Mechanisms of Anthelmintic Resistance
Nick Sangster
Faculty of Veterinary Science
19871991
2003
1999
1995
Prevalence estimates of resistance(% NSW sheep farms with treatment failure)
OP one isolate
Benzimidazoles 90%
Levamisole 80%
BZ and Lev 60%
MLs (eg. IVM) 10%
Closantel 25%
Resistance Summary
Drug
Genus
BZ LEV BZ +
LEV
ML (resistance to IVM)
Ostertagia
Teladorsagia
Common Common Common Common in WA, other states emerging
Trichostrongylus Common Common Common Rare, but some cases in NSW & QLD (MOX also)
Haemonchus Common Rare Rare Rare, but emerging in NSW & QLD
FECR % against Cyathostomins
PropertyOxibendazole Morantel Ivermectin
11 8686 100100 - -
33 9696 9696 100100
55 9494 9999 100100
66 8989 9797 100100
1010 5454 8989 100100
1212 6666 9898 100100
1313 5959 100100 100100
New Zealand (per Bill Pomroy)
• Little data collation since 1995, but notionally• Sheep:
– BZs: Nematodirus spathiger , H,O,T, very common– Lev: Reports in O and T– MLs: developing in Ostertagia (serious in goats)
• Cattle:– ML: Common in Cooperia oncophora – BZs: Common? in Cooperia oncophora, some O. ostertagi
• Horses:– BZs: common in cyathostomines
Anthelmintic-resistance• PIGS
– Oesophagostomum spp.• pyrantel• ivermectin• benzimidazoles
• HORSES– Small strongyles
• benzimidazoles• piperazine• pyrantel
• HUMANS– Schistosomes
• hycanthone
• SHEEP– Trichostrongylids
• benzimidazoles• levamisole (rare in
Haemonchus)
• macrolactones• closantel
– Fasciola hepatica• closantel• benzimidazoles
• CATTLE– Cooperia spp.
• benzimidazoles• macrolactones
Aspects of anthelmintic resistance• Resistance is now common.• In nematodes of ruminants and horses, Fasciola• Resistance to all drug classes but with gaps in the
matrix• Why it is so serious in sheep?
– Lambs have poor immunity, so heavy reliance on drugs– Merinos highly susceptible to infection– Arid climate helps select for resistance– Haemonchus is highly pathogenic– Resistance to all chemical classes including Moxidectin– Some farms have no available drug choices
Anthelmintic modes of action
Class example MOA
Benzimidazoles Albendazole Tubulin binding and cellular disruption
Tetrahydropyrimidine Levamisole Nicotinic-like agonists
Organophosphates Dichorvos Acetylcholine esterase inhibitors
Piperazines Piperazine GABA agonists
Macrocyclic lactones Ivermectin GluCl- potentiators
Praziquantel Enhance Ca++ permeability
Salicylanilides Closantel Proton ionophores
Methods to study resistance
• In vivo assays (egg count)• In vitro development, migration• Drug/receptor binding assays• Muscle contraction assays• Patch clamp, single channel analysis• Gene sequence analysis• Maintain sheep infected with each isolate of three
species
Resistant isolates kept in sheep
Resistant to
Genus
Susc BZ LEV ML (IVM)
Ostertagia
Teladorsagia
McMO - - WAPRO
Trichostrongylus MT VRSG - MOX
Haemonchus MH LAWES LAWES CAVR
TechniquesLarval Development Assay
• 96-well plates, containing AMs at halving concentrations
• DrenchRite protocol for LDA (egg to L3 development)
• Calculate % undeveloped (eggs, L1, L2) /total including L3
• Assume action relates to inhibition of feeding
increasing concentration
diff
eren
t A
M’s
Inheritance
Parent F1 F2
Rfm linem line eggs, L3, adulteggs, L3, adulteggs, L3, adulteggs, L3, adult
Rm
Smp linep line eggs, L3, adulteggs, L3, adulteggs, L3, adulteggs, L3, adult
Sf
Benzimidazoles
HN
S
N
C
C
C
C C
C
C C
C
C
T h i a b e n d a z o l e
BZ resistance
