Transcript CARS Benzimidazole Resistance Workgroup

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
investigation of resistance-associated mutations using realtime PCR
Jan Schwenkenbecher, Marco Albonico, Quentin Bickle, Ray M.
Kaplan
Mol 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
Genetic analysis of a relationship between macrocyclic lactone
and benzimidazole anthelmintic selection on Haemonchus
contortus
de 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!)
P-glycoprotein selection in strains of Haemonchus contortus
resistant to benzimidazoles
William J. Blackhall, Roger K. Prichard and Robin N. Beech
Vet 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”?
The role of polymorphisms at b-tubulin isotype 1 codons 167
and 200 in benzimidazole resistance in cyathostomins
J.E. Hodgkinson, H.J. Clark, R.M. Kaplan, S.L., Lake and J.B.
Matthews
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 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
Fasciola hepatica expresses multiple a- and b-tubulin isotypes
Louise 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 Johnston
Mol 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
Absence of three known benzimidazole resistance mutations in
Trichostrongylus tenuis, a nematode parasite of avian hosts
Lucy M.I. Webster, Paul C.D. Johnston, Aileen Adam, Barbara
K. Mable, Lukas F. Keller
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 Trichostrongylus axei in sheep:
Long term monitoring of affected sheep and genotypic
evaluation of the parasite
Chrystele Palcy, Christine Sauve, Jacques Cortet and Jacques
Cabaret
The 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
Genotyping of benzimidazole susceptible and resistant alleles
in different populations of Haemonchus contortus from
Himalayan and sub-Himalayan regions of North-West India
R. Garg & C.L. Yadav
Trop 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
High-throughput detection of highly benzimidazole resistant
allele E198A with mismatch primers in allele-specific real-time
polymerase chain reaction
C. Chen, W. Zheo, Y. Wang, Y. Chen, H. Li and M. Zhou
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
Haemonchus contortus using real time PCR and
pyrosequencing
G. von Samson-Himmelstjerna, T.K. Walsh, A.A. Donnan, S.
Carriere, F. Jackson, P.J. Skuce, K. Rohn and A.J.
Wolstenholme
Parasitol 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?
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 gene
J. Hoglund, K. Gustafsson, B-L. Ljungstrom, A. Engstrom, A.
Donnan and P. Skuce
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 b-tubulin codon 200 polymorphism in Trichuris
trichuria and Ascaris lumbricoides
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. Prichard
PLoS 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
Tetra primer ARMS-PCR for identification of SNP in b-tubulin of
Botrytis cinerea, responsible of resistance to benzimidazole
Claudio 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
Real-time PCR assays for monitoring benzimidazole
resistance-associated mutations in Ancylostoma caninum
Jan M. Schwenkenbecher and Ray M. Kaplan
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 vitro selection of Haemonchus contortus for benzimidazole
resistance reveals a mutation at amino acid 198 of b-tubulin
Lucien Rufener, Ronald Kaminsky and Pascal Maser
Mol Biochem Parasitol
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 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
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 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)
Outcome
200-PHE-Susc
200
200-TYR-Res
allele frequency
1.00
Selection of F2 with
albendazole indicates
dominant role of 198 vs 200 in
Bz resistance
in F2 population
0.75
0.50
0.25
0.00
198
allele frequency
1.00
198-GLU-Susc
198-ALA-Res
Haplotype analysis:
0.75
– no 198A/200Y double
homozygotes
0.50
0.25
W
al
la
ng
ra
20
M
cM
03
as
te
r1
F2
93
1
un
se
le
F2
ct
ed
al
be
nd
az
ol
e
0.00
Population genotyped
- 198A/200F under +ve
selection in ABZ-selected F2 cf
198E/200Y – work in progress!
Parasites of Cattle
“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
Egg and Larval Count Reduction Test
Reduction in Percent
120%
100%
egg reduction -unselectedlarval reduction -unselectedegg reduction -selectedlarval reduction -selected-
80%
60%
40%
20%
0%
0,75
1,5
2,25
mg ABZ/kg BW
3
- SSCP
- SNPs
• BZ-R mechanism?!
