UNIVERSITÉ FRANÇOIS – RABELAIS DE TOURS

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UNIVERSITÉ FRANÇOIS – RABELAIS DE TOURS

ÉCOLE DOCTORALE SSBCV Equipe multirésistance et pouvoir pathogène des nématodes

THÈSE présentée par :

Caroline CHYLINSKI

soutenue le : 19 septembre 2014

pour obtenir le grade de : Docteur de l’Université François – Rabelais de Tours

Discipline/ Spécialité : Science de la vie et de la santé

Qu’est-ce qui fait le succès des nématodes gastro-intestinaux chez leur

hôte ? Etude du rôle des nématodes, des moutons et des éleveurs

What makes a gastrointestinal nematode successful in their sheep host?

Exploring the role of the nematode, the sheep host and the farmer

THÈSE dirigée par :

M CABARET Jacques Directeur de recherche, INRA Val de Loire - Tours

Co-encadré par :

Mme BLANCHARD-LETORT Alexandra Chargé de recherche INRA Val de Loire - Tours

RAPPORTEURS :

Mme MARTIN Coralie Chargé de recherche, HDR, Muséum national d’histoire naturelle Paris M STEAR Mike Professeur, Université Glasgow Ecole vétérinare

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JURY :

M JACQUIET Philippe Professeur, Ecole national vétérinaire de Toulouse

Mme PRUNIER Arnelle Directeur de recherche, INRA Rennes

M CHANDENIER Jacques Professeur, Faculté de Médecine université de François Rabelais

Tours

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Dedicated to anyone who takes the time to read this thesis

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Acknowledgements Cheers everyone!

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Table of contents

Acknowledgments……………………………………………………………………………..4

Abstract………………………………………………………………………………………..7

Résumés de these français……………………………………………………………………..8

Introduction…………………………………………………………………………………...33

Setting the scene……………………………………………………………………………...36

Gastrointestinal nematode

1. Introduction……………………………………………………………………………43

2. Parasitic gastrointestinal nematodes coping with chemotherapy, resistant hosts and

unfavourable climatic environments: An experimental evaluation……………………50

3. Resistant sheep select for increased fitness in their parasitic nematodes

(Teladorsagia circumcincta): experimental evidence…………………………………76

4. Desiccation tolerance of gastro-intestinal nematode third stage larvae: exploring the

effects on survival and fitness………………………………………………………….92

5. Storage of gastrointestinal nematode infective larvae for species preservation and

experimental infection………………………………………………………………..110

6. Discussion…………………………………………………………………………….123

Sheep host

1. Introduction………………………………………………………………………….128

2. Exploring immune gene expression relative to sheep resistance against Haemonchus

contortus: A story of sex? ………………………………………………………….132

3. Disentangling the relative contribution of the parasite and the host to consequences of

Haemonchus contortus infection in sheep…………………………………………..148

4. Do gastrointestinal nematode infections induce pain in their sheep host?.................159

5. Discussion…………………………………………………………………………...178

Farmer

1. Introduction………………………………………………………………………….182

2. Trade-off between farmers’ autonomy and control of sheep parasitic gastro-intestinal

nematodes in conventional and organic farms………………………………………184

3. Exploring the limitations of pathophysiological indicators used for targeted selective

treatment in sheep experimentally infected with Haemonchus contortus…………..199

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4. Discussion…………………………………………………………………………225

General Discussion………………………………………………………………………..227

References…………………………………………………………………………………229

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Abstract The success of gastrointestinal nematodes in their sheep hosts is so extensive that they present one

of the leading threats to ruminant health and production throughout the globe. This thesis research

identified three key factors which influence their success including the gastrointestinal nematode

biology, the sheep host protective response and the farmers control decisions. Using Haemonchus

contortus as a model species, we demonstrated that the success of GIN biology is aided by their

capacity to overcome numerous selective pressures that target both parasitic and free-living stages

in their life cycle. This was achieved by amplifying life-history traits following challenge to recoup any

costs in survival and reproduction. In turn, high levels of fitness were maintained and they remained

stable in the face of numerous selective pressures. Sheep have the capacity to exert almost perfect

control over GIN success by blocking their life cycle through via protective responses. Harnessing this

capacity is of great interest to the future of GIN control. This research has advanced current

understanding of sheep resistance by identifying sheep sex as an integral limiting factor to the

expression of sheep resistance. A novel biomarker to predict sheep resistance against H. contortus

was identified in sheep body temperature which could be adapted to aid selective breeding and

monitoring of sheep flock health. The relative costs of resistance and susceptibility were explored

with pathophysiological indicators and findings support any negative effects of resistance are far

outweighed by the costs of an H. contortus infection. With the right tools and knowledge, farmers

are also able to greatly reduce the success of gastrointestinal nematodes within their flock. However

studies in this work reveal a breakdown in knowledge dissemination between science and farmers is

preventing optimum control over gastrointestinal success. The inter-disciplinary approach used here

to explore gastrointestinal nematodes has highlighted the inter-dependent relationship between the

worm, the sheep host and the farmer to enabling and/or preventing their success.

Keywords: gastrointestinal nematode, sheep resistance, farmer, success, fitness, life history

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RESUMES FRANCAIS

Qu’est-ce qui fait le succès des nématodes gastro-intestinaux chez leur hôte ?

Etude du rôle des nématodes, des moutons et des éleveurs.

Introduction

Le titre de la thèse concernant le succès des nématodes peut sembler extremement vaste. Notre

projet était pourtant bien d’avoir une vue d’ensemble de l’ensemble des acteurs, à savoir les

nématodes parasites du tube digestif mais également l’hôte et certains aspects de sa réponse, et

enfin l’éleveur qui est celui qui décide du mode d’élevage et des traitements antiparasitaires. Nous

avons essentiellement travaillé en conditions expérimentales et sur une espèce de ces strongles. En

ce qui concerne les traits de vie des nématodes plusieurs grands phénomènes conditionnent leur

succès : a) leur capacité assez variable selon les isolats à infester un hôte, b) l’interaction entre

résistance aux antiparasitaires et cette capacité à infester, c) enfin leur aptitude à survivre aux stress

climatiques au cours de leur phase non-parasitaire. Cet ensemble de mesures, sur des isolats de

nématodes bien caractérisés n’a jamais été réalisée et est donc nécessaire pour mieux comprendre

les raisons de ce succès des nématodes gastro-intestinaux à l’échelle de la planète. Le rôle de l’hôte,

tant lors de sélection génétique classique ou assistée par marqueurs, ses réactions de protection

innée ou immune ont été largement étudiées. Nous avons voulu savoir si une co-évolution existait

entre acquisition de résistance des ovins et capacité des nématodes à infester les ovins. Enfin les

ovins ont des aptitudes à résister à l’infestation par ces nématodes de façon variable, tant au niveau

individuel que selon les races. Cette résistance de type génétique s’exprimera de manière plus ou

moins efficace et nous avons voulu accéder à cette information en comparant au sein d’une race

considérée comme résistante, les performances de protection selon le sexe. Nous avons également

tenté de visualiser l’expression différentielle de gènes impliqués dans la résistance à l’infestation.

Nous avons aussi tenté pour la première fois de rechercher une relation entre la température

corporelle des ovins (indicateur du niveau de métabolisme individuel) et leur niveau d’infestation. En

regard des réactions pathophysiologiques connues, les infestations semblent constituer une atteinte

au bien-être de l’animal et un frein à sa productivité ; pour la première fois nous avons désiré savoir

si ces infestations causent une douleur réelle chez l’ovin, à l’instar de ce qui est mentionné, assez

rarement, chez l’homme. L’éleveur enfin, constitue une étape fondamentale en ce qui concerne le

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succès des nématodes : il décidera des éléments à prendre en compte pour les traitements

antiparasitaires, mais aussi de nombreux autres modes de gestion des ovins qui auront des

répercussions sur l’infestation. Nous prendrons pour exemple les traitements stratégiques ciblés

(une partie seulement du troupeau est traitée, selon des critères proposés par la recherche, en

particulier un indice d’anémie pour H. contortus. Notre objectif sera de vérifier en conditions

contrôlées quelle est sa valeur comme prédicteur précoce pour un orienter vers les animaux qui

nécessitent un traitement. Dans un ultime travail nous tenterons d’estimer l’importance des

pratiques d’éleveurs dans des domaines proches de la gestion du parasitisme (de l’alimentation au

traitement), sous l’angle de l’autonomie de l’éleveur. L’autonomie est un trait du métier d’éleveur

qui permet d’appréhender si des propositions biotechniques ont des possibilités d’être utilisée ou

non.

Ma thèse considère donc trois ensembles dans le succès des parasites gastro-intestinaux : le

parasite lui-même, l’hôte, et l’éleveur. De très nombreux travaux ont été réalisés sur le

deuxième point, quelques uns sur le premier, et très peu sur le dernier. Le sujet est

extrêmement vaste et les point un et deux sont recentrés sur une espèce de strongle

(Haemonchus contortus). Nous avons recherché, en particulier sur les points 1 et 2, ce qui

pouvait nécessiter des études complémentaires pour comprendre le succès des nématodes

gastro-intestinaux. Je ne présente dans cette version Française que les articles ou projets

d’articles avec les principaux résultats discutés. Le nombre d’article est élevé et la matière

est constituée par mes propres travaux mais aussi par des travaux réalisés en collaboration

avec d’autres unités de l’INRA mais aussi d’autres organismes.

Parasite / « point du vue » du parasite

1) S’adapter à la résistance génétique de l'hôte

2) S’’adapter aux anthelminthiques ET à la résistance de l’hôte

3) S’adapter à des conditions climatiques extrêmes

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Des moutons résistants qui sélectionnent leurs nématodes parasites (Teladorsagia

circumcincta) pour leur meilleure fitness : preuves expérimentales

Contexte et objectifs :

La sélection génétique de moutons résistants aux nématodes gastro-intestinaux offre une possibilité

prometteuse pour le contrôle de ces parasites en élevage. Cependant, la résistance hôte est aussi

considérée comme une pression de sélection définissant l'évolution des nématodes gastro-

intestinaux. Le modèle Teladorsagia circumcincta a été utilisé dans cette étude comme espèce

modèle pour explorer comment les traits d'histoire de vie (établissement, fécondité, développement

des œufs en larves infestantes) et la fitness globale des nématodes gastro-intestinaux sont affectés

après plusieurs générations de sélection sur moutons résistants en comparaisons avec des passages

sur moutons sensibles. Nous avons ensuite examiné l'impact de la sélection de lignées sur mouton

résistant sur la fitness du parasite lors d'un retour sur mouton sensible.

Matériel et méthodes :

Moutons résistants : Romanov (obtenus sur une sélection divergente sur 3 générations)

Moutons sensibles : ile de France

Passage d'une population de T. circumcincta sur 3 générations sur moutons résistants ou sensibles.

Les deux populations issues des ces passages sont nommées TcirS et TcirR.

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La fitness et les traits d'histoire de vie sont suivis à chaque passage pour les deux lignées R/S pour

comparer l'effet du fond génétique hôte résistant/sensible sur l'évolution des populations de T.

circumcincta.

Est-ce que la fitness est modifiée entre les deux lignées R/S ?

Pour cela, après sélection, les lignées sont passées sur mouton sensible ile de France traités aux

corticoïdes pour homogénéiser la réponse de l'hôte à l'infection. Les paramètres des lignées de

nématodes (fitness et traits d'histoire de vie) ont été suivis.

Résultats

Les résultats montrent que les passages sur mouton résistant réduisent significativement la fitness

du parasite en impactant négativement les traits d'histoire de vie examinés, incluant les stades libres

du parasite. Lorsque ces parasites sélectionnés sont passés sur mouton sensible, leur fitness est

considérablement augmentée. Ceci démontre que les parasites se sont adaptés très rapidement, en

seulement 3 générations de sélection. Les moutons résistants sélectionnent des parasites de T.

circumcincta avec de meilleures capacités de survie que ce soit au niveau des stades parasitaires

(taux d'installation) ou des stades libres (taux de développement des œufs en L3). Par contre il n'y a

pas d'impact sur la fécondité. Par conséquent, l'adaptation des nématodes gastro-intestinaux aux

hôtes résistants, pourrait représenter une menace forte pour les races de mouton plus sensibles au

parasitisme et surtout présentent un potentiel de transmission augmenté liée directement à

l'augmentation de leur fitness.

2) Évolution de la fitness d’H. contortus

Le passage en en série d’H. contortus chez des ovins sensibles et résistants ne modifie pas sa

valeur adaptative

Contexte et objectifs

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Le nématode T. circumcincta, sélectionné sur l’excrétion des œufs dans les fèces a montré

son aptitude à s’adapter à des hôtes résistants. Inversement une littérature abondante

fondée sur le même type de sélection aboutit à l’absence d’adaptation du parasite H.

contortus. Nous avons désiré vérifier si cette absence d’adaptation se mesurait sur la fitness

(valeur adaptative) du parasite, qui est le critère évolutif (contrairement à un trait de vie

isolé) qui peut être changé au cours d’une sélection.

Matériels et méthodes

Dix ovins de la race Martinik Blackbelly (population de Guadeloupe) ont été soumis à une

infestation de 10000 larves infestante L3 (4 passages) et ensuite 5000 L3 pour les deux

derniers passages. Les larves obtenues pour chaque animal resservaient pour sa propre

infestation. Lorsque le nombre produit était insuffisant pour assurer une infestation de

10000 ou 5000 L3, des larves d’un autre animal peu producteur étaient utilisées. Les

animaux extrêmes en sixième génération on servi a constituer les lignée résistante (chez des

animaux à faible production) et sensibles (chez des animaux à forte production. Une phase

d’amplification des deux lignées a été réalisée sur des Berrichons du Cher sensibles. Les

larves obtenues ont ensuite été évaluées chez des races résistante (10 MBB) ou sensible (10

Romane).

Résultats

La variabilité des animaux utilisés pour la sélection a été importante et la production de

larves infestantes au cours de la période va de 1 à 30. Aucune différence significative n’est

remarquée chez les animaux ayant servis à l’amplification. Il en est de même chez les

animaux tests.

Discussion

La fitness n’est pas modifiée. Ce résultat est cohérent avec les études précédentes sur les

traits de vie chez H. contortus. Il est par contradictoire avec ce que nous savons pour T.

circumcincta. Ces deux trichostrongles ont des traits de vie très différents, en particulier leur

fertilité : H. contortus pond 10 à 15 fois plus d’œufs que T. cirumcincta. Les variations de

fitness entre animaux sont très fortes chez H. contortus. Pour cette espèce il semblerait que

le devenir des populations repose sur quelques infestations très réussies alors que chez T.

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circumcincta, la réussite est plutôt populationnelle. Cette variabilité chez H. contortus ne

s’est peut-être pas exprimée chez les animaux que nous avons infestés en test, car leur

fréquence est sans doute faible.

3) Anhydrobiose

Tolérance à la dessiccation des larves infestantes de nématodes gastro-intestinaux:

exploration des effets sur la survie et la fitness

Article publié dans Parasitolgy Research (2014)


Les larves L3 des nématodes gastro-intestinaux sont les larves infestantes libres qui sont ingérées sur

la pâture par les animaux hôtes. Ces larves ont la capacité de tolérer des conditions climatiques

extrêmes comme la dessiccation mais peu de choses sont connues sur les conséquences de cet état

d'anhydrobiose transitoire sur la fitness du parasite.

Cette étude avait deux objectifs :

1- explorer l'effet de la dessiccation des larves L3 d'Haemonchus contortus sur leur fitness absolue

2- évaluer la capacité de sortie d'anhydrobiose des larves L3 en fonction du temps de dessiccation et

de l'âge des larves chez différentes espèces de nématodes gastro-intestinaux (H. contortus,

Teladorsagia circumcincta, Trichostrongylus colubriformis).

Matériel et Méthodes

Expérimentation 1 : effet de la dessiccation sur la fitness des larves L3 d'H. contortus

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9 agneaux de race ile de France (sensible aux infections à NGI), âgés de 3 mois, ont été inoculés per

os avec 10000 larves L3 d'H. contortus isolat Weybridge âgées de 6 mois.

Les larves témoins ont été inoculées directement. Les larves testées ont été déposées sur une lame

de verre pendant 24h à température ambiante pour obtenir un dessèchement total. Ces larves ont

été réhydratées avec de l'eau pendant 3h avant d'être inoculées aux animaux.

Les matières fécales ont été récoltées pour la réalisation d'un suivi coproscopique de l'excrétion des

œufs à J+15, 20 (période prépatente) et J+ 21, 22, 23, 26, 28, 29 et 33.

Les matières fécales récoltées à J+28, 29 et 33 ont été utilisées pour la réalisation de coprocultures

(évaluation du taux de développement des œufs jusqu'au stade infestant L3).

A l'autopsie (J+33), les vers contenus dans la caillette (lieu d'installation d'H. contortus) ont été

récoltés et dénombrés. La recherche de stade L4 a également été réalisée par incubation des

caillettes à 37°C pendant 4 heures pour mettre en évidence un éventuel retard de développement le

cas échéant.

La fécondité, correspondant au nombre total d'œufs produits/jour/femelle, a été évaluée à J+33.

La fitness absolue a été calculée en multipliant le taux de développement des œufs en larves par la

quantité de matière fécale émise par jour (J+28 et J+29), divisé par le nombre de larves inoculées

(10000).

Le succès d'installation des larves desséchées a été déterminé pour évaluer la capacité des larves à

faire un cycle complet et une nouvelle génération et comparé aux mêmes paramètres dans le cas de

larves contrôles.

Les paramètres mesurés : établissement, fécondité, développement larvaire, pathogénicité

(hématocrite)

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Expérimentation 2 : effet de différents temps de dessiccation et de l'âge des larves sur leur survie,

chez différentes espèces de GIN (H. contortus, Teladorsagia circumcincta, Trichostrongylus

colubriformis).

