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CARS Benzimidazole Resistance Workgroup Update 2009 Philip J. Skuce, Moredun Research Institute, Edinburgh, UK

CARS Benzimidazole Resistance Workgroup Update 2009 Philip J. Skuce, Moredun Research Institute, Edinburgh, UK. Outline. Literature review, CARS 2007-present ~60 publications on “benzimidazole resistance” - highlight papers of technical/parasitological interest(?)

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CARS Benzimidazole Resistance Workgroup Update 2009 Philip J. Skuce, Moredun Research Institute, Edinburgh, UK

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  1. CARS Benzimidazole Resistance WorkgroupUpdate 2009Philip J. Skuce,Moredun Research Institute,Edinburgh, UK

  2. Outline • Literature review, CARS 2007-present • ~60 publications on “benzimidazole resistance” - highlight papers of technical/parasitological interest(?) • Ongoing research on BZ-R in parasites of: - Sheep - Cattle - Horses - Man

  3. Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCRJan Schwenkenbecher, Marco Albonico, Quentin Bickle, Ray M. KaplanMol Biochem Parasitol 156:167-174 (2007) • Ancylostoma duodenale & Necator americanus • Mass drug administration with BZ anthelmintics • SNPs at codons 167 & 200 in b-tubulin implicated in other spp. • Cloned isotype-1 genes from A.caninum + the two human spp. • Highly conserved, similar genomic structure in all three • Designed real-time PCR assays to detect 167 & 200 SNPs • Pemba Island schoolchildren with sub-optimal response to MBZ – no evidence of association with 167 or 200 SNPs

  4. Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortusde Lourdes Mottier M. and Prichard R.K.Pharmacogenet Genomics 18(2): 129-140 • Lab strain of Haemonchus contortus – repeated IVM treatment in vivo selected for TTC to TAC mutation in b-tubulin, previously implicated in BZ-R • Examined 17 different field & lab H.contortus isolates with known treatment history and IVM- and/or BZ-R status • Repeated IVM or MOX treatment selects for F167Y & F200Y or E198A • Haplotypes? - 167Y & 200Y associated with V or L368, whereas 167F and 200F associated with I or V368 • ML-BZ “cross-talk” i.e. ML use may predispose parasitic nematodes to BZ-R (if they’re not BZ-R already!)

  5. P-glycoprotein selection in strains of Haemonchus contortus resistant to benzimidazolesWilliam J. Blackhall, Roger K. Prichard and Robin N. BeechVet Para 152: 101-107 (2008) • H.contortus – BZ-R associated with selection on b-tubulin; ML-R associated with selection on p-glycoprotein(Pgp) • Genetic changes in Pgp correlated with BZ-R in nematodes? • RFLP of BZ-S v BZ (cambendazole)-R isolate revealed significant difference in Pgp allele frequency • SSCP analysis revealed same allele at high frequency in an independently derived thiabendazole-selected field isolate • More evidence of BZ-ML “crosstalk”?

  6. The role of polymorphisms at b-tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostominsJ.E. Hodgkinson, H.J. Clark, R.M. Kaplan, S.L., Lake and J.B. MatthewsInt J Parasitol 38: 1149-1160 (2008) • Cyathostomins primary parasitic pathogens of horses • BZs used over 40 years, widespread BZ-R in the field • Comparison of b-tubulin genes in BZ-S v BZ-R isolates revealed consistent differences at codons 167 & 200 (isotype-1) • Highly significant allele frequency differences for F167Y (FBZ) and F200Y (OBZ) by Pyrosequencing • No individuals found to be homozygous at both 167Y & 200Y – lethal combination? • No significant differences in mRNA expression level between FBZ-S and FBZ-R at either isotype 1 or 2

  7. Fasciola hepatica expresses multiple a- and b-tubulin isotypesLouise A. Ryan, Elizabeth Hoey, Alan Trudgett, Ian Fairweather, Marc Fuchs, Mark W. Robinson, Emma Chambers, David J. Timson, Eimear Ryan, Theresa Feltwell, Al Ivens, Geoffrey Bentley and David JohnstonMol Biochem Parasitol 159: 73-78 (2008) • Identified 5 a-tubulin and 6 b-tubulin isotypes expressed in adult • 3 of the isotypes had tyrosine (Y) at codon 200, 2 had phenylalanine (F) and 1 had leucine (L) • All had F at 167 and glutamic acid (E) at 198 • Comparison between Cullompton (TCBZ-S) and Sligo/Oberon (TCBZ-R) isolates – all residues conserved