• BZ’s effect to depolymerise microtubules lost in resistant worms
• Reduced binding of BZs to worm tubulin
• Resistance develops in two steps– Selection for worms with resistant tubulin allele
with one amino acid change– Loss of second tubulin gene
Muscle transmitters
Excitatory, Acetylcholine
Inhibitory, GABA
Glutamate gated
LEV
PIPERAZINE
AVM,
MLB
Effect of GABA on ACh-induced contraction (with Cl- )
ACh
time
ACh
ACh
GABAACh
GABA + AChGABA & ACh
Effect of GABA on ACh-induced contraction (No Cl-)
ACh
ACh
GABA
GABA + ACh
time
ACh
ACh
GABA & ACh
Levamisole resistance
• LEV is a cholinergic agonist (acts like acetylcholine to cause contraction)
• Resistance shared with other cholinergic drugs including acetylcholine
• Binding studies show changes in binding affinity and number of binding sites
• Genetic studies fail to find difference in gene sequence• Single channel studies suggest changes in
– Expression of channel components– Differences in phosphorylation or desensitisation
[3H]MAL binding sites in H. contortus and C. elegans
High affinity site Low affinity siteKD(nM) Bmax(pmol/mg) KD(M) Bmax
(nmol/mg)
H.contortussusc. 2.8 38 2.4 21res. 2.9 58 4.6 63
C. elegans3.0 13.3 fmol/mg
Avermectin/milbemycin Resistance
Mechanisms of resistance to IVM in arthropods
Resistance CO potato House ..Spider
Mechanisms Beetle Fly mite
Penetration ++ +
Excretion + ++
Oxidativemetabolism ++ ++ +
EsteraticMetabolism/ + +sequestration
Altered target NA ++ NA
GST conjugation +
from: Clarke et al. 1994, Annu. Rev. Entomol. 40:1
IVM receptor expressing cells
Trichostrongylus Trichostrongylus colubriformiscolubriformis
Caenorhabditis Caenorhabditis eleganselegans
Pharyngeal muscle physiology
+
EXCITATORY
INHIBITORY
pm4
X+
Cl-
INTERNEURONS
M3
IVM
A
G
mouth
meta corpus
terminal bulb
ML potency on R and S H. contortus
L1 L3 Adult
Pharynx ~1nM not 0.12nMRF 5-17x feeding 100-177x
Muscle 30nM >600nM 10nMRF ? 2.5-20x ~10x
in vivo RF - - 30-100x
Rank potency of macrolactones (H. contortus)
L1 (LDA) L3 (motility) Adult (efficiency)
AVM B1 AVM B1 AVM B1IVM IVM (IVM)AVM B2 AVM B2 AVM B2IVM AG IVM MSIVM MS IVM AG
Gill et al. 1995Gill et al. 1991 Fisher & Mrozik, 1989
Research into IVM-R
• Genes– P-glycoprotein– GluCl– GABA
• No accepted mechanisms of resistance
• Studies of sites of action and resistance
The Parasites
Haemonchus contortus
Ostertagia (Teladorsagia) circumcincta
Trichostrongylus colubriformis
The AM-resistant isolatesIsolate/Species Efficacy of 0.2 mg/kg
IVM MOX
CAVR-S Haemonchus* 0% 96%
WAMIRO Ostertagia 0% ~95%
MOX Trichostrongylus* 0% 0%
*F1 crosses of these isolates indicate “dominant” resistance to IVM but “partially recessive” resistance to MOX.
Why we want to understand the action of AM’s
• Resistance to the AMs is emerging and better tests are required
• There is conflicting evidence for two sites of action: – muscle of pharynx– body muscle
• The aim is to clarify the target organ(s) for the AMs and describe how they change with resistance
• Sites of action and resistance may differ between parasite species
• This will allow us to compare sites of resistance with localisation of expression of putative resistance genes
Avermectin/Milbemycins
• Avermectins– IVM
– IVM B1a
– IVM B1b
• Milbemycins– Milb A3
– Milb A4
– Moxidectin
TechniquesLarval Development Assay
• 96-well plates, containing AMs at halving concentrations
• DrenchRite protocol for LDA (egg to L3 development)
• Calculate % undeveloped (eggs, L1, L2) /total including L3