4 isolats ont été utilisés pour l'espèce H. contortus, 2 isolats pour T. colubriformis, 3 isolats pour T.

circumcincta.

Les larves utilisées dans cette étude ont été séparées en deux groupes :

Jeunes : extraites moins de six mois avant l'expérimentation

Âgées : extraites 6 à 12 mois avant l'étude

Les larves L3 ont été incubées 24h à températures ambiantes avant l'expérimentation (conservation

au froid). Cinq fois 10µL de chaque solution contenant les larves ont été déposées sur une lame de

verre (environ 20 L3/goutte). Le nombre exact de larve par goutte a été compté. Les lames ont

ensuite été placées dans une boîte en plastique contenant un papier humide pendant 1h, 24h, 1

semaine et 1 mois. A près dessiccation, les larves ont été réhydratées avec 10µL d'eau. Après 30 min,

les larves L3 ayant survécu ont été dénombrées.

Résultats

Expérimentation 1 :

Nous avons montré au cours de cette étude que la dessiccation affecte de manière significative la

survie des larves L3 avant l'infestation. Par contre, les larves qui survivent, lorsqu'elles sont inoculées

à un mouton, présentent une fitness normale. L'état d'anhydrobiose affecte de façon positive la

fécondité des femelles 58,6 œufs/gMF/femelle/jour contre 30,3 œufs/gMF/femelle/jour pour les

contrôles. Enfin, les moutons ayant reçu des larves desséchées puis réhydratées présentent une

anémie plus importante.

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Expérimentation 2 :

Les larves infestantes d'aucune des espèces testées ne sont capables de survivre à une dessiccation

d'un mois. Une diminution de la survie est observée des la première heure de desséchement et

diminue avec le temps. Après une semaine, les larves de T. colubriformis sont celles qui survivent le

mieux (40%), celles de T. circumcincta moyennement (22%) et celles d' H. contortus survivent très

mal (2%).

La plus grande faculté de survie a été mise en évidence pour T. circumcincta suivi de T. colubriformis

puis H. contortus. Pour chaque espèce, des différences significatives ont été observées en fonction

de l'âge des larves : les larves les plus anciennes sont celles qui survivent le moins au processus

d’anydrobiose.

4) Fécondité densité-dépendante

5) Age des larves infestantes

Stockage des larves infestantes de nématodes gastro-intestinaux pour la conservation des espèces et

les infestations expérimentales

Article à soumettre à Parasitology Research


Les techniques pour preserver les larves infestantes de nematodes gastro-intestinaux sont d'une

importance considérables pour la conservation des espèces rares et pour maintenir une source

stable de parasites pour la réalisation d'infestations expérimentales.

Dans cette étude, nous avons listé les avantages et les inconvénients des deux techniques principales

de cryopréservation et de conservation au froid en comparant comment ces deux techniques

influencent les paramètres de l'infestation (firness et trais d'histoire de vie) en fonction du temps

chez le nématode gastro-intestinal Haemonchus contortus.

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Matériel et méthodes

- Moutons

La comparaison des paramètres d'infestation entre les larves cryogénisées et les larves conservées au

froid a été réalisée sur des agneaux de race ile de France âgés de 3 mois.

La comparaison des différents temps de conservation des larves a été réalisée sur 15 béliers de race

Romane âgés de 9 mois.

Les deux races sont sensibles aux infestations à H. contortus.

- Cryopréservation des larves L3 :

Les larves infestantes, après avoir été débarrassées de leur cuticule, sont lavées 4 fois dans du PBS

10X (pH 7,2). Puis elles sont reprises dans du PBS 1X pour la cryopréservation. Celle-ci se déroule par

diminution graduelle de 1,4°C/min dans de l'azote gazeux durant 30 min. Les larves sont ensuite

plongées dans l'azote liquide.

Avant utilisation, les larves sont décongelées et diluées dans du PBS 1X.

- Réfrigération des larves

Après coproculture, les larves sont passées en Baerman puis conservée en boîte de culture dans de

l'eau, à 4°C. Les larves sont conservées au minimum 30 jours avant d'être utilisées. Avant infestation,

les larves sont incubées 24h à température ambiante puis repassées en Baerman avant d'être

inoculées (de manière à séparées les larves vivantes des mortes).

-Dose de larves et isolates d'H. contortus

Tous les moutons sont infestés per os avec une dose de 10000 L3.

Pour l'étude visant à comparer l'installation et la fécondité des nematodes cryopréservés versus

conservés à 4°C, nous avons utilisé deux isolats : ISE, sensible aux trois familles majeurs

d'anthleminthiques, et KOK (Kokstadt), un isolat résistant à ces trois même familles.

Pour l'évaluation de l'impact du temps de conservation sur la fitness d'H. contortus, nous avons

utilisé les isolats ISE et ISE-BB (isolat sensible ISE passé sur mouton résistant Martinik Black Belly).

-Mesure des paramètres de l'infestation

Un suivi coproscopique a été réalisé dans chaque expérimentation : à J+28 et 35 pour la comparaison

larves cryogénisées versus conservées au froid ; à J0 puis J+ 21, 24, 28, 32 et 35 pour l'évaluation de

la fitness en fonction du temps de conservation des larves.

Les adultes présents dans la caillette à l'autopsie ont été dénombrés pour évaluer le taux

d'installation. Les caillettes ont été ensuite placées à 37°C pendant 4h pour isoler d'éventuels stades

L4 dans le cas d'un retard dans le cycle de développement certaines larves.

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La fécondité correspond à la quantité d'œufs excrétés déterminée au dernier comptage divisé par le

nombre de femelles comptées à l'autopsie.

Le taux de développement des œufs en L3 a été évalué à J+28, 32 et 35 par mise en culture de 5X5g

de matière fécale à 23°C, 70% humidité pendant 10 jours.

Résultats

La cryopréservation des larves infestantes induit une diminution de leur capacité d'installation par

rapport aux larves conservées au froid. En revanche, la cryopréservation induit une augmentation de

la fécondité des nématodes installés.

La fitness des nématodes n'est pas affectée par une conservation au froid même après 16 mois. Par

contre la durée de conservation affecte la capacité d'installation suivie d'une augmentation de la

fécondité des larves installées.

Cette étude montre clairement des biais qui peuvent être induits par le mode et la durée de

conservation des larves dans les expérimentations.

Hôte / « point de vue » de l'hôte

Différents aspects de la résistance

1) Effet sexe

Exploration de l'expression des gènes de l'immunité relative à la résistance des moutons à

Haemonchus contortus: une histoire de sexe?

Contexte et Objectifs

La lutte contre les nématodes gastro-intestinaux repose essentiellement sur l'utilisation

d'anthelminthiques. L'émergence de plus en plus fréquente de populations de nématodes résistants

aux drogues a conduit à de nombreuses études sur la sélection génétique d'animaux résistants. Les

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études menées jusqu'ici montrent que de nombreux facteurs influencent le statut de résistance des

animaux : la race, la variabilité inter-individus, l'âge, l'alimentation, le statut reproductif. Il semblerait

que le sexe puisse également avoir une forte influence. En effet, de nombreuses études montrent

qu'il existe un effet sexe dans la capacité des hôtes à résister aux infections parasitaires, dans

lesquelles les mâles sont le "sexe faible". Dans cette étude, nous avons utilisé des moutons infectés

par le nématode gastro-intestinal Haemonchus contortus pour voir si cet effet sexe était également

observé dans notre modèle et si cet effet pouvait être attribué à des différences d'expression de

gènes associés à la réponse immunitaire.


49 Martnik Black Belly (24 brebis et 25 béliers) âgés de 18 mois (adultes) ont été infestés avec 10000

L3 d'H. contortus per os.

Les matières fécales (MF) ont été collectées à J+ 0, 14, 19, 21, 25, 28, 32, 39, 42 et 53 pour la

réalisation du suivi coproscopique de l'excrétion d'œufs.

Des échantillons de sang ont été collectés à J+0, 6, 19, 25, 32, 42 et 53 pour le suivi de l'hématocrite

et la mesure de pepsinogène.

A J+63, les animaux ont été mis à mort et les caillettes ont été isolées pour extraire les parasites

adultes. Des prélèvements de tissus (2x2cm) au niveau de la caillette (zone pylorique et fundique)

ainsi que des nœuds lymphatiques mésentériques (3g) ont été prélevés et stockés dans du RNA later

à 20°C pour la réalisation de RT- PCR quantitative. Le choix des gènes a été fait sur la base des

travaux publiés par Terefe et al. 2007 et Sallé et al., 2014. Dans les nœuds lympahtiques

mésentériques, ont été suivis les gènes : TNF-α, OX40, INF-γ, CXCL14, CCL26, Galectin 15, Interlectin

2, IL-4, IL5 et IL-13. Dans les fragments de muqueuse, les candidats suivis ont été : TFF3n Galectin 15,

Interlectin 2, IL-4 et IL5. 8 animaux (4 mâles et 4 femelles) extrêmes ont été suivis en RT-PCRq.

Résultats

Les mâles présentent un faible niveau d'infestation (756 œufs/gMF en moyenne) mais les femelles

ont un niveau de résistance quasi-total (<1 œuf/gMF). Ces résultats confirment ce qui a d'ores et déjà

été observé dans d'autres modèles d'infection parasitaire.

20

Dans cette étude, nous avons cherché à comprendre les raison de ces différences par l'analyse de

l'expression de gènes précédemment décrits comme impliqués dans les mécanismes de résistance

ainsi que dans la réponse immune de l'hôte. Les résultats de RT-PCRq sur les animaux les plus

extrêmes montrent que les mâles ont une plus forte expression de cytokines TH2 (IL-4, IL-5, IL-13)

classiquement associées à la résistance. Les femelles quant à elles ont une expression plus

importante des gènes associés à l'immunité des muqueuses (interlectin-2, galectin-15) et à la

réparation épithéliale (TFF3). De plus les gènes suivis sont plutôt sur exprimés dans les nœuds

lymphatiques chez les mâles alors que les sur expressions chez les femelles sont plutôt observées

dans la muqueuse.

Ces résultats suggèrent que les femelles pourraient être plus résistantes aux infections à H. contortus

du fait de leur immunité mucosale innée plus efficace.

2) Bio-marqueurs de la résistance (ex: valeur prédictive de la température corporelle)

3) Coût de la résistance

Les nématodes gastro-intestinaux composant avec les traitements chimiques, les hôtes

résistants et les conditions climatiques difficiles : une évaluation expérimentale

Le contrôle des nématodes gastro-intestinaux repose essentiellement sur l'utilisation

d'anthelminthiques depuis les années 60. Actuellement, l'apparition et l'émergence de résistances

aux trois familles majeures d'anthelminthiques au sein des populations de nématodes, remet en

cause l'efficacité des traitements. L'acquisition relativement rapide des résistances, parfois multiples,

souligne les fortes capacités d'adaptation dans nématodes gastro-intestinaux. A l'avenir, l'utilisation

en élevage de races de moutons résistants semble une voie prometteuse. Néanmoins, aucune étude

n'a été menée sur le devenir des populations de nématodes actuelles, comportant de nombreuses

résistances aux anthelminthiques, au sein d'hôtes résistants. Ainsi l'objectif de cette étude a été de

comparer les coûts de la résistance aux anthelminthiques, en termes de fitness, sur des génotypes de

moutons sensibles et résistants afin d'évaluer les capacités d'évolution des nématodes gatro-

intestinaux.

21


- Isolats d'H. contortus

3 isolats ont été utilisés au court de cette étude : ISE, sensible aux anthelminthiques ; RHS6 =

Borgstet (BOR), résistant au lévamisole ; Kokstadt-ISE (KOKISE), partiellement résistant aux trois

familles majeurs d'anthelminthiques et introgressée avec l'isolat sensible ISE.

Chaque mouton a été infesté per os avec 10000 larves L3.

- génotypes de mouton :

Au cours de cette étude, 12 agneaux de race berrichon du cher (sensibles à Haemoncus contortus ;

âgés de 4 mois) et 18 béliers de race Martinik Black Belly (MBB) (résistants ; âgés de 18 mois) ont été

utilisés.

Trois groupes de 4 berrichon du cher et 3 groupes de 6 MBB ont été infestés avec les 3 isolats d'. H

contortus.

-paramètres mesurés chez l'hôte :

Des échantillons de sang ont été prélevés à J+0 et J+53 pour la mesure de l'hématocrite. La charge

parasitaire a été évaluée grâce au comptage des œufs / g de matière fécale à J+0, 19, 21, 27, 29, 30,

34, 40, 42, 58 et 61. Les matières fécales récoltés à J+27, 29 et 30 ont été utilisées également pour la

réalisation de coprocultures (évaluation du taux de développement des œufs jusqu'au stade

infestant L3). A l'autopsie, les vers adultes ont été dénombrés pour évaluer l'installation des vers et

isolés pour la réalisation des expériences transcriptomiques.

Les études transcriptomiques ont été menées par cDNA-AFLP sur des pools de 10 vers adultes pour

chaque isolat d'H. contortus développé sur mouton résistant Black Belly.

La fitness des nématodes a été évaluée selon 3 paramètres :

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- la fitness absolue : nombre de larves de seconde génération produit à partir d'une dose initiale de

larves infestantes (10000).

- performance des isolats à chaque étape du cycle de développement

- différences transcriptomiques (cDNA-AFLP)

Résultats

Les analyses transcriptomiques ont clairement montré une régulation de l'expression de certains

gènes en fonction du statut de résistance des isolats. Sur les trois isolats testés et sur la partie du

transcriptome analysée (seuls les profils entièrement exploitable chez les trois isolats à la fois ont

été pris en compte), KOKISE est le plus divers en termes de fragments exprimés, ISE est médian et BOR

est le moins divers. Ces résultats sont une première évaluation des régulations pouvant intervenir en

relation avec le statut de l'isolat. L'analyse complète des profils en comparaison avec ceux qui seront

générés pour ces mêmes isolats sur la race de mouton sensible permettra de mettre en évidence

l'impact du fond génétique résistant sur les différences d'expression des gènes ainsi que certains

gènes spécifiquement exprimés sous pression de sélection hôte (gènes potentiellement impliqués

dans la virulence).

D'un point de vue de la fitness, les deux isolats résistant aux anthelminthiques ont une fitness réduite

par rapport à l'isolat sensible ISE (fitness ISE> KOKISE >BOR). Ceci suggère un coût associé au statut de

résistance du parasite que ce soit face à un hôte sensible ou résistant.

Le fond génétique hôte résistant n'influence pas la fitness relative de chaque isolat, mais affecte la

performance des isolats en modifiant certains traits de vie.

Enfin, le stress thermique semble réduire la fitness de tous les isolats d'H. contortus.

a- coût physiopathologique

23

Démêler la contribution relative du parasite et de l'hôte dans les conséquences d'une

infection à Haemonchus contortus chez le mouton


Les infections à nématodes gastro-intestinaux tells que Haemonchus contortus sont marquées par

des dommages sévères induits chez les hôtes et en conséquence des pertes de production

substantielles. De manière générale, les symptômes sont attribués exclusivement au parasite avec

une corrélation directe entre l'intensité de l'infection et la sévérité des symptômes. Pourtant les

hôtes résistants ne sont pas indemnes de symptômes. L'objectif de cette étude a été de définir les

contributions respectives de l'hôte et du parasite dans la physiopathologie. Pour cela, les symptômes

liées à une infection à H. contortus ont été caractérisés dans différents fonds génétiques hôtes.


Trois groupes de moutons avec des statuts de résistance à H. contortus différents ont été utilisés :

-Très résistants : 24 brebis de race Martinik Black Belly de 18 mois

- Moyennement résistants : 24 béliers de race Martinik Black Belly de 18 mois

- Sensibles : 15 béliers de race Romane de 7 mois

Chaque mouton a été infesté per os avec 10000 larves infestantes (L3) d'H. contortus isolat ISE

Au cours de l'infestation, différents paramètres permettant de caractériser la physiopathologie ont

été suivis :

- Excrétion d'œufs de parasites dans les matières fécales (J+21, 25 et 32)

- Hématocrite (J+0, 21, 25, 32)

- Pepsinogène (J+0, 14, 21, 25 et 32)

- Poids des animaux (J+0 et 32)

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Résultats

Les résultats coproscopiques ont confirmé le statut de résistance des animaux.

Cette étude montre que la réponse protectrice hôte influence fortement la sévérité et le timing

d'apparition des symptômes d'anémie. Les groupes voient leur hématocrite diminuer à différents

temps après l'infestation et surtout avant les activités d'alimentation en en sang des adultes. Ce qui

signifie que l'hôte lui-même est responsable en partie de la diminution de son hématocrite.

L'augmentation du pepsinogène qui reflète la sévérité des dommages au niveau de la caillette est

visible uniquement sur les moutons de statut de résistance moyen. Peu de changements ont été

observés chez les moutons résistants et sensibles. Ce qui suggère une étroite "coopération" entre

l'hôte et le parasite pour le développement de symptômes sévères.

Ces travaux montrent que le parasite seul n'est pas responsable des symptômes observés

(diminution de poids, anémie, augmentation du pepsinogène sanguin) dans le cas d'une infection à

H. contortus. Les moutons présentant les symptômes les plus sévères sont ceux qui ont un statut de

résistant moyen.

b- douleur

Est-ce que infections par les nématodes gastro-intestinaux induisent de la douleur chez leur

hôte ?


25

Les nématodes gastro-intestinaux sont réputés pour les dommages physiopathologiques qu'ils

induisent chez leur hôte et qui provoquent potentiellement de la douleur. Si tel est le cas, les

conséquences sur la production et sur le bien-être animal sont à prendre en compte.

Dans cette étude, nous avons cherché à savoir si les infections à nématodes gastro-intestinaux

entrainent réellement une douleur chez les petits ruminants dans le modèle mouton/Haemonchus

contortus.

