Transcript IVM

Mechanisms of Anthelmintic
Resistance
Nick Sangster
Faculty of Veterinary Science
2003
1995
1991
1999
1987
Prevalence estimates of resistance
(% NSW sheep farms with treatment failure)
OP
Benzimidazoles
Levamisole
BZ and Lev
MLs (eg. IVM)
Closantel
one isolate
90%
80%
60%
10%
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
Rare
Rare
Rare, but emerging in
NSW & QLD
Common
FECR % against Cyathostomins
Property
Oxibendazole
1
3
5
6
10
12
13
86
96
94
89
54
66
59
Morantel
Ivermectin
100
96
99
97
89
98
100
100
100
100
100
100
100
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
Closantel
Proton ionophores
Salicylanilides
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
Ostertagia
Teladorsagia
Susc
BZ
LEV
ML (IVM)
McMO
-
-
WAPRO
Trichostrongylus MT
VRSG
-
MOX
Haemonchus
LAWES
LAWES
CAVR
MH
Techniques
Larval 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
Inheritance
Parent
F1
F2
Rf
m line
eggs, L3, adulteggs, L3, adult
p line
eggs, L3, adulteggs, L3, adult
Rm
Sm
Sf
Benzimidazoles
S
C
H
N
C
C
C
C
C
C
C
C
C
N
Thiabendazole
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
Glutamate
gated
AVM,
LEV
MLB
Excitatory,
Acetylcholine
Inhibitory,
GABA
PIPERAZINE
Effect of GABA on ACh-induced
contraction (with Cl- )
ACh
GABA & ACh
GABA + ACh
GABA
ACh
ACh
ACh
Effect of GABA on ACh-induced
contraction (No Cl-)
tim
e
& ACh
GABAGABA
+
ACh
GABA
ACh
ACh
ACh
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 site
KD(nM) Bmax(pmol/mg)
KD(mM)
H.contortus
susc.
2.8
res.
2.9
38
58
C. elegans
3.0
13.3 fmol/mg
2.4
4.6
Bmax (nmol/mg)
21
63
Avermectin/milbemycin
Resistance
Mechanisms of resistance to IVM in
arthropods
Resistance
Mechanisms
CO potato
Beetle
Penetration
Excretion
Oxidative
metabolism
..Spider
mite
++
+
+
++
Esteratic
Metabolism/
sequestration
+
Altered target
NA
GST conjugation
House
Fly
++
++
+
+
++
NA
+
from: Clarke et al. 1994, Annu. Rev. Entomol. 40:1
IVM receptor expressing cells
Trichostrongylus
colubriformis
Caenorhabditis
elegans
Pharyngeal muscle physiology
EXCIT AT O RY
+
meta
corpus
X
A
+
pm4

