1. Mechanisms of Anthelmintic Resistance
Nick Sangster
Faculty of Veterinary Science

2. 2003
1995
1991
1999
1987

3. 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%

4. Resistance Summary Drug Genus BZ LEV BZ + ML (resistance to IVM)
Ostertagia
Teladorsagia
Common
Common in WA, other states emerging
Trichostrongylus
Rare, but some cases in NSW & QLD (MOX also)
Haemonchus
Rare
Rare, but emerging in NSW & QLD

5. FECR % against Cyathostomins
Property Oxibendazole Morantel Ivermectin

6. 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

7. Anthelmintic-resistance
SHEEP
Trichostrongylids
benzimidazoles
levamisole (rare in Haemonchus)
macrolactones
closantel
Fasciola hepatica
CATTLE
Cooperia spp.
PIGS
Oesophagostomum spp.
pyrantel
ivermectin
benzimidazoles
HORSES
Small strongyles
piperazine
HUMANS
Schistosomes
hycanthone

8. 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

9. 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

10. 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

11. Resistant isolates kept in sheep
Resistant to
Genus
Susc BZ
LEV
ML (IVM)
Ostertagia
Teladorsagia
McMO -
WAPRO
Trichostrongylus MT
VRSG
MOX
Haemonchus MH
LAWES
CAVR

12. 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
different AM’s

13. Inheritance Parent F1 F2 Rf m line eggs, L3, adult eggs, L3, adult RmSm
p line eggs, L3, adult eggs, L3, adult Sf

14. Benzimidazoles

15. 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

16. Muscle transmitters AVM, MLB LEV PIPERAZINE Glutamate gated
Excitatory, Acetylcholine
Inhibitory, GABA
PIPERAZINE

17. Effect of GABA on ACh-induced contraction (with Cl- )
time
GABA & ACh
GABA + ACh
GABA
ACh
ACh
ACh

18. Effect of GABA on ACh-induced contraction (No Cl-)
time
GABA + ACh
GABA & ACh
GABA
ACh
ACh
ACh
ACh

19. 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

20. [3H]MAL binding sites in H. contortus and C. elegans
High affinity site Low affinity site
KD(nM) Bmax(pmol/mg) KD(mM) Bmax (nmol/mg)
H.contortus
susc
res
C. elegans
fmol/mg

21. Avermectin/milbemycin Resistance

22. Mechanisms of resistance to IVM in arthropods
Resistance CO potato House ..Spider
Mechanisms Beetle Fly mite
Penetration
Excretion
Oxidative
metabolism
Esteratic
Metabolism/
sequestration
Altered target NA ++ NA
GST conjugation
from: Clarke et al. 1994, Annu. Rev. Entomol. 40:1

23. IVM receptor expressing cells
Trichostrongylus colubriformis
Caenorhabditis elegans

24. Pharyngeal muscle physiology+

25. ML potency on R and S H. contortus
L1 L3 Adult
Pharynx ~1nM not nM
RF x feeding x
Muscle 30nM >600nM 10nM
RF ? x ~10x
in vivo RF x

26. Rank potency of macrolactones (H. contortus)
L1 (LDA) L3 (motility) Adult (efficiency)
AVM B1 AVM B1 AVM B1
IVM IVM (IVM)
AVM B2 AVM B2 AVM B2
IVM AG IVM MS
IVM MS IVM AG
Gill et al Gill et al Fisher & Mrozik, 1989

27. Research into IVM-R Genes No accepted mechanisms of resistance
P-glycoprotein
GluCl
GABA
No accepted mechanisms of resistance
Studies of sites of action and resistance

28. Ostertagia (Teladorsagia) circumcincta Trichostrongylus colubriformis
The Parasites
Haemonchus contortus
Ostertagia (Teladorsagia) circumcincta
Trichostrongylus colubriformis

29. 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.

30. 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

31. Avermectin/Milbemycins
Avermectins
IVM
IVM B1a
IVM B1b
Milbemycins
Milb A3
Milb A4
Moxidectin

32. 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
different AM’s

33. 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
different AM’s

34. LDA - Haemonchus EC50 (nM) DRUG S R IVM 1.45 4.42 B1a 0.97 3.08
B1b
MOX
Mil 4A

35. LDA - Ostertagia
RF=
3.5
RF=
1.3

36. LDA - Trichostrongylus

37. LMA – Haemonchus IVM vs MOX
EC50 (mm)
DRUG S R RF
IVM
MOX

38. Ostertagia LDA vs LMA
RF=
3.5
RF=
8.9
RF=
1.3
RF=
~15

39. LMA – Trichostrongylus IVM analogues
RF=
4.7
RF=
1.9
RF=
13.6

40. So… AMs - All species resistant in LDA except
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