Plusieurs paramètres ont été mesurés : les paramètres physiologiques (hématocrite, éosinophilie,

taux de cortisol, température corporelle), l'examen clinique (réponse d'échappement, vocalises après

percussion et palpation de la région infectée), les lésions (pepsinogène) et la performance (poids).

Un analgésique a également été utilisé pour vérifier les résultats.

Mis à part les dommages induits sur les tissus hôtes, aucun indicateur mesuré n'a permis de mettre

en évidence de la douleur associée à l'infection.


16 béliers Romane âgés de 9 mois ont été utilisés. Cette race de mouton est sensible à Haemonchus

contortus.

Les béliers ont été répartis en 4 groupes de 4 : contrôle non infecté, contrôle non infecté recevant un

analgésique, infecté avec 2500 L3 d'H. contortus, infecté avec 2500 L3 d'H. contortus et traité avec un

analgésique.

A J+25, les moutons ont été traités à l'ivermectine pour les déparasiter, puis laissés 10 jours. Ils ont

ensuite été réinfestés avec le même nombre de larves. Des études ont montré qu'après avoir subit

des dommages tissulaires une première fois, l'animal est plus sensible dans cette même région.

Les animaux ont été mis à mort à J+25 soit 60 jours après la première infestation. Les vers contenus

dans la caillette ont été extraits et dénombrés.

Les groupes recevant des analgésiques ont été traités à J+1, 2, 3, 4, 9 et 10 avec un mélange de

butorphénol 1% et des microdoses de kétamine 1g administrés en intramusculaire.

Des contrôles coproscopiques ont été réalisés pour valider l'infestation.

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Des échantillons de sang ont été prélevés à J-3, J+3, 24, 35, 51, 60 pour le suivi des paramètres

physiologiques (pepsinogène, hématocrite, éosinophilie, taux de cortisol). Les animaux ont

également été pesés à différents temps (J-3, J+28, 32, 51 et 60).

La température corporelle a été suivie grâce à des transpondeurs administrés par voie orale

(prototype MEDRIA).

Pour valider une diminution de la température en fonction de la douleur, nous avons du induire une

douleur dans la caillette au préalable et faire un suivi de température. Deux brebis Berrichon du Cher

âgés de 2 ans ont reçu un transpondeur 3 jours avant l'administration de 5mL de Tabasco

directement injectés dans la caillette (échographie). Les deux moutons ont été mis à mort le

lendemain et les caillettes examinées.

Résultats

Dans un premier temps, nous avons observés que les moutons infestés ayant reçu un analgésique

présentent une excrétion d'œufs/g MF plus élevée que le groupe infecté non traité. La deuxième

infestation n'a en revanche pas fonctionnée sur les moutons infestés préalablement. Ce qui signifie

qu'ils se sont immunisés à la première infestation.

Aucune différence entre les groupes infectés n'a été montrée en considérant le niveau sanguin de

pepsinogène, le poids des animaux, le taux d'hématocrite, l'éosinophilie, le taux de cortisol sanguin,

ou la température corporelle.

De plus, nous n'avons pas pu montrer de relation entre les variations de températures et l'induction

de la douleur (Tabasco) chez les animaux testés.

Cette étude ne nous a pas permis de mettre en évidence de douleur associée à une infection à H.

contortus chez les ovins avec les paramètres que nous avons mesurés.

Éleveurs / point de vue des éleveurs

27

Transfert des résultats scientifiques et meilleures connaissances des pathologies liées aux

nématodes gastro-intestinaux

Les éleveurs disposent d’outils pour gérer le parasitisme par les nématodes gastro-

intestinaux (NGI). Certains sont anciens comme l’évaluation du nombre d’œufs de ces

parasites dans les matières fécales. La méthode est recommandée par les chercheurs et

certains vétérinaires, mais reste très peu utilisée sur le terrain, en raison du coût de ces

examens, supérieur à celui d’un traitement anthelminthique. Ce moyen d’évaluation

concerne l’ensemble des NGI et ne cible pas particulièrement H. contortus L’utilisation des

TST (targeted selective treatment : traitement ciblé contre les GIN et chez les animaux les

plus infestés ou qui manifestent le plus de symptômes) a été de plus en plus proposée au

cours de ces dernières années et a même fait l’objet d’un projet européen (PARASOL :

parasite solutions) ce qui a permis d’évaluer son intérêt sur le terrain. L’idée fondamentale

était que les éleveurs ou leurs conseils techniques puissent s’emparer de ces méthodes

(Famacha ou indice d’anémie développé en Afrique du Sud ou Disco indice de diarrhée.

Développé en France). Le Famacha en ciblant l’anémie consécutive aux infestations par les

NGI hématophages est finalement relié à l’effet de l’infestation par H. contortus. Les TST

bien que validés sur le terrain (TST contre traitement mensuel de tous les individus par

exemple) ne nous ont pas totalement convaincu car une expérimentation et une

modélisation de traitement sélectifs (20% des animaux traité au hasard ou bien l’ensemble

du troupeau, tous les mois) à laquelle j’ai participé permettait un contrôle convenable du

parasitisme par NGI. J’ai donc désiré vérifier en conditions expérimentales si le TST, fondé

sur le Famacha permettait de recenser les animaux les plus sensibles qui nécessitaient un

traitement.

1) Outils de détection : TST

28

Exploration des limitations des indicateurs pathophysiologiques utilisés pour les traitements sélectifs

ciblés chez des ovins expérimentalement infestés par Haemonchus contortus.

Contexte et objectif

Le Famacha ou indice d’anémie a été spécialement développé pour les infestations par H. contortus.

Testé sur le terrain avec tous les facteurs de confusion que l’on peut, rencontrer, nous avons désiré

l’évaluer dans des conditions expérimentales. Nous avons choisi de le tester chez des ovins réputés

résistants (Martinique Blackbelly) ou sensibles (Romane). Nous avons également souhaité utiliser des

isolats du nématode qui ont des caractéristiques différentes.


Vingt-quatre brebis Martinik Blackbelly- MBB (très résistantes), 24 béliers MBB (partiellement

résistants) et 15 béliers Romane (sensibles) ont été infestés en une seule fois par 10000 larves

infestantes d’H. contortus. Quatre isolats d’H. contortus ont été utilisés, deux sensibles aux

traitements anthelminthiques et deux résistants au lévamisole. L’expérimentation a duré 32 jours.

Les paramètres suivants ont été mesurés : Famacha, Disco, poids, excrétion des œufs du nématode,

anémie selon l’hématocrite sont mesurés individuellement une fois par semaine.

Résultats

Les quatre isolats n’influencent pas significativement les valeurs du Famacha bien que leurs aptitudes

à l’infestation soient très différentes. Le famacha a varié significativement entre les trois groupes de

sensibilité ovins ; ce sont ceux qui ont les infestations intermédiaires qui ont des Famacha les plus

bas. Les estimateurs de TST n’étaient pas reliés entre eux significativement (sauf l’hématocrite et le

Famacha, deux mesures d’anémie). Les seuils habituels de Famacha retenus pour les traitements

anthelmintiques ne permettaient de traiter qu’un seul animal.

Discussion

Le Famacha, comme cela était attendu se révèle un bon indicateur d’anémie. Il ne se révèle pas

cependant efficace pour détecter les animaux les plus anémiés, qui ont eu des pertes de poids ou

des excrétions d’œufs de parasites les plus élevés. Il ne se révèle pas, contrairement à ce que nous

attendions, comme un indicateur précoce d’infestation ou des effets de l’infestation. Cela peut

provenir du fait que les animaux dans les conditions de ces expériences ont reçu une alimentation de

29

très bonne qualité, que leurs conditions de logement était également favorables à leur bien être. Nos

observations n’ont de sens que dans la période du premier mois d’infestation et dans des conditions

d’élevage favorables. Cela doit quand même alerter sur l’utilisation sans contrôle du Famacha.

L’autonomie de l’éleveur dans son élevage est considérée en élevage biologique comme une

nécessité qui permet à l’éleveur de rester un paysan et non pas un employé assujetti à des

contraintes venues d’ailleurs pour des objectifs qui ne sont pas obligatoirement les siens. En élevage

conventionnel de ruminant cette autonomie est également revendiquée. L’autonomie concerne

pratiquement toutes les activités liées à l’élevage et non pas seulement l’usage des traitements

antiparasitaires. Nous avons voulu vérifier si l’autonomie en termes généraux permettait un

meilleur contrôle du parasitisme par les NGI (H. contortus inclus) dans plusieurs conditions d’élevage.

2) L’autonomie des éleveurs est-elle un facteur de stabilisation de l’infestation par les NGI chez

les ovins ?

Compromis entre l’autonomie de l’éleveur et le contrôles des nématodes gastro-

intestinaux des ovins dans des exploitations conventionnelles ou biologiques.


L’autonomie est prisée en élevage des ruminants, elle correspond à une image de

l’éleveur qui a le pouvoir de décision et qui l’exerce en connaissance de cause. L’un

d’entre nous a déjà remarqué que certains élevages biologiques, menés en autonomie

ne montrait pas des résultats encourageant au plan du parasitisme. Nous avons donc

décidé d’évaluer cette autonomie et de la resituer face aux intensités d’infestation par

les NGI (H. contortus inclus).


30

Vingt fermes conventionnelles en Algérie (région de Batna), et 16 en France (Auvergne),

dont 9 conventionnelles et 7 biologiques ont été visitées. Un questionnaire relatif à

l’autonomie a été rempli sur place. Les examens d’œufs de NGI dans les fèces pour

évaluer le niveau parasitaire sont classiques. Le degré d’autonomie dans les diverses

catégories de préoccupations ‘agriculture, élevage, nourriture des animaux,

thérapeutique, commercialisation, acquisition des savoirs) des éleveurs est de type

qualitatif (de 0 à 3).

Résultats

L’Algérie constitue le site le moins infesté sans doute pour des raisons de surface

disponible pour les animaux. Les espèces présentes sont également peu prolifiques et

diminuent artificiellement le niveau d’infestation. Pour cette région c’est l’autonomie sur

la nourriture qui présente une relation avec l’infestation. Pour les fermes biologiques

Françaises, l’infestation est liée à l’autonomie thérapeutique ; aucune autonomie n’est

reliée à l’infestation pour les fermes conventionnelles.

Discusssion

L’autonomie n’est pas une garantie de contrôle effectif du parasitisme par les NGI. Cela

peut provenir du fait que les éleveurs n’ont pas, seuls, les bonnes pratiques. Cela

indique que le conseil d’experts peut être utile, s’il est réaliste et accepté par l’éleveur.

Le rôle de l’éleveur par ses façons de faire est central dans la détermination de l’intensité

du parasitisme. Leurs pratiques sont construites, ils savent pourquoi ils font ce qu’ils font

comme nous avons pu le montrer dans une présentation récente qui n’a pas encore

donné lieu à un projet de publication. Le modèle Santé- Croyance développé par les

psychologues nous semble un cadre conceptuel très adapté. Les questions se

décomposent en : 1) ce problème sanitaire est-il important dans l’exploitation, 2) Existe-

t-il des solutions efficaces pour le régler s’il est important ? 3) Ces mesures correctives

sont-elles contraignantes et si oui, cela vaut- il la peine de les entreprendre en regard des

31

résultats escomptés ? La réponse à ces questions ne peut se faire seul, ni pour l’éleveur,

ni pour celui qui propose des solutions techniques nouvelles.

Discussion générale et conclusions

Les rôles des trois acteurs de l’infestation et de son succès sont relativement nets. Le fait

que les isolats d’H. contortus aient des propriétés différentes ne surprend pas au plan

théorique. Les peuplements de nématodes sont extrêmement nombreux et diversifiés

grâce à leurs hôtes domestiqués qui se comptent en milliards. Ils peuvent aussi coloniser

des zones géographiques extrêmement variées. La colonisation actuelle du Nord de l’Europe

par H. contortus qui est considéré comme un parasite « tropical » montre bien cette

capacité. Ces capacités sont aussi façonnées par les traitements antiparasitaires qui ont

amené le phénomène de résistance aux anthelminthique à une prévalence élevée. Cette

acquisition pouvait se faire avec ou sans coûts sur les autres aptitudes. Je montre qu’il

existe un coût à cette acquisition de résistance qui peut être dans certaines conditions très

élevées. Toutefois la mesure intégrée de tous les traits d’histoire de vie montre que cette

différence est moins grande qu’il n’y parait. Cette mesure intégrée n’a jamais été réalisée

auparavant (elle était concentrée sur la phase parasitaire uniquement) et montre bien que

des interactions fortes existent entre les traits de vie de la phase parasitaire et de la phase

libre. Ces différences existent toutefois et montrent que des solutions « universelles » ne

sont pas obligatoirement adaptées, qu’il faudra les vérifier dans un ensemble de conditions

du terrain.

Une des solutions alternative proposée dans le monde de l’élevage, en raison de la montée

catastrophique de la résistance des nématodes aux anthelminthiques, est la sélections

d’hôtes résistants. Nous avons noté que cela peut avoir une incidence sur les capacités de

l’isolat de nématodes. Ces changements sont toutefois assez variables :une évolution forte

est notée chez un nématode gastro-intestinal proche d’H. contortus (T. circumcincta) alors

que nous n’avons pas vu d’évolution des capacités du parasite pour H. contortus. Cela tient

pour beaucoup aux traits de vie essentiels pour chaque espèce. La sélection génétique

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permet un contrôle modéré de l’infestation et elle est modulée selon le sexe par exemple

comme nous avons mis en évidence au sein d’une race résistante, avec des expressions de

gène différentes et dans des localisations elle mêmes différentes. Ces modulations font que

des indicateurs phénotypiques ont tout leur intérêt. C’est le cas de la température corporelle

mesurée en continu qui nous a permis de mettre en évidence une relation entre les

températures élevées et la capacité à résister à l’infestation.

L’éleveur va utiliser des indicateurs de type pathophysiologiques pour réaliser ses

traitements en particulier lorsqu’il cible les animaux les plus atteints. L’un d’entre eux a été

largement utilisé sur le terrain en régions tropicales ou chaudes, sans que des estimations

absolues puissent être retirées de ces pratiques qui ont connu un engouement certain. Nous

avons montré que son intérêt est limité, au moins comme indicateur précoce. La

pénétration de ces méthodes de traitement ciblé peut être faible soit parce que les

contraintes sont trop fortes pour l’éleveur, soit parce que la méthode n’est pas excellente.

Nous avons donc cherché à comprendre comment l’autonomie de l’éleveur (dans divers

domaines) pouvait interférer sur le succès des nématodes. L’autonomie jour un rôle

modeste dans la construction d’une gestion efficace du parasitisme, et ce n’est pas

obligatoirement l’autonomie thérapeutique qui est la plus importante. Cela revient à dire

que cette autonomie nécessite un accompagnement technique, qui ne pourra se mettre en

place qu’en co-construction entre l(éleveurs, ses conseillers et les chercheurs qui

investissent les méthodes de gestion des parasites comme thème d recherche.

L’ensemble des trois point de vue montre bien la nécessité d’une intégration des

connaissances et des pratiques pour arriver à une gestion acceptable des parasites gastro-

intestinaux du mouton, comme H. contortus.

33

INTRODUCTION

Opening a can of worms

The severe and pervasive effects of gastrointestinal nematode (GIN) infections span both medical

and veterinary sciences. Their socio-economic impacts are felt globally. Understanding the factors

that contribute to their continued success is of the upmost interest to biological and applied

sciences. The success of theses parasites is easily evidenced in medicine; with 50% of the worlds

population infected resulting in 39 million disability-adjusted life years (DALYs). This is more than

comparable to the 35.7 million DALYs lost to malaria which receives exceptionally more funding for

research and development than GIN (Moran 2011). Despite advances in control with medicine and

sanitation, the prevalence of GIN has showed remarkable stability having remained unchanged in

over 50 years (Stepek et al. 2006). Likewise in veterinary medicine, GIN prevalence is virtually

inescapable for global livestock production with associated pharmaceutical costs alone proposed to

run into the tens of billions of dollars worldwide, not including production costs (Roeber et al. 2013).

Finding an answer to the question ‘what makes a gastrointestinal nematode successful’ is thereby a

worthy question to ask with far reaching interests. For the purpose of this research, the success of

GIN will be studied specifically in the context of sheep production systems.

How do you define GIN success?

In order to answer the question as to what makes a GIN so successful, we must first define what we

mean by success before we can determine who or what is responsible for it. The most obvious means

to translate success would be to look at their extensive prevalence, which is virtually ubiquitous to

sheep production systems throughout the globe. Clearly, the well adapted biological traits of GIN

have provided them with tools they needed to attain such a global foothold. Yet defining GIN success

based purely on prevalence is problematic as we cannot definitively separate their numeric success

from the opportunities presented to them in terms of sheep host. In 2010, global production

presented them with 2 billion sheep host opportunities (FAO Statistical Year Book 2013). GIN

prevalence would therefore not have such resounding success in terms of prevalence had man not

provided them with so many hosts. Thus, the GIN alone are not responsible for their own success,

but have had an inadvertent helping hand from man. There are two strong factors that actively

regulate GIN success. On an individual level, the success of a GIN can be helped or hindered

depending on the host in which they find themselves. Sheep have co-evolved alongside GIN over

evolutionary history to develop tactics of their own to reduce parasitic exploitation by means of a

34

protective response. The efficacy of this response can vary to the extremes, from susceptible sheep

in which the GIN can maximize fitness, or resistant sheep which can stop the GIN dead in its tracks.

More often than not, the sheep protective response falls somewhere in the middle of these

extremes. Nonetheless, the role of the sheep host is another contributing factor to the relative

success of GIN, and will be considered. On a population level, the GIN can also be helped or hindered

depending on the choices made by farmers regarding both husbandry and control decisions. Many

modern farming systems for example maintain high stocking densities which serve to facilitate GIN

transmission. On the other hand, GIN control choices made by the farmer also have the power to

strongly negate success. There are some instances in the past whereby control over GIN reigned

almost supreme, until the remarkable biological traits of the GIN once again corrected the balance in

their favour. It is thereby the view of this research that there are three main factors integral to the

success of the GIN: I) The GIN and their associated biology; II) The sheep host and their associated

resistance; III) The farmer and their approach to GIN management.

