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 ???
