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 Benzimidazoles 90% Levamisole 80% BZ and Lev 60% MLs (eg. IVM) 10% Closantel 25%

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

FECR % against Cyathostomins Property Oxibendazole Morantel Ivermectin 1 86 100 - 3 96 96 100 5 94 99 100 6 89 97 100 10 54 89 100 12 66 98 100 13 59 100 100

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 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

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 CAVR

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

Inheritance Parent F1 F2 Rf m line eggs, L3, adult eggs, L3, adult Rm Sm p line eggs, L3, adult eggs, L3, adult Sf

Benzimidazoles

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 AVM, MLB LEV PIPERAZINE Glutamate gated Excitatory, Acetylcholine Inhibitory, GABA PIPERAZINE

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

Effect of GABA on ACh-induced contraction (No Cl-) time GABA + ACh GABA & 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) Bmax (nmol/mg) H.contortus susc. 2.8 38 2.4 21 res. 2.9 58 4.6 63 C. elegans 3.0 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 + ++ Oxidative metabolism ++ ++ + Esteratic Metabolism/ + + sequestration Altered target NA ++ NA GST conjugation + from: Clarke et al. 1994, Annu. Rev. Entomol. 40:1

IVM receptor expressing cells Trichostrongylus colubriformis Caenorhabditis elegans

Pharyngeal muscle physiology +

ML potency on R and S H. contortus L1 L3 Adult Pharynx ~1nM not 0.12nM RF 5-17x feeding 100-177x Muscle 30nM >600nM 10nM RF ? 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 B1 IVM IVM (IVM) AVM B2 AVM B2 AVM B2 IVM AG IVM MS IVM MS IVM AG Gill et al. 1995 Gill et al. 1991 Fisher & Mrozik, 1989

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

Ostertagia (Teladorsagia) circumcincta Trichostrongylus colubriformis 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 different AM’s

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

LDA - Haemonchus 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 RF= 3.5 RF= 1.3

LDA - Trichostrongylus

LMA – Haemonchus IVM vs MOX EC50 (mm) DRUG S R RF IVM 88.06 176.1 2 MOX 39.27 957 24.4

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

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

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