Deep amplicon sequencing of preselected isolates of Parascaris equorum in β-tubulin codons associated with benzimidazole resistance in other nematodes

Particle and Fibre Toxicology

Johan Höglund¹  Olof Karlberg²  Johan Dahlberg²  Eva Tydén¹ 

[1] Department of Biomedical Sciences and Veterinary Public Health, Division of Parasitology, Swedish University of Agricultural Sciences, Uppsala S-750 07, Sweden;Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Uppsala S-751 44, Sweden

关键词: Illumina HiSeq;    Fenbendazole;    SNP;    β-tubulin;    Anthelmintic resistance;    Parascaris equorum;   

Others  :  1150671
DOI  :  10.1186/1756-3305-7-410

 received in 2014-06-16, accepted in 2014-08-18,  发布年份 2014

【 摘 要 】

Background

The development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles now being the most widely used substance to control Parascaris infections. However, over-reliance on one drug class is a risk factor for the development of AR. Consequently, benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the β-tubulin genes. The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. This study investigated whether these SNPs exist in the P. equorum population and tested the hypothesis that BZ resistance can develop from pre-existing SNPs in codons 167, 198 and 200 of the β-tubulin isotype 1 and 2 genes, reported to be associated with AR in strongylids. The efficacy of the oral paste formula fenbendazole on 11 farms in Sweden was also assessed.

Methods

Two isotype-specific primer pairs were designed, one on either side of the codon 167 and one on either side of codons 198 and 200. A pool of 100 000 larvae was sequenced using deep amplicon sequencing by Illumina HiSeq. Faecal egg count reduction test was used to assess the efficacy of fenbendazole.

Results

No SNPs were observed in codons 167, 198 or 200 of the β-tubulin isotype 1 or 2 genes of P. equorum, even though 100 000 larvae were sequenced. Faecal egg count reduction testing of fenbendazole showed that this anthelmintic was still 100% effective, meaning that the likelihood of finding high allele frequency of SNPs associated with benzimidazoles resistance in P. equorum was low. Unexpectedly, the allele frequencies observed in single worms were comparable to those in pooled samples.

Conclusions

We concluded that fenbendazole does not exert selection pressure on the β-tubulin genes of isotypes 1 and 2 in P. equorum. The fact that no pre-existing SNPs were found in codons 167, 198 and 200 in P. equorum also illustrates the difficulties in generalising about AR mechanisms between different taxonomic groups of nematodes.

