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(?)

• Ongoing research on BZ-R in parasites of:- Sheep - Cattle- Horses - Man

Characterization of beta-tubulin genes in hookworms and Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-investigation of resistance-associated mutations using real-

time PCRtime PCR

Jan Schwenkenbecher, Marco Albonico, Quentin Bickle, Ray M. Jan Schwenkenbecher, Marco Albonico, Quentin Bickle, Ray M. KaplanKaplan

Mol Biochem Parasitol 156:167-174 (2007)Mol Biochem Parasitol 156:167-174 (2007)

• Ancylostoma duodenale & Necator americanus

• Mass drug administration with BZ anthelmintics

• SNPs at codons 167 & 200 in -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

Genetic analysis of a relationship between macrocyclic lactone Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on and benzimidazole anthelmintic selection on Haemonchus Haemonchus

contortuscontortus

de Lourdes Mottier M. and Prichard R.K.de Lourdes Mottier M. and Prichard R.K.Pharmacogenet Genomics 18(2): 129-140Pharmacogenet Genomics 18(2): 129-140

• Lab strain of Haemonchus contortus – repeated IVM treatment in vivo selected for TTC to TAC mutation in -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!)

P-glycoprotein selection in strains of P-glycoprotein selection in strains of Haemonchus contortusHaemonchus contortus resistant to benzimidazolesresistant to benzimidazoles

William J. Blackhall, Roger K. Prichard and Robin N. BeechWilliam J. Blackhall, Roger K. Prichard and Robin N. BeechVet Para 152: 101-107 (2008)Vet Para 152: 101-107 (2008)

• H.contortus – BZ-R associated with selection on -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”?

The role of polymorphisms at The role of polymorphisms at tubulin isotype 1 codons 167 tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostominsand 200 in benzimidazole resistance in cyathostomins

J.E. Hodgkinson, H.J. Clark, R.M. Kaplan, S.L., Lake and J.B. J.E. Hodgkinson, H.J. Clark, R.M. Kaplan, S.L., Lake and J.B. MatthewsMatthews

Int J Parasitol 38: 1149-1160 (2008)Int 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 -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

Fasciola hepaticaFasciola hepatica expresses multiple expresses multiple - and - and -tubulin isotypes-tubulin isotypes

Louise A. Ryan, Elizabeth Hoey, Alan Trudgett, Ian Louise A. Ryan, Elizabeth Hoey, Alan Trudgett, Ian Fairweather, Marc Fuchs, Mark W. Robinson, Emma Chambers, Fairweather, Marc Fuchs, Mark W. Robinson, Emma Chambers,

David J. Timson, Eimear Ryan, Theresa Feltwell, Al Ivens, David J. Timson, Eimear Ryan, Theresa Feltwell, Al Ivens, Geoffrey Bentley and David JohnstonGeoffrey Bentley and David Johnston

Mol Biochem Parasitol 159: 73-78 (2008)Mol Biochem Parasitol 159: 73-78 (2008)

• Identified 5 -tubulin and 6 -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

Absence of three known benzimidazole resistance mutations Absence of three known benzimidazole resistance mutations in in Trichostrongylus tenuisTrichostrongylus tenuis, a nematode parasite of avian hosts, a nematode parasite of avian hosts

Lucy M.I. Webster, Paul C.D. Johnston, Aileen Adam, Barbara Lucy M.I. Webster, Paul C.D. Johnston, Aileen Adam, Barbara K. Mable, Lukas F. KellerK. Mable, Lukas F. Keller

Vet Para 158: 302-310 (2008)Vet 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

Benzimidazole resistance in Benzimidazole resistance in Trichostrongylus axeiTrichostrongylus axei in sheep: in sheep: Long term monitoring of affected sheep and genotypic Long term monitoring of affected sheep and genotypic

evaluation of the parasiteevaluation of the parasite

Chrystele Palcy, Christine Sauve, Jacques Cortet and Jacques Chrystele Palcy, Christine Sauve, Jacques Cortet and Jacques CabaretCabaret

The Veterinary Journal doi:10.1016/j.tvjl.2008.09.012 (2008The 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 -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

Genotyping of benzimidazole susceptible and resistant alleles Genotyping of benzimidazole susceptible and resistant alleles in different populations of in different populations of Haemonchus contortusHaemonchus contortus from from

