GENETIC VARIATION OF A P-GLYCOPROTEIN GENE IN UNSELECTED AND IVERMECTIN- AND

MOXIDECTIN-SELECTED STRAINS OF E4EMONCHUS CONTORTUS

HA0 YUAN LIU

INSTM'UTE OF PARASITOLOGY

McGILL UNIVERSITY, CANADA

A thesis subdtted to the Faculty of Gnduate Studies and R d in partiai folnllment of

the requirements for the degne of

O HA0 YUAN LIU, Aplurt, 1998.

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Canada

SUGGESTED SHORT TITLE:

Genetic variation of a Pgp gene in Haemonchus contoms

Abstract

Anthelmintics, antiparasitic agents, have been developed as a main weapon to

control parasitic nematodes of domestic rwniaants. Uoforhmately, the intensive use of

anthelmintics leaâs to the development of dnig resistance in parasite populations.

AntheLmintic nsistance has compromised the control of nematode parasites and bas becom

a major probkrn in many counûies of the world. Resistance to the newest antheimintics

such as ivermectin 0 and nlated anthelmintics in Huemonchus contortus in sheep has

been developing rapidly in recent years. The development of dnig cesistance is an

evolutionaty process that leads to genetic changes in parasite populations in nsponse to

dmg exposure. However, the mcchanism of ivermectin nsistance in nematode parasites is

unknown. P-glycoprotein (Pgp) has been well documented in mamrnalian cek as a

membrane transporter by actively extruding a variety of stnicturaliy and fuactionaily

unrelateci hydrophobie cytotoxic dnigs out of the ceil. This study was to determine whether

there is an association between spccific deles at the Pgp locus and IVM or moxidectin

(MOX) selection in H. contortus, by investigathg the genetic variation of the Pgp

homologue in unselected and IVM- and MOX- selected s a s of H. contortus. nine

laboratory strains. derived h m the same unselected parent population, nrM-selected

(IVF17). M O X - s e I d (MOF17) (passageci for 17 generations with IVM or MOX

treatment respectively) and unselected (PF17) strains (passaged for 17 gemrations without

any dmg aamient) of H. contomrr were surveyed in this study. Fony individuai aduit

male w o m were investigated for each strain in this study. Using restriction fkagment

length polymorphism (RFLP) malysis, we found that Pgp is highly poiymo~hic in H.

contortus. Alleiic variation is extremely high ai this locus amnig thme strains. Tbe allek

ri#iuenciics at this locus in IVM- and MOX-selected süains w m sigeincantly diffemt

fiom the uoselected strain (p

Les anthelmintiques, des agents antiparasitaires, ont ét6 développés comme outil

p ~ c i p a i à la lutte contre les nématodes parasitaires chez les ruminants.

Mdheureusement, l'utilisation intensive de ces produits a engendrt chez les popdations

de parasites le d6veloppement d'une réustance i ceux-ci. Cette rbsistance aux

anthelmintiques compromet la lutte contre les parasites et de ce fait est devenue un

probltme majeur dans plusieunr pays. Chez le parasite du mouton, Haemonchus

contor?us, la résistance i de nouveaux produits tel que l'ivemectin (IVM) et autres anthelmintiques de la meme famille s'est développée rapidement durant les dernières

années. Le développement de la résistance est un processus évolutif des populations de

parasites face i l'exposition aux mCdicommts se traduisant par des changements

ghdtiques. Cependant, le mécanisme de la résistance i I'lVM chez les parasites est

inconau. La P-glycoptot6ine (Pgp) est bien connue dans les cellules de mammiféns.

Ellle agit comme ûansportau au niveau de la membrane et expulse une vuiét6 de

médicaments cytotoxiques hydrophobes diffkents de part leur structure et leur fonction.

L'objectif de cette étude est de déterminer si une association existe entre des allèles

spécifiques du locus Pgp et la résistance P lPIVM et au moxidectin (MOX) chez

ff-op~cchus cmtwt~. Pour ce fun, ia -ation ghétique de l'homologue Pgp dans les lignées sensibles et résistantes i lSIVM a au MOX d'Haemonchus contortta a Cté étudiée. Créées en laboridoin, 3 iignées dérivées de la m&me souche parentale sensible

d'H ccmtorlus ont été utilisées, soit une résistante i l'MM (MW), une rdsistante au MOX (MûF17) (17i6me génération de parasite traitée i chaque fois avec IVM et MOX), a une lignée sensible (PF17) (17idme génération n'ayant jamais été exposée aux anthelmintiqua). Quamnte mflcs Haemonchils ont &6 ChidiCs pour chaque lignée. Grâce la technique de polymorphisme de taille des Eiagments de restriction (PTFR), il

est démontré que h Pgp d'R. c0lltOQtlGT eit hautement polymorphe. Une grande variation

der alléles est obravCa i ce locus pumi les 3 lignées. La tiéquenœ des ailéles i ce locus

chez ler lignées distantes i i'NM a ru MOX est st&tiquement ciiffiCrante de la lignbe

sensible (p

TABLE OF CONTENTS

Suggested short titie ............... ............................................................o.i . O Abstract.. .................................................................................. *.........IL

Abreg6 ................................................................. ..............................iv ................................................................................. Table of contents vi

List of figures. ....................................................................~.............. ix List of tables ...................................................................................... x i

Thesis office statement ....................................................................... xii Acknowledgments ............................................................................ xiv

List of abbreviations .......................................................................... xv

CHAPTER 1. INTRODUCTION ..................................................... i 1. General introduction ...........t............t...Ct........,......C............ 1

2. Speciîic objectives of thls study ................... .... ................. 4

CHAPTER 11. LITERATURE REVIE W. ....................................... .. 1. PARASITIC NEMATODE INFECTIONS .,.. .. ...............O..........

1. Life cycle of Haemonchus contortus ......................................... ........................... 2. Distribution and control of nematode infections

II. ANTHELMINTICS ................................................................ 1. Benzimidazole class ............................................................

................................................... 2. Levamiso~e/Morantel class ...................................................... 3. Macrocyclic lactone class 11

A. Chemisay and production ....................et................... 1 1

. ....... B Mode of action .. .............................................. 12 III . ANTHELMINTIC RESISTANCE .......................................... 14

........................ ......O........* 1 . Macrocyclic lactone resistance ... 15 2 . Mechanisms of anthelmintic resistance ......... ...-.. ............... . 16

A . BenWmidazole resistance .......................................... 17 ..*..*......*......... ..**...... B . Levamisole resistance ... .... . 18

C . Macrocyclic lactone nsistance .................................... 19 3 . Detection of anthelmintic resistance .......................O............... 20

IV . GENETIC VARIATION AND TEE DEVELOPMENT OF .....*..*.......... .*.........*. DRUG RESISTANCE ..... ................... 21

............................................................ V . P-GLY COPROTEIN 23 1 . Structure and function of P-glycoproteins ................... .. ....... .. 24 2 . Cornlation of P-glycoprotein with dmg

resistance in parasitic diseases ...................... ............. 27 3 . Rationale of Pgp involved in macrocyclic lactone

resistance in nematodes .................. .... .............................. 29

............... CHAPTER m . MATERIALS AND METHODOLOCY 31 ...................... 1 . EXPERIMENTAL DESIGN .................. ..... 31

II . MATERIALS AND METHODS .............................................. 32 1. Parasitic nematode strains ................... ,....,................... 32 2 . Preparation of parasites ..................................................... 32 3 . Extraction of genomic DNA ................................................. 33 4 . Sequences of the primers used in PCR amplification .................... 33 5 . Polymersse Chain Reaction (KR) amplification ......................... 34

vii

...... 6 . Restriction enzyme digestion and identifcation of ailele patterns 35 7 . AUele fkequencies and statistical analysis .............................. 35 8 . Cloning of the Pgp alleles .................................................. 36

A . Purification of DNA ................. ........... .................. 36 B . Ligation .................... ... ................................ 36 C . Transformation ............................................*...... 36 D . Mini-Preparation ................................................... -37 E . Identification of clones .............................................. 38

9 . Sequencing of the alleles ................................................... 38

CHAPTER IV . RESULTS OF EVALUATION OF RESTRICTION FRAGMENT LENGTH

POLYMORPHISM ................................................ .... 40 1 . Restriction enzyme digestion analysis .................................... 40 2 . Cornparison of allele frequencles between unselected

and IVM- and MOX-selected strains .................. ..., ....... ..,..... 42 3 . Hardy-Weinberg equilibrium analysis ........................ ............. 43

CHAPTER V . RESULTS OF CLONING AND ..................................................................... SEQUENCING 61

1 . Cloning of the alleles ................... .... ............................. 61 2 . Sequenclng of the alleles ...................................................... 62

. ................... CHAPTER VI DISCUSSION ..........~.....C............... 64 REBERENCES .............................................................................. 70

viii

LIST OF FIGURES

Figure 1. Amplified fragment and the position of PCR primers ........................ 45

Fipre 2. DdeI digestion pattems fiom individual worms of the three H. contortus saains: unselected suain (PF17). ivennectin selected (IVF17) and moxidectin selected

(MOF17) strains .................... ..................... ................ . . 46

Fipre 3. A M digestion patterns fiom individual worms of the i lme different H. contortus strains: PF17, IVF17 and MOF17 .................... ........... ..... ............ . . 48

Figure 4. Hinji digestion pattems fiom individual worms of the t h e If'. contortus strains: PF17, IVF17 and MOF17 ........... . ....... ..... 50

Figure 5. RsaI digestion pattems nom individual worms of the three H. c o n t o m strains: PF17, NF17 and MOF17 ..................... .. 52

Figure 6. Total DdeI digestion patterns fiom the three strains of worms .............. 54

Figure 7. Total AluI digestion pattems fiom the three strains of worms .............. 55

Figure 8. Total Hinfl digestion patterns h m the thm strains of wonns ............. 56

Figure 9. Total RwI digestion pattems h m the three strains of worms .............. 57

Figure 10. Allele ftequencies .................... ....o...~..o....o.t....t.~....... . . 58

CHAPTER V

Figure 1. Sequence cornparison of aiieîes A. B, P and x ................... .. ....... 63

LIST OF TABLES

CHAPTER IV

Table 1 . Allele classification ........................ ... ............................ 59 Table 2 . Genotypes of different straïns of wonns ................... ...... 60

Thesis Office S tatement

The foliowing statement is a regdation ftom the Facuity of Graduate Studies and

Research, McGiiI University concerning thesis preparation:

" Candidates have the option of including, as part of the thesis, the text of a paper(s)

submitted or to be submitted for publication, or tbe clearlyduplicated text of a published

paper(s). These texts must be bound as an integral part of the thesis.

