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Mechanisms of Anthelmintic Resistance Nick Sangster Faculty of Veterinary Science

Mechanisms of Anthelmintic Resistance

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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 one isolate Benzimidazoles90% Levamisole80% BZ and Lev60% - PowerPoint PPT Presentation

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Page 1: Mechanisms of Anthelmintic Resistance

Mechanisms of Anthelmintic Resistance

Nick Sangster

Faculty of Veterinary Science

Page 2: Mechanisms of Anthelmintic Resistance

19871991

2003

1999

1995

Page 3: Mechanisms of Anthelmintic Resistance

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%

Page 4: Mechanisms of Anthelmintic Resistance

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

Page 5: Mechanisms of Anthelmintic Resistance

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

Page 6: Mechanisms of Anthelmintic Resistance

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

Page 7: Mechanisms of Anthelmintic Resistance

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

Page 8: Mechanisms of Anthelmintic Resistance

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

Page 9: Mechanisms of Anthelmintic Resistance

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

Page 10: Mechanisms of Anthelmintic Resistance

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

Page 11: Mechanisms of Anthelmintic Resistance

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

Page 12: Mechanisms of Anthelmintic Resistance

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

Page 13: Mechanisms of Anthelmintic Resistance

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

Page 14: Mechanisms of Anthelmintic Resistance

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

Page 15: Mechanisms of Anthelmintic Resistance

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

Page 16: Mechanisms of Anthelmintic Resistance

Muscle transmitters

Excitatory, Acetylcholine

Inhibitory, GABA

Glutamate gated

LEV

PIPERAZINE

AVM,

MLB

Page 17: Mechanisms of Anthelmintic Resistance

Effect of GABA on ACh-induced contraction (with Cl- )

ACh

time

ACh

ACh

GABAACh

GABA + AChGABA & ACh

Page 18: Mechanisms of Anthelmintic Resistance

Effect of GABA on ACh-induced contraction (No Cl-)

ACh

ACh

GABA

GABA + ACh

time

ACh

ACh

GABA & ACh

Page 19: Mechanisms of Anthelmintic Resistance

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

Page 20: Mechanisms of Anthelmintic Resistance

[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

Page 21: Mechanisms of Anthelmintic Resistance

Avermectin/milbemycin Resistance

Page 22: Mechanisms of Anthelmintic 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

Page 23: Mechanisms of Anthelmintic Resistance

IVM receptor expressing cells

Trichostrongylus Trichostrongylus colubriformiscolubriformis

Caenorhabditis Caenorhabditis eleganselegans

Page 24: Mechanisms of Anthelmintic Resistance

Pharyngeal muscle physiology

+

EXCITATORY

INHIBITORY

pm4

X+

Cl-

INTERNEURONS

M3

IVM

A

G

mouth

meta corpus

terminal bulb

Page 25: Mechanisms of Anthelmintic Resistance

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

Page 26: Mechanisms of Anthelmintic Resistance

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

Page 27: Mechanisms of Anthelmintic Resistance

Research into IVM-R

• Genes– P-glycoprotein– GluCl– GABA

• No accepted mechanisms of resistance

• Studies of sites of action and resistance

Page 28: Mechanisms of Anthelmintic Resistance

The Parasites

Haemonchus contortus

Ostertagia (Teladorsagia) circumcincta

Trichostrongylus colubriformis

Page 29: Mechanisms of Anthelmintic Resistance

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.

Page 30: Mechanisms of Anthelmintic Resistance

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

Page 31: Mechanisms of Anthelmintic Resistance

Avermectin/Milbemycins

• Avermectins– IVM

– IVM B1a

– IVM B1b

• Milbemycins– Milb A3

– Milb A4

– Moxidectin

Page 32: Mechanisms of Anthelmintic Resistance

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

Page 33: Mechanisms of Anthelmintic Resistance

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

Page 34: Mechanisms of Anthelmintic Resistance

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

Page 35: Mechanisms of Anthelmintic Resistance

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

Page 36: Mechanisms of Anthelmintic Resistance

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 ]

Page 37: Mechanisms of Anthelmintic Resistance

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

Page 38: Mechanisms of Anthelmintic Resistance

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

Page 39: Mechanisms of Anthelmintic Resistance

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

Page 40: Mechanisms of Anthelmintic Resistance

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

Page 41: Mechanisms of Anthelmintic Resistance