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