If this option is chosen, coanecting texts that provide logical bridges between

the Memat papers are mandatory. The thesis must be Wfitten in such a way that it

is more than a mere collection of manuscripts; in other words, results of a series of papers

mut be integrateâ.

The thesis must stüi conform to ali other nquirements of the "Guidclines for thesis

Preparation". The thesis must include: A table of Contents, an abstract in English and

French, an i n ~ u c t i o n which clearly States the rationaie and objectives of the study, a

comprehensive nview of the Literaîure, a final conclusion and summary, and a thorough

bibliography or reference list.

Additionai xliate!riai must be pmvided where appmpnate (e.g. in appendices) and in

sacient detail to ailow a clear and precise judgmnt to k made of the importance and

onginality of the research repoacd in the thesis.

In the case of manuscripts CO-puthored by the candidate and others, the candidate i s

cequird to make an expikit statement in the thesis as to who contributcd to

mch work and to what extent. Supervisors must attest to cbe accuracy of such

statements at the doctoraî oral defense. Since the task of the examiners is nude more

difECUIt in 1&se cases, it is in the candidate's intemst to maicc p e r f i y c1ear the

responsibilities of ail the authors of the CO-authoreâ papers. Under no circumstances

caa a CO-author of any component of such a thesis serve as an examiner Cor

tbat thesis.

Acknowledgments

First, 1 would Wre to express my deepest gratitude to my supervisor, Dr. Roger

Richard who gave m the opportunity to study in the Institute of Parasitology of McGill

University, and bis financial support, and àeepest th& to both my supervisors: Dr.

Richard and Dr. Robin Beech, for k i r consistent support, advice, helpful suggestions,

patience, and encouragement at every stage of my studying.

I would iike to thank my thesis cornmittee mmbers: Dr. P. Riberio aad Dr. Ming

Xu for their advice and help during the course of this study.

1 would also like to thank BU Blackhall for bis tremndous help, support,

especiaily his patience to answer m countless questions in the lab. Many th& to

Chcistiane Trudeau for prepacing the parasites for m and for tmslating the abstract. My

th& also go to Marcello Molento and many other people in Dr. Prichard's and Dr.

Beech's lab wbo gave m many different belp and support during my studying. 1 also

wouid like to thank them for their fiiendship.

Thanks to the professors, students and other m&rs at the ïnstitute of

Parasitology for their always smiüag and fiiendship. 1 wiii always remmber the tim 1

spent in the Institute of Parasitology!

Many thanks to Dr. Cathryn Nagler-Anderson at Harvard University for her

support and help during my thesis wciting.

Finaiiy, but very importantîy, 1 give special thenl

LIST OF ABBREVIATIONS

Adenine nucleotiâe Binding Cassette

Albendazole

B e d d a m l e

BZ-mistance

BZsusceptibility

Cambendazole

Faecd egg count reduction test

Fenbendazole

Garnma-aminobutyric acid

Ivermectin

Levamisole

Larval developrnent assay

Mebendazole

Morante1

Moxidec tin

Muitidnig mistance '

Nicotlliic acetylcholipie receptor

Menciamle

P-giycoprotein

Phosphate-buffend saline

PoIymerase Chain Reaction

PVrantei

Resûiction -nt length pdymorpaism

Thiabedamle

ABC

AB2

BZ

BZ-R

BZ-S

CBZ

FECRT

FBZ

GABA

IVM

LEV

LDA

MBZ

MOR

MOX

MDR

nAChR

OEZ

Pm PBS

PCR

PYR

RFLP

TB2

This work is dedicated to my parents.

xvi

Chapter 1

Introduction

1. General introduction

Parasitic nematodes are a major cause of morbidity and mortality in humans and also

cause widespread loss of food production by infection of livestock throughout the world

(Holden-Dye et al., 1997). The cost of intemal parasitism in the high winter rainfall areas

aione has been estimated at $89 million annuaily in a n b a l s in Austraiia (MacLeod et al.,

1992). In order to reduce the economic loss, at least for the foreseeable biture, to control and

to pnwnt the parasitic infection, and to treat nematode infections in man and animals, will

depend on the use of chemotherapeutic agents (He~essy, 1994). such as anthelmintic drugs,

which were âeveloped as a major mthod for conhohg the parasites of domestic nuninam.

Even with the introduction of narmw or broad spectnim helminth vaccines, or the breediag of

parasite-resistant hosts. tacticai (and in many cases, strategic) parasite control WU requin

chernical intervention (Hennessy, 1994).

Current coutrol of important veterinery parasitic nematodes relies mainly on the use of

three major classes of bmad spectrum aatbeimintics: benzimidazole class, levamisole class

and the macrocycüc lactoac class. Ivenncctin and moxidectin (MOX) anthelmiotics,

belong to the newest class - macrocyciic lactones. They aie commoniy used for the control of nematodes in domestic animpls. Ivermcctin can biid to and open the alpha subunit of a

glutamate-gated chioride charme1 of~litmatnAL? cd mcmbrane (Cuiiy et al., 1996) remlting in

the hyperpolarization of neuromuscular tells and to evenhial paralysis of the worms.

Unfortunately, their muent use has caused selection for dnig resistance. The development

of resistance to anthelmintics is widespread in nematode parasites of sheep. goats. horses,

cattk and swine, and has becorne a mjor impediment to the tnatment and conml of diseases

of parasitic origin in many countries of the world (Richard, 1994). If resistance to a psrticular

anthelmintic occurs, it is lürely that anothet anthelmintic that bas the same mode of action will

also be ineffective (cross-resistance) (Martin et al., 1997). So far, &stance has ben

reported in nematodes to ai i thne broad spectnun anthelmintic classes (Richard, 1990).

Resistance in sheep and goat nematodes to ivermecth is spreading rapidly in the field

(Van Wyk & Malan, 1988; Craig & Miller, 1990, Le Jambre, 1993). The emergence and

rapid development of IVM resistmce in important nematode v i e s now loom large as a

major international k a t (Waller, 1994). This is of particuiar concem to the sheep industry.

especiaily in the major sheep-raising countnes of the southem hemisphere. For example, in

New Zealand and South Afnca, anthelmintic nsistance is of major concem. In South America

and Africa that lack hancial or manpower mources, anthelmintic mistance is almost cestain

to be widespd (Wailer, 1994). Among the gastrointestinal nematdes, Haemonchui

contortus develops resistance most rapidly (Echevarria & T ~ d a d e , 1989). In cecent years,

anthelmintic resistance has becorne a significaat and growing problem causing loss of

production in animal industries in many countries of the world (Bj$rn, 1994).

However. the mechanisms by which macrocyciic lactone mistance is induced are not

yet known. The possiiiiity that IVM mistance is due to allucd binding to its receptor was

investigated. Rohrer et al in 1994 showed that ivermctia cesistance was not due to an

alteration in its bindiog to the glutamate-gated chloride channcl receptor. Cumntly. Blackhall

et al (1998b) found that change in allele fiequeIlCies of the alpha subunit of a glutamstepated

chloride charme1 is associsted with IVM and MOX mistancc in H. contortirr, but how s m g

the relationship between this chloride c h e l and IVM nsistance is still a question. The

alteration of IVM effective concentration may aiso cause the dmg cesistance; transport of the

dnig by a mechanism simüar to multidnig resistance (MIX, see below) could d u c e the local

dnig concentration resulting in nsistance. In recent years. P-glycoprotein (Pgp) has been

investigaîeâ to detemine whether it is involveci in I V ' cesistance in H. contortus (Xu et al.,

1998; Blackhail et al., 1998a).

P-glycopmteins (Pgps), a group of integral transmernbrane proteins, were initially

identifid to cause multidrug mistance in mammalian hunor ceiis (Juliano & Ling, 1976;

Gottesman & Pastan, 1993). Its overexpression in som f o m of dmg-resistant cancer ceus

confus the multidrug resistaace phenotype, when additional Pgp molecules are able to pump

out a range of structurally and hiactionally unrelated anti-caucer dmgs (van der Biiek &

Borst, 1989) fmm inside of the cells. Endem that IVM is a good substrate for Pgp coms

from the fact that disniption of the mouse mdda Pgp gene leads to a severe increase in the

toxicity of M M in mice (SchinLel et ai., 1994). Furthermon, Didier & Loor (1996) and

Pouliot et ai (1997) have demonstrated biochemically that IVM is an excekt substrate for

Pgp in msmmaiian ceUs and that it is aiso a multidnig resistance teversing agent. Recently, a

fuli length Pgp cDNA h m H. contortus has k e n cloaed and sequenced (Xu et al., 1998).

Expression of this P-glycoprotein mRNA wos higher in ivermcctin-selected strains compared

with uoselected strains of H. contortus (Xu et al., 1998). DEereat restriction patterns have

aiso been identifieci between resistant and susceptible strains through Southem blot aoalysis

with this cDNA clone (Xu a al.. 1998). These rcsuits suggest that Pgp may bind and

transport TVM h m the lipid biayer and cytoplasm to outsi& of the ceils before lVM can get

into the h i d e of the d i s to exert its toxicity, and ibe alleles at ihis lacus may cespond to

selection with ivertmctin.

