Abstract
The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL.
Highlights
The anthelmintic resistance (AR), defined by Prichard et al (1980) as the increase in the frequency of anthelmintic tolerance in a nematode population, is currently one of the biggest concerns faced by sheep and goat farmers worldwide.The AR is widespread, and the occurrence of resistance and even multidrug resistance has been reported for the main pharmacological groups of anthelmintics used for gastrointestinal nematode control, such as benzimidazoles (BZs), macrocyclic lactones (MLs) and imidazothiazoles (Lamb et al, 2017; Cazajous et al, 2018)
In the larval development test (LDT), larval development inhibition for IVM ranged from 6.96% (± 5.74%) to 100% (0.02-50 ng/mL) (Figure 1A)
The predominance of the single nucleotide polymorphisms (SNPs) F167Y (69.08%), with the second highest percentage for F200Y (26.24%), and the absence of E198A codon polymorphism corroborate the findings of Santos et al (2017), who evaluated the pattern of BZs resistance of H. contortus from 20 sheep farms in Ceará state
Summary
The anthelmintic resistance (AR), defined by Prichard et al (1980) as the increase in the frequency of anthelmintic tolerance in a nematode population, is currently one of the biggest concerns faced by sheep and goat farmers worldwide.The AR is widespread, and the occurrence of resistance and even multidrug resistance has been reported for the main pharmacological groups of anthelmintics used for gastrointestinal nematode control, such as benzimidazoles (BZs), macrocyclic lactones (MLs) and imidazothiazoles (Lamb et al, 2017; Cazajous et al, 2018). Monepantel (MPTL), one of the most recently released anthelmintic on the market, already has reports of resistance (Van den Brom et al, 2015; Cintra et al, 2016). Diagnosis of AR is essential and has been made using phenotypic techniques, such as the egg hatch test (EHT), larval development test (LDT) and fecal egg count reduction test (FECRT), and genotypic techniques based on polymerase chain reaction (PCR) described for BZs and, most recently, levamisole (LEV). The resistance mechanism to BZs is the more elucidated so far, and its molecular based diagnosis in trichostrongylids relies on the detection of single nucleotide polymorphisms (SNPs) in the coding gene for β-tubulin isotype 1. Three SNPs were described, with the first one located at codon 200 (F200Y), the second at codon 167 (F167Y) and the third one at codon 198 (E198A) (Kwa et al, 1994; Silvestre & Cabaret, 2002; Ghisi et al, 2007)
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