Himalayan and sub-Himalayan regions of North-West IndiaHimalayan and sub-Himalayan regions of North-West India

R. Garg & C.L. YadavR. Garg & C.L. YadavTrop Anim Health Prod Trop Anim Health Prod

doi 10.1007/s11250-008-9292-5 (2008)doi 10.1007/s11250-008-9292-5 (2008)

• Allele-specific PCR used to diagnose F200Y in -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

High-throughput detection of highly benzimidazole resistant High-throughput detection of highly benzimidazole resistant allele E198A with mismatch primers in allele-specific real-time allele E198A with mismatch primers in allele-specific real-time

polymerase chain reactionpolymerase chain reaction

C. Chen, W. Zheo, Y. Wang, Y. Chen, H. Li and M. ZhouC. Chen, W. Zheo, Y. Wang, Y. Chen, H. Li and M. ZhouPest Manag Sci 65: 413-419 (2009)Pest 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

Molecular detection of benzimidazole resistance in Molecular detection of benzimidazole resistance in Haemonchus contortusHaemonchus contortus using real time PCR and using real time PCR and

pyrosequencingpyrosequencing

G. von Samson-Himmelstjerna, T.K. Walsh, A.A. Donnan, S. G. von Samson-Himmelstjerna, T.K. Walsh, A.A. Donnan, S. Carriere, F. Jackson, P.J. Skuce, K. Rohn and A.J. WolstenholmeCarriere, F. Jackson, P.J. Skuce, K. Rohn and A.J. Wolstenholme

Parasitol 136: 349-358 (2009)Parasitol 136: 349-358 (2009)

• Investigated -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?

Anthelmintic resistance in Swedish sheep flocks based on a Anthelmintic resistance in Swedish sheep flocks based on a comparison of the results from the faecal egg count reduction comparison of the results from the faecal egg count reduction

test and resistant allele frequencies of the test and resistant allele frequencies of the -tubulin gene-tubulin gene

J. Hoglund, K. Gustafsson, B-L. Ljungstrom, A. Engstrom, A. J. Hoglund, K. Gustafsson, B-L. Ljungstrom, A. Engstrom, A. Donnan and P. SkuceDonnan and P. Skuce

Vet Para 161: 60-68 (2009)Vet 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

Assays to detect Assays to detect -tubulin codon 200 polymorphism in -tubulin codon 200 polymorphism in Trichuris Trichuris trichuriatrichuria and and Ascaris lumbricoidesAscaris lumbricoides

A. Diawara, L.J. Drake, R.R., Suwillo, J. Kihara, D.A.P. Bundy, M.E. Scott, C. A. Diawara, L.J. Drake, R.R., Suwillo, J. Kihara, D.A.P. Bundy, M.E. Scott, C. Halfpenny, J.R. Stothard and R.K. PrichardHalfpenny, J.R. Stothard and R.K. Prichard

PLoS Negl Trop Dis 3(3): e397. doi; 10.1371/ journal.pntd.0000397 PLoS Negl Trop Dis 3(3): e397. doi; 10.1371/ journal.pntd.0000397 (2009)(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

Tetra primer ARMS-PCR for identification of SNP in Tetra primer ARMS-PCR for identification of SNP in -tubulin of -tubulin of Botrytis cinerea, responsible of resistance to benzimidazoleBotrytis cinerea, responsible of resistance to benzimidazole

Claudio Munoz, Sebastian Gomez Talquenca & Claudio Munoz, Sebastian Gomez Talquenca & Melisa Lanza Volpe Melisa Lanza Volpe

J Microbiol Methods 78: 245-246 (2009)J Microbiol Methods 78: 245-246 (2009)

• Competitive PCR – Tetra primer Amplification Refractory MMutation SSystem (ARMS) PCR adapted to identify SNP in -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

Real-time PCR assays for monitoring benzimidazole Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in resistance-associated mutations in Ancylostoma caninumAncylostoma caninum

Jan M. Schwenkenbecher and Ray M. KaplanJan M. Schwenkenbecher and Ray M. Kaplan

Exp Parasitol 122: 6-10 (2009)Exp 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

In vitroIn vitro selection of selection of Haemonchus contortusHaemonchus contortus for benzimidazole for benzimidazole resistance reveals a mutation at amino acid 198 of b-tubulinresistance reveals a mutation at amino acid 198 of b-tubulin