MOX, an agent belonghg to the milbernycin famüy of macrocyclic laaone

anthelmintics. has a similac maaocyclic lactone structure as IVM. Shoop et d (1993) have

suggested that MOX and IVM may shan a common mechanism of action and of resistance.

Developmnt of anthelmintic resistance is an evolutionary process that leads to genetic

changes in parasite populations in nsponse to dmg exposure. It depends on the pnsence of

aileles which can confer resistance. Prior to the introduction of a particular h g , the relevant

resistance aüeles are usually present ai a low hquency in the susceptible population and

presumably they have no advantage to compete with the susceptible alleles. Following the

introduction of a new dmg, smali number of wonns can survive. and deles in the small

portion of population that swvived cm then be canied to the next generatioo and give more

and more individuals carrying this resistance allele. Over several generations, the fiequency of

alleles conferring resistance hcreases and the mistance aliele(s) wilI then be over represented

in the offspring. Thenfore, the appeacance of dmg resistance reflects changes in the

composition of a parasite population gene pool (Beech et al., 1994).

Up to now the prevaleace of IVM resistance has reniained low, but the number of

cases bas hcreased during ment yem (Geerts, 1995). Thcnfore, understanding the

mechanisms and dynamics of the developmnt of resistance. at both molecular and population

genetic levels, is needed in order to âetect the developmnt of resistance in H. contortus

populations at an early stage and to develop adequate control programs.

2. Specific objectives of this study

1). To investigate the geiwtic vatiation of a Pgp gene in u~lselected and TVM- and . MOX-selccted H. contorttcr populations.

2). To determine the alleles of the Pgp gene in H. contortus and to test for a iinkage

between Pgp and IVM and MOX nsistance in H. contortus.

3). To i&ntify alleles which are associated with IVM or MOX resistance and

susceptibility in H. contortus.

4). To develop a d e r for detection of early stage IVM and MOX resistance in H.

contortus populations.

This genetic study aiiows an understanding of the evolutionary response of the

parasite to h g treatment and. therefore to better use anthelmintics to control parasites. This

will have important practical implications in preventing the spread of dnig resistance and in

the control of parasitic infections and in assisting with the design of more effective dnig

administration stratepies for farmers that minimize selection for IVM and MOX resistance.

Chapter II

Literature review

1. Parasitic nematode infections

Diseases caused by parasitic nematades pose a considerable medical and veteriaary

health problem* Interna1 parasites or wonns cause economic and production losses to animal

producers in many countries of the world. In western countries problems consist mainly of

wide spread infections in domestic livestock. Nematodes that feed on blood cause Som of

the most important parasitic diseases of humans and dornestic animals (Aberejola et al., 1979;

Georgi, 1980; Preston & Aiionby, 1979; Warren, i988). Anemia is a major problem which

can lead to acute disease and death or chronic disease in the parasiîkd host. Costs of

treaanent and loss of production in sheep and cattie in Australia are estimateci to range

between Aus$200 to Au&WO mülon amuaüy. Many of the nematode parasites that derive

nutrition fiom host blood an gastmintestinai mmatodes, which belong to the order

Strongyfida. ûne of the most important parasites is the stomach worm of Nminants such as

Haemonchus. Haemonchus contortus, one of the most pathogenic gastrointestinal parasitic

nematodes of sheep and goats, is widespnad thmughout the world. It results in 3 0 4 %

mortality in lambs not treateâ with anthetmintics. Although the disease may occur in all age

groups, animals 2-24 months 01d are mon comrnonly affected. Ol&r sheep may have an

acquired resistance to the parasite because of eariier exposurr to hm,

1. Lifè cycle of Haenionchus confortus

The Me cych of H. contortirs consists of several stages as is shown in figure 1.

Haemonchus contortus d a s not requirc an intermediate host. Adult male and f e d e W O ~ ~ S

live in ihe abomasum (or me stomach) of ruminant animais. Tbe female can lay 5,000 to

10,000 eggs per day which pass out of the host with the feces. The eggs in the feces sin

develop into h t . second and third stage larvae. Eggs and newly k h e d lame (LI and L2)

cemain in the feces whae ihey feed on bacteria. The larvae in these stages are not nsistant to

cold and desiccation. Third stage larvae do not f d and an highly mistant to drying and cold

temperatures. Tbey c h b to the tips of blades of gram in moist conditions and the definitive

host becomes infectecl by ingcsthg tbird stage lmae on herbage. AAer infection, the young

w o m pass into die a b o m where they burrow into du mucosa and develop into forth

stage larvae. It talces about 48 hours. They then begin to feed on b l d of the infected host

and develop to adulthood. Mating of adults occurs and egg production commences 15 days

laiu. Enormous numbers of larvae may accumulate on heavily grazed pastures.

Figure 1. Life Cycle of Haemonchus contortus

The lambs are most severely affected by the parasite. Even relatively low infection

levels alrrady a&ct growth rate, weight gain md wool production. When large numbcrs of

larvae infect sheep, death can occur suddedy, while the sheep still appear to be in good

health, and cause signifiaut losses in production (Gibbs, 1985; Janssens et al., 1989).

Therefore, H. contortus is one of an economicaüy important group of gastmintestind

nematodes of sheep which are closely nlaîed taxonomically, epidemiologically and

pathologically (Soulsby, 1982). It is regarded as one of the most pathogenic hehhths in

domesticated animals (Soulsby, 1982).

2. Distribution and control of nematodes infections

H. contortus is found worldwide. Larval development of H. contortus occurs

optimaîly at relatively high tempetanues. Hue~nchosis is primarüy a disease in warm,

humid climates wherever sheep are raised for food or wool (Soulsby, 1982). This is the most

important wonn iofection in the areas of the tropics and subtropics such as Australia, Brazil,

the Middle East and Nigeria

Diffennt stratepies have ken developed to control these parasitic nematde infeaions.

Anthelmintics, the antiparasitic agents, have been discovcred as a major mthod to control

nematodes of domestic animais, but the emrgence of drug resistance is becoming a serious

pmblem. Progress dso continues to be made with nonchernothetapeutic appwhes such as

vaccination (Emery, 1996). breediiig for hosts that display increased tolemce or mistance

CO the parasite (reviewed by Gray & GU, 1993; Gill et al., 1994) and biological conml

( 3 n v o l d et al., 19%). but pmcticai application has been slow to develop.

II. Anthelmintics

Anthelmintics prevent, contml and &eat many types of parasitic nematodes in both

humans and domtic animais. Factors tbat favored the use of these anthelmintic drugs

include the fkt that these drugs usually act against a broad spectnim of different nematode

species, arr d y available and an relatively cheap (Bird, 199 1). Anthelmintics currently on

the market can be groupeâ into thrce distinct modem broad-specmmi classes: the mcrocyclic

lactone class; benzimidamle class and levamisole/mormtel class. They WU continue to be

used until effective vaccines are produceci andor hygiem standards have been improveâ

(Martin et al., 1997).

1. Benzimidazole class

Benzimidazole (BZ) antheîmintics, iiiaoduced in the 1960s. are the most common

chemotherapeutic agents used to remove intestinal helminihs (mmatodes, cestodes and

trematodcs) from animals. Various d o g s ais0 display distinct antifimgal activity. The

members of the class include thiabenâazole (TBZ), albendamle (ABZ), fenbendazole (FBZ),

mbendazole (MBZ), cambendazole (CBZ), and oxfendazoe (OEZ). TBZ was the nrSt broad

spectnun anthelmintic which is highly effective against a wide range of parasite species

(Brown et al., 1961; McKellar & Scott, 1990). Since the introduction of the fmt BZ, new

derivatives bave appeand ngularly. Investigations of the mode of action of BZ dmgs in

parasitic nematodes have indicated that BZs induce disintegration of the micmtubullar

fiamework in nematode c d s (Borges & De Nolh, 1975; Bughio et al., 1994). They are

believed to internt with the cytoskeletal protein tubului and act by specitically binding with

high affinity to nematode ptubulin which altcrs the tubulin-microtubule equilBrim that

preveats polymerizatioa during rnicmtubuie assembly and causes depolprization of

microtubules in intestinal celis of nematodes (Sangster et al., 1985; Lacey, 1988). Tubulh is

a soluble protein found in most euLaryotic ceh that binds to the dmg colchicine. a- and B- tubulins are tightly associated fomiing a heterodher, and are the main compents of

microtubules. Microtubules. built of tubulin, are critical to cûromosomal movemnt during

c d division. Usually microtubules depolymerize into stable ap dimers (tubulin). Tubului

does not dissociate into a and B monomers d e s s denaturing agents are added (Darnell et al., 1990).

2. Levamisole/Morantel class

This group of anthelmintics have been in use since the mid 1960s. In addition to

levamisole and morantel (MOR), pyrantel (PYR) also ùelongs to ihis class. They are

cholinergie agonists with a seleaive pharmacology for nematode receptors. These cfaemicais

are stnicturauy related and act on the sam site on nematode muscle (Coles et ai.. 1975;

Harrow & Gration, 1985). LEV is a potent anklmintic which acts on nkotinic

acetylchohe ceceptors (nAUiR) of nematodes, thereby dimpting neuromuscuiar

transmission resuiting in piualysis of worms. Paraiysis is due to sustained muscle contraction

(Coles et al., 1975; Harrow & Gration, 1985). RAChRs are weii characterized multi-subunit

cation channels activated by binding of the neuroeansmitter acetylcholine (Hoekstra et al.,

1997). in vertebrates, the muscle nAaiRs are composed of fout di&rent types of subunits

forming a pntameric structure, wkreas the neuronal nAChRs consist of oniy two types of

subunits: a- and non-a-subunits. The a-subunits contniute the major part of a binduig site

for ac*ylcholine (Conti-Troncoui et al.. 1994). The nAChR subunits are encoded by

muitigenc fiimiües wûh a homology betmen subunits and across species Ieaàing to a

fiuictional diversity of nACW (Le Novert & Chaageux, 1995; Hoekstra et al., 1997). LEV

also inhibits the migration of third stage lame of susceptible and resistant isolates of H.

contortus (Sangster et al., 1988).

