Drug Resistance in Nematodes Populations Matter !!! Ray M. Kaplan, DVM, PhD, DipEVPC Department of Infectious Diseases College of Veterinary Medicine University of Georgia Athens, Georgia, USA An Inconvenient Truth Anthelmintic resistance is an inevitable consequence of anthelmintic treatment “It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is the most adaptable to change.” An Inconvenient Truth Anthelmintic resistance is highly prevalent in parasites of livestock worldwide Multiple-drug resistance and “total anthelmintic failure” are common Resistance in all important worm species of all livestock hosts Problem worst in small ruminants Becoming increasingly severe in horses, cattle, farmed deer, camelids, exotic ungulates (zoos) Resistance in human parasites and dog heartworm is a major concern Anthelmintic Classes Nematocides Benzimidazoles fenbendazole (FBZ), oxibendazole (OBZ), albendazole (ABZ), mebendazole (MBZ), others Avermectin / Milbemycins ivermectin (IVM), eprinomectin (EPR), doramectin (DRM) moxidectin (MOX), others Imidazothiazoles / Tetrahydropyrimidines levamisole (LEV), pyrantel (PYR), morantel others (MOR), Risk of Having No Effective Anthelmintics is Real Large drug companies invest in drugs with very large profit potential little investment in new animal drugs Avermectins set a new unrealistic bar Reverse pipeline for anthelmintics Veterinary medicine is primary market In past cattle market was greatest Now dog heartworm market is by far the largest Must be inexpensive to synthesize Risk of Having No Effective Anthelmintics is Real New drug classes introduced every decade during 50’s, 60’s, 70’s, & 80’s Less than 20 years between thiabendazole and ivermectin No new drug classes for use in livestock introduced (into US market) since the avermectins (ivermectin) in 1981 “We have what we have” Where Are The New Drugs ? Emodepside -- cats only (2005) Monepantel (2010) Derquantel-Abamectin (2010) Amino-acetonitrile derivative (AAD) Introduction in the US – soon ??? Spiroindole Only for sheep (NZ, Australia) Can New Drugs Solve the Problem ? Resistance is very likely to outpace the introduction of new anthelmintics 13 years from first published report of cyclodepsipeptide as a new anthelmintic to marketing of a product New anthelmintics will be much more expensive Anthelmintic Resistance The ability of worms in a population to survive drug treatments that are generally effective against the same species and stage of infection at the same dose rate Caused by changes in allele frequencies of “resistance” genes Resistance Genes = alleles of relevant genes that confer resistance Result of drug selection Slow evolutionary process that takes years to develop Where Do Resistant Worms Come From ??? Nematodes have great genetic diversity & large population sizes High mutation rates and rapid evolution Haemonchus contortus 5000 eggs per female/day 500 female worms/animal 50 animals approx 1 billion eggs/week Where Do Resistant Worms Come From ??? “Resistant” worms seem to exist within populations prior to the introduction of a drug Some worms, in the population, are able to live without this target protein or with a modified target or other biological process and be resistant Same allele seen in wide variety of resistant lines R-allele arose once and spread as neutral allele Initial allele frequency is very low Relative changes in allele frequencies rather than appearance of new alleles Development of Resistance Treatment eliminates parasites whose genotype renders them susceptible Parasites that are resistant survive and pass on their “resistant” alleles Worm populations don’t really become resistant, rather they lose susceptibility High level of animal movement guarantees dispersal of resistant worms Detection of Drug Resistance Resistant alleles accumulate but are undetected As drug resistance develops further, more worms survive until treatment failure finally occurs Clinical definition: <95% or 90% reduction Normal therapeutic dose - no longer fully effective Recognized clinically as a phenotypic trait BUT – at its core resistance is a genetic trait Genotypic resistance occurs long before phenotypic resistance Percent of Worms that Are Resistant Changes in “Resistance Genes” in Response to Drug Selection Clinical detection level Diagnostic detection level