Approach to answering the question of GIN success

Gastrointestinal nematode biology, sheep resistance and farmer control measures are factors which

individually have received varying degrees of research attention in the past. Substantial efforts have

gone into understanding both GIN biology and the sheep hosts protective response, but the

influential role of the farmer on GIN control has been relatively neglected. But virtually no work

could be found that considers each of the three topics collectively and how they interact to form the

basis of GIN success. This has provided the present thesis research with an entirely novel approach to

answering a longstanding question.

The research within this thesis explores the success of the GIN biology from an evolutionary ecology

point of view. The selective pressures the GIN have experienced over thousands of millions of years

have shaped their biological success through natural selection. However, over the last 60 years, a

mere blink in evolutionary time, the biological stability of GIN has been tested like never before.

Environmental selective pressures have changed with the expanding geographical distribution of

sheep production systems. The host selective pressures have changed not only in sheer volume, but

with the diversity of breeds and protective responses they encounter. And they now face an entirely

new selective pressure like never before, the interference of man with his chemical and biological

attempts at GIN control. This presents an exciting moment in evolutionary history to study the

fortitude of the GINs biology. The research within this thesis will explore how some of todays most

influential selective pressures are shaping our GIN of tomorrow.

35

The interactions between a parasite and a host are considered to be so intricate, that considering

one in the absence of the other is to consider only part of a whole (Coombes 2001). Exploring the

reaction of the sheep host to parasitism by GIN was thereby a fundamental step to understanding

GIN success. Throughout this thesis research, the host is largely considered in the context of its

resistance to infection, and thus, their capacity to regulate success. The natural occurrence of GIN

resistance in sheep populations, and the heritability of the trait, has lead to this trait being exploited

for control in selective breeding programs. The prospect of manipulating the sheep hosts

evolutionary adaptations to counter the GINs evolutionary success presents exciting advancement in

GIN control. Yet the rarity that host resistance occurs in nature raises questions as to whether it is a

rational endpoint to aim for with our sheep. The research in this thesis addresses sheep host

resistance relative to integral factors determining its expression, the associated negative effects of

resistance and potential biomarkers for the trait. This research will help understand the extent to

which sheep host resistance can be relied upon to control the success of GIN.

The relative role of the farmer shall be considered in term of their contribution to GIN success and

the barriers that prevent him from controlling them more effectively. A definite disconnect exists

between the work being done to tackle the problem of GIN in the laboratory and the application of

the results in the field. Even if researchers were to present farmers with the proverbial silver-bullet

for GIN control, we cannot necessarily assume they we use it. The transfer of knowledge is more

complicated than that. This research addresses some of the obstacles that are preventing the

optimum use of GIN controls by examining the farmers’ beliefs, tools and expectations regarding GIN

infection. This research highlights some of the steps scientists need to take if they hope to see their

results applied on the field.

The objective of this thesis was to contribute to the scientific understanding of each of these three

topics individually and to highlight their interdependent relationship as they contribute to GIN

success to be discussed collectively in the discussion. The most important practical implications of

the research will be extracted and their relevance will be discussed in the context of improving GIN

experimental research, what the results can tell us about GIN populations in the field, considerations

for selectively breeding sheep for resistance, the importance of knowledge transfer from the

laboratory to the field and vice versa and finally, the virtues of using an inter-disciplinary approach in

trying to understand GIN success with suggestions for advancing the future of control.

36

SETTING THE SCENE

We have identified a triumvirate regime that influences the success of the GIN consisting of the GIN

itself, the sheep host and the farmer. This chapter will set the scene as to what we know about each

of these three major players, their role in GIN success and how this thesis research will progress upon

the knowledge. We recognize that each of these subtopics could individually be the focus for several

research theses. We thereby do not intend to be exhaustive in our exploration of each. Our aim is to

identify current gaps in the knowledge as they pertain to the research question at hand and to

respond to them. Moreover, we aim to draw attention to the interdependent relationship of these

factors as they relate to GIN success with the express intention of contributing to the advancement

of GIN control in sheep farming.

Gastrointestinal nematodes in sheep farming

Among the diseases that constrain the survival and productivity of sheep and goats, gastrointestinal

nematode infection ranks highest on a global index (Perry et al. 2002). They can be found in all major

sheep production systems throughout the world (Saddiqi et al. 2011), showing remarkable

adaptability in their climatic tolerance. The term GIN extends to include a number of species which

are known to cause significant morbidity and mortality in small ruminants including Haemonchus

spp., Trichostronglyus spp., Teladorsagia spp., Cooperia spp., Nematodirus spp., and

Oesophagostomum spp. (Saddiqi et al. 2011). Their phylogenetic classification in can be viewed in

Fig. X. For the purpose of this thesis research, studies will focus almost exclusively on Haemonchus

contortus in sheep as a model to explore GIN success.

Haemonchus contortus as a model GIN

Of all the GIN species, H. contortus is considered the single most important (Perry et al. 2002). There

are several reasons for this. Firstly, H. contortus is the most prevalent of the GIN species (Cabaret

2000). While it is most concentrated in the tropics/subtropics, H. contortus is increasingly

encroaching into cooler climates reaching as far North as the Polar Circle (Lindqvist et al. 2001).

Haemonchus contortus is also considered the most pathogenic of the GIN species (Saddiqi et al.

2011). Located in the host abomasum, H. contortus are a highly voracious blood sucker that provoke

significant pathophysiological damage in the sheep host, particularly in young lambs (Saddiqi et al.

2011). Typical symptoms include haemorrhages, anorexia, depression, severe chronic anaemia, loss

37

of condition and eventually death of the affected animal (Allonby 1975; van Wyk and Malan 1988;

Overend et al. 1994; Miller et al. 1998; Amarante et al. 1999a; Gauly et al. 2002; Notter et al. 2003).

The combination of this species’ prevalence and pathogenicity inflict a substantial economic impact

on sheep production loss throughout the world (Barger and Cox, 1984; Larsen et al. 1995; Campos et

al. 2009).

Haemonchus contortus also has certain biological features that make them a favourable model for

research. They are a highly fecund species with each female laying about 4000 eggs per day and

reproduction is sexual (Redman et al. 2008). Given H. contortus maintain large population sizes,

these traits lend themselves to producing high levels of genetic variation within the species. Greater

genetic variability provides the foundation for a greater adaptive potential, the cornerstone for

success. Further, H. contortus is known to maintain unusually high levels of genetic diversity, so much

so that differences found between individuals of the same isolate could be as high individuals of two

different mammalian species that do not interbred (Otsen et al. 2001).

The extent of H. contortus diversity has been expressed morphologically in the female worms which

exhibit four differing cuticular morphs for the vulvar flaps. The frequency each of these vulvar

morphs occurs varies depending on the geographic location as a function of the local host and

microclimate interaction (Das and Whitlock 1959). The ratio in which these vulvar morphs are found

is used a means of identifying different subspecies within H. contortus (Le Jambre and Whitlock

1976).

Another advantage of choosing H. contortus as a model system is the sheer amount of knowledge

available on this species compared to others. A simple search on Web of Science for the top three

most common GIN of sheep showed H. contortus to be the most studied of all: Haemonchus 13,628

articles; Trichostrongylus 9,763 articles; Teladorsagia 1,697 articles. The H. contortus genome project

is also one of the most advanced of the parasitic nematodes providing an excellent source for

advancing genetic studies.

The life cycle of H. contortus provides another biological point of interest in trying to understand

their success. It entails both a parasitic and a free-living phase meaning they have to face the

selective pressures of both the host and external climatic environment. The parasitic phase starts

when the third stage larvae (L3) are consumed from the pasture by grazing sheep. The L3 undergo a

developmental period in the host abomasal mucosa to exit as L4 a couple of weeks later to remain in

the abomasal lumen thereafter. Maturation is complete about 21 days post infection (dpi) when

females commence egg laying (Veglia 1915). The eggs are passed out with the host faeces onto

38

pasture where they undergo successive larval moults before attaining the infective L3 stage to

complete the life cycle. The life cycle is illustrated in Fig. X.

A brief history of GIN research

The report of worms in veterinary practice started to make a consistent appearance in scientific

records immediately following the creation of the world’s first veterinary colleges in France; first in

Lyon in 1762 shortly followed by on in Alfort Paris in 1765. Although several references to their

existence date back well before this time, it wasn’t until this point that their potentially harmful

nature was considered. Philippe Chabert, Director of the veterinary college in Alfort, became

particularly concerned with the worm induced losses in domestic animals. Naturally, this was met

with attempts at control and Chabert created the famous “empyreumatic oil” that remained in use

as an anthelmintic up until the mid 19th century. The preparation for the “Traité des maladies

vermineuses des animaux domestiques1” was as follows ; “Au début il faut distiller de la corne de

pied de cheval ou de pied de bœuf jusqu’a l’obtention d’une huile noire, puis cette huile noire est à

nouveau distillée et on y ajoute de la térébenthine…2ˮ (Chabert 1782). Perhaps somewhat

unsurprisingly, this did not prove to be a magic elixir in worm control and research continued. But it

wasn’t until the turn of the 19th century that great strides forward were made with numerous works

accruing into this new science from several active countries. By 1886, Alcide Railliet from the

veterinary college in Alfort, coined the term “parasitologie” to be universally accepted throughout

the literature (Trouratier 1989). The studies carried out thereafter up until the late 1960s provided

much of the basic parasitological data that we still rely on today; from characterization of their

anatomy and life-cycles (Veglia 1915 H. contortus) to the ecological interactions shaping populations

(see works by Michel 1950s). With the discovery of DNA 60 years ago, our understanding of GIN was

magnified to the level of molecular biology, genetics and proteomics to massively progress our

knowledge on the parasites. Yet the continued advancement of biological technologies and research

has occurred rather at the exclusion of some of the more classical, but highly relevant, parasitological

studies. The research carried out in this thesis ‘picks up the slack’ to extend our knowledge on what

makes gastrointestinal nematodes successful from an evolutionary ecology point of view.

1 Treatment of worm diseases for domestic animals

2 At first one must distil the hoof of a horse or an ox until obtaining a black oil, then distil this black oil

once again and add turpentine

39

An evolutionary ecology approach to understanding GIN success

An evolutionary ecology approach permits us to study how H. contortus interact with their

environment to enable their success. The measure of success in this discipline is based on a central

concept in evolution and natural selection, that of fitness. Fitness encompasses all efforts made by

an organism to survive and reproduce within a single generation to pass on their alleles to their

offspring. Anything which contributes to greater fitness will be naturally selected over evolutionary

time where genotypes with greater fitness leave, on average, more offspring than less fit genotypes.

Alleles which promote greater fitness will thereby be over-represented in succeeding generations to

increase in frequency until the fitter genotypes are formed. Any challenges which negate the

organisms survival or reproduction will ultimately reduce fitness. This thesis research challenges the

fitness of H. contortus with various selective pressures targeting both parasitic and free-living stages,

that are likely to be experienced on modern farms today. The use of a universal measure of fitness

allows us to draw direct fitness comparisons between challenges to determine the relative effects of

each on the success of H. contortus. By targeting our challenges at different life-stages it may reveal

those that are more susceptible to challenge than others, which would have great interest in a

control context. While the measure of fitness provides an excellent measure of success, it is slightly

precarious in its generality. Gastrointestinal nematodes demonstrate remarkable plasticity with their

response to potential threats whereby negations at one life-stage can be compensated via increased

effort at consequent life stages (Poulin 1998; Cherheresa et al. 1997). While life history traits can

fluctuate drastically, this plasticity in response may enable the GIN to recoup costs at one life history

stage to maintain similar levels of fitness overall. Our studies thereby compare the success of GIN on

the level of overall fitness in a single generation and by examining their relative success at some of

the most important life-traits pertaining to survival and reproduction including establishment,

fecundity and the developmental capacity of egg to larvae. With this approach, we can identify how

the success of each life-trait is affected, and whether this provokes an increase or decrease in fitness.

The sheep host

For the all the attempts at control man has tried, the sheep host themselves have developed their

own barriers against GIN infection. There is a well known arms race exists in parasite-host systems in

which both are involved in a series of escalating mutual counter-adaptations to exploit or inhibit

exploitation (Behnke and Barnard 1990). In response to the prevalence and pathogenicity of GIN,

sheep have naturally evolved resistance against them. Resistance can be defined as the ability to

suppress the establishment and / or subsequent development of infection (Albers et al. 1987) and

40

involves eliciting an innate and / or adaptive protective response to infection (Cousteau et al. 2000).

The problem with resistance is that not all sheep have it. The existence of naturally resistant sheep

breeds is more common to indigenous breeds in tropical climates such as the Martinik Blackbelly of

the French West Indies, compared to recently imported breeds (Preston and Allonby 1978, 1979;

Mugambi et al. 1996; Baker et al. 1999). Yet even within a breed the extent to which a protective

response is initiated is dependent upon various factors such as; individual variation – GIN have highly

aggregated distributions where most of the worms are in few of the hosts (Good et al. 2006); age –

lambs are slower to develop adaptive immunity (Kosi & Scott, 2001); reproductive status – ewes are

more susceptible to infection around parturition (Sykes 1994); nutrition – sheep in good nutritional

condition with a high protein supply show greater resistance to infection (Wallace et al., 1996; Coop

& Kyriazakis, 2001) .

To maximize on the number of resistant sheep in a flock selective breeding has come into place to

take advantage of their heritability of resistance, with a heritability value of about 0.3 (Axford et al.

2000). In the wake of anthelmintic resistance, it has rapidly garnered support as a cenral control

measure of GIN and is currently being applied to sheep flocks in Australia and New Zealand (Bishop

and Morris 2007; Karlsson and Greeff 2012). Breeding for enhanced disease resistance offers a

number of advantages over other methods of control. It can be inexpensive and relatively simple way

to improve animal health, welfare and productivity. As the GIN have evolved resistance to the drugs

that control them (Nicholas 1987) as the cost of treatment and veterinary care increase faster than

the value of the animals, then breeding for disease resistance appears more and more attractive. The

endorsement of selective breeding however has not been unanimous and theoretical doubts have

been cast regarding the sustainability, feasibility and desirability of the approach (Stear et al. 2001).

Sustainability refers to the prospect that the GIN will counter the resistance of sheep by developing

their own adaptations to overcome them. Given what we know about the extent of genetic diversity

within H. contortus, questioning whether this doubt has some grounding is merely responsible

procedure for its application. Perhaps if similar investigations had occurred for anthelmintics the

shelf-life of this control measure would be extended. The effect of resistant sheep on H. contortus

fitness is explored in the GIN chapter.

Feasibility refers to the heritability of the trait or traits used to measure GIN resistance as well as the

amount of variation among animals. As mentioned in the proceeding paragraph, there are numerous

factors that interact with resistance i.e. age, nutrition, breeding status etc., but most of these factors

can be altered to some degree i.e. lambs will mature, feed can be supplemented, reproduction cycles

change. Even the variation between breeds and within individuals can be controlled for in selective

41

breeding programs. Yet one factor that cannot be controlled is the sex of the offspring produced.

Rams are apparently notorious among stockmen and stud breeders for their greater susceptibility

than ewes to diseases caused by excessive infection to parasitic nematodes, yet there exists but one

study documenting this in the literature (Barger 1993). We thereby explore the possible contribution

of sex in limiting the expression of resistance against H. contortus in a resistant breed of sheep.

The desirability of selective breeding refers to the potentially unfavourable consequences this may

impart on the sheep (Stear et al., 2001). This has been explored in the context of resistance against

GIN increasing susceptibility to other diseases, either via immune biases (Carson and Philips 1981) or

immune suppression and nutrient loss (van Houteret and Sykes, 1996; Stear et al., 1997a, Coop and

Kyrizakis 1999). And desirability has also been studied in how it may impact desired production traits

with studies showing both favourable (Bishop et al. 1996; Bouix et al. 1998) and unfavourable

(McEwan et al. 1995; Eady et al., 1998; Shaw et al., 1999) results. The research done in this thesis

rather focuses on the desirability of being resistant from the sheep’s point of view. It has long been

known that sheep naturally resistant to infection do not necessarily remain symptom free (Clunies-

Ross, 1932) and there are increasing references to immune mediated pathology of eliciting an

effective protective response (Greer 2008; Williams et al. 2008).On the other hand, acceding an H.

contortus infection is known to inflict substantial pathophysiological damage to the host, especially

during the developmental phases of the life cycle (Bueno et al. 1984) and we have already made

reference to the life threatening effects heavy infection may result in for the host (Saddiqi et al.

2011). We thereby try and understand the relative desirability for a sheep to be resistant to

infection, weighing up the potential costs of resistance against susceptibility with a view on sheep

health. After all, healthy sheep equal healthy profits.

The Farmer

With the right tools, farmers have the capacity to exact very effective control over the success of

GIN. With the wrong tools, or wrong knowledge, certain husbandry choices can lead to the farmer

inadvertently facilitating the GIN success. For example, a combination of high stocking densities in

confined areas are ideal GIN transmission conditions inevitably resulting in high GIN infection

intensities. Yet it is fairly safe to assume that farmers are in favour of reducing GIN success rather

than supporting it. We then have to question where the farmers can obtain the right tools and the

right knowledge for effective GIN control. The answer to that points back to us; to science and

research. Much of the research done in veterinary parasitology of livestock is done with the express

purpose of increasing production for the farmers. The efficacy of the research is then entirely

42

dependent on its correct application by the farmers. Thus, like the intricate relationship between a

parasite and its host, there is also an inter-dependent relationship between farmers and scientists.

