Post on 19-Dec-2015
Drug Resistance in NematodesPopulations Matter !!!
Ray M. Kaplan, DVM, PhD, DipEVPC
Department of Infectious DiseasesCollege of Veterinary Medicine
University of GeorgiaAthens, 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 (MOR),
others
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) Amino-acetonitrile derivative (AAD) Introduction in the US – soon ???
Derquantel-Abamectin (2010) 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
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
Where Do Resistant Worms Come From ???
Nematodes have great genetic diversity & large population sizesHigh mutation rates and rapid evolutionHaemonchus 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
Changes in “Resistance Genes” in Response to Drug Selection
Arbitrary Time Units(Worm Generations exposed to repeated
treatment)
Perc
ent
of
Worm
s th
at
Are
R
esi
stant
Clinical detection level
Diagnostic detection level
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
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. 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 nematode5. 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 frequency10. drug dose rate11. drug pharmacokinetic profile - persistence12. 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 Resistance ??? -- Small Ruminants
Country/Continent BZ LEV IVM MOX Spp.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
What is the Prevalence of Resistance ?? -- Cattle
Country/Continent BZ LEV IVM MOX
Brazil +++ + ++++ +
Argentina ++ - +++ +
New Zealand +++ + ++++ +
US and Europe ? ? ? ?
Resistant Genera Ostertagia Cooperia
HaemonchusTrich
Oesoph
Ostertagia Cooperia
Haemonchus
Cooperia Haemonchus
Oesophagostomum Ostertagia
Trichostrongylus
In past few years – rapid increases in level and spectrum of resistance
What is the Prevalence of Resistance ??? -- Horses
Drug Cyatho-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
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 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
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 refugiaDecrease treatment frequency
Must treat selectively
Resistance is InevitableWhat Can We Do ???
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
EACH WORM =
100 EPG
courtesy of Rose Nolen-Walston, DVM, DACVIM
} refugia
courtesy of Rose Nolen-Walston, DVM, DACVIM
0 50 100 150 200 2500
200
400
1000
3000
5000
Individual Horse
FEC
(ep
g)
Distribution of FEC on 12 Horse Farms in Georgia, USA
High Egg Shedders:
27% of Horses 83% of Total Egg OutputModerate Egg Shedders:
18% of Horses 13% of Total Egg Output
Low Egg Shedders:
55% of Horses 4% of Total Egg Output
What Happens if We Apply Selective Treatment ? ? ?
Assume Treatment Reduces FEC by 99.9%
Treat horses with FEC > 200 EPG
What Happens if We Apply Selective Treatment ? ? ?
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
Controlled efficacy studies
Fecal egg count reduction tests
In vitro bioassays
Molecular assays
Diagnosis of Anthelmintic ResistanceQualitative or Quantitative ???
Diagnosis of Anthelmintic Resistancein 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
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 AssaysLDA, EHA, LMIA, LFIA
Laboratory Diagnosis of Resistance in vitro tests
Larval Development Assay
L2
L3L1
Drug
X X XL3
Laboratory Diagnosis of Resistance
LDA - DrenchRiteOnly one test needed per group or can
be performed on individual animalAll 3 major drug classes plus
moxidectin tested in a single assayOnly for small ruminants and zoo ungulatesAvailable as a diagnostic service in my lab
Haemonchus contortusDose Response: Larval Development Assay
DrenchRite LDADose-response for ivermectin/moxidectin
-7.5 -5.0 -2.5 0.0 2.5 5.0 7.5
0.0
0.2
0.4
0.6
0.8
1.0
WtMyJsCsC2C1
Ln Ivermectin Concentration
Pro
po
rtio
n A
ffec
ted
Ivermectin Sensitive
Ivermectin Resistant
Moxidectin Resistant
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
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
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