【 授权许可】

   
2014 Tydén et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Kaplan RM: Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol 2004, 20:477-481.
• [2]Reinemeyer CR: Anthelmintic resistance in non-strongylid parasites of horses. Vet Parasitol 2012, 185:9-15.
• [3]Coles GC, Jackson F, Pomroy WE, Prichard RK, Von Samson-Himmelstjerna G, Silvestre A, Taylor MA, Vercruysse J: The detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 2006, 136:167-185.
• [4]Craig TM, Diamond PL, Ferwerda NS, Thompson JA: Evidence of ivermectin resistance by Parascaris equorum on a Texas horse farm. J Equine Vet Sci 2007, 27:67-71.
• [5]Lyons ET, Tolliver SC, Ionita M, Collins SS: Evaluation of parasiticidal activity of fenbendazole, ivermectin, oxibendazole, and pyrantel pamoate in horse foals with emphasis on ascarids (Parascaris equorum) in field studies on five farms in Central Kentucky in 2007. Parasitol Res 2008, 103:287-291.
• [6]Reinemeyer CR: Diagnosis and control of anthelmintic-resistant Parascaris equorum. Parasit Vectors 2009, 25(2 Suppl 2):S8.
• [7]Beech RN, Skuce P, Bartley DJ, Martin RJ, Prichard RK, Gilleard JS: Anthelmintic resistance: markers for resistance, or susceptibility? Parasitology 2011, 138:160-174.
• [8]Kwa MSG, Veenstra JG, Roos MH: Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation in beta tubulin isotype 1. Mol Biochem Parasitol 1994, 63:299-303.
• [9]Elard L, Comes AM, Humbert JF: Sequences of β-tubulin cDNA from benzimidazole-susceptible and -resistant strains of Teladorsagia circumcincta, a nematode parasite of small ruminants. Mol Biochem Parasitol 1996, 79:249-253.
• [10]Silvestre A, Humbert JF: A molecular tool for species identification and benzimidazole resistance diagnosis in larval communities of small ruminant parasites. Exp Parasitol 2000, 95:271-276.
• [11]Schwab AE, Boakye DA, Kyelem D, Prichard RK: Detection of benzimidazole resistance-associated mutations in the filarial nematode Wuchereria bancrofti and evidence for selection by albendazole and ivermectin combination treatment. Am J Trop Med Hyg 2005, 73:234-238.
• [12]Silvestre A, Cabaret J: Mutation in position 167 of isotype 1 β-tubulin gene of Trichostrongylid nematodes: role in benzimidazole resistance? Mol Biochem Parasitol 2002, 120:297-300.
• [13]Ghisi M, Kaminsky R, Maser P: Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes. Vet Parasitol 2007, 144:313-320.
• [14]Hodgkinson JE, Clark HJ, Kaplan RM, Lake SL, Matthews JB: The role of polymorphisms at β tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int J Parasitol 2008, 38:1149-1160.
• [15]Gilleard JS, Beech RN: Population genetics of anthelmintic resistance in parasitic nematodes. Parasitology 2007, 134:1133-1147.
• [16]Slivestre A, Humbert JF: Diversity of benzimidazole-resistance alleles in populations of small ruminant parasites. Int J Parasitol 2002, 32:921-928.
• [17]Blouin MS, Yowell CA, Courtney CH, Dame JB: Host movement and the genetic structure of populations of parasitic nematodes. Genetics 1995, 141:1007-1014.
• [18]Lyons ET, Tolliver SC, Collins SS, Drudge JH, Granstrom DE: Transmission of some species of internal parasites in horses born in 1993, 1994, and 1995 on the same pasture on a farm in Central Kentucky. Vet Parasitol 1997, 70:225-240.
• [19]Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics 2009, 25:1754-1760.
• [20]McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010, 20:1297-1303.
• [21]Anonymous: Manual of Veterinary Parasitological Laboratory Techniques. London: Ministry of Agriculture Fisheries and Food; 1986:24.
• [22]Blackhall WJ, Kuzmina T, Von Samson-Himmelstjerna G: β-Tubulin genotypes in six species of cyathostomins from anthelmintic-naive Przewalski and benzimidazole-resistant brood horses in Ukraine. Parasitol Res 2011, 109:1199-1203.
• [23]Diawara A, Drake LJ, Suswillo RR, Kihara J, Bundy DA, Scott ME, Halpenny C, Stothard JR, Prichard RK: Assays to detect beta-tubulin codon 200 polymorphism in Trichuris trichiura and Ascaris lumbricoides. PLoS Negl Trop Dis 2009, 3:e397.
• [24]Diawara A, Halpenny CM, Churcher TS, Mwandawiro C, Kihara J, Kaplan RM, Streit TG, Idaghdour Y, Scott ME, Basáñez MG, Prichard RK: Association between response to albendazole treatment and β-tubulin genotype frequencies in soil-transmitted helminths. PLoS Negl Trop Dis 2013, 30:e2247.
• [25]Tydén E, Engström A, Morrison DA, Höglund J: Sequencing of the β-tubulin genes in the ascarid nematodes Parascaris equorum and Ascaridia galli. Mol Biochem Parasitol 2013, 190:38-43.
• [26]Hansen TVA, Nejsum P, Olsen A, Thamsborg SM: Genetic variation in codons 167, 198 and 200 of the beta-tubulin gene in whipworms (Trichuris spp.) from a range of domestic animals and wildlife. Vet Parasitol 2013, 193:141-149.
• [27]Webster LM, Johnson PC, Adam A, Mable BK, Keller LF: Absence of three known benzimidazole resistance mutations in Trichostrongylus tenuis, a nematode parasite of avian hosts. Vet Parasitol 2008, 158:302-310.
• [28]Schwenkenbecher JM, Albonico M, Bickle Q, Kaplan RM: Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCR. Mol Biochem Parasitol 2007, 156:167-174.
• [29]De Lourdes MM, Prichard RK: Genetic analysis of relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus. Pharmacogenet Genomics 2008, 18:129-140.
• [30]Lindgren K, Ljungvall Ö, Nilsson O, Ljungström BL, Lindahl C, Höglund J: Parascaris equorum in foals and in their environment on a Swedish stud farm, with notes on treatment failure of ivermectin. Vet Parasitol 2008, 151:337-343.
• [31]Lind EO, Christensson D: Anthelmintic efficacy on Parascaris equorum in foals on Swedish studs. Acta Vet Scand 2009, 51:45. BioMed Central Full Text
• [32]Niranjan TS, Adamczyk A, Bravo HC, Taub MA, Wheelan SJ, Irizarry R, Wang T: Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis. Genome Biol 2011, 28:R93.
• [33]Grossmann V, Roller A, Klein HU, Weissmann S, Kern W, Haferlach C, Dugas M, Haferlach T, Schnittger S, Kohlmann A: Robustness of amplicon deep sequencing underlines its utility in clinical applications. J Mol Diagn 2013, 15:473-484.
• [34]Von Samson HG, Walsh TK, Donnan AA, Carriere S, Jackson F: Molecular detection of benzimidazole resisatnce in Haemonchus contortus using real-time PCR and pyrosequencing. Parasitol 2009, 136:349-358.
• [35]Tsibris AM, Korber B, Arnaout R, Russ C, Lo CC, Leitner T, Gaschen B, Theiler J, Paredes R, Su Z, Hughes MD, Gulick RM, Greaves W, Coakley E, Flexner C, Nusbaum C, Kuritzkes DR: Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo. Plos One 2009, 25:e5683.
• [36]Tydén E, Morrison DA, Engström A, Nielsen MK, Eydal M, Höglund J: Population genetics of Parascaris equorum based on DNA fingerprinting. J Infect Genet Evol 2013, 13:236-241.