Tailoring science and research to the needs of agriculture really took-off at the end of the Second

World War. The food shortages and low farm productivity highlighted a need to modernize

agriculture and husbandry to increase production. In France, agricultural structures formed such as

the Groupes d’Exploitants Agricoles (GAEC) to help strategize farming approaches, and the

Coopérative d’Utilisation du Materiel Agricole (CUMA) took charge of advancing agricultural

machinery (Nicourt 2013). Combined, these organizations effected huge transformations in French

farming systems and the management of animals (Nicourt 2013). Farming management choices were

refined further still with the development of research institutes such as l’Institut National de la

Recherche Agronomique (INRA) (established in 1946) which considerably advanced understanding of

animal and plant production. The resulting technical propositions however were delivered to farmers

in a very top-down manner. They were highly specific in subject matter i.e. GIN control and they had

little consideration for other farm planning. Little has changed in this approach since and without the

appropriate communication of tools and knowledge, farmers will continue to lack both.

Any flaws in the communication and knowledge transfer routes were largely hidden with the

development of anthelmintics in the 1960s to 1980s (McKellar and Jackson 2004). During this time

the GIN control message was clear to all farmers, treat with anthelmintics as much as needed. This

approach was cheap, easy and effective for the farmers to apply, thus readily complied with. But now

with spread of anthelmintic resistance (AHR), proposed control strategies are more numerous,

complicated and labour and cost intensive. Now more than ever, there is a need for effective

knowledge transfer between science and farmers.

With the exception of a few notable studies (Cabaret et al.2011; Bouihol et al. 2011; Sadiqqi et al.

2012) the communication of GIN control is a virtually uncharted area in research that requires much

attention. The issues addressed in this research include aspects such as the farmers understanding of

GIN control, how current practices may influence infection intensities and how effective some of the

tools science has already provided them are proving. By understanding the farmers’ attitudes and

knowledge and approach to GIN control, we can work towards developing an accessible and realistic

model for GIN control for the farmers.

43

GASTROINTESTINAL NEMATODES

The measure of success

Exploring the interactions between an organism and its environment has been at the forefront of

evolutionary ecology ever since Darwin’s (1959) realization that the latter played an important role in

shaping the former. Natural selection over evolutionary time can be used to explain all

manifestations of an organismal phenotype. The currency of measuring evolution in different

contexts is fitness (Fisher 1930; Haldance 1932; Pianka 1970). Fitness is a function of life-history, that

is, the age specific schedule of fecundity and mortality (Crow and Kimura 1970). The evolution of life

histories across taxa has previously received much attention in the context of natural selection

(Fisher 1930; Hamilton 1966; Charlesworth 1973, 1980). The deft observation that no organism has

maximized their life-history to the point of world domination leads inexorably to the conclusion that

life histories must involve compromise between what selection can achieve (adaptation) and what

selection is prevented from achieving (constraint) (Gould & Lewontin 1979; Charlesworth 1990;

Parker & Maynard Smith 1990; Partridge & Sibly 1991; Stearns 1992; Barton & Partridge 2000;

Barnes and Partridge 2003). The dominant constraint on life history evolution is the idea that an

organism is limited by the amount of resources it can obtain from its environment (Barnes and

Partridge 2003). The life histories have largely been compartmentalized into categories such as

‘growth’, ‘maintenance’ and ‘reproduction’, each of which is conceptualized as competing with the

others for resources. Fitness can be maximized by adjusting resource allocation between these

(Levins 1968; Calow 1979).

The fluidity of life-history traits represents an excellent manner in which to overcome environmental

challenges; losses of fitness at one life history trait could be recuperated by increased effort at

consequent life history traits. Gastrointestinal nematodes are an excellent example of this whereby

life history traits are altered to overcome challenges (Chehresa et al. 1997; Poulin et al. 1998;

Skorping and Read 1998). Comparisons of life-history traits across nematode species has highlighted

just how much they can vary in size, development rates, longevity and reproductive patters (Levine

1980; Wharton 1986; Skorping 1991). Studies comparing how life history traits alter within a single

GIN species are limited. Only one study could be found which studied several life-history traits

simultaneously; the mouse GIN Heligimosomoides polygyrus was observed to incur differences in life-

history as a function of experimental serial passage (Chehresa et al. 1997). The research in this

chapter comprises the most comprehensive work available that documents how H. contortus

respond to different selective pressures by altering their life history traits, and how this affects their

overall fitness. The precise definition of fitness as used in the following studies is a calculation of the

44

number of L3 offspring produced daily, divided by the initial infective dose given. We hypothesized

that flexibility of their life history response to challenges was a central factor contributing to the

fitness and success of H. contortus. The selective pressures selected represent common threats that

the ‘average’ GIN on the field would likely be exposed to. These threats target aspects of both the

parasitic and free-living stages of H. contortus. By varying which life trait was the focus of each

selective pressure, it enabled us to determine whether all the life stages were equally responsive to

challenges, or whether some were more vulnerable than others. The selective pressures targeting

the parasitic phases include:

Chemical attack from anthelmintics

Biological attack from the sheep host protective response

Density dependent limitations due to intra-species interactions

The selective pressures targeting the free-living stages included

Exposure to climatic variables

Temporal affects on L3 survival and preservation

A hypothetical example of flexible life traits in H. contortus

The initial use of anthelmintics, prior to the development of resistance, provides a good example for

us to speculate on how life history traits could potentially have changed for H. contortus.

Anthelmintics have the potential to affect the GIN from the moment of L3 ingestion to the adult

stages. However, given the developmental L3 and L4 stages reside in the abomasal tissue, it is

unlikely they were exposed to the same intensity of pressure from the anthelmintics. Differences in

the physiology and metabolic activity of the developmental and adult stages also likely had an

influence on the vulnerability of the life stages to anthelmintics (Sangster 1996). Regardless,

anthelmintics had the capacity to clear already established adult populations. Studies have suggested

that if adult life expectancy is targeted, natural selection should favour parasites which mature

earlier (Medley 1994; Poulin 1998). Once they start reproducing, the anthelmintic pressure would

ease on the consequent free-living stages and an increased reproductive effort should be favoured to

maximize fitness (Stearns 1992). However, variations in the larval phase may increase pathogenicity

by increasing host lesions, inflammatory responses and other pathological complications associated

with migrating larvae (Anderson 1992). Likewise, an increased reproductive output may increase

pathogenicity owing to a greater need to extract resources from the host to invest in the offspring

(May and Anderson 1983). This hypothetical outcome thereby produces a GIN that is effectively

45

controlled in the sheep, but increases its force of infection with offspring that have greater

pathogenicity.

Selective pressure 1: Anthelmintic resistance

This research does not study the effects of anthelmintics on H. contortus life history and fitness. That

would provide only a very retrospective view on what has already happened. Instead, anthelmintic

resistant (AHR) H. contortus populations are the reality on the field and the effect of developing this

AHR status on the H. contortus life history and fitness was contrasted against susceptible

populations. This question has been the topic of some interest in the past, but with a view to

determine if reversion to anthelmintic susceptibility would be possible in the absence of treatment. If

there were significant fitness costs associated with AHR, than theoretically there would be a reduced

frequency of AHR alleles in a population making reversion to susceptibility possible. Yet there is little

evidence to support that true reversion would occur in their field (reviewed by Leathwick et al. 2001;

2014). The study here differs from previous studies in two important ways. Firstly, the use of life

history traits and fitness to determine the effects of AHR provides a much more complete picture of

how the GIN are affected compared to prior studies focused on a select few life history traits.

Secondly, the study questions for the first time whether AHR confers any advantages to the H.

contortus in the face of other common selective pressures. We selected two of the most probable

selective pressures that H. contortus are likely to face on the field in the near future; one that targets

the parasitic stages – resistant sheep; and the other that will challenge the free-living stages –

temperature stress.

Sheep selectively bred for resistance are increasingly looked towards as the GIN control method of

choice for the future (Waller and Thamsborg, 2004) owing to both their supposed efficacy and

sustainability. While mechanisms of host resistance are undoubtedly more numerous and complex

than those of anthelmintics, we cannot exclude the possibility that there may be an interaction

between the two. As far as could be determined, many of the past experimental studies have largely

explored sheep resistance following infection with anthelmintic susceptible GIN isolates (Gruner et

al. 1994; Lacroux et al. 2006; Terefe et al. 2008). In the event of an interaction between AHR and

their fitness in resistant sheep, either positive or negative, this will have important implications for

the responsible use of selectively breed sheep in the future.

Secondly, the free-living stages of sheep GIN are strongly affected by climate. Extremes of heat and

cold are detrimental to development and survival, while within tolerable limits, increasing

temperatures generally accelerate development but increase mortality (Morgan and Van Dijk 2012).

46

With the unequivocal effects of climate change looming (Gauly et al. 2013) we consider whether AHR

confers any advantages to the survival of free-living stages under heat and cold temperature stress.

This is the first known study to consider the possible interactions between different selective

pressures H. contortus will face on the field in the future to provide some predictive value. This study

not only uses the phenotypic demonstration of the H. contortus fitness in different AHR isolates, it

relates this back to the diversity of transcriptomic diversity within the isolates to assess their

adaptive potential.

Selective pressure 2: Sheep resistance

As a control method, the use of selective breeding appears to overcome many of the current

problems associated with anthelmintics; the spread of resistance, cost, availability in developing

countries and limited scope in many pastoral systems (Saddiqi et al., 2011). Additionally, there is

increasing evidence to suggest breeding for cross-resistance to other GIN species is attainable

(Windon, 1990; Gray et al., 1992; McEwan et al., 1992) making it even more compatible with the

poly-parasitic reality of farming. Some reports have gone as far to refer to selectively breeding as the

“ultimate tool in sustainable parasite control” (Waller & Thamsbourg, 2004). Where resistance is

truly effective, the benefits may even extend into improved animal welfare, reduced environmental

contamination by drugs, delayed development of further drug resistant parasites and improved

return on the investment of time and money (Stear et al. 2012). But as is the cynical nature of

science, the optimism of this control has breed skepticism with regards to its sustainability.

The argument in favour of the sustainability of resistant sheep is compelling, with both experimental

and field studies to support it. Resistance against GIN is heritable, with an approximate value of 0.3

(Axford et al. 2000). Greater resistance has been attributed to certain breeds, and can be heightened

further still in certain individuals (Axford et al. 2000). Thus with the right tools to identify resistant

sheep, resistance is relatively accessible to manipulation in selective breeding programs. Additionally,

numerous studies have demonstrated the polygenic nature of host resistance (Dominik 2005;

Crawford et al. 2006; Davies et al. 2006; Beraldi et al. 2007), suggesting the control should be more

sustainable than the presumably less numerous mechanisms that chemical anthelmintics operate

with. In the 1970s, several breeds of sheep were identified to be resistant, a capacity they

presumably and before experimental studies and a capacity they maintain today. Examples include

the Martinik Blackbelly from the French West Indies (Courtney et al. 1985); Red Maasai of Kenya

47

(Preston and Allonby 1978, 1979a; Miller et al. 1995), Scottish Blackface sheep in the UK and (Altaif

and Dargie 1978); Florida Natives from the US (Radhakrishnan et al. 1972) among others. Stear et al.

(2001) argued that if resistance wasn’t sustainable then they would not maintain their resistant

status today.

Nonetheless, numerous studies have questioned the sustainability of resistant sheep. These studies

largely, and in my opinion rightly, acknowledge the ecological relationship that exists between the

parasite and host. Even when presented with a ‘resistant’ host the parasite cannot be expected to

remain constant or stagnant following an encounter. After all, by very definition of selective

breeding, the host response will essentially become increasingly homogenized to represent a more

consistent selective pressure to the GIN. Consequently, several experimental studies have attempted

to explore whether repeated exposure to resistant sheep will result in H. contortus adaptation to

overcome the host defenses for up to 10 generations of serial passage (Adams 1988; Albers and

Burgess, 1988; Saulai et al., 2001). All of them failed. No demonstration of adaptation occurred.

Rather than adding to the list of studies that ultimately support the sustainability of resistant sheep,

we designed a similar study but addressed a slightly different question and used slightly different

methods. Instead of asking whether adaptability to overcome sheep resistance was possible, we

addressed how the selective pressure of resistant sheep would shape consequent generations of

GIN, be it positively or negatively. And instead of relying on the sole measure of FEC, or in the case of

Saulai et al. (2001) FEC and fecundity, to understand adaptive potential of the GIN, we once again

looked at all of the life history traits to gain an overall perspective of how fitness was being affected.

Understanding how control measures influence consequent generations is integral to their ultimate

success, especially if we are to avoid nullifying high potential controls such as what happened with

anthelmintcs. Here we present two studies that explore the effects of resistant sheep on GIN success.

The first study monitors the life-history and fitness response of H. contortus to six generations of

serial passage in resistant Martinik Blackbelly (MBB) sheep. Accounting for the three prior studies

which observed no adaptation in H. contortus, we further wanted to verify whether the lack of

adaptation was specific to the species, or Trichostrongyles in general. We thereby conducted a

second study exploring the effects of three generations of Romanov sheep, selectively breed for

resistance, on the life history and fitness of the GIN Teladorsagia circumcincta.

Selective pressure 3:Density dependence

Due to the prolific nature of H. contortus, sheep kept in close contact can experience very high

infection intensities. Densities above a certain threshold can modulate population parameters in GIN

48

with previous studies demonstrating density dependent effects on GIN egg production, body length

and fecundity (Michel 1965). Experimental studies have shown that the size of the initial infective

inoculum can also regulate these parameters (Fleming 1988). The study had a wide range of

inoculums from 3000 to 30000 L3 and it will be interesting to study the density dependence effect on

more usual range of infective doses (5000 to 10000). The study presented here explores whether the

highly fecund nature of H. contortus can also present a self-limiting role in the event of medium

densities by focusing specifically on the effects on fecundity.

Selective pressure 4: Desiccation

Temperature and moisture are vital determinants in the success of free-living stages, but they do not

affect all life stages equally. The most vulnerable stages are the egg up until the development of the

L3. Their survival, development, and phase length operate within specific temperature and moisture

ranges that largely underpin the seasonal patterns and geographical distribution of H. contortus

(reviewed in Smith and Grenfell 1994; Kao et al. 2000; O’Conner et al. 2006). By and large, the

climatic conditions of tropical and subtropical regions seem to be optimum for H. contortus

development into L3 (O’Connor et al. 2006). However on developing into L3 their tolerance to

climatic stress increases dramatically owing to the development of an external protective sheath

(Selvan et al. 1993). This feature is likely a contributing factor that has enabled H. contortus to extend

their geographic range into much more extreme environments than they are traditionally associated

with such as the Polar Circle (Lindqvist et al. 2001) or the deserts of Mauritania (Jacquiet et al. 1995).

As we have come to expect from H. contortus, they have maximized their survival in such regions by

drastically altering their life history stages. Survival in colder climates such as France, Sweden,

Denmark and the Netherlands (Waller et al. 2004) has been attributed to their capacity to undergo

hypobiosis, or arrested development at the L4 stage following ingestion to survive the cold of winter

temperatures with shorter free-living phases (Connan 1975; Waller and Thomas 1975). In the deserts

of Mauritania, life history strategies changed by extending the survival time of the adult females in

the host and limiting reproductive efforts to coincide with rainy seasons (Jacquiet et al. 1995). But we

also know that the L3 aren’t completely invincible to climatic stress either. Their external protective

sheath is somewhat of a double edged sword, while it increases climatic tolerance for the L3 it also

prevents them from feeding meaning they have to survive on a limited energy budget (Selvan et al.

1993; Medica and Sukhedo 1997). Both heat (Vlassoff et al. 2001) and desiccation (Siamba et al.

2011) conditions have been shown to accelerate this depletion of their energy reserves. What we

don’t know is how well the L3 can recuperate from such a direct attack on their energy budget with

49

consequent life history traits and fitness. This study thereby further explores the effects of

desiccation on H. contortus fitness and their capacity to resist desiccation in comparison to other

common GIN species. Extending our understanding of the epidemiology of gastrointestinal

nematodes is central to improved control and management.

Selection pressure 5: Ageing of L3

The successful transmission of H. contortus L3 faces both temporal and spatial challenges. These L3

are unable to travel great distances and they consequently must wait in hope that a grazing sheep

comes there way. As we have established, H. contortus L3 stages have a limited energy budget and

the length of time they are able to wait on the filed will be a direct function of the climatic variables

they are exposed to. It would thereby be interesting to determine how age effects influence H.

contortus life history and fitness in constant temperatures. This experiment was designed to explore

this, but rather from an experimental point of view. The physiology of the H. contortus L3 make them

amenable to preserving stocks in the laboratory, either via refrigeration or cryopreservation. All of

the studies conducted in this thesis used stocks maintained at 4°C. However, the length of time that

had been maintained in the fridge varied. It was thereby our aim to determine if the length of time

stocks had been maintained would influence the infection outcome of H. contortus on the premise

their resources may have depleted. We further compared this against the relative merits of using

cryopreserved stocks in infections. These results were not only important to determine the

comparability of the studies done within this research, but to determine whether this could be an

influential factor in comparing H. contortus studies throughout the literature.

Key findings

All the selective pressures examined negatively affected one-life history trait

This was compensated for by increased effort at consequent life-history traits to

maintain fitness

AHR was the only exception: LEV-resistance negatively affected the life-history

stages to result in reduced fitness.

The use of resistant sheep is likely a sustainable. The results however question its

efficacy in control.

50

Parasitic gastrointestinal nematodes coping with chemotherapy, resistant hosts

and unfavourable climatic environments: An experimental evaluation

Chylinski. C, Blanchard-Letort A, Neveu C, Cortet J, Cabaret J.