  8. Absence of three known benzimidazole resistance mutations in Trichostrongylus tenuis, a nematode parasite of avian hostsLucy M.I. Webster, Paul C.D. Johnston, Aileen Adam, Barbara K. Mable, Lukas F. KellerVet Para 158: 302-310 (2008) • BZ-R in T.tenuis – nematode parasite of red grouse • BZs used in this system >15 years but no reports of BZ-R as yet • Used PCR-RFLP to screen 1530 individuals from total of 14 populations at isotype-1 codon 200 and 167 and isotype-2 codon 200 • No BZ-R genotypes found

  9. Benzimidazole resistance in Trichostrongylus axei in sheep: Long term monitoring of affected sheep and genotypic evaluation of the parasiteChrystele Palcy, Christine Sauve, Jacques Cortet and Jacques CabaretThe Veterinary Journal doi:10.1016/j.tvjl.2008.09.012 (2008) • First report of BZ-R in Trichostrongylus axei in sheep in France • Post-treatment worm counts • Sequencing isotype-1 b-tubulin from adult T.axei recovered post mortem revealed only one non-synonymous SNP i.e. F200Y • Allele-specific PCR revealed r allele frequency = 0.63% • Seven years after BZ treatment ceased, T.axei still resistant – no reversion to susceptibility

  10. Genotyping of benzimidazole susceptible and resistant alleles in different populations of Haemonchus contortus from Himalayan and sub-Himalayan regions of North-West IndiaR. Garg & C.L. YadavTrop Anim Health Prod doi 10.1007/s11250-008-9292-5 (2008) • Allele-specific PCR used to diagnose F200Y in b-tubulin • Adult worms & larvae collected from sheep under different managemental practices and different geo-climatic zones • AS-PCR revealed frequency of resistant (r) alleles was: • - significantly higher at Sub-Himalayan>subtropical>temperate • - significantly higher under intensive v extensive management

  11. High-throughput detection of highly benzimidazole resistant allele E198A with mismatch primers in allele-specific real-time polymerase chain reactionC. Chen, W. Zheo, Y. Wang, Y. Chen, H. Li and M. ZhouPest Manag Sci 65: 413-419 (2009) • E198A SNP responsible for high level BZ-R in plant pathogenic fungus, Sclerotinia sclerotiorum • Allele-specific nucleotide PCR (ASPCR) and allele-specific quantitative real-time PCR (ASQPCR) used widely for its detection • ASPCR not suitable for high throughput; ASQPCR high background amplification • Have developed rapid, high-throughput genotyping method using mis-match primers (ASQPCR-MP) – mismatches in penultimate 3’ bases cf ASPCR • ASQPCR-MP took <6hr to complete and was suitable for large-scale epidemiological studies

  12. Molecular detection of benzimidazole resistance in Haemonchus contortus using real time PCR and pyrosequencingG. von Samson-Himmelstjerna, T.K. Walsh, A.A. Donnan, S. Carriere, F. Jackson, P.J. Skuce, K. Rohn and A.J. WolstenholmeParasitol 136: 349-358 (2009) • Investigated b-tubulin isotype-1 sequences of 18 H.contortus isolates with varying levels of resistance to TBZ • Only SNP to change significantly in BZ-R isolates was F200Y, drug sensitivity decreased with increasing frequency of TAC • Good agreement between RealTime and Pyrosequencing, both more sensitive than EHT and less time-consuming than current in vivo or in vitro tests – realistic option for resistance testing?

  13. Anthelmintic resistance in Swedish sheep flocks based on a comparison of the results from the faecal egg count reduction test and resistant allele frequencies of the b-tubulin geneJ. Hoglund, K. Gustafsson, B-L. Ljungstrom, A. Engstrom, A. Donnan and P. SkuceVet Para 161: 60-68 (2009) • FECRT Survey conducted during grazing season 2006-2007 = 1330 samples from 90 flocks on 45 farms with >20 ewes per farm • L3 identified morphologically from pooled cultures then used as source of genomic DNA for 2 molecular tests (i) PCR-based test for H.contortus and (ii) Pyrosequencing assay for F200Y SNP • Teladorsagia & Trichostrongylus dominant species but Haemonchus diagnosed in 37% of flocks (100% agreement with morphological ID) • Pyrosequencing assay detected BZ-R allele frequencies of >40% in Haemonchus +ve farms and high r allele frequencies in clinically most resistant farms

