedsalih et al, 2020;Redman et al, 2015;von Samson-Himmelstjerna et al, 2009). These beta-tubulin alleles represent promising markers of BZ resistance across parasitic nematode populations.…”