Macrocyclic lactones, a group of stnrairally related anthelmintic compound, include

the avermectins such as ivennectin 0, abamectin. doramecth and the milbemycins such

as moxidectin (MOX). They are widely used for the ûeaûnent of both interna1 and extemai

parasites, specificaiiy nematodes and arthropods in humans, domstic animals and plants.

They are highly effective against different stages and species of susceptible strains of most

gastrointestinal nematodes of sheep (Benz et al., 1989; Bisset et al.. 199 1).

A. Chemistry and production

Maaocyclic lactones are the most widely used compounds and derived from

fermentation products of soildwelling bacteria of the genus Streptomyces. They are a group

of chemicaiiy nlatcd 16-mmbtred macrocyclic lactones with a similar ring system and an

acaricidal, insecticidal, and nemaficida1 (Steel, 1993; Clark et al., 1994). The averrnectins

wen first isolateci h m a soi1 acrinomycete microorgaaism, Streptomyces uvemititis (Burg

et al., 1979), and were discovend in the mid 19709 b u s e of their potent antheimintic

activity (Egeaon et d, 1979; Chabaia et al., 1980) and marketcd foc veteniiary use in 198 1.

Avennectins bave bcen trcmendously s u c d antheimintic agents because of their ability to

parasites withwt affectiag the host organism (Osthci et ai, 1979; Riner et al., 198 1) .

IVM is the mostly uJed dtug in the avcrmctin gmup.

IVM, a semi-synthetic member of the avenriech p u p of dnigs, is the 22, 23-

dihydn, derivative of avennecth B 1. It was the h t broad spectrum endectocide with a wide

margin of safety against a wide range of parasites (Campbell et al., 1983; Eguton et al.,

1980). k was released for registratioa in 1981, and has k a approved for use in over 60

countries forcontrol of parasites in humaris, cattk, sheep, horses, goats, swine, dogs, and

other mammals (Di Netta, 1989; Campbell et al., 1983). It is particuiarly effective against

arrested and founh larvai stages of economically important gastrointestinal nematodes in

sheep (McKelier & Marriner, 1987; Echevarria et al., 1992) and cade (Barth & Preston,

1987). 1t has also b a n shown to be effective against both benwmidazole and

levamisole/morantel resistant nematodes in sheep (Wailer & Donaici, 1983). in general. IVM

administered at a dose of 02 mgkg orally to sheep results in efficacies of 97- 100% against

addt and fourth stage larval forms of H. contortw (Campbell & Benz, 1984).

MOX is stcucturauy nlated to ivermectin. It is a chemicaîly modifieci denvative of a

macrocyclic lactone and is isolatcd from Smptoniyces cymeogreseus noncyanogenus. It has

becn shown to have a prolongai period of activity in sheep (Rendeîi & Callinan, 1996). At

0.2 mgkg, nematocidal concentrations of IVM wen maintained for 28 days and of MOX for

42 &y s in sheep (Taylor et al., 1997).

B. Mode of action

The mechanism of action of ivermcctin and related dmgs on nematodes is not entirely

elucidated. It is thought to spccincally bind to and open glutamate-gateâ chloride channels

located on muscles of the pharynx and, possibly, the somatic musculahin ( M y et al., 1996;

Sangster, 1996) in the nematode and arthropoàs œîi membrane, increasing the Ci-

petmcability and lcaâing to the hypezpolatization of the target neuromuscuîar al1 (Cully et al.,

1994; Arena et al., 1995); eventually paralyzing or stacving the nematodes (Sangster, 19%).

However, the identity of the target ion

glutamate-gatd chloride chsinne1 results in a flaccid paralysis of affected parasites followed by

their death or expulsion (Courtaey & Robenon, 1995).

Studies on tbe mecbanism of action of moxidectin were conducted by measuring the

changes in membrane conductance in leg muscle fibrae of the common shore crab using

standard micro-elecüode techniques. It was concluded from these expuimnts that

moxidectin and ivennectin share a common mechanism of action (Shwp, 1993).

III. Anthelmintic resistance

The extensive use of these enthelmintic dmgs has led to wide spread h g resistance

causing a serious threat to the effective contrd of nematode infections throughout the world

(Coles et al., 1994). Resistance to ail of the broad spectnun macrocyclic lactone compounds

has been encountered in most regions of the world where the compounds are used routinely in

animal production. It is common for parasitic nematodes to develop resistance to

anthelmintics, especially when they are used in continuous, intensive treahnent progmns

(Pankavich et al., 1992). It is now over 30 years since cesistance to thiabendazole, the h t

mie broaâ spectnun anthelmintic, was reprted by Dmdge et al (1964) in H. contoms

infections of sheep in îhe USA. Anthelmintic resistance in parasitic nematodes of sheep and

goats is becoming an iacreasingly important subject in vete~ary, parasitologicd and farming

circles in many counuies. There is no question of the seriousmss ofthe paobkm in Australia,

South M c a and the humid semi-tropical regions of South Amrica where 300 million sheep

are raised, and the scient* Literatwe is weli served with âetailed reviews of tbe pnvalence,

the rate of spread and the increase in magnitude of the &tance (Martin, 1986; Waiier,

1986, 1987; Richard, 1990; Bomy et ut., 1990). It is a continuhg probltm for conad of

nemaiodes in sheq (ChRstuu et al., L992). Adelmintic tesistance has compromised tk

control of parasitic nematodes in several laimal based industries. In the United Kingdom.

Austraüa, and New W a n d field sweys have documnted developmnt of hg-resistant

strains of H. contom and Ostemg& circtuncirttu (Christine et ai., 1992). There are also

extensive reports on anthelmintic resistance in the USA, Europe, South Africa, Kenya,

Indian, Malaysia, Latjn American. In fact, Waially whenver it has been investigated

anthelmintic resistance has been found.

In addition to resistance to thiabendazole reported in the eatly 1960s (Dnidge et al.,

1964; Conway, 1964; Shelton, 1968), si& resistance to other beddazoles rapidly

occurred (Hambry et al., 1986; Miller & Craig, 1988; Craig & m e r , 1990). Benzimidazole

resistance in H. contortus bas k e n extensively reported in Australia (Smal et al.. 1968;

Webb & McCuîiy, 1979; Rühin et al., 1984; Edwards et al., 1986). New Zealand (Vlassoff

& Kettle, 1980; Kettie et al.. 198 1) and the USA (Craig & Milkr, 1990). Resistance to the

anthelmintic levamisole has also been reported (Santiago & Costa, 1979; Green et al., 198 1;

Kettle et al., 1981; Edwards et al., 1986). Resistance to the benzimidazole,

levamisole/morantel group, ivermectin, closantel and multiple resisbnce have been found in

gastrointestinal nematodes of sheep, goats, caitle and horses in many anas of the world

when repeated treatment with anthelmintics is common.

1. Macrocyclic Iactone resistance

Resistance to IVM arose very rapidy. H. contortus is the parasite most commonly

identified as resistant although other isolates of parasitic mistance to IVM are emrging too

(Sangster, 1996). Clinical mistancc rcmains rare in most countries but has been nported in

South Afnca and Lath A m e h hug &stance to W, has kcom a widespread problem,

particulariy in nematodes of sbeep, goats and c a t k In some pans of the world, the suzylval

of commercial animal production is tbreatened by the development of anthelmintic resistance.

The fmt indication of IVM mistance by H. contortus in sheep under field conditions

occuned in 1986 in South Afnca (Van Wyk & Malan, 1988). 33 moaths after the dnig was

inûoduc@ then (Carmichael et al.. 1987). In 1988. four additional isolates of H. contortus

fmm sheep in South M c a were reported to be resistant to i v e m t i n (Van Ayk & Malan.

1988). By 1989. a M e r five were reported (Van Wyk et al.. 1989). IVM resistance has also

been reported h m other regions of the world. An ivennectin-resistant saain fiom sheep

raised on a pastoral expetimental station was isolatecl in Southem Brad (Echevacria &

Trindade. 1989; Vieim et al.. 1992). and Craig and Miller (1990) isolated an ivermectin-

resistant sûain h m an angora goat flock in Southem Texas. IVM resistance was also been

nporicd in New Zealmd (Badger & McKema, 1990) and Scotiand (Jackson et al., 1992).

Uuàer laboratory conditions, nematodes that are resistance to ivennectin rnay also show

reâuced sensitivities to moxidectin (Conder et al.. 1993).

2. Mechanisms of anthelmintic resistance

To interfere with parasite reproduction, a dnig must h d its target within the parasite.

AAer the dmg associates with the target, the parasite mwt be suniciently incapacitated to be

killed by the host defense or to die spontaneously. Each of these steps provides the parasite

with opportuaities to intetfere with drug action, resuiting in dnig rrsistance.

HistoricaIly, IVM-resistant worms. wbich have sunRved I V ' trea- cmain

susceptible to MOX. Agsiast nematodes MOX is a mom potent anthelmintic(Sangster, 1995).

Pankavich et al (1992) and Craig a d (1992) showed thrt moxidectin was effiitive against

ivemecth-resistant strains of contortus* The mode of action of MOX is thought to be

similac to that of MM. It acts against nematodes at a relatively low dose-0.2 mglkg or less. 1t

is likely that milbemycias and avermectins act on helminths in the same way although tbey do

differ in potency and spectnun (Sangster, 19%).