Arbitrary Time Units (Worm Generations exposed to repeated treatment) Development of Resistance: Nematodes Vs. Non-Metazoan Organsims Nematodes reproduce sexually R-offspring must infect a new host Resistant worms cannot directly multiply themselves No direct infection from 1 host to the next All helminth parasites have a free-living (non-parasitic) stage or utilize an intermediate host Eggs shed from resistant worms are greatly diluted by those of susceptible worms New hosts are infected 1 worm at a time Development of Resistance: Nematodes Vs. Non-Metazoan Organsims With nematodes, re-infection and drug selection must occur over many life-cycles to increase the frequency of resistant worms to clinically important levels In early stages, large majority of worms are not resistant – chances of R x R matings is low Resistance occurs slowly over years This contrasts greatly with organisms that can reproduce clonally 1 surviving resistant organism can replicate itself and repopulate the host with a “pure” resistant strain What Governs The Rate of Selection For Drug Resistance ??? Some Species/Drugs Have a Much Greater Propensity to Develop Resistance Than Others Biological Factors Affecting Anthelmintic Resistance Selection 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. extent of genetic polymorphism in the population initial frequency of ‘resistance’ alleles (which already exist) number of genes involved and complexity of resistance mechanism(s) the biology of the nematode whether resistance gene(s) dominant or recessive the extent of refugia treatment coverage the relative reproductive fitness of the wild-type (susceptible) and resistant genotypes in the absence of treatment treatment frequency drug dose rate drug pharmacokinetic profile - persistence drug potency Prevalence of Resistance on Sheep & Goat Farms in SE USA (2002-2006) Based on evaluation using DrenchRite LDA Anthelmintic (Data 2002 – 2006) Prevalence of Resistance (%) Benzimidazole 98 Levamisole 54 Ivermectin 76 Moxidectin 24 MDR – all 3 classes 48 MDR to all 3 classes + Moxidectin 17 What is the Prevalence of ??? -- Small Ruminants Resistance Country/Continent United States (S/G) (G) Brazil Australia BZ LEV IVM ++++ ++ +++ ++++ ++ ++++ ++++ ++++ ++++ ++++ ++++ +++ New Zealand +++ Europe +/++ +/++ ++ MOX ++ +++ ++++ + Spp. Hc, Tcol Hc Hc, Tcol, Tcirc ++ + Tcirc, Tcol, Nem +/++ + Tcirc, Tcol, Nem Production of small ruminants is threatened in tropical/subtropical climates. Total anthelmintic failure increasingly common What is the Prevalence of ?? -- Cattle Resistance Country/Continent BZ LEV IVM MOX Brazil +++ + ++++ + Argentina ++ - +++ + New Zealand +++ + ++++ + ? ? ? ? US and Europe Resistant Genera Ostertagia Ostertagia Cooperia Cooperia Cooperia Haemonchus Haemonchus Haemonchus Oesophagostomum Trich Ostertagia Oesoph Trichostrongylus In past few years – rapid increases in level and spectrum of resistance What is the Prevalence of ??? -- Horses Drug Resistance BZ Cyathostomins ++/++++ Strongylus Parascaris O equi spp. equorum Habronema ? +/- PYR +/+++ ? + - IVM +/- ? +++ + MOX +/- ? +++ + Ivermectin and moxidectin resistance in cyathostomins appears to be emerging Overall trends toward higher prevalence and spectrum of resistance What About Human Parasites ??? Elimination -- Eradication programs for Onchocerciasis and Lymphatic filariasis raise concerns May inadvertently also select for resistance in STH Will it occur ??? Depends largely on genetic diversity and levels of selection pressure If low diversity there may be no resistance alleles to select It is folly to assume it won’t occur -- molecular assays for resistance detection are needed to monitor for this Genetics of Resistance May Vary Depending Upon Selection Pressures Heavy drug pressure – few survivors May decrease genetic diversity If one allele can confer resistance, only a single gene will appear to be responsible Low dose selection – many survivors Likely to select for all the alleles on all of the genes that can contribute to resistance Analyses of these strains may reveal all potential resistanceassoc genes, but will fail to distinguish which gene(s) are most important in field isolates Field selection – some survivors Several genes selected simultaneously Breeding between different generations of survivors Resistance is Inevitable What Can