Lucien Rufener, Ronald Kaminsky and Pascal MaserLucien Rufener, Ronald Kaminsky and Pascal Maser

Mol Biochem Parasitol Mol Biochem Parasitol doi: 10.1016/j.molbiopara.2009.07.002 (2009)doi: 10.1016/j.molbiopara.2009.07.002 (2009)

• Used novel in vitro selection/in vivo 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 -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

Ongoing BZ-R Research

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)

Observations on role of Observations on role of -tubulin 198 / 200 in -tubulin 198 / 200 in Haemonchus contortusHaemonchus contortus

Peter Hunt, Andrew Kotze et al. 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)

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OutcomeOutcome

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!

Parasites of Cattle

““Selection for BZ-resistance in Selection for BZ-resistance in Ostertagia ostertagiOstertagia ostertagi””

•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=0.12gTBZ/ml• Validated by FECRT & adult/larval reductions

Stefan Pachnicke, Bill Blackhall, Georg v. Samson-Himmelstjerna, Stefan Pachnicke, Bill Blackhall, Georg v. Samson-Himmelstjerna, University of Veterinary Medicine, Hannover, GermanyUniversity of Veterinary Medicine, Hannover, Germany

Egg and Larval Count Reduction Test

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20%

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100%

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egg reduction -unselected-

larval reduction -unselected-

egg reduction -selected-

larval reduction -selected-

Genetic comparisons

• beta-tubulin SNPsbeta-tubulin SNPs

• PgpA & C: PgpA & C:

- RealTime PCR- RealTime PCR

- SSCP- SSCP

- SNPs- SNPs

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100%150%200%

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• BZ-R mechanism?!BZ-R mechanism?!

  Effic. D7 Effic. D21

2006 72 50,0

2007 83,3 45,7

2008 72,3 21,2 

Cooperia oncophora

• The isolate is highly resistant to ivermectin but fully susceptible to BZ and levamisole

Analysis of Analysis of -tubulin SNPs in a ML-R field -tubulin SNPs in a ML-R field isolate of isolate of Cooperia oncophoraCooperia oncophora

Abdel El-Abdellati & Peter Geldhof, University of Abdel El-Abdellati & Peter Geldhof, University of Gent, BelgiumGent, Belgium

Results to dateResults to date

• Changes in candidate IVM-R genes – what about -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

Parasites of Horses

Determination of genomic DNA sequences for Determination of genomic DNA sequences for beta-tubulin isotype 1 from multiple species of beta-tubulin isotype 1 from multiple species of

cyathostomin and detection of resistance alleles cyathostomin and detection of resistance alleles in third-stage larvae from horses with naturally in third-stage larvae from horses with naturally

acquired infectionsacquired infections

Lake SL, Matthews JB, Kaplan RM and Hodgkinson JE*

Lake et al., 2009, in press

• 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!)

ResultsResults

• Two arrangements of the beta tubulin isotype 1 gene exist in cyathostomin species

ResultsResults

Cya cat CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGMGGAGTATCCTGATAGAATCAT Cyc nas CAGGGYTTCCAGYTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGSGAGGAGTATCCTGATAGAATCAT Cyc ins ---------------------------------GGTACCGGATCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cyc ash CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGTTCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cyd bic CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cyc cal ----------------------------------GTACCGGWTCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT D L3 -----------------------------------TACCGGATCGGGTATGGGMACTCTCCTCATCTCYAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cys gol CAGGGCTTCCARCTAACTCACTCACTTGGAGGAGGTACCGGWTCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT ILPH L3 --------------------------------AGGTACCGGWTCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT JC L3 ------------------------------GGGAGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cyc lep CAGGGCTTCCAGCTAACTCACTCACTCGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cys lon CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT Cys min CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT WK L3 ---------------------------------GGTACCGGWTCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT 167 Con CAGGGYTTCCARYTAACTCACTCACTYGGAGGRRGTACCGGWTCGGGTATGGGMACTCTCCTCATYTCYAAAATTCGSGMGGAGTATCCTGATAGAATCAT Cya cat GTSCTCRTWCTCCGTTGTTCCCTCACCAAAGGTY Cyc nas GTCCTCGWWNTCSGTTGTTCCCTCACCAAAGGTC Cyc ins GTCCTCRTTCTCYGTTGTTCCCTCACCAAAGGTC Cyc ash GTCCTCGTTCTCCGTTGTTCCCTCACCAAAGGTC Cyd bic GTCCTCTTTCTCCGTTGTTCCCTCACCAAAGGTC Cyc cal GTCCTCGWWCTCSGTTGTTCCCTCACCAAAGGTC D L3 GTCCTCGTWYTCYGTTGTTCCCTCACCAAAGGTC Cys gol GTCCYYSKWCTCCGTTGTTCCCTCACCARAGGTY ILPH L3 GTCCTCGWWCTCSGTTGTTCCCTCACCAAAGGTC JC L3 GTCCTCRTWCTCCGTTGTTCCCTCACCAAAGGTC Cyc lep GTCCTCGTTCTCTGTTGTTCCCTCACCAAAGGTC Cys lon GTCCTCATWCTCCGTTGTYCCYTCACCAAAGGTC Cys min GTCCTCGTACTCCGTTGTTCCCTCACCAAAGGTC WK L3 GTCYTCRTWCTCSGTTGTTCCCTCACCAAAGGTC 167 Con GTSYYYVDWNTCBGTTGTYCCYTCACCARAGGTY