Cl
terminal
bulb
G
M3
-

INHIBIT O RY
IVM
IN T E R N E U R O N S
mouth
ML potency on R and S H.
contortus
L1
L3
Adult
Pharynx
RF
~1nM
5-17x
not
feeding
0.12nM
100-177x
Muscle
RF
30nM
?
>600nM
2.5-20x
10nM
~10x
in vivo
RF
-
-
30-100x
Rank potency of macrolactones (H.
contortus)
L1 (LDA)
L3 (motility)
Adult (efficiency)
AVM B1
IVM
AVM B2
IVM AG
IVM MS
AVM B1
IVM
AVM B2
AVM B1
(IVM)
AVM B2
IVM MS
IVM AG
Gill et al. 1995
1989
Gill et al. 1991
Fisher & Mrozik,
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 isolates
Isolate/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
Techniques
Larval 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
Techniques
Larval Migration Assay
• 24-well plates, containing
AMs at ~1:3 dilutions
• L3, 24h in drug followed by
24h migration thru 25mm
• Calculate % not migrating
(L3 left in sieve/total L3)
• Assume action relates to
inhibition of motility
increasing concentration
% not developing
120
110
100
90
80
70
60
50
40
30
20
10
0
-10
-11
LDA - Haemonchus
H.c. McM
H.c. CAVR
-10
-9
-8
110
100
90
80
70
60
50
40
30
20
10
0
-7 -11
-10
-9
-8
-10
-9
-8
-7
log [ IVM B1b ]
log [ IVM B1a ]
log [IVM ]
110
100
90
80
70
60
50
40
30
20
10
0
-11
-7
% not developing
EC50 (nM)
110
100
90
80
70
60
50
40
30
20
10
0
-11
H.c. McM
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
H.c. CAVR
-10
-9
log [ MOX ]
-8
-7
110
100
90
80
70
60
50
40
30
20
10
0
-10.5 -10.0 -9.5
-9.0
-8.5
log [ Mil A4 ]
-8.0
-7.5
-7.0
% not developing
LDA - Ostertagia
110
100
90
80
70
60
50
40
30
20
10
0
-11
110
100
90
80
70
60
50
40
30
20
10
0
-11
O.c. McM
O.c. WAM
RF=
3.5
-10
-9
-8
-7
-6
log [ IVM ]
-10
-9
-8
-7
-6
110
100
90
80
70
60
50
40
30
20
10
0
-11
-10
%not developing
O.c. WAM
O.c. McM
RF=
1.3
-10
-9
-8
log [ MOX ]
-7
-6
110
100
90
80
70
60
50
40
30
20
10
0
-11
-10
-9
-8
log [ Mil A3 ]
-8
-7
-6
log [ IVM B1b ]
log [ IVM B1a ]
110
100
90
80
70
60
50
40
30
20
10
0
-11
-9
-7
-6
110
100
90
80
70
60
50
40
30
20
10
0
-11
-10
-9
-8
log [ Mil A4 ]
-7
-6
% not developing
LDA - Trichostrongylus
110
100
90
80
70
60
50
40
30
20
10
0
-10
-10
T.c. McM
T.c. MOXR
-9
-8
-7
-6
110
100
90
80
70
60
50
40
30
20
10
0
-10
-9
% not developing
T.c. McM
T.c. MOXR
-9
-8
log [ MOX ]
-7
-6
-9
-7
-6
110
100
90
80
70
60
50
40
30
20
10
0
-10
-9
-8
log [ Mil A3 ]
-8
-7
-6
-7
-6
log [ IVM B1b ]
log [ IVM B1a ]
log [ IVM ]
110
100
90
80
70
60
50
40
30
20
10
0
-10
-8
110
100
90
80
70
60
50
40
30
20
10
0
-10
-10
-7
110
100
90
80
70
60
50
40
30
20
10
0
-6 -10
-9
-8
log [ Mil A4 ]
% not migrating
LMA – Haemonchus
IVM vs MOX
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
H.c. McM
H.c. CAVR
-8
-7
-6
-5
-4
-3
-2
-8
-1
-7
-6
-5
-4
log [ MOX ]
log [ IVM ]
EC50 (mm)
DRUG
S
R
RF
IVM
88.06
176.1
2
MOX
39.27
957
24.4
-3
-2
-1
110
100
90
80
70
60
50
40
30
20
10
0
-11
O.c. McM
O.c. WAM
% not migrating
% not developing
Ostertagia LDA vs LMA
RF=
3.5
-10
-9
-8
-7
-6
%not developing
log [ IVM ]
110
100
90
80
70
60
50
40
30
20
10
0
-11
RF=
1.3
-9
-8
log [ MOX ]
-7
O.c. McM
O.c. WAM
RF=
8.9
-8
-7
-6
-6
-5
-4
-3
-2
-1
log [ IVM ]
110
100
90
80
70
60
50
40
30
20
10
0
-10
O.c. WAM
O.c. McM
-10
110
100
90
80
70
60
50
40
30
20
10
0
-10
RF=
~15
-8
-7
-6
-5
-4
log [ MOX ]
-3
-2
-1
% not migrating
LMA – Trichostrongylus IVM analogues
T.c. McM
T.c. MOXR
110
100
90
80
70
60
50
40
30
20
10
0
RF=
4.7
-8
-7
-6
-5
-4
-3
-2
-1
log [ IVM ]
110
100
90
80
70
60
50
40
30
20
10
0
-10
RF=
1.9
-8
-7
-6
-5
-4
-3
log [ IVM B1a ]
-2
-1
110
100
90
80
70
60
50
40
30
20
10
0
RF=
13.6
-8
-7
-6
-5
-4
-3
log [ IVM B1b ]
-2
-1
• AMs -
So…
– 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