Abstract

The gastrointestinal nematode Haemonchus contortus is the one of the leading threats to the health

and production of ruminant farming systems throughout the globe. The overuse of anthelmintics in

their control has lead to anthelmintic resistant populations now being the norm on the field. This

study questioned how being anthelmintic resistant affected the adaptive potential of H. contortus

against two of the most common selective pressures they will likely face on the field in the near

future: sheep selectively bred for resistance and climatic extremes. Three different H. contortus

isolates were compared which represented and anthelmintic susceptible isolate, a levamisolae

resistant isolate and a multi-anthelmintic resistant isolate i.e. levamisole, benzimizadole and

avermectins. Their adaptability was compared using phenotypic measures based on fitness in one

complete generation and their performance at individual life history traits, and gene expression

between the lines was also measured using cDNA Amplified Fragment Length Polymorphisms (AFLP)

to explore the isolate transcriptome. The results show the anthelmintic susceptible isolate had the

greatest fitness and was able to alter its life history traits to cope with the stress of resistant sheep to

maintain similar fitness levels as in susceptible sheep. The levamisole resistant isolate had the

poorest fitness under all conditions tested and showed minimal flexibility in their life traits. The

multi-resistant isolate had an average fitness, but the greatest transcriptomic expression. This was

followed by the anthelmintic susceptible isolate and lastly by the levamisole resistant isolate. In

conclusion, anthelmintic resistance conferred H. contortus fitness costs that made them more

susceptible to the selective pressures of resistant sheep and climatic stress.

51

Introduction

Parasitic gastrointestinal nematodes (GIN) present one of the greatest threats to the productivity and

sustainability of ruminant production systems throughout the globe (Perry et al. 2002). The

development of anthelmintics drugs between the 1960s and 1980s was a landmark in GIN control.

During this time, three different anthelmintics were produced each with a different mode of action

including: Benzimidazoles (BZ) which bind nematode tubulin to inhibit the formation of microtubules

and provoke cell lysis (Lacey 1988; McKellar & Jackson 2004); Levamisole (LEV) which acts as a

cholinergic agonist at nicotinic neuromuscular junctions and cause spastic paralysis (Robertson &

Martin 1993); macrocyclic lactones (Avermectins AVM) which act on glutamate gated ion channels

correlated to nematocidal activity (Brownlee et al. 1997; Feng et al. 2002) to cause a loose paralysis.

However, almost as soon as these anthelmintics were introduced reports of resistance emerged

(Kaplan 2004). Anthelmintic resistance (AHR) has since spread globally to become the norm in field

populations, including cases of multi-resistance against two or more of the compounds (Kaplan

2004).

The relative rapidity in which resistance developed against each of the three anthelmintics and the

rate in which it spread highlights the remarkable adaptive potential existing within GIN populations.

But how has being AHR affected their consequent adaptive potential in the face of new selective

pressures? This study explores how AHR GIN respond to selection pressures that are likely to be

present on farms in the near future, namely sheep selectively breed for GIN resistance and

temperature stresses associated with climate change. While substantial research has been carried

out in each of these areas individually i.e. AHR (Neveu et al. 2007, 2010; Cabaret et al. 2009; Boulin

et al. 2009) sheep resistance (Buitkamp et al. 1996; Beh et al. 2002; Davies et al. 2006; Kemper et al.

2011; Hassan et al. 2011; Sallé et al. 2012, 2014) and epidemiological consequences of climate

change (Morgan & van Dijk 2012; Gauly et al. 2013), no previous studies have considered the

interactions between all three factors.

Using the GIN Haemonchus contortus was as a model species this study addressed three main

questions: I) Is there AHR associated fitness costs in the absence of anthelmintic treatment? II) Does

AHR confer an advantage to survive in resistant sheep? III) Does AHR enable greater tolerance to

temperature stresses? Haemonchus contortus was selected as a model species for numerous

reasons. It arguably represents the greatest threat of all the common GIN species, with the highest

levels of AHR, prevalence (Cabaret 2000), prolificacy (Redman et al. 2008) and pathogenicity (Sadiqqi

et al. 2011). Furthermore, their prolificacy and large populations sizes have contributed to greater

52

genetic diversity in this species compared to other GIN (Blouin et al.1995; Troell et al. 2006; Gilleard

& Beech 2007; Redman et al. 2008).

The adaptability of three different H. contortus isolates with varying AHR capacities were compared

using phenotypic and gene expression measures. Phenotypic measures are based on fitness, a

concept central to natural selection and evolution. Fitness provides an indication of an organisms

combined survival and reproductive success in a single generation. This provides a means to draw

direct comparison between the isolates examined under different conditions. We further compare

the performance of the isolates at differing life-history traits to better understand how survival and

reproduction are affected by the interactions between AHR, sheep resistance and temperature

stress. The relative gene expression between the isolates is also compared using cDNA-AFLP to

provide an indication of the relative genetic divergence between them, and how this affects their

fitness. The results of this study provide new insight into how our current GIN populations are being

shaped by selective pressures in the context of GIN control.

Materials and methods

Haemonchus contortus isolates

Three different H. contortus isolates were selected for fitness comparison: i) ISE: an isolate

susceptible to all classes of anthelmintics (see Roos et al. 2004 for line history); ii) RHS6 aka BOR: an

isolate that had been selected experimentally for high levels of resistance against LEV (see Hoekstra

et al. 1997 for line history); iii) KOKISE: an isolate originating from the KOK isolate which is partly

resistant against the three main anthelmintic classes i.e. LEV, AVM, benzimizadole, originally

obtained from a farm in South Africa and kindly donated by J. Van Wyk in 2000. The levels of

resistance against each of these anthelmintics in the KOK isolate was determined previously based

on reductions in worm establishment (n = 6) compared to untreated control sheep (n = 6) (Table 2)

(Van Wyk unpublished data). The KOK isolate was introgressed with ISE in attempt to homogenize the

genetic backgrounds of the two isolates with the exception of their AHR status to produce KOKISE.

This was done in several stages. Firstly, sheep were infected with a dose of H. contortus consisting of

50% ISE and 50% KOK. The larvae produced from this infection were then used to supply 80% of the

consequent infective dose combined with 20% of the original KOK isolate. The sheep were then

treated with LEV and the surviving larvae collected for consequent passage. This was done over four

generations to produce KOKISE. The precise degree of LEV resistance in the all the isolates used in this

study was determined before the present infection by carrying out faecal egg count reduction tests

(FECRT) before and after LEV treatment on several occasions. The BOR isolate was also introgressed

53

with ISE i.e. BORISE but due to insufficiently low levels of L3 needed for this study, the BOR isolate was

used instead. None of the isolates had previously been exposed to resistant sheep prior to this study.

Sheep

The study used 12 Berrichon du Cher lambs and 18 Martinik Blackbelly (MBB) rams (originating from

populations bred in Bourges, France) based on their established susceptible and resistant status’

respectively to H. contortus infection (Terefe et al. 2008). The Berrichon du Cher lambs were four

months of age at the start of the experiment while the MBB rams were 18 months. Given young

lambs are known to be more susceptible to GIN infection (Kosi & Scott, 2001), the use of lambs

against adult sheep increased the probable susceptible and resistance status of the sheep groups. All

the sheep were maintained indoors prior to the start of the experiment and had no prior experience

of GIN infection as validated by negative faecal egg counts (FEC) at 0 days post infection (dpi). The

FEC indicator is the best available proxy to gauge GIN intensity in a sheep based on the number of

eggs it excretes.

Infection

Each of the H. contortus isolates was tested on four Berrichon du Cher lambs and six MMB rams.

Each sheep was infected with a single dose of 10,000 infective L3 larvae from their respective isolate

per os. The infection lasted 61 days after which time the sheep were necropsied following

electronarcoses and exsanguination. The 61 day period enabled ample time to collect parasitological

data and observe the stability of the isolates with time.

Sheep parameters

Blood samples were collected from all sheep prior to (0 dpi) and at the end of infection (53 dpi) to

determine the overall losses in haematocrit. Samples were drawn from the jugular vein of the sheep

into two dry tubes using a 10 ml gauge syringe. Haematocrit percentage was determined using a

manual Hawksely micro-haematocrit reader. The haematocrit level at 53 dpi was subtracted from 0

dpi to determine the total haematocrit loss. This was then divided by the number of worms counted

at necropsy to provide the haematocrit loss/worm as an indication of isolate pathogenicity. The

sheep were weighted using walk-on scales on 0 and 61 dpi.

Faecal egg counts

Faecal samples were collected from all sheep on 0, 19, 21, 27, 29, 30, 34, 40, 42, 58, 61 dpi to carry

out fecal egg counts (FEC) using a modified McMaster technique (Raynaud 1970) in a sodium chloride

flotation solution, accurate to 50 eggs per gram (EPG) of faeces.

54

Egg to L3 larvae development

The faeces collected on 27, 29 and 30 dpi were also used to test the capacity of the eggs to develop

into infective third stage larvae (L3). These days represent a moment when fitness should be at its

peak i.e. after the pre-patent period but before the consequent decline in FEC that is known to occur

with time (Fleming 1988).This was done for all four Berrichon du Cher lambs but due to the labour

intensive nature of this technique this was carried out on only three of the six MBB. The three rams

with the highest FEC were selected and thereby represented the maximum potential egg to L3

development capacity for that isolate. The same three rams were used for each of the three days

tested.

Egg to larval development assays were done by culturing 3 x 3 gram(g) faecal samples for each sheep

in three different climatic conditions: i) those deemed optimal for H. contortus development

(Rossanigo 1994) i.e. 23°C, 70% Humidity (H), 10 days ii) heat stress i.e. 28°C, 80% H, 5 days; iii) cold

stress i.e. 18°C, 60% H, 15 days. Exploring the capacity of L3 to develop under different climatic

conditions reflects their flexibility to respond to different conditions which is likely a favourable trait

for fitness, especially in view of the increasing effects of climate change. The L3 were then extracted

from each 3g sample of faecal matter using the Baermann funnel technique over a 24 h period at

room temperature (Baermann 1917) and counted under a microscope to obtain the number of L3

developed per 3 g of faeces. Only living L3 were included in the count. The number of L3 counted for

each 3 g sample was then divided by 3 to obtain the number of L3 per 1g of faeces. This was then

multiplied by the total quantity of faecal matter produced by the individual sheep in the day, as

calculated by QFM equation (see below).

Establishment

The capacity of the L3 to establish in their sheep host was determined by counting the number of

adult worms found in the abomasum at necropsy (61 dpi) following the procedure described by Gaba

et al. (2006). It was previously observed that resistant sheep can slow the developmental rate of H.

contortus with greater number of fourth stage larvae (L4) observed in the abomasal mucosa after the

21 day pre-patent period (Dawes 1975). Although the presence of L4 was unlikely as late as 61 dpi

such as in this study, the possibility was excluded by following measures normally used to extract L4.

In brief, the abomasal mucosa was incubated in water at 37°C for four hours, then washed

thoroughly in water which was saved, and then put through a 30µm mesh sieve to collect any L4.

Fecundity

55

The fecundity of the adult female worms was evaluated as described in Cabaret and Ouhelli (1984).

In brief, the total daily egg output per sheep was determined. Given the FEC only provide a measure

for 1g of faeces, the FEC on 61 dpi (just prior to necropsy) was multiplied by the total quantity of

faecal matter (QFM) produced in a day. The QFM was calculated using the following formula (back-

transformed from logarithm) developed specifically to the conditions of this study, based on a linear

regression between the logarithm of the weight of faecal excretion collected over a 24h period and

the logarithm of the metabolic weight (W0.75) of Berrichon du Cher lambs and MBB rams:

QFM= 0.041W0.75 p=0.00; r=0.95

Where W is the weight of the individual lamb (kg).

The total daily egg output (EPG x QFM) was divided by the number of female adult worms found at

necropsy to give the number of eggs produced per female per day.

Absolute Fitness

Absolute fitness for the control and desiccated H. contortus groups was determined to reflect their

capacity to survive and reproduce. This was done by multiplying the egg to L3 larvae development

ratios (i.e. number of L3 per 1g of faeces) by the QFM to provide the total number of living L3

produced in a day. This was done for three different days for all Berrichon du Cher lambs (n = 4 per

isolate), and the MBB rams selected for use in the L3 development assays (n = 3 per isolate) on 27,

29, 30 dpi. The number of L3 produced in a day was then divided by the number of L3 in the infective

dose i.e. 10,000 to provide the daily absolute fitness value for the H. contortus in each sheep.

Calculations of fitness were made based on egg to L3 development data under optimum, heat and

cold stress conditions.

Statistical analyses

Isolate fitness and life-traits were compared using a general linear model (GLM) with SPSS software.

The FEC and fitness variables were log transformed prior to analyses to normalize variance. The role

of the H. contortus isolate, susceptible and resistant sheep breeds, and their interaction were

evaluated. The principal component analysis( PCA) is a multivariate analysis used when variables are

quantitative (here the life-traits). It was modified since we took the sheep as variables (columns of

the data matrix) and their life-traits as lines of the data matrix .All the life-traits were centered

(difference from the average value) and standardized (this difference being divided by the standard –

deviation of the above difference) in order to give equal weight to the different life-traits. The axes

and graphic interpretation are similar to the correspondence analysis described below. The

56

significance of each axis was obtained by simulations (Lebart et al. 1982): for example a data matrix

of 10x20 similar to ours the first axis is significant at p0.05 when 30% of inertia are obtained, and

25% for the second axis

RNA extraction and double strand cDNA synthesis on H. contortus adult males

RNA extractions and consequent cDNA-AFLP analyses were carried out on five H. contortus isolates:

ISE, BOR and KOKISE following infection on resistant MBB sheep, and BORISE/C (introgressed with ISE)

and KOKISE/C were selected as controls from a prior study following infection on susceptible sheep.

For each H. contortus isolate, the RNA from 10 adult males which had previously been stored at -80°C

in RNAlater was homogenized in Trizol reagent (Life Technologies) and the total RNA was isolated as

per the manufacturer’s instructions. RNA pellets were dissolved in 50 µL of RNAse free water and

stored at -80°C. Each sample was treated with Dnase I (Promega), according to the manufacturer

instructions, 30min at 37°C, to avoid genomic DNA contaminations.

The synthesis of the first strand cDNA was performed in a mix containing 5 µg of total RNA, 5µM of

d(T)25 primer 0.5mM of each dNTP and a sufficient quantity of of RNAse free water to complete at

13µL. Each sample was heated for 10 min at 70°C and set on ice. A 1x superscript buffer, 40U of

RNasin (Promega), 5µM of dithiothreitol (DTT) and 200U of superscript II (Invitrogen) were then

added. The mix was incubated at 42°C for 1h, then at 70°C for 15min and stored at -20°C until use.

The second cDNA strand was synthesized in a mix containing 20µL of the first strand cDNA, 1x second

strand buffer (NEB2 buffer), 0.2mM of each dNTP,20µM of DTT, 40U of E. coli DNA Polymerase I

(NEB), 15U of E. coli DNA ligase (NEB), 6U of RNAse H (NEB) in 150µL of sterile distilled water. The

mix was incubated at 16°C for 2h. The double stranded cDNA was then purified using

phenol/chloroform/isoamyl alcohol (25:24:1) and resuspend in 40µL of sterile distilled water.

cDNA-AFLP experiments

The cDNA samples were digested with HindIII/MseI (NEB), restriction fragments were ligated

with their corresponding adapters (Semblat et al.2000) and pre-amplification was carried out during

30 cycles (94°C 30s, 55°C 45s, 72°C 60s) using primers without selective nucleotide (H+0: 5'

GACTGCGTACCAGCTT 3', M+0: 5' GATGAGTCCTGAGTAA 3'). PCR was then performed with selective

primers (detailed below) and a trace amount of [γ33P]-labelled 5' primer using the following program:

94°C 2 min, 14 cycles (94°C 30s; 60°C – 56°C 30s (the temperature decreasing slightly after each cycle

to reach 56°C by the 14th cycle) 72°C 1min), 24 cycles (94°C 30s, 56°C 30s,72°C 1min), 72°C 5 min. The

amplification products were separated on a 5% polyacrylamide gel and analyzed after exposure to X-

ray film overnight.

57

Several combinations of primers were used for cDNA-AFLP but only 16 were selected for consequent

analyses on the basis they provided the clearest band profiles. These included HA + M12, M14, M15,

M16, M17, M18 M19, M20, M24, M26; HT + M15, M16, M19; HT + M16, M23, M25. Details of these

primers can be viewed in Table 1. All together, 689 bands were compared. Polymorphic fragments

were coded in binary by their presence or absence for each isolate. The closer the isolates are

positioned on the graph, the greater their genetic similarity. The inertia of each axis corresponds to

the part of variance explained by the axis. Each axis corresponds to a synthetic variable which result

from combination of variables in such a way that it maximize the inertia. Each axis is independent

from the others and thus brings its own information. Since the number of bands was important, the

explanatory value of each of the three axes was high and they were significant (Lebart and Fenelon,

1982).

Table 1. Primer sequences used in cDNA-AFLP analyses

Primer name 5' - 3'

M0 GATGAGTCCTGAGTAA

H0 GACTGCGTACCAGCTT

H+A GACTGCGTACCAGCTTA

H+T GACTGCGTACCAGCTTT

M12 GATGAGTCCTGAGTAAAC

M14 GATGAGTCCTGAGTAAAT

M15 GATGAGTCCTGAGTAACA

M16 GATGAGTCCTGAGTAACC

M17 GATGAGTCCTGAGTAACG

M18 GATGAGTCCTGAGTAACT

M19 GATGAGTCCTGAGTAAGA

M20 GATGAGTCCTGAGTAAGC

M23 GATGAGTCCTGAGTAATA

M24 GATGAGTCCTGAGTAATC

M25 GATGAGTCCTGAGTAATG

M26 GATGAGTCCTGAGTAATT

Results

Level of anthelmintic resistance within H. contortus isolates

58

The level of anthelmintic resistance in the original KOK isolate was established by van Wyk based on

worm count reductions in treated sheep compared to untreated controls (unpublished data). Clear

resistance was observed against Fenbendazole, Ivermectin (IVM) and macrocyclic lactones, and to a

lesser extent, LEV and Moxidectin (Table 1). This KOK isolate was then further selected for LEV

resistance (Table 2) and considered to be multi-resistant to all thee major classes of anthelmintic.

