2. 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

3. 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.”

4. 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

5. 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 (MOR), others

6. 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

7. 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”

8. Where Are The New Drugs ?  Emodepside -- cats only (2005)  Monepantel (2010)  Amino-acetonitrile derivative (AAD)  Introduction in the US – soon ???  Derquantel-Abamectin (2010)  Spiroindole  Only for sheep (NZ, Australia)

9. 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

10.  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 Anthelmintic Resistance

11. 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

12. 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

13. 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

14. 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

15. Changes in “Resistance Genes” in Response to Drug Selection Arbitrary Time Units (Worm Generations exposed to repeated treatment) Percent of Worms that Are Resistant Clinical detection level Diagnostic detection level

16. Development of Resistance: Nematodes Vs. Non-Metazoan Organsims  Nematodes reproduce sexually  Resistant worms cannot directly multiply themselves  R-offspring must infect a new host  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

17. 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

18. What Governs The Rate of Selection For Drug Resistance ??? Some Species/Drugs Have a Much Greater Propensity to Develop Resistance Than Others

19. Biological Factors Affecting Anthelmintic Resistance Selection 1. extent of genetic polymorphism in the population 2. initial frequency of ‘resistance’ alleles (which already exist) 3. number of genes involved and complexity of resistance mechanism(s) 4. the biology of the nematode 5. whether resistance gene(s) dominant or recessive 6. the extent of refugia 7. treatment coverage 8. the relative reproductive fitness of the wild-type (susceptible) and resistant genotypes in the absence of treatment 9. treatment frequency 10. drug dose rate 11. drug pharmacokinetic profile - persistence 12. drug potency

20. 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 (%) Benzimidazole98 Levamisole54 Ivermectin76 Moxidectin24 MDR – all 3 classes48 MDR to all 3 classes + Moxidectin17

21. What is the Prevalence of Resistance ??? -- Small Ruminants Country/ContinentBZLEVIVMMOXSpp. United States (S/G) (G)++++ ++ +++ ++++ ++ +++ Hc, Tcol Brazil++++ Hc Australia++++ ++++ Hc, Tcol, Tcirc New Zealand+++++ + Tcirc, Tcol, Nem Europe+/++ + Tcirc, Tcol, Nem  Production of small ruminants is threatened in tropical/subtropical climates. Total anthelmintic failure increasingly common

22. What is the Prevalence of Resistance ?? -- Cattle Country/ContinentBZLEVIVMMOX Brazil+++++++++ Argentina++-++++ New Zealand+++++++++ US and Europe???? Resistant Genera Ostertagia Cooperia Haemonchus Trich Oesoph Ostertagia Cooperia Haemonchus Cooperia Haemonchus Oesophagostomum Ostertagia Trichostrongylus  In past few years – rapid increases in level and spectrum of resistance

23. What is the Prevalence of Resistance ??? -- Horses DrugCyatho- stomins Strongylus spp. Parascaris equorum O equi Habronema BZ++/++++?+/-- PYR+/+++?+- IVM+/-?++++ MOX+/-?++++  Ivermectin and moxidectin resistance in cyathostomins appears to be emerging  Overall trends toward higher prevalence and spectrum of resistance

24. 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

25. 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 resistance- assoc 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

26.  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 Resistance is Inevitable What Can We Do ???

27.  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 What Causes Resistance To Dewormers ?? Lack of Refugia

28. EACH WORM = 100 EPG courtesy of Rose Nolen-Walston, DVM, DACVIM

29. } refugia courtesy of Rose Nolen-Walston, DVM, DACVIM

30. Distribution of FEC on 12 Horse Farms in Georgia, USA High Egg Shedders: 27% of Horses 83% of Total Egg Output Moderate Egg Shedders: 18% of Horses 13% of Total Egg Output Low Egg Shedders: 55% of Horses 4% of Total Egg Output

31. What Happens if We Apply Selective Treatment ? ? ? Assume Treatment Reduces FEC by 99.9% Treat horses with FEC > 200 EPG

32. What Happens if We Apply Selective Treatment ? ? ?

33. Only horses with FEC > 200 EPG were treated with a drug that has 99.9% efficacy Treated horses shedding 2% of eggs Untreated horses now shedding 98% of eggs = REFUGIA Total egg shedding decreased by 96% !! Change in Distribution Following Targeted Selective Treatment

34.  Controlled efficacy studies  Fecal egg count reduction tests  In vitro bioassays  Molecular assays Diagnosis of Anthelmintic Resistance Qualitative or Quantitative ???

35. Diagnosis of Anthelmintic Resistance in vivo tests  Only real tool available for most hosts/parasites  Reduction in worm numbers – requires slaughter  Reduction in fecal egg counts (FECRT)  FECRT - anthelmintic trial  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

36.  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 In vitro Assays LDA, EHA, LMIA, LFIA

37. Laboratory Diagnosis of Resistance in vitro tests  Larval Development Assay L2L2 L3L3 L1L1 Drug X X X L3L3

38. Laboratory Diagnosis of Resistance  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

39. Haemonchus contortus Dose Response: Larval Development Assay

40. DrenchRite LDA Dose-response for ivermectin/moxidectin Ivermectin Sensitive Ivermectin Resistant Moxidectin Resistant

41.  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 Molecular Assays

42. 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

43.  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 ??? The Future of Parasite Control