A. Benzimidazole resistance

Molecuiar shidies at both the protein (Lubega & Pricbard, 199 Ib) and the DNA Ievel

(Roos, 1990; Roos et al., 1990; Beech et al., 1994) of the mechanisrn of BZ resistance in

nematodes provide evidence that BZ resistance is associated with an alteration in &tubulin

genes which ceduces or abolishes the high affinity binding of BZs for tubulin in the

organisms, and not in a-tubuiin. This decreases the ptubuiin-BZ interaction. In this way. the

resistant parasite is able to tolerate higher dnig concentration. So far. two distinct &tubulin

isotype classes have been reporteci in H. contortu (Roos et al., 1990; Geary et al., 1992).

They can be discriminateci by their amino acid sequences at ihe carboxy-terminus and also by

their DNA sequence, because probes constructeci h m tbese genes do not cross-react in a

Southeru Mot due to a ciifference in codon usage (Geary et al, 1992). Using (fragments of)

these genes as probes to screen H. contorru~ populations by restriction fiagrnent length

polymorphism (RFLP), a clear Merence in RFLP pattern W e e n susceptible and resistant

populations was obtained (Roos et al., 1990. Lubega & Richard, 1990; 1991a). It was

suggested that Ptubulin isotype 1 locus has muitiple aüeks (Roos et al., 1990; Roos. 1990)

and this was p m n by Beech et uf (1994). h type 2 fhubulin genes are lilcely coded by a

different locus fmm isotype 1. These shidies on the sheep parasite H. contortus have shown

that mistance to benn'mi-le dmgs is comIated with rlection for individuais in the

population possessing a specific fLtubulin isotype 1 gene (Lacey, 1988; Roos, 1990; Rom et

al.. 1990, Kwa et al., 1993a; Kwa et al., 1993b). Sequence aaalysis of this P-nibUiin locus in

both BZsusceptible (BZS) and BZ-resistant (BZ-R) H. contortu populations showed three

amino acid diffennces at positions 76,200 and 368 (Geary et al., 1992; Kwa et al., 1993b).

The Phe 200 (BZS) to Tyr 200 (BZR) change is of importance, since it is commonly

conserved between different BZS and BER H. contorrur populations and functional analysis

of p-tubulin genes from H. contortu showed that the single Phe to Tyr mutation at position

200 in p-tubulin isotype 1 coupled with either on alteration in isotype 2 w b u h or the

deletion of the isotype 2 &tubulin gene may coder BZ-resistance in H. contortus (Kwa et

aL, 1994; Lubega & Richard, 199 la; Beech et al., 1994) .

B. Levamisole resistance

Levamisole is a cholinergie agonist. The phamiacological evidence showed that an

alteration in acetylcholine receptor is associateci with LEV resistance in H. contortus. LEV

resistance is thought to be associated with nduction in the numbet of nicotinic acetylchoiine

receptors or a change in the binding athity of these receptors (Sangster et al.. 1988;

Hoekstra et al., 1997). The mistant nematodes either have fewet acetylcholw binding sites

or their afiiaity for LEV is nduceâ (Sangster, 1990). Although several genes encoduig

nAChR subunits have alnady ken isolated fiom the C. eleguns, it remeins to be elucidai#l

which genes art associated with levamisole mistance in parasitic nematodes in the field

(Hoekstra et al., 1997).

C. Macrocyclic lactone cesistance

IVM appears to be a glutamate agonist. So far, the mechanism of macmcyclic lactone

nsistance in H. contom*r is not clear. There are several possibilities: (1) alteration of the

macmcyclic ktone binding site (Coles, 1989) such as tht glutamate-gated chioride channei

receptor could cause conformation changes which prevents binding or decreases affinity of

ivecmectin to the nceptor. In vitro studies of M M nsistant nematodes suggest that IVM

receptors are locateâ on pharyageai and somatic muscle; (2) the alteration of ivermectin

effective concentration also cm cause the nsistance. transport of the drug by a mechanism

simiiar to MDR couid &ce the local dmg concentration nsulting in resistance; (3) Coles

(1989) also hypothesized that hydrdysis of the lactone ring could probably cause avemtin

resistance. For the reason outlined below the second of these alternatives appears to have

occuned in ivennectin resistant He contorhrs.

S o m snidies showed that MOX is effective agakt IVM-resistant H. contortus (Craig

et al., 1992; Pankavich et ale, 1992). They suggested that this is due to either different modes

of action of these two dmgs or ciifferences in rrsponse to a resismce mechanism. Some

studies showed that IVM and MOX sbare CO-mistance and concludeâ that apparent efficacy

of MOX against IVM-mistant parasites may be dosedependent (Conder et al., 1993; Shoop

et al., 1993). Neveaheless, smictural similarity. the sam taqget site and a concentration-

dependent cross-resistance suggest that iverniectin and moxidectin may s h common

mechanisms of resistance in nematdes. However. fectors which modulate drug concentration

at the site of action mpy determine dative expression of &stance between ive- and

moxidec t h .

3. Detection of anthelmintic tesistance

In general, the reduction in anthelmintic potency is used to detect resistmce. The= are

no quick effective methods to test ivemtin and moxidecth resistance in an H. cu~ttutur

population. The in vivo fecal egg couat reduction test (FECRT) is widely used and considered

the test of choice for sheep parasites in the field. It is a simple and relatively noninvasive

methoci, In this case, nsistance is àefined as the failure of a commercially recommeaded dose

of anthelmintic to duce egg counts by >95%; but the= were falst positive LEV-tesistance

and faise negative IVM-resistance diagnostic probkms, because NM can suppress egg laying

so chat wonns mistant to this class of dmgs appear to be susceptible in egg count reduction

tests (Sangster, 1996). The eggs of the different species involved in resistance are

morphologicaliy similar, so tests based on egg counts lack species specificity (Sangster,

1996). This test has poor sensitivity and may faii to detect mistance even when it affects 25%

of the wom population (Martin et al., 1989). In vitro tests are prefemd and ideally

pexformed on eggs or lame and produce results quickly. The larvai development assay

(LDA), proôably the best in vitro test currently available, works weii with BZ, LEV and

MOR, but some of the assays have problems with IVM (Bj$m. 1994). Eggs and larvae can

be nadily recovered or gmwn h m experimentally-infected animals or h m those in the field

and incuôated in the presence of a range ofco~ntrations of solubiiized h g . The effects on

developmnt or motility are then measured (Sangster, 1996). In vivo tests such as worm

count nduction tests are available. Infécted hosts are treated with h g and wonn numbers

compared with untreated coatrols, but it is an expensive approach and severai animafs have to

be slaughtered for each way; dtematives are more common (Sangster, 1996). These

meth& are insufncieatly sensitive to monitor the developmnt of resismce before it is oven

Ifa way could be found to ddcct nsistance when gene fkquencies ate low, then altemative

measuns (such as the use of a nmw-spectnun dmg) could be used to kill the specific

resistance population and extend the life of the broad-spectnim anthelmintic classes.

IV. Genetic variation and the development of drug resistance

Genetics is the study of how physicai and physiological featwes of an organisms are

passed dom to their offspring. ûenetic variation is expressed as focms of a gene with

slightly different sequences, which are known as aileles. Mviduals with two different aileles

at a particuiar locus an said to be heterozygous, and with two identical alleles are

homozygous. Although any one individual can have a maximum of two difYerent alleles at a

locus, the number of different alleles in a population can be much gnater than two. h g

resistance has been defirvd as 'a change in the aiiele fnqueacy of a population thac is

produceci by drug selection whereby more drug is reqwred to exact sorne effect than was

quired prior to selection' (Shoop, 1993). Selective removal of susceptible memben in a

population by dnig toxicity leaves nsistant members to contribute the gceater share of genes

to the next generation. Apparendy, resistance has arisen as a nsult of selection of parasites

during therapeutic treatment (Martin, 1990; Jackson, 1993). Survivon of a treatment, king

the most nsistant compoaeut of the population, carry resistance geiies which they pass on to

their offspring (Sangster. 1996).

During the selection process nsuiting in resistance, there must k Som genetic r

variation in the population gene pool. An absolute pre-cequisite for a genetic nsponse to a

dmg selective pressure is genetic variation within the population unda selection. Befose a

new dmg is nrSt used, the aUele(s) which are associated with resistance are usually present at

a very low fkquency in tbe suscepuille population, and pieSumabIy they have no advantage

compare with the susccptiible alleies and aUek variation should be highcr in a swcepti'ble

population compare to dmg nsistant $trains (Roos et al., 1990; Beech et al., 1994). The

evolution of resistance wu proceed rapidly when the initial population is very variable. When

the deles which are present in the mistant straia also exist in the susceptible strain, the

Cesistance may arise rapidly. However, the speed with which dnig resistance àevelops

depends on the change in fnquency of worms carcying nsistance alleles. The rate at which

cesistance àevelops an dependent on the amount of genetic variation in the population and the

intensity of selection (May, 1993). It is impossible for any dnig to be 100% efficacious.

against 100% of parasite species, 100% of the tirne. Therefore, on each occasion when an

anthelmintic is use& the small number of surviving worms, which are the nsistance portion

of the population, have an incxeased opportunity to contribute to the next generation. Thus,

one of the existing alleles may give a few individuais carrying this allele a survival advantage.

Over several generations, aileles conferring resistance accumulate, so that more worms in the

population survive tmitment (Craig, 1993). This ailele wili be over represented in the

offspnng. Selection for mistance removes the susceptible alleles h m the population and

evennially ody resistant deles nmain. This is consistent with the mode1 which proposes that

a susceptiile population is genetically and phemtypically heterogeneous (McKenPe, 1985)

and. import~litly, dots not requVe that mistance arises as a resuit of a new mutationai event

(Beech et al., 1994). The evolution of nsistance will proceed rapidly when the initiai

popuiation is very variable.

