Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus
Introduction
Resistance to the benzimidazole anthelmintics has been widely observed around the world, and reduces the ability of farmers to control nematode parasites in small ruminants. Haemonchus contortus, a hematophagic parasite occurring in ruminants, is very commonly resistant to benzimidazoles (Wolstenholme et al., 2004, Kaplan, 2004, Jabbar et al., 2006). The molecular basis of benzimidazole resistance is reasonably well understood. Mutations called SNPs (single nucleotide polymorphisms) in the β-tubulin gene have been linked to drug selection in H. contortus. The β-tubulin protein is directly involved in the mechanism of action of the benzimidazoles; the drug binds to the protein and blocks the polymerisation of tubulin and consequently the formation of microtubules (Lubega and Prichard, 1990, Lubega and Prichard, 1991a, Lubega and Prichard, 1991b, Coles et al., 1995). Mutations in the β-tubulin isotype 1 gene have been shown to encode amino acid changes that result in a poor binding of benzimidazoles (Kwa et al., 1995, Lubega and Prichard, 1990). These mutations can be used as markers to detect benzimidazole resistance in H. contortus. Originally, the mutation at position 200 was associated with resistance (Kwa et al., 1994). Recently, two other mutations, at codon 167 (Silvestre and Cabaret, 2002) and codon 198 (Ghisi et al., 2007), have been found linked to benzimidazole resistance. Benzimidazole susceptible parasites harbour a TTC codon, coding for phenylalanine at positions 200 and 167 in the β-tubulin isotype 1 protein. Resistant parasites can have a TAC codon, coding for tyrosine at either codon 200 or 167 in β-tubulin; however the mutation at codon 200 is most commonly associated with benzimidazole resistance (Silvestre and Cabaret, 2002, Mottier and Prichard, 2008, von Samson-Himmelstjena et al., 2009). Both mutations are recessive (Elard et al., 1996, Silvestre and Cabaret, 2002). The third mutation, located at position 198, causes a change from GAA (glutamate) in susceptible populations to GCA (alanine) in some resistant H. contortus (Ghisi et al., 2007).
The technique of pyrosequencing, based on the principle “sequencing by synthesis” has been found to be rapid, and suitable for testing multiple SNPs (von Samson-Himmelstjena et al., 2007). The samples obtained from an albendazole dose–response study on an isolate of H. contortus, that was moderately resistant to benzimidazoles (Entrocasso et al., 2008, Alvarez et al., 2010) provided an opportunity to investigate the effect of increasing dose rate on the genotype of treatment survivors.
Section snippets
Animals and parasites
Twenty-four non parasitized Corriedale lambs (34.3 ± 6.3 kg) were experimentally infected with a field isolate of benzimidazole resistant H. contortus. The isolate was from a sheep Experimental Unit (Reserva 8, Instituto Nacional de Tecnología Agropecuaria, Balcarce, Argentina) with a parasite control program based on the intensive use of anthelmintics over many years. The use of different benzimidazole compounds (mainly albendazole and oxfendazole) several times a year over many years had been
Drug efficacy
There was no evident change in the behaviour of treated lambs (food and water consumption, activity, etc.), even in the animals treated at the highest dose rate. The adult H. contortus counts and resultant efficacy data obtained for the different experimental groups are shown in Table 2. The low efficacy level (16%) observed for the recommended dose rate (5 mg/kg) indicates the presence of worms resistant to albendazole. An efficacy of 59% was observed for the 15 mg/kg treatment group. The
Discussion
Albendazole at the highest dose rate (45 mg/kg) was found to be 94% effective against the benzimidazole-resistant H. contortus; no adverse host events were noted. However, both the recommended (5 mg/kg) and the 3× recommended (15 mg/kg) dose rates failed to adequately control this parasite.
The isolate from the field had been previously classified as benzimidazole resistant (egg count reduction after the recommended dose < 95%). The results obtained for the drug efficacy at the recommended dose rate
Conclusions
The work described here demonstrates a strong association between SNPs at codons 167 and 200 in β-tubulin and the survival of H. contortus individuals at a high dose rate of albendazole. The genotype (Phe/Phe)167–(Tyr/Tyr)200 was consistently observed in worms exhibiting high levels of resistance. Heterozygosity at both codons 167 and 200 conferred resistance with treatments up to three times the recommended albendazole dose rate. Routine tests for evaluation of benzimidazole resistance using
Acknowledgements
The study was supported by CONICET, Argentina, NSERC, Canada and the Québec Centre for Host-Parasite Interactions/FQRNT. The technical assistance of Kathy Keller and constructive suggestions Dr Robin Beech are greatly appreciated. The authors would also like to thank Dr Georg von Samson-Himmelstjerna for his advice on the pyrosequencing analysis.
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