Table 2. Level of resistance against various anthelmintics, formulations and routes of administration

for the H. contortus KOK isolate (based on unpublished results of J. van Wyk). The % worm reductions

compared establishment in six infected control sheep against six infected sheep treated with

anthelmintics. Level of LEV resistance denoted as high*** (≥ 10% worm reduction) medium** (10-

30% worm reduction) and low*(30-100% worm reduction). Isolates highlighted in bold are those

used in the fitness experiments.

Anthelmintic Administration % worm count reductions Level of

resistance

Fenbendazole Per os < 10% ***

Levamisole Per os 82% *

Macrocyclic lactones Sub-cutaneous < 30% **

Ivermectin (7 formulations) Sub-cutaneous < 30% (all 7) **

Moxidectin Per os 93% *

Subcutaneous 89% *

All the isolates used in this study were tested for their level of resistance against LEV based on fecal

egg count reduction tests (FECRT) which compare FEC before (n = 2) and after (n = 3) LEV treatment

(Table 3). The KOK had an above average resistance against LEV but this resistance was lost in the

KOKISE introgressed isolate. The ISE isolate was completely susceptible to LEV.

Table 3. Level of LEV resistance based on FECRT, denoted as high*** (> 10% FEC reduction)

medium** (10-25% FEC reduction) and low*(25-100% FEC reduction) resistance. Isolates highlighted

in bold are those used in the fitness experiments.

H. contortus

isolate

No. of sheep LEV

selected in

FEC before LEV

treatment

% FEC reduction

after treatment

Level of resistance

against LEV

ISE 4 8587 100 *

BOR 1 7983 -16 ***

KOK 3 8755 18 **

59

KOKISE 4 21275 78 ***

Genetic differentiation of H. contortus isolates

Analyses of the partial transcriptome selected from the combinations of primers that provided the

clearest banding profiles presented 689 bands aka transcriptome derived fragments (TDFs). The

number of TDFs present in each isolate was as follows: KOKISE = 459 > KOK + 418 > ISE = 401 > BOR =

390. Correspondence analyses comparing the pattern of transcriptomic expression between the

isolates showed that the three isolates used in this study were significantly distinct genetically (Fig.

1). The three-dimensional axes reflect a linear combination of all variables examined (i.e. bands)

representing a similar value of inertia (37, 35 and 28% of the total variance). There was significant

genetic differences between ISE (susceptible) BOR (LEV resistant) and KOKISE (multi-resistant) isolates.

However KOKISE exhibited similar transcriptomic expression to the original KOK isolate prior to

introgression.

Fig. 1. Correspondence analysis to represent the transcriptomic patterns of the H. contortus isolates

based on cDNA-AFLP analyses.

Fitness performance of H. contortus isolates

The fitness measure reflects the relative success of the isolate in one generation i.e. the number of

L3 offspring produced divided by the initial L3 infective dose. Fitness was examined for the three

60

isolates under varying combinations of potential stress. First by exposing the parasitic stages (L3-

adult) to either susceptible or resistant hosts, and then by exposing the free-living stages (egg-L3) to

differing temperatures, including those which are optimal for development, and those which were

both colder and hotter than optimum. Thus, isolate fitness was examined under six different

conditions i.e. 3 climatic conditions (optimum, heat and cold stress) x 2 sheep conditions (susceptible

and resistance). Isolate fitness varied significantly between the isolates under all three temperature

conditions (Table 4). Resistant sheep only significantly impacted isolate fitness in cold stress

conditions (Table 4). The ISE isolate had the greatest fitness in 5/6 of the cases and the worst in 0/6.

KOKISE performed the best in 1/6 cases and the worst in 2/6. BOR performed the best in 0/6 cases and

the worst in 4/6 (Table 5). Thus globally, ISE had the greatest fitness, followed by KOKISE then lastly by

BOR. In all the isolates, fitness was significantly reduced when the free-living stages were exposed to

both heat and cold stress compared to optimal conditions (Table 4 and 5).

Influence of the host genetics on H. contortus isolate fitness

The isolates varied significantly in their performance at each life-history trait examined in susceptible

sheep. Exposure to resistant sheep changed their fitness profiles completely (Table 5). In susceptible

sheep ISE had the best establishment capacity and the worst fecundity and egg-L3 development in

optimal temperature and cold stress. In resistant sheep, ISE had the greatest fecundity and the

greatest egg-L3 development in all three temperature conditions. In susceptible sheep KOKISE had the

greatest fecundity, but in resistant sheep they had the greatest establishment. In both susceptible

and resistant sheep they had low capacity for egg-L3 development under heat stress. Finally BOR had

the greatest egg-L3 development in susceptible sheep under all three temperatures, but consistently

performed worse than the other isolates at all life-history stages in resistant sheep.

H. contortus isolate pathogenicity

The BOR isolate consistently provoked the greatest drop in host haematocrit/worm levels in both

susceptible and resistant sheep, despite their very low establishment (Table 4). For both BOR and ISE,

the haematocrit loss increased from susceptible to resistant sheep from 21.1 to 31 in BOR and 0.3 –

18.1 in ISE. KOKISE provoked comparatively little losses going from 1.7 in susceptible sheep to 1.4 in

resistant.

61

Table 3. Fitness and life-trait results for each H. contortus isolate in both susceptible and resistant sheep. ISE = anthelmintic susceptible; BOR = LEV-resistant,

KOKISE = multi-AH resistant. Based on analysis from General Linearized Models where p > 0.05. Number of repeated samples from which means were made

:fitness n = 9; L3 development n = 9; FEC n = 10. All others, n = 1.

Life history trait Susceptible sheep Resistant sheep Statistics ISE BOR KOK ISE BOR KOK P

value Breed Isolate Breed*Isolate

Fitness Optimum 91.2 ± 62.6

19.5 ± 27.8

58.2 ± 8.6 90.8 ± 136.6

26.5 ± 20.7

35.9 ± 21.7

0.170 0.84 0.08 0.41

Heat stress 18.2 ± 21 10 ± 7.6 0.9 ± 1.2 8.8 ± 2.2 9 ± 8.8 6.5 ± 4.5 0.020 0.13 0.03 0.08 Cold stress 5 ± 3.1 1.8 ± 3.2 1.7 ± 0.9 27.6 ± 28.8 9.2 ± 7.2 33.6 ±

31.4 0.000 0.00 0.04 0.5

Parasitic Establishment 43.1± 25.3

1.2 ±0.8 12.6 ±8.1 4.7±6.3 1.4 ± 2.5 6.8 ±5.8 0.000 0.007 0.001 0.101

Fecundity 1633 ± 1620

15122 ± 21280

16554 ± 18701

13543 ± 17512

1141 ± 1094

6584 ± 12568

0.291 0.078 0.773 0.290

Female ratio 48 ± 3 40 ± 10 46 ± 8 32 ± 24 57 ± 12 53 ± 12 0.066 0.643 0.269 0.047 Free-living

Egg-L3 - Optimal 505 ± 246 4655 ± 7029

3235 ± 4949

14668 ± 15349

2971 ± 3347

1638 ± 778

0.190 0.252 0.384 0.081

- Heat stress 129 ± 136 2495 ± 1929

24 ± 24 4522 ± 7254

501 ± 288

153 ± 83 0.005 0.075 0.010 0.049

- Cold stress 30 ± 17 448 ± 798 76 ± 106 5385 ± 4415

813 ± 583

1440 ± 1354

0.002 0.000 0.943 0.247

Virulence Haematocrit/worm 0.3 ± 8.6

12.1 ± 8.6 1.7 ± 8.6 18.1 ± 7.1 31.0 ± 7.0

1.4 ±7.7 0.000 0.02 0.000 0.044

Other FEC 11299 ± 6989

2940 ± 4959

4290 ± 936

1765 ± 1513

216 ± 320

517 ± 386 0.000 0.001 0.00 0.811

62

Table 4. Visual flow chart of fitness and life-traits for each H. contortus isolate in both susceptible and resistant sheep (adapted from Table 3). ISE =

anthelmintic susceptible; BOR = LEV-resistant, KOKISE = multi-AH resistant. Life-trait and fitness colour-coded according to strength i.e. black – high, grey –

medium; white – low value.

Life history trait Susceptible sheep Resistant sheep Statistics

ISE BOR KOK ISE BOR KOK P value Breed Isolate Breed*Isolate

Fitness Optimum 0.170 0.84 0.08 0.41

Heat stress 0.020 0.13 0.03 0.08

Cold stress 0.000 0.00 0.04 0.5

Parasitic stages Establishment 0.000 0.007 0.001 0.101

Fecundity 0.291 0.078 0.773 0.290

Female ratio 0.066 0.643 0.269 0.047

Free-living stages L3 develop - Optimal 0.190 0.252 0.384 0.081

- Heat stress 0.005 0.075 0.010 0.049

- Cold stress 0.002 0.000 0.943 0.247

Virulence Haematocrit/worm 0.000 0.02 0.000 0.044

Other FEC 0.000 0.001 0.00 0.811

63

Relationship between life-traits vs H. contortus isolates and sheep (susceptible and resistant)

The two PCA graphs show the relationship between the life-traits in relation to the isolates (Fig. 2)

and to the sheep (susceptible and resistant) (Fig. 3). For both graphs, the first axis was a synthetic

variable; to the left haematocrit represents host damage, and to the right FEC, establishment with

male and female worms and egg to L3 development under heat stress conditions. The second axis

represents high egg to L3 development under optimum and cold stress conditions at the top of the

graph, and to a lesser extent, worm fecundity in the lower part of the graph. Some traits were

therefore related. Male and female establishment with FEC and egg to L3 development under heat

stress conditions. Fecundity was not related to FEC nor to egg to L3 development in optimum and

cold stress. Haematocrit (representing host damage) was not related to establishment of male and

female worms.

The PCA of the H. contortus isolates (Fig. 2) shows the highest haematocrit (host damage) was

associated with BOR, although it had lower establishment of males and female worms. The ISE

isolate demonstrates a highly variable performance in the different life-traits compared to the other

two graphs. The KOKISE isolate was located near the origin of the graph demonstrating well balanced

capacities in all the traits.

The PCA of the sheep (susceptible and resistant) (Fig. 3) show the susceptible sheep had good

establishment of male and female worms, good development of egg to L3 under heat stress, but not

under optimum or cold stress. The resistant sheep experienced greater haematocrit loss (host

damage) than the susceptible, although they had less worms.

64

Fig. 2. PCA relationship between H. contortus isolates and life-traits. Data centred and standardized.

Axes represent 60% of variance.

65

Fig. 3. PCA relationship between sheep (susceptible and resistant) and H. contortus life-traits.

Discussion

Four key finding emerged from the results of this study: I) The three H. contortus isolates expressed

different patterns of transcript derived fragments (TDFs) with the greatest number in KOKISE > ISE >

BOR; II) The three isolates differed significantly in their fitness which was greatest in ISE > KOKISE >

BOR; III) Resistant sheep did not influence the relative fitness of each of the isolates, but they did

change the isolates performance at each of the life-history traits; IV) Temperature stress greatly

reduced fitness of all H. contortus isolates. These results shall be discussed in turn.

The cDNA-AFLP results demonstrate the three isolates had truly divergent transcriptomes associated

with their different AHR status. From the section of the transcriptome examined with the primers in

this study, a total of 689 TDFs were expressed across all three isolates. Of these, the KOKISE isolate

expressed the most (459 TDFs), followed by ISE (401 TDFs) and then BOR (390 TDFs). Given the AHR

isolate KOKISE and BOR both experienced significant reductions in fitness compared to the AH

susceptible isolate ISE, we may have expected to see associated decreased expression of the

transcriptome in the AHR isolates. Studies exploring the transcriptomic response of a multi-AHR T.

circumcincta isolate in response to IVM exposure found significant reductions in the expression of

66

genes linked to metabolism, nematode specific proteins associated with parasite establishment and

maintenance, and vitellogenin production compared to those not exposed to IVM (Dicker et al.

2011). The study proposed a reduction in metabolism may have been an attempt by the IVM

exposed T. circumcincta to stop or down regulate any non-essential cellular processes; the reduction

in nematode specific proteins may reduce parasitic behavior; and the reduction in vitellogenin

suggests a reduction in egg production (Dicker et al. 2011). We could speculate that this would also

lead to a reduction in fitness for the IVM treated T. cirmcumcincta. Interestingly in this study the AHR

isolates represented the extremes of transcriptome expression; while the multi-AHR KOKISE expressed

the greatest number of TDFs, the LEV-R BOR expressed the least. There are two possible explanations

for this.

Firstly, it is possible that resistance against each of the different anthelmintics does not have an

equal impact on the transcriptome of H. contortus. While the original KOK isolate had resistance

against LEV, following introgression with ISE to form KOKISE they exhibited much higher susceptibility

to LEV (78% FECRT following LEV treatment). We do not know how this affected resistance against

either BZ or AVM in KOK. BOR on the other hand maintained potent levels of LEV resistance (-16%

FECRT following LEV treatment). It is possible that resistance against LEV specifically is limiting to

transcriptomic expression. It is important to highlight however that the portion of the transcriptome

explored in this study was limited to 689 TDFs. Results from an RNA-sequencing study analyzing gene

expression in H. contortus males identified 15,569 genes are significantly expressed at this time

(Laing et al. 2013). Had we compared the complete transcriptome of all three isolates, the results

may differ.

Secondly, differences in the transcriptomic expression between the isolates may be impacted by

their different experimental history in the laboratory. While ISE had been maintained in laboratory

conditions optimal for survival for several years, the lack of challenge from selective pressures may

have reduced transcriptomic expression. Comparison of alloenymes between field and laboratory T.

circumcincta isolates demonstrated losses in genetic diversity following laboratory maintenance

(Gasiner et al. 1992). The BOR isolate experienced a strong founder effect after initial experimental

selection for LEV resistance reduced the population to 1000 adult worms. Although the BOR had

since been amplified in several sheep over several generations to recover some of its genetic

variation through sexual reproduction, we cannot exclude that this may have contributed to the

isolates reduced TDF expression and reduced fitness. The KOK isolate obtained its multi-AHR status

on the field and likely represent a more ‘normal’ variability in H. contortus gene expression.

67

Interestingly, the introgression of KOK with ISE to produce KOKISE resulted in greater number of TDF

expression for this isolate than either of the parent isolates.

The fitness of the three H. contortus isolates was tested in six different conditions: exposing the

parasitic stages to susceptible or resistant sheep then exposing the free-living stages to different

climatic conditions i.e. optimum, heat or cold stress. The ISE anthelmlmintic susceptible isolate had

the greatest fitness in 5/6 of these conditions. The KOKISE isolate arguably had greater fitness than

BOR in the most of these conditions. These results suggest a fitness cost as a function of AHR. Several

other studies have noted reductions in GIN life-history traits as a function of AHR i.e. reduced

establishment and egg viability in BZ resistant H. contortus (Maingi et al. 1990); reduced egg – L3

development in BZ, ivermectin and salicylanilides resistant H. contortus (Scott & Armour 1991);

reduced egg-L3 development in IVM resistant H. contortus (Echevarria et al. 1993); reduced

establishment in BZ resistant Trichostrongylus colubriformis (Maclean et al. 1987). Only one example

to the contrary was found whereby BZ resistant H. contortus resulted in increased establishment and

egg-L3 development (Kelly 1978).

A novel element to this research was to explore whether there was an interaction between AHR

isolates and their capacity to survive in resistant sheep. If AHR conferred greater survival in resistant

sheep, this would bode ill for the future use of selectively breed sheep facing AHR populations of

GIN. This however was not found to be the case in this study. Yet neither do the results provide

strong support for the efficacy of resistant sheep in controlling infection. While the resistant sheep

did significantly reduce establishment of all three H. contortus isolates compared to susceptible

sheep, their overall fitness was not affected in optimum or heat stress conditions. This demonstrates

the capacity for GIN to alter life-history traits in the face of selective pressures to maintain overall

fitness (Poulin 1998). In turn this would suggest that selectively breeding resistant sheep based on

reduced GIN establishment may not have an overwhelming impact on the GIN populations owing to

their capacity to compensate later.

Exposure to resistant sheep decidedly changed the isolates’ performance at each of life-history traits.

For example, ISE appeared to exploit the lack of protective response in susceptible sheep by attaining

high levels of establishment (43%) which was greatly reduced in resistant sheep (4.7%). However,

following the challenge of resistant sheep on their establishment capacity, ISE experienced an 8-fold

increase in fecundity and a 29-fold increase in egg to L3 development under optimum conditions

(with similar increases under heat and cold stress) compared to their performance in susceptible

sheep. This contrasted sharply with what was observed for the AHR isolates. Firstly, BOR and KOKISE

68

had a significantly reduced capacity to establish compared to ISE, with 1.2% and 12.6% respectively

in susceptible sheep. Resistant sheep reduced the establishment capacity of KOKISE (6.8%), but not

BOR (1.4%). Unlike ISE, neither BOR nor KOKISE responded to resistant sheep by altering their

consequent life-history traits. Instead, BOR had a 13-fold reduction in fecundity and a 1.5-fold

reduction in egg-L3 development (optimum conditions) and KOK had a 2-fold reduction in fecundity

and almost a 2-fold reduction in egg to L3-development (optimum conditions).

These results suggest ISE had greater plasticity to respond phenotypically to the selective pressure of

resistant sheep. Phenotypic plasticity is the ability of a single genotype to give rise to different

phenotypes in different environments (Bradshaw 1965) allowing organisms to respond rapidly to

changing environmental conditions without the time lag required for natural selection (Zhou et al.

2012). Molecular studies have shown that the reactions of phenotypic plasticity are not just passive

responses of genetic-physiological machinery, but are highly specific and coordinated by an array of

regulatory genes acting at different hierarchical levels (Pigliucci 1996). It is possible that the AHR

isolates BOR and KOKISE had reduced expression of transcripts which enable plasticity.