When dnig pressure is applied npeateâly and intensively, successive populations

move directionally towards less and less susceptiibilty, because the fnquency of the

resisbnce aüele increases through drug seleaion and the fkquency of other alleles decreases.

Overall, developmnt of cesistance is an evolutionary response.

For an antbeimintic to initiaüy be highly efficacious, the frosuency of nxistaace alleles

must be low in the population. Rwistance ailcles WU be initiaily low in a population because

those alleles are less advantageous, or at ieast confer no selective advantage, in the absence of

anthelmintic pressure. Under anthelmintic pressure. they do confes a selective advantage and

will increase in fnquency. If tk antbelmintic pressure is withdrawn for a long period, one

couid expect that the frquency of the mistance alleles would decnase if they were less

advantageous than susceptibility aileles in the absence of anthelmintic pressure. Thecefore. if a

population of worms is no longer exposed to that anthelmintic, one would expect a revenion

to susceptibiiity. However. the tate of reversion wilI be fast or slow in relation to the selective

disadvantage of the resistance dieles in the absence of the anthelmintic pressure.

P-glycoprotein was initiaiiy identifieci 20 years ago through its ability to confer

muitidnig resistance in m a m m a h tumor cells by Juliano and Ling (1976). It is a high

molecular weight (approximately 140-170kDa) polytopic membrane glycoprotein which is

located in the plasma membrane at the ceii surface consistent with its huiction as a multidnig

transport protein (Wiigham et al., 1987). It is recognjzed that the Pgpencoding genes

klong to a superfamüy of genes that encode ATP-binding membrane transport proteins

[Adenine Nuckotide Binding Cassette (ABC)] in diverse species (Childs & Ling, 1994). The

importance of Pgps for drug cesistance in organisms ranging h m bacteria to human celis has

also been found in many other organisms, such as the invertebrates Drosophila rnelanogaster

and C. ekguns (Wu et al., 199 1; L k k e et al., 1992; Gerrard et al.. 1993; Broeks et al.,

1995) and parasitic pmtozoa Leishmartia tamtoloe, Plasmodium falcipaa~n (Foote et al.,

1989; Wilson et al., 1989; ûrelktte et al., 1990, Samuelson a al., 1990; Borst & Ouellette,

1995). Entanurebu @escoteaux et al., 1992).

The cloning of Pgp or MDR cDNA has revealed that Pgps are encoded by small gene

f d e s : thete are two members in h m - M D R 1 and MDR3 (also known as MDR2), three

membem in redents-mouse d r 1 a (mdr3). &l b (mdrl) and mdr2, rat pgp 1, pgp2 (mdrl b)

and pgp3 and hamster pgpl, pgp2 and pgp3. Transfection experiments have shown that these

genes are functionally different (Gros et al., 1986% Ueda et al., 1987; Schinkel & Borst,

199 1). The humas MûR1, mouse mdrla and mdrl b, hamster pgpl and pgp2, rat pgpl and

pgp2 can cause multidnig nsistance (Chen et al., 1986; Gros et al., 1986b; Devault & Gros,

1990) while human MDR3, mouse mdr2. hamster pgp3 and rat pgp2 and pgp3 cannot

(Schinicel& Borst, 1991; Gros et ai., 1988), but the latter Pgps have an essential role in the

secretion of phosphatidylcholhe iato bile and it bas been hypothesized that they may be

phospholipid transport proteins or phospholipid flippases (Smith et al.. 1993). Amplification

or overexpression of the Pgp gene(s) has been described in some clinicaily resistant isolates,

and transfection of some parasite Pgp genes confened resistance in wild-type parasites

(OuelIette et ai., 1994).

The multidnig nsistance phenotype mdiated by Pgp cm be reversed by a large

member ofcompounds (Georges a al., 1990) calleci chemosensitizers (or reversing agents).

They are noncytotoxic agents. These compounds can bind to Pgp by coxnpeted with drugs,

thus they block the binding of cytotoxic dmgs to Pgp and enhance the cytotoxicity of drugs in

multidrug resistant ceîls. The binding of these agents will result in reversal of multidnig

resistance ( Pearce et al., 1989).

1. Structure and Lnction of P-glycoproteins

Pgp has bem found to ôe causdve of the MDR phenotype in many human cancers

and parasitic diseases by actively extruding out of celis a variety of stnicturaiîy and

functioaally unrelated hydrophobic amphipathic molecules which aie not negatively charged

(Ambudkar et al., 1995). Pgp contaias more than 120 amino acids (Chen et al., 1986).

Based on a cornputer-assisted amino acid sequence cornparisan with prokaryotic transport

proteins, a structural mode1 was pndicted. No X-ray crystal structure is available at present.

It was known that Pgp consists of two tandem dupiicated homologous halva, each half

contaias six predicted hydrophobic transmembrane domains and one inûaceiluiar hydrophilic

ATP binding site (Chen et al., 1986; Gros et al., 1986b). The ATP binding domains nside in

the cytoplasm and provide the energy by hydrolysis for Pgp to remove dnigs. It is believed to

beloag to the ATP-binding cassette superfamily. Therefore, these ATP-binding sites are very

important in exrniding drugs fiom ceils, because binding and hydmlysis art essential for

substrate transport.

P-glycoproteia bas been shown to actively transport a wide variety of hydrophobic

agents, including vinca allraloids, colchicine. antibiotics and anthracyclines, out of cells by an

ATPdependent mechanism. thus reducing their cytosolic concentration (Gottesman & Pastan,

1993; b i g e & Sharom. 1992; Nielsen & Skovsgaard, 1992). Pgp is expressecl in some

chemotherapy-mistant tumors (Pastan & Gottesman, 1991) and in many normal tissues

(Thiebaut et al., 1987; Cordon-Cardo et al., 1989; Buschman et al., 1992). in which its

physiological hction still remains unclear in spite of suggested d e s in detoxification by

excluding toxins h m normal cells and secretion of metaboiites into bile, urine and the

gastrointestinal hact (Thiebaut et al., 1987; Cordon-Caido et al., 1989; Smith et al., 1993).

MDRl Pgp is found in epitheliai ceiis of the liver, kidney, pancreas, d and large

intestines, in ceUs of the adrenal cortex, and in endothelid cells of the brain and testis, as weli

as in the placenta (Chin & Liu, 1994). The role of Pgp as an energydependent efflux pump

has been pmposeâ h m its structurai homology to the bacteriai transport protein.

Very high levels of Pgp were found in braia capillaries (Cordon-Cardo et al.. 1989;

Jette et al., 1993). where it interacts with various dmgs (Jette et al., 1995) and appears to play

a criticai role in host defease agaht ceriain lipophüic tonins (Schinkel et al., 1994).

Immunohistological snidies have suggested that Pgp is locaüÿed in the luminal membrane of

endothelial cells f o h g brain blood vessels (Sugawara et al., 1990, Tanaka et al*, 1994).

High levels of P-gp in the luminal membranes of brain capillary endothelial cells suggests that

it may play an important role in l i m i ~ g the access of anticmcer drugs to the brain (Beaulieu

et al., 1997). It may thus contribute to multidnig resistance in brain tumors by expelling dmgs

h m the endothelial ceils into the circulation in addition to extruding them fkom tumoral celis

(Henson et al., 1992) and may also contribute greatly to the properties of the blood-bnin

barrier, where blood vessels are less permeable than in other tissues such as the heart and

lungs. High expression levels of Pgps in the a h n d gland mebaut et aL, 1987; Croop et

a . , 1989) and in the endometriun of the gravid mouse utenas (Arceci et al., 1988) have

suggested involvement in transport of steroid hormones. The expression study of pgp-1 and

pgp-3 in the nematode C. ekguns shows that Pgp is tigbtly controlied and restricted to

intestinal cels. This indicates that the presence of Pgps in the digestive tract is an

evolutionarily conserved fature of these pmteins (Lincice et al., 1993). These pmteins (Pgps)

are thenfore thought to provide a protective mechanism to the body by eliminatllig exogenous

toxins or their toxh metattolites before they have a chance to exert their cytotoxic effects.

It is known that drug resistance is caused by the abiiity of Pgps to extrude drugs

against a concentration gradient, resulting in a decrease of tbe intraceiluiar dnig concentration

in contact with tk dmg target. There are two ways for Pgp to nmve substrates h m ceils

redting in demased uptake and inaeased eMux. ûne way is by binding of substrates

intracellulady to specific domains of the Pgp and tbe substrates are released on the

exttsbceiilulac si& of the membrane (resuiting in an increa9e in enlux of drugs). Amtber way

is by removing substrates b t l y h m the lipid bilayer befon they get into the ceils (nsulting

in decnased accumulation of dnigs).

In independent drug-resistant clones, multiple copies of the transfected plasmid are

associated with high levels of expression of mdr cDNA. These msults establish a dinct

nlationship between the expression of the cloned mdr cDNA and the multidnig resistant

phenotype (Gros et al., 1986a). These suggest tbat overexpression andlor amplification of

Pgp gene in mammnlian cells confer multidmg resistance.

2. Correlation of Poglycoprotein with drug resistance in parasitic

diseases

P-glycoprotein is widely represented in the animal kingdom and parasites (Ouellette et

al., 1994; Upcroft, 1994), but only a srna minoiity of these Pgps have been found in a wide

range of some parasitic protozoans selected for h g resistance. However, the hinctions of

most of these Pgps are not howa Ampüfication of Pgp-encoding genes ha9 been associated

with mistance to a specmim of hydrophilic agents in Leishmania (Ouelette et al., 1990,

Ellenberger & Beverley, 1989). Two geaes rrlaied to P-glycoprotein have been describecl:

Leishmania pgpA and mdrl gene. Lekhntaniiz pgpA is frcquently ampiiflied in mnite-

resistant mutants (ûueliette et al., 1991; Grondin et al., 1993), and trmsfection of the gene

indicated t h it was involved in nsistance to oxyanions such as arsenite and antirnonite

(Papadopoulou et aL, 1994). Sequence andysis of d r l gene in Leisimanria has show that

it is closely related to the mamnwiian P-glycopmteins and transfectioa experimeats indicated

that it was conferring resistance to dmgs included in the multidmg resistance spectnun

(Hendemn et al., 1992; Chow et aL, 1993).