We Do ??? Resistance is a natural biological consequence of drug treatment Rate of resistance development is within our control and can be greatly reduced Aim of resistance control is to delay the accumulation of resistance alleles – reduce drug selection pressure Goal = Preserve drug efficacy for as long as possible Increase refugia Decrease treatment frequency Must treat selectively What Causes Resistance To Dewormers ?? Lack of Refugia Refugia = the proportion of the worm population that is not selected by drug Tx Worms in untreated animals Eggs and larvae on pasture Provides pool of sensitive genes Dilutes resistant genes Considered the most important factor in the development of drug resistance Treatment frequency also important EACH WORM = 100 EPG courtesy of Rose Nolen-Walston, DVM, DACVIM refugia courtesy of Rose Nolen-Walston, DVM, DACVIM Distribution of FEC on 12 Horse Farms in Georgia, USA High Egg Shedders: 5000 FEC (epg) 3000 27% of Horses 83% of Total Egg Output 1000 Moderate Egg Shedders: 18% of Horses 13% of Total Egg Output 400 200 0 0 50 100 Low Egg Shedders: 55% of Horses 4% of Total Egg Output 150 Individual Horse 200 250 What Happens if We Apply Selective Treatment ? ? ? Treat horses with FEC > 200 EPG Assume Treatment Reduces FEC by 99.9% What Happens if We Apply Selective Treatment ? ? ? Change in Distribution Following Targeted Selective Treatment Untreated horses now Treated horses shedding shedding 98% of eggs 2% of eggs = REFUGIA Total egg shedding decreased by 96% !! Only horses with FEC > 200 EPG were treated with a drug that has 99.9% efficacy Diagnosis of Anthelmintic Resistance Qualitative or Quantitative ??? Controlled efficacy studies Fecal egg count reduction tests In vitro bioassays Molecular assays Diagnosis of Anthelmintic Resistance in vivo tests Only real tool available for most hosts/parasites FECRT - anthelmintic trial Reduction in worm numbers – requires slaughter Reduction in fecal egg counts (FECRT) can be performed by a veterinarian in the field requires large groups (>10) for accurate results labor-intensive high variability – potential for errors in interpretation if performed or analyzed incorrectly In vitro Assays LDA, EHA, LMIA, LFIA Where in vitro assays have been validated Tend to be quite host and nematode species specific Are labor intensive Require a high level of technical expertise Level of resistance is often quantifiable Availability is extremely limited Narrow scope of host/species/drug for which validated assays exist Few laboratories offer this service to livestock producers Laboratory Diagnosis of Resistance in vitro tests Larval Development Assay L1 X X L3 L2 X L3 Drug Laboratory Diagnosis Resistance of LDA - DrenchRite Only one test needed per group or can be performed on individual animal All 3 major drug classes plus moxidectin tested in a single assay Only for small ruminants and zoo ungulates Available as a diagnostic service in my lab Haemonchus contortus Dose Response: Larval Development Assay DrenchRite LDA Proportion Affected Dose-response for ivermectin/moxidectin 1.0 C1 C2 Cs Js My Wt Ivermectin Resistant 0.8 0.6 Ivermectin Sensitive 0.4 0.2 Moxidectin Resistant 0.0 -7.5 -5.0 -2.5 0.0 2.5 5.0 Ln Ivermectin Concentration 7.5 Molecular Assays Requires knowledge of molecular mechanisms and/or genetic markers linked to a resistant genotype Exist only for benzimidazole drugs Beta-tubulin mutations in codons 167, 198, 200 Necessary for resistance – but is it the only mechanism ?? How does the genotype correlate with the phenotype ??? Critical need for molecular assays for all drug classes But will still require extensive field study to correlate with phenotype The Future of Parasite Control Frequent broad-scale application of anthelmintics is no longer a viable approach for livestock Effective anthelmintics must be thought of as extremely valuable and limited resources Strategies for preservation of efficacious anthelmintics must be implemented Development of anthelmintic resistance is almost sure to outpace the development of new drugs The Future of Parasite Control Anthelmintic resistance is now redefining how parasite control should be practiced An evidence-based approach based on medical need is required Reduced-chemical and non-chemical approaches are needed Strategies must be sustainable Vaccines ???