167fs 167seq

167r

Cya cat TCYGACACTGTTGTGGAGCCATACAATGCTACCCTATCCGTTCATCAGYTGGTTGAAAATACAGACGWMACTTWCTGTATTGACAATGAAGCTCTYTAY Cyc nas WCCGAYACYGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGYTGAAAATACAGACGAGACTTWCTGTATTGACAATGAAGCTCTKTAT Cyc ins TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTTTAT Cyd bic TCCGACACCGTTGTGGAGCCATACAATGCTACCCTTTCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCGCTTTAT Cyc cal TCCGACACCGTTGTGGAGCCRTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTWCTGTATTGACAATGAAGCTCTGTAT Cor cor TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTGTAT D L3 TCCGACACCGTTGTGGAGCCRTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGARACTTTCTGTATTGACAATGAAGCTCTYTAY Cyc elo TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGAC---------------------------- Cys gol TCYGACACYGTTGTGGAGCCRTACAATGCTACCCTRTCCGTTCATCARTTGGTTGAAAATACAGACKWGACTTTCTSTATTGACAATGAAGCTCTKTAT ILPH L3 TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTTTAT JC L3 TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTWCTGTATTGACAATGAAGCTCTTTAT Cys lon TCCGAYACHGTTGTGGAGCCRTACAATGCYACCCTWTCCGTTCAYCAGTTGGTTGAAAATACAGRCGARACTTWCTGTATTRACAAYGAAGCTYTHTAT Cya pat TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTGTAT WK L3 TCCGACACCGTTGTGGAGCCWTACAATGCYACCYTWTCCGTYCAYCWRTTGGTTGAAAATACAGACGARACTTWCTGTATTGACAAYGAAGCTYTNTAT 200 Con WCYGAYACHGTTGTGGAGCCDTACAATGCYACCYTDTCCGTYCAYCWRYTGGYTGAAAATACAGRCKWVACTTWCTSTATTRACAAYGAAGCKYTNTAY Cya cat GATATTTGCTTCCGCACYYTKAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtragcrayatkcsayyrctgagcytkgtrgaattysc Cyc nas GATATTTGCTTCCGCACYTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggkgrgcaatatgygattgstgagcttggtggaatttgc Cyc ins GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtgagcaatatgcgattgctgagcttggtggaatt--- Cyd bic GATATTTGCTTCCGCACCTTGAAACTTACAAACCCAACTTATGGAGATCTGAATCATCTTggt------------------------------------ Cyc cal GATATTTGCTTCCGCACTTTRAAACTCACGAACCCAACTTATGGTGATCTAAATCATCTTggtaagtaaccatgccattggtgagcttgtcagat---- Cor cor GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTT----------------------------------------------------------- D L3 GATATTTGCTTCCGCACCCTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtragcgaymwycsmysakykrrymkwrtagaatagtt Cyc elo --------------------------------------------------------------------------------------------------- Cys gol GATATNTGCTTCCGCACYYTGAAACTCACGAACCCAACWTATGGAGATCTGAATCATCTTggkragcratatgcgattgctgagcttggtggaatttgc ILPH L3 GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtgagcaatatgcgattgctgagcttggtgaatwscyw JC L3 GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtgrgcaatatgcgattgctgagcttggtgcaattgct Cys lon GATATTTGCTTCCGCACYYTGAAACTYACRAACCCAACYTAYGGAGATBTGAATCATCTTggtragcrataygsratygynvrvyhbsdbndwdkrbmy Cya pat GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAAC------------------------------------------------------------- WK L3 GATATTTGSTTCCGCACYYTRAAACTYACRAACCCAACHTATGGWGATYTRAATCATCTTggtragbrrhmhkbyhhhdbdwkhdyhbbwndvwwdvhh 200 Con GATATNTGSTTCCGCACYYTDAAACTYACRAACCCAACHTAYGGWGATBTRAATCATCTT--------------------------------------- Cya cat kaatttktyyrawtwtcwa-------------GTGTCTGTAACAATGTCTGGWGTCACYACRTGTCTTC Cyc nas taatttttwcaaatwtcta-------------GTGTCTGTAACAATGTCTGGWGTCACTACATGYCTTC Cyc ins --------------------------------------------------------------------- Cyd bic --------------------------------------------------------------------- Cyc cal --------------------------------------------------------------------- Cor cor --------------------------------------------------------------------- D L3 --------------------------------------------------------------------- Cyc elo --------------------------------------------------------------------- Cys gol taatttttwcaaatatcta-------------GTGTCTGTAACAATGTCTGGWGTCACTACATGTCTTC ILPH L3 atatatttaccatctttcccttttgaaacaat------------------------------------- JC L3 a-------------------------------------------------------------------- Cys lon rwhhhmbkmwwytgtctgattttca-------GTGTCTGTAACAATGTCTGGTGTCACYACATGYCTTY Cya pat --------------------------------------------------------------------- WK L3 mwwwyktwttggtattta--------------GTGTCTGTAACAATGTCTGG----------------- 200 Con --------------------------------GTGTCTGTAACAATGTCTGGWGTCACYACRTGYCTTY