PgpA
L3
*
eg
gs
eg
gs
ed
ed
se
le
ct
ed
s
s
L3
wo
rm
se
le
ct
ed
ad
ul
t
wo
rm
*
un
se
le
ct
ed
ad
ul
t
eg
gs
700%
600%
500%
400%
300%
200%
100%
0%
un
se
le
ct
un
se
le
ct
ed
L3
eg
gs
ed
300%
250%
200%
150%
100%
50%
0%
se
le
ct
ed
un
se
le
ct
se
le
ct
ed
s
s
L3
wo
rm
wo
rm
se
le
ct
ed
ad
ul
t
un
se
le
ct
ed
ad
ul
t
- RealTime PCR
un
se
le
ct
se
le
ct
ed
Genetic comparisons
• beta-tubulin SNPs
• PgpA & C:
*
*
PgpC
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
2006
2007
2008
Effic. D7
72
83,3
72,3
Effic. D21
50,0
45,7
21,2
Cooperia oncophora
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
Parasites of Horses
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 infections
Lake 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
Results
• Two arrangements of the beta tubulin isotype 1 gene exist
in cyathostomin species
•
Results
Consensus sequence was generated for regions flanking
codon 167 and codon 200 from 91 and 76 sequences,
representing 13 and 10 species, respectively
•
Cya cat
Cyc nas
Cyc ins
Cyc ash
Cyd bic
Cyc cal
D L3
Cys gol
ILPH L3
JC L3
Cyc lep
Cys lon
Cys min
WK L3
167 Con
Degenerate PCR and pyrosequencing primers sited as
shown:
CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGMGGAGTATCCTGATAGAATCAT
CAGGGYTTCCAGYTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGSGAGGAGTATCCTGATAGAATCAT
---------------------------------GGTACCGGATCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGTTCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
----------------------------------GTACCGGWTCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
-----------------------------------TACCGGATCGGGTATGGGMACTCTCCTCATCTCYAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGCTTCCARCTAACTCACTCACTTGGAGGAGGTACCGGWTCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
--------------------------------AGGTACCGGWTCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
------------------------------GGGAGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGCTTCCAGCTAACTCACTCACTCGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGCTTCCAGCTAACTCACTCACTTGGAGGAGGTACCGGATCGGGTATGGGCACTCTCCTCATCTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
---------------------------------GGTACCGGWTCGGGTATGGGCACTCTCCTCATYTCCAAAATTCGGGAGGAGTATCCTGATAGAATCAT
CAGGGYTTCCARYTAACTCACTCACTYGGAGGRRGTACCGGWTCGGGTATGGGMACTCTCCTCATYTCYAAAATTCGSGMGGAGTATCCTGATAGAATCAT
TCYGACACTGTTGTGGAGCCATACAATGCTACCCTATCCGTTCATCAGYTGGTTGAAAATACAGACGWMACTTWCTGTATTGACAATGAAGCTCTYTAY
WCCGAYACYGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGYTGAAAATACAGACGAGACTTWCTGTATTGACAATGAAGCTCTKTAT
TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTTTAT
TCCGACACCGTTGTGGAGCCATACAATGCTACCCTTTCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCGCTTTAT
TCCGACACCGTTGTGGAGCCRTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTWCTGTATTGACAATGAAGCTCTGTAT
TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTGTAT
TCCGACACCGTTGTGGAGCCRTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGARACTTTCTGTATTGACAATGAAGCTCTYTAY
TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGAC---------------------------TCYGACACYGTTGTGGAGCCRTACAATGCTACCCTRTCCGTTCATCARTTGGTTGAAAATACAGACKWGACTTTCTSTATTGACAATGAAGCTCTKTAT
TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTTTAT
TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTWCTGTATTGACAATGAAGCTCTTTAT
TCCGAYACHGTTGTGGAGCCRTACAATGCYACCCTWTCCGTTCAYCAGTTGGTTGAAAATACAGRCGARACTTWCTGTATTRACAAYGAAGCTYTHTAT
TCCGACACCGTTGTGGAGCCGTACAATGCTACCCTATCCGTTCATCAGTTGGTTGAAAATACAGACGAGACTTTCTGTATTGACAATGAAGCTCTGTAT
TCCGACACCGTTGTGGAGCCWTACAATGCYACCYTWTCCGTYCAYCWRTTGGTTGAAAATACAGACGARACTTWCTGTATTGACAAYGAAGCTYTNTAT
WCYGAYACHGTTGTGGAGCCDTACAATGCYACCYTDTCCGTYCAYCWRYTGGYTGAAAATACAGRCKWVACTTWCTSTATTRACAAYGAAGCKYTNTAY
Cya cat
Cyc nas
Cyc ins
Cyd bic
Cyc cal
Cor cor
D L3
Cyc elo
Cys gol
ILPH L3
JC L3
Cys lon
Cya pat
WK L3
200 Con
GATATTTGCTTCCGCACYYTKAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtragcrayatkcsayyrctgagcytkgtrgaattysc
GATATTTGCTTCCGCACYTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggkgrgcaatatgygattgstgagcttggtggaatttgc
GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtgagcaatatgcgattgctgagcttggtggaatt--GATATTTGCTTCCGCACCTTGAAACTTACAAACCCAACTTATGGAGATCTGAATCATCTTggt-----------------------------------GATATTTGCTTCCGCACTTTRAAACTCACGAACCCAACTTATGGTGATCTAAATCATCTTggtaagtaaccatgccattggtgagcttgtcagat---GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTT----------------------------------------------------------GATATTTGCTTCCGCACCCTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtragcgaymwycsmysakykrrymkwrtagaatagtt
--------------------------------------------------------------------------------------------------GATATNTGCTTCCGCACYYTGAAACTCACGAACCCAACWTATGGAGATCTGAATCATCTTggkragcratatgcgattgctgagcttggtggaatttgc
GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtgagcaatatgcgattgctgagcttggtgaatwscyw
GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAACTTATGGAGATCTGAATCATCTTggtgrgcaatatgcgattgctgagcttggtgcaattgct
GATATTTGCTTCCGCACYYTGAAACTYACRAACCCAACYTAYGGAGATBTGAATCATCTTggtragcrataygsratygynvrvyhbsdbndwdkrbmy
GATATTTGCTTCCGCACTTTGAAACTCACGAACCCAAC------------------------------------------------------------GATATTTGSTTCCGCACYYTRAAACTYACRAACCCAACHTATGGWGATYTRAATCATCTTggtragbrrhmhkbyhhhdbdwkhdyhbbwndvwwdvhh
GATATNTGSTTCCGCACYYTDAAACTYACRAACCCAACHTAYGGWGATBTRAATCATCTT---------------------------------------
GTSCTCRTWCTCCGTTGTTCCCTCACCAAAGGTY
GTCCTCGWWNTCSGTTGTTCCCTCACCAAAGGTC
GTCCTCRTTCTCYGTTGTTCCCTCACCAAAGGTC
GTCCTCGTTCTCCGTTGTTCCCTCACCAAAGGTC
GTCCTCTTTCTCCGTTGTTCCCTCACCAAAGGTC
GTCCTCGWWCTCSGTTGTTCCCTCACCAAAGGTC
GTCCTCGTWYTCYGTTGTTCCCTCACCAAAGGTC
GTCCYYSKWCTCCGTTGTTCCCTCACCARAGGTY
GTCCTCGWWCTCSGTTGTTCCCTCACCAAAGGTC
GTCCTCRTWCTCCGTTGTTCCCTCACCAAAGGTC
GTCCTCGTTCTCTGTTGTTCCCTCACCAAAGGTC
GTCCTCATWCTCCGTTGTYCCYTCACCAAAGGTC
GTCCTCGTACTCCGTTGTTCCCTCACCAAAGGTC
GTCYTCRTWCTCSGTTGTTCCCTCACCAAAGGTC
GTSYYYVDWNTCBGTTGTYCCYTCACCARAGGTY
Cya cat
Cyc nas
Cyc ins
Cyd bic
Cyc cal
Cor cor
D L3
Cyc elo
Cys gol
ILPH L3
JC L3
Cys lon
Cya pat
WK L3
200 Con
kaatttktyyrawtwtcwa-------------GTGTCTGTAACAATGTCTGGWGTCACYACRTGTCTTC
taatttttwcaaatwtcta-------------GTGTCTGTAACAATGTCTGGWGTCACTACATGYCTTC
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------taatttttwcaaatatcta-------------GTGTCTGTAACAATGTCTGGWGTCACTACATGTCTTC
atatatttaccatctttcccttttgaaacaat------------------------------------a-------------------------------------------------------------------rwhhhmbkmwwytgtctgattttca-------GTGTCTGTAACAATGTCTGGTGTCACYACATGYCTTY
--------------------------------------------------------------------mwwwyktwttggtattta--------------GTGTCTGTAACAATGTCTGG------------------------------------------------GTGTCTGTAACAATGTCTGGWGTCACYACRTGYCTTY
167r
Codon 167
200seq
200f
167seq
167fs
Cya cat
Cyc nas
Cyc ins
Cyc ash
Cyd bic
Cyc cal
D L3
Cys gol
ILPH L3
JC L3
Cyc lep
Cys lon
Cys min
WK L3
167 Con
Cya cat
Cyc nas
Cyc ins
Cyd bic
Cyc cal
Cor cor
D L3
Cyc elo
Cys gol
ILPH L3
JC L3
Cys lon
Cya pat
WK L3
200 Con
200rs
Codon 200
200r
Results – proof of principle
• Application of PCR and pyrosequencing primers to adults of
multiple species and L3s of unknown species
Codon 167
assay
Adults - 13 species of
cyathostomin
Codon 200
assay
Adults -10 species of
cyathostomin
Codon 167
assay
L3s of unknown species
•
Results
Resistance alleles at codon 167 in L3s from horses with
naturally acquired infections
- Two locations in USA, ‘before’ and ‘after’ FBZ treatment
90
80
Frequency (%)
70
60
50
Pre-treatment location 1
40
Post-treatment location 1
Pre-treatment location 2
30
Post-treatment location 2
20
10
0
TTC/TTC
TTC/TAC
Genotype
Location 1 n= 125 L3s
TAC/TAC
Location 2 n= 116 L3s
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
Parasites of Man
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
N.americanus
A.duodenale
• Very difficult to tell apart using standard parasitological
tests, requires purging and examination of adults
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
PCR Amplicon for MassARRAY
Speciation
SNPs
BZ
“Resistance”
SNPs
• Designed a PCR primer pair to span AA 181-219 of the btubulin 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
Unextended ASX primers
Extended Primers
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.)
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
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
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!