  14. Assays to detect b-tubulin codon 200 polymorphism in Trichuris trichuria and Ascaris lumbricoidesA. Diawara, L.J. Drake, R.R., Suwillo, J. Kihara, D.A.P. Bundy, M.E. Scott, C. Halfpenny, J.R. Stothard and R.K. PrichardPLoS Negl Trop Dis 3(3): e397. doi; 10.1371/ journal.pntd.0000397 (2009) • STHs Ascaris lumbricoides & Trichuris trichuria, major GI parasites of humans, especially children, BZs commonly used for mass treatment • Developed Pyrosequencing assay for TTC to TAC SNP in both spp. • Assay applied to samples from East Africa and Central America where mass treatment programmes have been implemented • All A.lumbricoides were TTC (BZ-S), however, found 63% of T.trichuria egg pools from treated people in Panama were TAC • Assays useful in assessing appropriate treatment strategies in areas of high prevalence and for monitoring BZ-R

  15. Tetra primer ARMS-PCR for identification of SNP in b-tubulin of Botrytis cinerea, responsible of resistance to benzimidazoleClaudio Munoz, Sebastian Gomez Talquenca & Melisa Lanza Volpe J Microbiol Methods 78: 245-246 (2009) • Competitive PCR – Tetra primer Amplification Refractory Mutation System (ARMS) PCR adapted to identify SNP in b-tubulin of pathogenic fungus, B. cinerea • All samples amplified PCR product of 372bp plus band of 154bp for wild type or 254 for mutant BZ-R strains – allele-specific multiplex PCR for BZ-R cf Silvestre et al? • Of 35 isolates analysed, 6 carried E198A SNP, no SNPs at codon 200 – all BZ-R strains

  16. Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in Ancylostoma caninumJan M. Schwenkenbecher and Ray M. KaplanExp Parasitol 122: 6-10 (2009) • Previously reported RealTime assay to detect codon 167 & 200 SNPs • Developed assay to detect BZ-R alleles in codon 198 • Used to screen hookworm specimens from dogs in Georgia • No elevated levels of polymorphism found

  17. In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of b-tubulinLucien Rufener, Ronald Kaminsky and Pascal MaserMol Biochem Parasitol doi: 10.1016/j.molbiopara.2009.07.002 (2009) • Used novel in vitro selection/invivo propagation to select BZ-R in Haemonchus contortus • 8 generations of selection with TBZ produced an in vitro resistance factor of 1000, BZ-R phenotype confirmed in vivo • Cloning and sequencing b-tubulin genes from TBZ-R isolate revealed all isotype-1 alleles, and some of the isotype-2 alleles, to carry the mutation E198A • Allele-specific E198A PCR assay developed

  18. Ongoing BZ-R Research

  19. Parasites of Sheep • Moredun/Glasgow/Calgary – survey of ~120 sheep farms in UK, species prevalence & BZ-R status (WAAVP CS30.1 & CS30.2) • Maria Martinez-Valladares (University of Leon, Spain) “Study of the beta-tubulin gene and resistance against macrocyclic lactones in Teladorsagia circumcincta” (WAAVP CS49.5)

  20. Observations on role of b-tubulin 198 / 200 in Haemonchus contortus Peter Hunt, Andrew Kotze et al. • Genotyping resistant populations shows: • - R200 always present • - R198 sometimes present • R / S crossing experiment: • - susceptible (McMaster 1931) x resistant (Wallangra2003) • - recovered F1 larvae • - reinfected sheep (F1 adults worms) • - collected larvae from faeces (= F2 larvae) • - reinfected sheep (F2 adult worms) • - treated some sheep with ABZ, left others untreated • - euthanased animals, collected adult worms & genotyped • (Sequenome)

  21. Outcome Selection of F2 with albendazole indicates dominant role of 198 vs 200 in Bz resistance in F2 population Haplotype analysis: – no 198A/200Y double homozygotes - 198A/200F under +ve selection in ABZ-selected F2 cf 198E/200Y – work in progress!

  22. Parasites of Cattle

  23. “Selection for BZ-resistance in Ostertagia ostertagi” Stefan Pachnicke, Bill Blackhall, Georg v. Samson-Himmelstjerna, University of Veterinary Medicine, Hannover, Germany Objective – to select a BZ-R isolate of O.ostertagi by sequential sub-therapeutic dosing with ABZ 8 rounds of selection produced isolate with EHT EC50=0.12mgTBZ/ml Validated by FECRT & adult/larval reductions

  24. Genetic comparisons • beta-tubulin SNPs • PgpA & C: - RealTime PCR - SSCP - SNPs PgpA PgpC • BZ-R mechanism?!

  25. Analysis of b-tubulin SNPs in a ML-R field isolate of Cooperia oncophora Abdel El-Abdellati & Peter Geldhof, University of Gent, Belgium • The isolate is highly resistant to ivermectin but fully susceptible to BZ and levamisole Cooperia oncophora