200f 200seq

200r

200rs

• 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

Results – proof of principleResults – proof of principle

• Application of PCR and pyrosequencing primers to adults of multiple species and L3s of unknown species

Adults - 13 species of cyathostomin

Codon 167 assay

Codon 200 assay

Adults -10 species of cyathostomin

Codon 167 assay L3s of unknown species

ResultsResults• Resistance alleles at codon 167 in L3s from horses with Resistance alleles at codon 167 in L3s from horses with

naturally acquired infections naturally acquired infections

- Two locations in USA, ‘before’ and ‘after’ FBZ treatment- Two locations in USA, ‘before’ and ‘after’ FBZ treatment

Location 1 n= 125 L3s

Location 2 n= 116 L3s

Conclusions and further workConclusions 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

Parasites of Man

An assay to quantitate Benzimidazole An assay to quantitate Benzimidazole resistance-associated SNPs in resistance-associated SNPs in

N.americanus N.americanus and and A.duodenaleA.duodenale

Ranbir Sarai, Alan Robertson, James McCarthy, Ranbir Sarai, Alan Robertson, James McCarthy, Institute of Institute of Medical Research, University of Queensland, AustraliaMedical Research, University of Queensland, Australia

N.americanus A.duodenale

• Very difficult to tell apart using standard parasitological tests, requires purging and examination of adults

ObjectiveObjective• 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

PCR Amplicon for MassARRAYPCR Amplicon for MassARRAY

Speciation SNPs

BZ “Resistance”

SNPs

• Designed a PCR primer pair to span AA 181-219 of the -tubulin gene (PCR product length of 119 nt)

• This encompassed 3 polymorphic sites upstream of the “drug resistance” SNPs enabling simultaneous speciation

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

Extended Primers

Unextended ASX primers

Example of quantitation of 3 alleles in H. contortus (standard curve generated in plasmid mixing expt.)

MassArray Platform enables quantitation of allele frequency in a worm (egg) population

Monitoring efficacy of anthelmintics Monitoring efficacy of anthelmintics for the treatment of Soil Transmitted for the treatment of Soil Transmitted

Helminths in humansHelminths 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

ObjectivesObjectives

• 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

AcknowledgementsAcknowledgements

• 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!