Genes homologous to the mammalian mdr genes have now been identified in

Plasmodium . Amplification of Pgp-encoding genes has been associated with chloroquine and rnefloquine resistance in Plasniodium folciparunt (Foote et al., 1989; Wilson et al., 1989). In

P. fdci'nun which causes human malaria, two mdr gene homologues p-1 and pfnul2

have been cloned and sequenced (Foote et al., 1989; Wilson et al., 1989; Zaüs et of., 1993).

m r l gene t a s found to be associated with dmg resistance. Resistance was associated with

a point mutation and gene amplification (Foote et al., 1990; Cowman et al., 1994; Peel et al.,

1994; Basco et al., 1996).

Only recently have Pgp homologs been investigated in nematdes. A family of Pgp

homologs have k e n described in the nematode, C. elegans. C. eleguns is a free living soi1

nematde. It cm dhctly contact toxins that an present in the nanual environment as weli as

those released through industry and agriculture (Leonard & Belgium, 199 1). In C. eleguns,

three fidl length Pgp gene and one partial Pgp gene hornologs, pgp- 1, 2, 3 and 4, have been

cloned, sequenced and mapped to chromosomes 1, IV and X (Lhcke et al., 1992). They were

identifid in both the apical membrane of excretory al and in the apical membrane of

intestinal ceiis (Lincke et al., 1992). Pgp- I , when deleted dong with a multidrug resistance-

associated protein homologue, mrp-1, confers hypersensitivity to heavy metals (Broeks et al.,

1996) and Pgp-3, whea deleted, confers sensitivity to colchicine and chloquine (Broeks et

al., 1995). The Pgp gene family of C. elegans sbares extensive structural homology with theu

mammalian counterparts (Lincke et al., 1992).

In 1993, Sangster et al suggested severai partial genes for Pgp in parasitic nematode

H. contortus, although sequence information was missing. In 1998, Xu e~ d have identifieci

a Pgp gene in H. contortus. They showed tbat the product en& by this gene may play a

d e in iverrœctin mistance in H. contortus. ' k y have also cloned and sequenced a fbil

length of dlNA encoding a Pgp homolog h m H. cor~onics. il^ anaiysis of this seQuence

showed 6165% homology to the other multidrug mistant protein sequences, found in mice,

humans and C. eleguns. Xu et al (1998) also showed that expression of this Pgp mRNA was

higher in eggs of an IVM-selected stralli than an unselected strain of H. contortus. Merent

restriction pattern have been identifïed between unselected saains and IVM- or MOX-

selected strains through Southem blot analysis.

3. Rationale of Pgp invotved in macrocyclic lactone resistance in

nematodes

Based on the above information. the nasons for choosing Pgp gene as a candidate

gene in macmcyclic lactone nsistance in the nematode H. contortus are surnmanzed as

foilows:

a Pgp has been shown to cause multidnig nsistance in mammalian tumor ceiis

(Gottesman & Pastan, 1993) and some parasitic diseases.

b. The overexpression of a Pgp homolog gene in IVM selected strains of H. contonus

was obscrved (Xu et al., 1998).

c. A fidi length of Pgp cDNA h m H. contortus has been cloneà and sequenced (Xu

et al., t 998).

d. Differcnt restriction patterns between unselected and LVM selected saaias in H.

contortus were also found (Xu et al., 1998).

e. In vivo experiments have shown that disnaption of the mouse d r l , a P-

glycoprotein gene, leads to an hpinœat in the blwd-brain banier and to inuePsed

sensitivity to IVM in these mice (Schinkel et al., 1994). Mice with deletion of mdrla were 50-

100 times more sensitive to ivermectin than normal mice.

f. IVM cm strongly interact with the Pgp drug binding site possibly due to its high

hydrophobicity (Pouliot et al., 1997). This evidence shows that IVM is a good substrate for

P ~ P -

g. Furthemore, the multidmg resistance ~versing agent verapamii can incnase the

efficacy of IVM and MOX against a MOX selected H. contortus strain (Xu et al., 1998).

This evidence shows that Pgp may contriiute to ivennectin and possibly moxidectin

resistance in the parasitic nematode H. contortzs. The present study, therefore, is designed to

invcstigate the genetic variation of the Pgp gene in the nematode H. contortus and its

association with resistance to IVM and MOX, using molecuiar biology techniques. The study

provicies important information to ktter understand the genetic basis of drug mistance in the

parasite, which can be used as a marker for diagnosis of IVM and MOX resistance at an eariy

stage and provide scientifk iaformation for development strategies for controliing the spread

of the drug resistance. This information may help to reduce the economic losses associated

with anthelmintic resistance and prdoag the life of antheimintic dnigs.

Chapter m

Materials and Methodology

1. Experimental Design

This study consisted of the foilowing experimental techniques: DNA extraction,

Pol ymerase Chain Reaction (PCR), restriction enzyme digestions, cloning and sequencing. In

the study. we designed the specific Pgp primers to look for the genetic variation of the Pgp

gene in H. contonus. Thne strains of adult H. contortus parasites were obtained from sheep

that were infectai with each saain of the infective stage larvae: PF17, IVF17 and MOF17

respectively. ûenomic DNA was extracteci fiorn forty individuai d e w o m of each svain

and PCR reactiom were perfolmcd to amplify an approximaîely 876 bp long fhgment by

using the specific Pgp primen. The PCR products were then digested with four different

restriction enzymes: DdeI, AluI, Hinfl and RsaI. The restriction patterns of four different

enzymes were observed and alleles of the t h strains at the Pgp locus were analyzed. The

alleles which are associated with susceptibility and IVM- and MOX-cesistance were detected

using analysis of fiequencies of the deles. The alleles that are related to IVM- and MOX-

resistance, or susceph'bility were then cloned and sequenced h m aii three strains of the

parasites.

II. Materials and Methods

1, Parasitic nematode strains

'Ihree laboratory seains of H. contortus were used in this study. AU of them were

derived h m the same parent isolate (it means that infective larvae 6 3 ) of H. contoriur were

originally sensitive to both i v emt in and moxidectin). The concentrations of individual

compounds were selected on the basis of adueving an efficacy of 80-958 in order to dow

sufncient egg pmduction for cultivation of U for subsequent passages. One strain is the

Cyanamid uaselected süain (PF17) which was obtained after 17 passages in sheep without

any anthelmintic exposure at Cyanami& one is Cyanamid ivennectin selccted strain (IVF17)

which was exposed to ivemectin for 17 passages. and another one is Cyanamid moxidectin

selected strain (MOF17) which was exposed to moxidectin for 17 passages of H. contortus.

PF17, M l 7 and MOF17 were passaged in p d e l . The dmg dosages in the fuial generation

were 0.15 mgkg for ïVM and 0.015 mgkg for MOX. At the twelfth generation, ED95 was

calcdated for each strain. It was 0.0 1 125 mglkg body weight for iVM and 0.00179 mgkg

body weight for MOX in PF12. The ED95 for IVFlZ was 0.10874 mg IVMkg body

weight, and for MOF12 was 0.00949 mg MOXkg body weight (Wang et al., 1995).

2. Prepantion of parasites

The larvæ of the above three strains of H. contortus were provided by Fort Dodge

Animal Health, Rinceton, NJ, USA. Sheep were inf'ted with 10,000 thkd infective stage

Iarvae. After about a month, the shap were Wed and duit maie worms were coiIected h m

the abmasum. W o m were then wpshed with steriîe phosphate-buffereâ saline (PBS) at

370C severai tims and were ready for extraction of DNA. if DNA was not isolateci

immediately after worms were collected, the worms were fkozen in liquid nitmgen and stored

at -800C until used.

3. Extraction of genomic DNA

H. contortus genomic DNA was extracted from fnsh or b z e n individual adult male

w o m by using standard procedures. Only adult male worms were used to isolate DNA to

avoid the possibility of DNA contamination from fertilized eggs or sperm present in fernale

samples. Worms wen washed with clean water, male worms were isolated under the

microscope, and then tnasferred into a 1.5 ml eppendod tube that containhg 200 pl STE

(0.1 M NaCl, 10 mM Tris-Cl, pH 8.0, 1.0 mM EDTA, pH 8.0). 10 pi p-mercaptoethanol,

10 pl of 20% SDS and 2.0 pi of protease K (20 mghl) , incubated at 370C for 2 hours to

completely digest the wom (Beech et al., 1994). DNA was extractcd with

phenoVchloroformli~amyl alcohol (2S:W 1) once and then precipitated with isopropanol:

ammonium acetate (2:l) following an addition of copocipitant with 4 pl of 2.5 rngiml linear

acrylamide (Gaillard & Sartrauss, 1990). The DNA pellet was washed with 70% alcohol once,

then air dned and msuspended in 100 pi TE buffer ( 10 mM Tris-HCI, pH 8.0, 1.0 mM

EDTA, pH 8.0) and s t o d at -200C.