  26. Results to date • Changes in candidate IVM-R genes – what about b-tubulin? • Pyrosequencing assay designed to target E198A and F200Y SNPs – analysed single individual L1s and pools - all C/C and T/T homozygotes i.e susceptible genotypes • F167Y SNP analysis in progress

  27. Parasites of Horses

  28. Determination of genomic DNA sequences for beta-tubulin isotype 1 from multiple species of cyathostomin and detection of resistance alleles in third-stage larvae from horses with naturally acquired infectionsLake SL, Matthews JB, Kaplan RM and Hodgkinson JE* • Objectives - to design degenerate Pyrosequencing assay to analyse the frequency of SNPs at codons 167 and 200 in populations of third stage larvae (L3) containing mixed species of cyathostomin (tribe of >50 species!) Lake et al., 2009, in press

  29. Results Two arrangements of the beta tubulin isotype 1 gene exist in cyathostomin species

  30. Results • Consensus sequence was generated for regions flanking codon 167 and codon 200 from 91 and 76 sequences, representing 13 and 10 species, respectively • Degenerate PCR and pyrosequencing primers sited as shown: Codon 167 Codon 200

  31. Results – proof of principle Adults - 13 species of cyathostomin Codon 167 assay Adults -10 species of cyathostomin Codon 200 assay Codon 167 assay L3s of unknown species Application of PCR and pyrosequencing primers to adults of multiple species and L3s of unknown species

  32. Results • Resistance alleles at codon 167 in L3s from horses with naturally acquired infections - Two locations in USA, ‘before’ and ‘after’ FBZ treatment Location 1 n= 125 L3s Location 2 n= 116 L3s

  33. Conclusions and further work • Now have an assay to detect SNPs at codon 167 and 200 of beta tubulin isotype 1 in L3 samples from naturally infected horses, irrespective of species composition • Allows robust statistical analyses of SNPs in large numbers of parasites • Detection of SNPs in L3 exposed to BZs - in vitro - in vivo

  34. Parasites of Man

  35. An assay to quantitate Benzimidazole resistance-associated SNPs in N.americanus and A.duodenale Ranbir Sarai, Alan Robertson, James McCarthy, Institute of Medical Research, University of Queensland, Australia A.duodenale N.americanus • Very difficult to tell apart using standard parasitological tests, requires purging and examination of adults

  36. Objective BZ-R-associated SNPs identified in other nematode species viz. F167Y, A198E & F200Y To develop a high throughput methodology enabling quantitive assay of these SNPs in pools of hookworm eggs collected from human stool To simultaneously assay for the species of hookworm (N.americanus vs A. duodenale) The MassARRAY platform uses mass spectrometry to measure the mass of short extension sequences of nucleic acids and, hence, the genotype

  37. PCR Amplicon for MassARRAY Speciation SNPs BZ “Resistance” SNPs • Designed a PCR primer pair to span AA 181-219 of the b-tubulin gene (PCR product length of 119 nt) • This encompassed 3 polymorphic sites upstream of the “drug resistance” SNPs enabling simultaneous speciation

  38. Primers for ASX Two assays were designed to identify the species and presence of the alleles associated with resistance The extension primer in red identifies the species The extension primer in green enables genotyping at the 198 and 200 SNPs

  39. Unextended ASX primers Extended Primers

  40. MassArray Platform enables quantitation of allele frequency in a worm (egg) population Example of quantitation of 3 alleles in H. contortus (standard curve generated in plasmid mixing expt.)

  41. Monitoring efficacy of anthelmintics for the treatment of Soil Transmitted Helminths in humans • WHO-sponsored project – inc. Jozef Vercruysse , Antonio Montresor, Marco Albonico, Andrew Kotze, James McCarthy and Jerzy Behnke • Aims: • To develop and validate a standard protocol to monitor efficacy of anthelmintics in populations with different exposure to anthelmintics –focus on hookworms - 6 study sites: Brazil, Cameroon, Ethiopia, India, Tanzania & Vietnam

  42. Objectives • To assess change in hookworm FEC in school age children 10-14d post-treatment with 400mg ABZ • Monitor efficacy by determining Cure Rate (CR) and Egg Reduction Rate (ERR) • To evaluate suitability of FECRT for monitoring efficacy • To compare relative performance of Kato Katz or other qualitative coprological techniques with McMaster egg counting method • To archive material for subsequent molecular analysis e.g. BZ genotyping

  43. Acknowledgements • Provision of slides/info - Jane Hodgkinson (UK), Ray Kaplan (USA), Georg von Samson-Himmelstjerna, Stefan Pahnicke (Germany), Peter Geldhof, Abdel El-Abdellati (Belgium), James McCarthy, Peter Hunt & Andrew Kotze (Australia) • Thanks for listening!

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