• Assume action relates to inhibition of feeding
increasing concentration
diff
eren
t A
M’s
TechniquesLarval Migration Assay
• 24-well plates, containing AMs at ~1:3 dilutions
• L3, 24h in drug followed by 24h migration thru 25m
• Calculate % not migrating (L3 left in sieve/total L3)
• Assume action relates to inhibition of motility
increasing concentration
diff
eren
t A
M’s
LDA - Haemonchus
-11 -10 -9 -8 -7-10
0102030405060708090
100110120
H.c. McM
H.c. CAVR
log [IVM ]
% n
ot
dev
elo
pin
g
-11 -10 -9 -8 -70
102030405060708090
100110
log [ IVM B1a ]
-11 -10 -9 -8 -70
102030405060708090
100110
log [ IVM B1b ]
-11 -10 -9 -8 -70
102030405060708090
100110
H.c. McM
H.c. CAVR
log [ MOX ]
% n
ot
dev
elo
pin
g
-10.5 -10.0 -9.5 -9.0 -8.5 -8.0 -7.5 -7.00
102030405060708090
100110
log [ Mil A4 ]
EC50 (nM)
DRUG S R
IVM 1.45 4.42
B1a 0.97 3.08
B1b 1.07 3.57
MOX 1.34 2.45
Mil 4A 0.45 3.64
LDA - Ostertagia
-11 -10 -9 -8 -7 -60
102030405060708090
100110
O.c. McM
O.c. WAM
log [ IVM ]
% n
ot
dev
elo
pin
g
-11 -10 -9 -8 -7 -60
102030405060708090
100110
log [ IVM B1a ]
-11 -10 -9 -8 -7 -60
102030405060708090
100110
log [ IVM B1b ]
-11 -10 -9 -8 -7 -60
102030405060708090
100110
O.c. McMO.c. WAM
log [ MOX ]
%n
ot
dev
elo
pin
g
-11 -10 -9 -8 -7 -60
102030405060708090
100110
log [ Mil A3 ]
-11 -10 -9 -8 -7 -60
102030405060708090
100110
log [ Mil A4 ]
RF=
3.5
RF=
1.3
LDA - Trichostrongylus
-10 -9 -8 -7 -6-10
0102030405060708090
100110 T.c. McM
T.c. MOXR
log [ IVM ]
% n
ot
dev
elo
pin
g
-10 -9 -8 -7 -60
102030405060708090
100110
log [ IVM B1a ]
-10 -9 -8 -7 -6-10
0102030405060708090
100110
log [ IVM B1b ]
-10 -9 -8 -7 -60
102030405060708090
100110
T.c. McM
T.c. MOXR
log [ MOX ]
% n
ot
dev
elo
pin
g
-10 -9 -8 -7 -60
102030405060708090
100110
log [ Mil A3 ]-10 -9 -8 -7 -6
0102030405060708090
100110
log [ Mil A4 ]
LMA – Haemonchus IVM vs MOX
-8 -7 -6 -5 -4 -3 -2 -10
102030405060708090
100110 H.c. McM
H.c. CAVR
log [ IVM ]
% n
ot
mig
rati
ng
-8 -7 -6 -5 -4 -3 -2 -10
102030405060708090
100110
log [ MOX ]
EC50 (m)
DRUG S R RF
IVM 88.06 176.1 2
MOX 39.27 957 24.4
Ostertagia LDA vs LMA
-8 -7 -6 -5 -4 -3 -2 -1-10
0102030405060708090
100110 O.c. McM
O.c. WAM
log [ IVM ]
% n
ot
mig
rati
ng
-8 -7 -6 -5 -4 -3 -2 -1-10
0102030405060708090
100110
log [ MOX ]
-11 -10 -9 -8 -7 -60
102030405060708090
100110
O.c. McM
O.c. WAM
log [ IVM ]
% n
ot
dev
elo
pin
g
-11 -10 -9 -8 -7 -60
102030405060708090
100110
O.c. McMO.c. WAM
log [ MOX ]
%n
ot
dev
elo
pin
g
RF=
3.5
RF=
8.9
RF=
1.3RF=
~15
LMA – Trichostrongylus IVM analogues
-8 -7 -6 -5 -4 -3 -2 -10
102030405060708090
100110 T.c. McM
T.c. MOXR
log [ IVM ]
% n
ot
mig
rati
ng
-8 -7 -6 -5 -4 -3 -2 -1-10
0102030405060708090
100110
log [ IVM B1a ]
-8 -7 -6 -5 -4 -3 -2 -10
102030405060708090
100110
log [ IVM B1b ]
RF=
4.7
RF=
1.9
RF=
13.6
So…• AMs - – All have dose responses and resistance develops to all, but not uniform– Drugs, especially IVM and MOX differ in resistance profiles– Have at least two sites of action in most cases
• All species resistant in LDA except– MOX for our Ostertagia isolate
• All resistant in LMA except– IVM for Ostertagia; IVM for Haemonchus (in our hands)
• Sites of action/resistance/drugs– Differ, eg. Trichs LDA-R to all 3 IVM analogues, – LMA-R to IVM1a, not 1b)
• Conclude– Sites of action and resistance differ between species, body sites and
drugs– There will not be a single mechanism of resistance across species or
even within species
• Next we will look at effects on adult worms