4. Sequences of the primers useà in PCR ampiiîication

PCR ceactions wen perfotmed to amplify a fragment appmximately 876 bp long

using specinc primrs designed fkcm the cDNA sequence of the H. c o n t o ~ Pgp gene. The

sense primer PGP2S (S'GAAATGACTCAAGCACAAG3') (&signai by Dr. M. Xu and

madined by Biîi BiacLhall in our lab) and the antiscnse p r h r MX-D

(S'AGACAAAGACAT'ïCAGAG3') (designed by Dr. M. Xu) and PGPlA (antisense primer

S'AGTGAACTAAGATGûGGTY) were used in the PCR reactions. Initial DNA

ampiification using PGP2S and MX-D p h e r s on DNA h m individual worms was not

efficient enough for restriction enzyme anaiysis. Thenfore, two rounds of K R action was

set up to obtain sufficient product for restriction emym digestion. nie fmt PCR reaction

was set up by using antisense primer PGPlA located M bp downstream fkom MX-D on the

Pgp cDNA with ffiPîS to produce a 200 bp longer fiagrnent ihaa that from the primes

PGP2S and MX-D, the finit PCR product was then used as templaîe to be reamplified with

primers ffiP2S and MX-D for a second round of PCR reaction. The PCR product obtained

with PGP2S and MX-D from pnomic DNA was approxllnately 876 bp in length.

5. Polymerase Chain Reaction (PCR) amplif'ication

The genomic DNA (approximately 2.0 pg) was used to ampiify an approximately 876

bp fiagrnent by PCR in the £inal volume of25 pl in the pcesence of 10X Taq buffer 2.5 pi, 25

m M MgC12 2.5 pi, 20 pM primrs PGPZS and MX-D 1.0. pi respectiveiy, Taq poiymerase 1

Unit and H20 up to 25 pl. The PCR amplification reaction was performed on an MJ

Rcsearch, Inc. PCR-100 Programmable Thermal Controller. Amplification conditions for the

tirst PCR reaction were 950C for 3 min 55 sec to &nature the tunplate DNA completely,

followed by 40 cycles of 950C for 10 sec, 53% for 20 sec, 720C for 1 min 30 sec. The final

extension temperature was 72% fot 5 min. 1 .O pi (appmximately 1 .O ng) of gewmic DNA

h m each individuai w o m was amplified in a total volume of 25 pl. The second PCR

amplification condition was similar to tbe first. e x a p 30 cycles were used instead of 40

cycles. After amplifîcatim and before restriction enzyme digestion, the ECR products wen

visualized on a 1.0% agarose gel containhg 0.5 pg/d of ethidium bromide with a base paÜ.

size marker (made by our own lab) un&r W illumination.

6. Restriction enzyme digestion and identification of allele patterns

The PCR product from genomic DNA was digested with four ciiffurnt restriction

enzymes-DdeI, AluI, HinfI and &a1 which were purchased from Romega The ftagments of

restriction e n y m digestion were separated by 6% non-denaturing polyacrylamide gel

(mixture of 45 ml of 6% acrylamide (0.5X TBE). 15 pi iI and 400 pl of 10%

ammonium persulphate) electrophoresis, where they Mgrare accordhg to size. The DNA

fragments were stained directly with 0.5 pg/d ethidiun bromide, and visualized under

ultraviolet light. The ideatity of the different alleles were deduced, where possible. by

ideniifying a homozygote, where the sum of the fragment sizes equal the size of the original

PCR fiagment, or by identifying those bands in heteroygotes which consistently occur

together (Beech et al., 1994).

7. Allele frequencies and statistical analysis

For the purpose of this study, the fiequency of an dele is caiculated shply as its

percentage of the total of ail alleles detected m g the samples examined. A Chi-square test

was used to analyze whether hquencies of alleies between TVM- and MOX-selecteâ strains,

beniveen iVM selected and unselected strains and benmen MOX selected and unselected

sadins were significantiy different. AUele classes which number less than 5 were pooied to

mske sure that there was a minimum expected numberof at least 5 pu class per saain.

8. Cloning of the Pgp alleles

A. Purification of DNA

Approximately 876 bp of bigment from genomic DNAs which contain the alleles that

are associated with IVM or MOX resistance and susceptibility were amplified by using the

same primers as we used k f o n (PGPZS and MX-D) in a large volume of PCR naction (50

or 100 p). The 50 or 100 of PCR product was loaded on a (hiclc 1% agarose gel, and the

band was excised under the UV iight and p u i f i d by using the Geneclean Kit (BIOICAN

Scientific), or DNA was purified with the 1-10 pl white plugged pipet tips by cutting the

bands into small pieces, putting the pieces into white plugged pipet tips, and placing the tips

into 1.5 ml tubes, then spinnhg at high speed for 30 sec. The DNA in the liquid at the bottom

of the tube was coliected and stored at -200C.

B. Ligation

The purüied DNA from abon was Ligated into the pGEM-T vector from the ligation

kit of bmega. In the ligation ceaction, the 5.0 pi of purified DNA was mixed with 0.5 @ of

pGEM-T vector, 1.0 pi of 10X Lgase b s e r and 1.0 pi of Ligase (3 U/pi), then H20 up to 10

pi was added and the reaction was ligated at 40C overnight.

C. Translormation

The resuiting recombinant plasmid h m the Ligation was then used for transformation

by following the protocol below. One tube of comptent cells (E. coli, prepared by ouiselves)

was thawed siowly on ice. 5.0 pi of ligwd p M d was mixed with 100 pi of competent

cells by gentie flicking. They are left on ice for 20 min, then heatd in a 420C water bath for

exactly 90 sec.; immdiotely replacing tbem on ice for 2 min. Traosformed ceb were

subsequentiy transfemd into a tube waich contained 3 4 times SOB (or SOC) mdium and

shaken at 370C for one hou. Appmximately 2ûû pi of cell culhin was mixed with 10 of

X-gai, 4.0 pi of D'TG and 2.0 pl of ampicillin (100 mgml). mixed gentiy and spread on the

LB Agar plate, and thca incubateci at 370C ovemight.

Twelve single white colonies of each sample fiom the above plates were picked up

and grown each in 5 ml of SB medium at 370C overnight in a shaker. The clones which

contained recombinant plasniiàs were then ideatüied by the miai-preparation. nie SB culture

was centriffiged at 2000 rpm for 10 min, and the supernatant was removed. The pellet was

resuspended with 200 pi of GTE. The resuspended solution was then transfemd Uito an new

Eppendorf tube; 400 pl of fnsh NaOH-SDS (M pl of 20% SDS, 910 @ of Hz0 and 40 pl of

5 N NaOH) were added, vortexed quicldy and placed on ice about 5 min. Then 300 pl of 7.5

M ammonium-=tate was added, and the mixture vortexed and spun for 2 min. The liquid

was üansfemd into a tube containing 5 0 pi of phuio~chlorofomirisoarnyl alcohol(25:24: 1).

vortexed and spun for 2 min. The top layer was subsequently transferred into a tube

containing the 600 pi of isopmponai, vortexed and spun for 10 min. The pellet was dried by

air and resuspended in the 50 pI of TE. 1 .O pl of the above plasmid DNA was nui on the 1%

Agarose gel with one plamid DNA which had no insert obtained h m the blue colony as a

negative control and one s k marker. The positive plamid DNA contalliing an insert bas a

largu size than the negative one dose. if the plasmid DNA was contaminated by RNA, L.0 pi

of Nase was added into each tube and digested at 370C for 1-2 hours.

E. Identification of clones

To identify the clones wbich contawd the alleles associated with iVM resistarre and

susceptib'ility, a PCR reaction was pdormed by using plasmid DNA which had a correct size

on the Agarose gel. 10X Taq reaction bufkr 5.0 pl, 2 mM dNTPs 5.0 pl, 25 mM MgCl?, 20

pM primen POPZS and MX-D 1.0 pl respeaively, Taq polymrase (10 CT) O. 1 pl, Plasmid

DNA 0.2 wl, Hz0 up to 50 pl. PCR amplification conditions w e n 950C for 5 min to

completely denatm the template DNA, foliowed by 35 cycles of 95oC for 15 sec, 530C for

20 sec, 720C for 1 min 30 sec. The final extension temperature was 720C for 5 min.

Restriction e n y m digestion reactions were then applied by using the same method . explained above to identQ the correct aiieles. Accorduig to the restriction pattern, clones rhai

coatained the alleles which associated with suscephiility and I V ' and MOX resistance were

found.

9. Sequencing of the alleks

The clones which contained the alleles of intexest were sequenced by the Sheldon

Biotech Center of McGiii University. A small part of the clones were sequenced in our lab.

Sequencing reactions were p e d o d by using the cycle sequencing pGEM Kit h m

Romega The following pmtocol was used: 2.0 jU of dlddNTP (A, C, O, T), 1 .O-2.0 pl of

DNA. 1 .O pf of [aJsS]dATP (10 pcilpl), 1.5 pl of 2 ph4 universal foward primer PGP2S

and reverse primer MX-D nspectively, 5.0 pi 5X sequencing buffer, 1 .O pl sequencing grade

Taq DNA po1ymefa~e, and Hz0 up to 16 pi of totai volume.

min; 950C. 30 sec; 42*C, 30 sec; 700C, 1 min. Aftex completion of the ceaction, 3.0 pi of

stop solution was added and tubes were beaied at 700C for anothcr 2 min or heated before

1-g on the sequenciag gel. Each sample was r u on a short gel and a long gel with 1X

TBE b a e r under 200 volts. The film was exposed for 3-5 days before reading the

sequeaces.

Chapter IV

Results of Evaluation of Restriction Fragment

Length Polymorphism

1. Restriction enzyme digestion analysis

Genetic variation at the Pgp locus was investigated in IVM- and MOX-selected and

unselected strains. Forty individual adult male w o m from each suain were surveyed. The

specific priwrs were designcd accordhg to the PGP-A cDNA sequence (Xu et al., 1998) for

PCR amplification of an approximately 876 bp Aagment fiom individual genomic DNA. The

position of the pprimers and the ampiifîed fkagment are shown in Figure 1. Insufficient PCR

product was obtaiaed to do restriction