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Mechanisms of Anthelmintic Resistance Nick Sangster Faculty of Veterinary Science.

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Presentation on theme: "Mechanisms of Anthelmintic Resistance Nick Sangster Faculty of Veterinary Science."— Presentation transcript:

1 Mechanisms of Anthelmintic Resistance Nick Sangster Faculty of Veterinary Science

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3 Prevalence estimates of resistance (% NSW sheep farms with treatment failure) OP one isolate Benzimidazoles90% Levamisole80% BZ and Lev60% MLs (eg. IVM)10% Closantel25%

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

5 FECR % against Cyathostomins PropertyOxibendazoleMorantelIvermectin

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 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

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 ClassexampleMOA BenzimidazolesAlbendazoleTubulin binding and cellular disruption TetrahydropyrimidineLevamisoleNicotinic-like agonists OrganophosphatesDichorvosAcetylcholine esterase inhibitors PiperazinesPiperazineGABA agonists Macrocyclic lactonesIvermectinGluCl - potentiators PraziquantelEnhance Ca ++ permeability SalicylanilidesClosantelProton 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 SuscBZLEVML (IVM) Ostertagia Teladorsagia McMO--WAPRO TrichostrongylusMTVRSG-MOX HaemonchusMH LAWES CAVR

12 Techniques L arval D evelopment A ssay 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 ParentF1F2 Rf m lineeggs, L3, adulteggs, L3, adult Rm Sm p line eggs, L3, adulteggs, 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 Excitatory, Acetylcholine Inhibitory, GABA Glutamate gated LEV PIPERAZINE AVM, MLB

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

18 Effect of GABA on ACh-induced contraction (No Cl - ) ACh GABA GABA + ACh tim e ACh GABA & 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 [ 3 H]MAL binding sites in H. contortus and C. elegans High affinity siteLow affinity site K D (nM) Bmax(pmol/mg)K D (  M) 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 targetNA++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 L1L3Adult Pharynx~1nM not 0.12nM RF5-17x feeding x Muscle30nM>600nM10nM RF ?2.5-20x~10x in vivoRF x

26 Rank potency of macrolactones (H. contortus) L1 (LDA)L3 (motility) Adult (efficiency) AVM B1AVM B1AVM B1 IVMIVM (IVM) AVM B2AVM B2AVM B2 IVM AGIVM MS IVM MSIVM AG Gill et al. 1995Gill et al. 1991Fisher & Mrozik, 1989

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

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

29 The AM-resistant isolates Isolate/SpeciesEfficacy of 0.2 mg/kg IVMMOX CAVR-S Haemonchus*0% 96% WAMIRO Ostertagia0%~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 L arval D evelopment A ssay 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 L arval Migration A ssay 24-well plates, containing AMs at ~1:3 dilutions L3, 24h in drug followed by 24h migration thru 25  m 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) DRUGSR IVM B1a B1b MOX Mil 4A

35 LDA - Ostertagia RF= 3.5 RF= 1.3

36 LDA - Trichostrongylus

37 LMA – Haemonchus IVM vs MOX EC50 (  m) DRUGSRRF 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 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

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