Abstract
BackgroundBenzimidazole (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.MethodsFaecal 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 third-stage 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.ResultsAlbendazole 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. EC50 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.ConclusionsTo 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.
Highlights
Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, in small ruminants
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
Albendazole efficacy based on faecal egg count and egg hatch test data Results of the faecal egg count reduction test (FECRT) and E C50 values in the EHT with 95% confidence intervals (CIs) are presented in Table 1, while Additional file 4: Table S3 shows mean egg count data with 95% CIs
Summary
Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, in small ruminants. The economic impact due to infection with these parasites, mainly gastrointestinal nematodes (GINs), is high, in livestock [2], e.g. estimated to be around 318 million dollars annually in Australia [3] This impact led to the routine use of anthelmintics in veterinary medicine over several decades [4]. As a consequence of frequent and occasionally indiscriminate use of anthelmintics, parasite populations have developed anthelmintic resistance (AR) This has become widespread in multiple parasites of animals, and threatens the efforts of parasite control in both human and veterinary medicine [5, 6]. Some of the limitations of the current in vivo and in vitro diagnostic tests for AR could be potentially overcome through the use of molecular techniques that detect specific mutations associated with the resistance phenotype. Such tests are potentially more sensitive, allowing the earlier detection of resistance [7]
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