A striking difference between the isolates was observed in their pathogenicity measured as a

function of haematocrit losses provoked per worm. Individual BOR worms induced significantly

greater losses than either ISE or KOKISE in both resistant and susceptible sheep suggesting BOR were

more pathogenic. Resistant sheep augmented the pathogenicity of BOR 2.5-fold and ISE 60-fold. This

massive increase in blood loss for ISE may result from an increased need for resources to enable their

increased fecundities. Pathogenicity of KOK was low in both susceptible and resistant sheep. Overall,

the LEV-resistant BOR isolate was unquestionably the most pathogenic. Studies by Kelly et al. (1978)

also found pathological changes to be worse in sheep infected with BZ-resistant H. contortus as

measured by haematocrit, plasma protein concentration and haemoglobin levels. It has been

suggested that greater virulence in strongylid nematodes such as H. contortus does not improve

transmission fitness (Medica & Sukhedo 2001) inferring another fitness cost for the BOR isolate.

The reduced capacity for the eggs to develop into L3 under both heat and cold stress negatively

impacted the fitness of all three isolates. There was however a limitation to these measurements.

Given our fitness measurements only followed one complete generation, from L3 (infective dose) to

L3 (offspring), this did not account for the possible compensatory response that may have followed

at consequent life-history traits. It would therefore be interesting to follow the isolates over two

generations. This would reveal whether the free-living stages could respond to selective pressures as

was seen for the parasitic stages i.e. ISE responded to reduced establishment in resistant sheep with

increased fecundity and egg to L3 development. It would also be interesting to determine if the

69

alterations in life-history traits observed in the isolates persisted into the second generation by

passaging the isolates from both the susceptible and resistant sheep on only susceptible sheep to

compare their responses.

In conclusion the results of this study imply that AHR H. contortus incur fitness related costs. Given

the multi-AHR KOKISE expressed the greatest number of TDFs, this suggests fitness was not directly

related to the section of the transcriptome examined in this study. Admittedly, it is difficult to strictly

separate whether the KOKISE isolates transcriptomic diversity and associated fitness was a result of its

AHR status, or the ISE introgression. Nonetheless, the AHR associated fitness costs would suggest this

is a promising moment to maximize on other control options while AHR GIN populations are on the

field. Caution should be applied should control include sheep selectively breed for resistance as it

appears their effect on H. contortus fitness was minimal, while the cost of infection was amplified in

the LEV-resistant BOR. Fitness costs in AHR isolates would suggest hope for reversion to

susceptibility. Yet others have found that with the co-existence of increasing selective pressure by

levamisole on one side and reversion of resistance due to reduce fitness of resistant worms

during the periods without treatment may result in a stable level of anthelmintic resistance as

observed in north-east Brazil. Thus Vieira and Cavalcante (1999) and later Bevilaqua et al.

(personnal communication, 2010) in the same state of Ceara obtained similar values for farms

presenting resistance. Both heat and cold stress were powerful controls on the fitness of H.

contortus ireespective of isolate. This suggests moments of heat or cold stress are particualrly

opportune moments to maximize on their reduced fitness with other GIN control practices such

as nematophagous fungi or grazing rotation strategies. The costs in terms of genetic expression

and fitness were most evident for LEV-resistant BOR. This may question the potential value of

intentionally continuing the spread of LEV-resistant populations to enable easier control and

reduced infectivity, although it risks greater pathogenicity.

Acknowledgments

C. Chylinski is a grateful recipient of Marie Curie ITN project NematodeSystemsHealth funding.

Authors would like to thanks Thierry Chaumeil and the PFIE team for thier assistance in

managing the animals.

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76

Resistant sheep select for increased fitness in their parasitic nematodes (Teladorsagia

circumcincta): experimental evidence

E. Schmidt1 a, b,, C. Chylinski1 b, c*, L. Gruner b, J. Cabaret b, c*

a Facultad de Ciencias Veterinarias, Producción animal, Universidad Nacional de La Pampa,

Calle 5 esq. 116 S/N, General Pico, Argentina

b INRA, UMR 1282 Infectiologie et Santé Publique, F-37380 Nouzilly, France

c Université de Tours, UMR 1282 Infectiologie et Santé Publique, F-37000 Tours, France

1These authors contributed equally to the work.

Abstract

Gastrointestinal nematode (GIN) species threaten the production and sustainability

of ruminant farming worldwide. Sheep selectively bred for resistance against GIN infection

offers a promising method of control however, this may also exert a strong selective

pressure on the GIN and provoke an adaptive response. This study explored whether the GIN

Teladorsagia circumcincta would adapt to the challenge of resistant sheep. Adaptation was

measured in terms of fitness i.e. their ability to survive and reproduce. Comparisons of

fitness using life-history biology traits were drawn between two lines of T. circumcincta; one

which had been passaged through three generations of highly resistant sheep and the other

which had been maintained in susceptible sheep. The results showed exposure to resistant

sheep significantly increased the relative fitness of the T. circumcincta. While those traits

pertaining to reproduction (fecundity) remained unchanged, the capacity to survive was

significantly augmented both in the parasitic phase (establishment of infective larvae) and

external free-living stages (egg to larval development ratio). The results of this study provide

the first known demonstration of GIN adapting to resistant sheep hosts. These results carry

important implications for selective breeding as a method of control.

77

Introduction

Gastrointestinal nematodes (GIN) are notoriously difficult to control. Given they

present a significant threat to the production and future sustainability of ruminant farming

systems worldwide, substantial efforts have been made to develop control methods.

Anthelmintic drugs have had the most success to date however, their extensive use has

driven adaptation in the GIN to the point where resistant populations are now widespread

(Kaplan, 2004). Alternative control methods have been proposed which target different life

stages of the GIN. Examples include predatory fungi (Waller and Thamsborg, 2005), grazing

management strategies (Barger, 1997), host nutritional supplementation (Coop and

Kyriazakis, 2001) and sheep selectively breed for resistance against GIN infection. Selective

breeding is emerging as one of the most promising control options and works on the

premise of harnessing heritable traits of GIN resistance which exist within, and between,

sheep breeds. It offers advantages such as increased production and welfare as well as

reduced pasture contamination rates and the need for chemical controls (Stear et al., 2012).

Nonetheless, this selection process would likely homogenize the genetic variability present

in a flock of sheep and exert a strong selective pressure on the GIN population. This in turn

may provoke an adaptive response in the GIN which have benefits such as extensive genetic

variability, short generation times and high fecundities (Gilleard and Beech, 2007) to draw

from. This study explored whether the challenge of resistant sheep would induce an

adaptive response in GIN using Teladorsagia circumcincta as a model species.

Several previous studies have tried, and failed, to demonstrate adaptation of GIN to

resistant sheep (Adams, 1988; Albers and Burgess, 1988; Kemper et al., 2009; Saulai et al.,

2010). However these studies all relied on the use of a single indicator to reflect adaptation,

faecal egg counts (FEC). This over-looked other important life-history traits which nematodes

are thought to alter in the face of challenge (Poulin, 1998). This study measured adaptation

in terms of fitness i.e. the ability to survive and reproduce. The aim was to compare the

fitness of two T. circumcincta lines, one which had been exposed to several generations of

highly resistance sheep and the other which had been maintained in susceptible hosts. The

life-history traits measured were divided into those pertaining to reproduction i.e. fecundity,

and those relative to survival i.e. adult establishment in the abomasum from the ingested

infective larvae (internal parasitic phase) and the development from the egg to infective

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larvae (external free living phase). We hypothesized that while the resistant sheep will

control the infection better than the susceptible sheep, they will simultaneously select for

populations of T. circumcincta with increased fitness.


Intensive divergent selection of susceptible and resistant sheep

Sheep highly resistant to T. circumcincta infection were obtained from a prior

intensive divergent selection process carried out over three generations on pasture (see

Gruner et al., 2004 for details). In brief, a flock of 100 Romanov (susceptible breed) 6 month

old rams were grazed on a T. circumcincta contaminated pasture for five months. Based on

faecal egg counts (FEC) expressed in egg per gram (EPG) taken at 22, 25 and 28 days post-

infection (dpi) the rams with the 5-lowest and 5-highest FEC were mated with unselected

Romanov ewes. From the resulting offspring, 96 ram lambs were once again challenged on

pasture as before. The rams with the 5-lowest and 8-highest FEC were mated with

unselected Romanov ewes. The resulting 43 ewe offspring were again grazed on

contaminated pasture and at the end of grazing season the two ewes with the lowest FEC

(i.e. resistant: 0-150 EPG) and the two ewes with the highest FEC (i.e. susceptible: 600-800

EPG) were selected to produce the first generation of susceptible and resistant T.

circumcincta (see below). Three more resistant ewes were kept for subsequent experimental

infection.

Experimental selection of susceptible and resistant T. circumcincta lines

The T. circumcincta eggs produced from the two susceptible ewes and from two of

the five resistant ewes from the intensive divergent selection process were collected and

cultured under conditions favourable to T. circumcincta development; 23°C, 80% humidity

for 10 days (Rossanigo, 1992). This produced the first generation of susceptible and resistant

T. circumcincta lines. These four ewes were then necropsied and their respective resistance

statuses verified by counting the number of adult worms in the abomasum (susceptible

ewes: 4100-4250 worms; resistant ewes: 200-400 worms).

The susceptible and resistant T. circumcincta lines were selected further with two

experimental passages through susceptible and resistant hosts. At each passage, the

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susceptible line was passaged through two naive Ile-de-France male lambs, three months of

age. Using new animals at each passage minimized the chance of the susceptible T.

circumcincta line encountering acquired resistance within their host. The resistant line on

the other hand, was passaged through the same three resistant ewes for both passages.

Prior to each infection, the resistant ewes were treated with the pro-benzimizidazole based

anthelmintic Netobimin following the manufacturers’ instructions and left for 15 days. The

nematodes did not present any resistance to benzimidazole treatment and were efficiently

removed before the next infection.

For both experimental selections, each animal received an infective does of 4000 L3

T. circumcincta larvae from their respective resistant and susceptible lines. For the first

experimental selection, L3 cultured from the intensive divergent selection process were

used. For the second experimental selection, L3 cultured from the first experimental

selection were used. For the final test on immunosuppressed lambs, L3 cultured from the

second experimental selection were used.

Comparison of fitness

The final third generation susceptible and resistant T. circumcincta lines were

compared by passaging them through 3 month old Ile-de-France male lambs. To homogenize

the host response to infection, the lambs were immunosuppressed using the long-term

action dexamethasone as per the manufacturers’ instructions, one day before and 8 days

after infection. This ensured any potential lamb immune responses were constrained for the

entire period (2 weeks) in which T. circumcincta could potentially establish. Six lambs were

infected with the resistant line; two received an infective dose of 2000 L3 larvae and the

remaining four received a dose of 1200 L3 larvae. These variations were due to the limited

number of eggs that were produced by the resistant sheep and cultured following the

second passage. The remaining four lambs were infected with the susceptible line, each

receiving 2000 L3 larvae.

Fitness measurements were taken for both the susceptible and resistant line during

the first and second experimental passage and for the test on immunosuppressed lambs.

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Faecal egg counts

Faecal egg counts were carried out at 21 and 50 days post-infection (dpi) for both the

experimental selections and for the test on immunosuppressed lambs. A modified McMaster

technique (Raynaud, 1970) was used in a magnesium sulphate flotation solution, accurate to

50 EPG of faeces. The FEC were log transformed (log x+1) prior to ANOVA comparisons to

stabilize the variance of the highly dispersed data.

Quantity of faecal matter

Several of the measurements in this study rely upon the individual sheep FEC to make

the calculations. However, as the FEC only provides a count for the number of eggs produced

in a 1 g concentration of faeces, it was necessary to calculate the total quantity of faecal

matter (QFM) produced by a sheep in a day to ascertain the daily FEC. This was done using

the following formula developed specifically to the conditions of this study:

QMF= 0.041W0.75 p=0.00; r=0.95

Where W is the weight of the individual sheep (kg).

Egg to larvae development ratio

The capacity of the eggs to develop into infective L3 larvae was tested under three

different climatic conditions to reflect local seasonal temperate climates as described by

Rossanigo (1992) including; winter: 4°C, 70% Humidity (H), 15 days; spring: 23°C, 70%H, 10

days; summer: 28°C, 60%H, 5 days. For each condition, 5 x 5g faecal samples were cultured

from the susceptible and resistant sheep 5first and second experimental selection) and from

the immunosuppressed lambs over a 10 day period (between 21-50 dpi). After the allotted

time period, the surviving L3 larvae were separated from the faecal matter using the

Baermann funnel technique (Baermann, 1917) and counted under a microscope to obtain

the number of larvae developed per 5 g of faeces. The number of larvae counted was then

divided by the respective FEC (multiplied to 5g) to ascertain the ratio of larvae developing

from the T. circumcincta eggs.

Establishment of infective larvae

81

The number of adult worms in the abomasum at necropsy (50 dpi) were counted

following the procedure described by Gaba et al. (2006). Fourth stage larvae were also

counted. These were extracted from the mucosa by leaving the abomasum in 37°C water for

four hours. The establishment rate was determined by dividing the number of adults and L4

larvae counted by the number of L3 in the infective dose.

Fecundity

The fecundity of the resistant and susceptible nematode lines was calculated using

the following formula:

Eggs per gram of faeces (50 dpi) x total quantity of host faecal matter

Number of females present in the host abomasum

Absolute (W) and relative fitness (w)

Absolute fitness (W) is a measure of the genotypes’ capacity to survive and

reproduce i.e. a culmination of their life traits. It refers to the number of reproductive units

created per certain genotype within a population. Thus, if one sheep produced 1000 L3

larvae from an original infective dose of 100 L3, the fitness is 10. Genotype refers to the

individuals with common genetic characteristics i.e. those T. circumcincta lines which have

been selected by the environmental pressures of a susceptible or resistant host. Thus, we

measured the performance of the susceptible and resistant lines of T. circumcincta after one

generation in the dexamethasone treated lambs.

W = Number of L3 larvae produced by sheep host from day 21-50 dpi (second generation)

Infective dose of L3 larvae (first generation)

Absolute fitness values (W) were determined for each of the three climatic conditions

tested (winter, spring, summer). In order to compare the two competing genotypes from the

susceptible and resistant hosts, the relative fitness (w) was determined. In this case the

susceptible genotype was normalized (w=1) and the fitness of the resistant genotype

measured in respect to that i.e.

w = W (r)/ W (s)

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Statistical Analysis

Using the statistical software package SPSS, analyses of variance (ANOVA) were

carried out to identify significant differences, followed by post-hoc Newman-Keuls

comparison of averages; confidence intervals were obtained using bootstraping (1000

samples) resampling technique with a Simstat statistical software (User’s guide, Montréal,

1996).

Results

Relative fitness

The fitness of the resistant T. circumcincta line was significantly reduced (ANOVA p ≤

0.05) during the first and second experimental passage compared to the susceptible line

under all three climatic conditions (Table 1). Following the test on the immunosuppressed

lambs, the situation reversed and the resistant line had significantly (ANOVA p ≤ 0.05)

increased fitness compared to that of the susceptible line under all three climatic conditions.

Table 1

Relative fitness ratio of the resistant T. circumcincta line compared to that of the susceptible

line

Relative fitness ratio and

confidence interval:

resistant line

Significance:

resistant vs.

susceptible

Winter

Expt. selection 1 0.28 ± 0.16a *

Expt. selection2 0.41 ± 0.22a *

Test immunosuppressed

lambs

1.17 ± 0.12b *

Spring




lambs

1.39 ± 0.27b *

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Summer




lambs

1.38 ± 0.17b *

Calculated by dividing the absolute fitness value of the resistant line by the susceptible line;

any ratios below 1 indicate a greater fitness in the susceptible line and any ratios above 1

indicate a greater fitness in the resistant line. The experimental selection of the resistant and

susceptible lines took place in resistant and susceptible sheep respectively. The test

evaluated both the resistant and susceptible lines in immunosuppressed lambs. Significant

differences between the resistant and susceptible line are denoted with * where ANOVA p ≤

0.05. Significant differences between the passages of the same line are indicated with

different letters in superscript where ANOVA p ≤0.05.

Fecundity

The fecundity of the resistant line was significantly (ANOVA p≤ 0.05) reduced

compared to the susceptible line by the second experimental selection (Table 2). However,

by the test on immunosuppressed lambs, this difference was diminished and there was no

significant difference between the lines. Thus, the life traits pertaining to reproduction did

not appear to contribute to the greater relative fitness observed in the resistant line

following the test in the lambs.

Table 2

Fecundity

Susceptible Resistant Significance

susceptible vs. resistant

Expt. selection 1 247a NA

84

Expt. Selection 2 476b 123a *

Test immunosuppressed lambs 214a 187a

The fecundity shows the mean number of eggs per female worm calculated by dividing the

mean FEC by the mean number of females observed at necropsy in the hosts (selection

passage 1, 2: susceptible n = 2, resistant n = 3; test on lambs: susceptible n = 4, resistant n =

6). The experimental selection of the resistant and susceptible lines took place in resistant

and susceptible sheep respectively. The test evaluated both the resistant and susceptible

lines in immunosuppressed lambs. Following the first infection it was not possible to

necropsy the resistant sheep hosts as they were needed for the next experimental selection,

thus no data is available i.e. NA. Significant differences between the resistant and

susceptible line are denoted with * where ANOVA p ≤ 0.05. Significant differences between

the passages of the same line are indicated with different letters in superscript where

ANOVA p ≤0.05.

Establishment

The establishment of the resistant line was significantly reduced (ANOVA p ≤ 0.05)

during the experimental selection in resistant sheep compared to the susceptible line (Table

3). Following the test on the immunosuppressed lambs, the situation reversed and the

resistant line had a significant (ANOVA p ≤ 0.05) 9% increased capacity to establish

compared to the susceptible line. This likely contributed to the increased relative fitness

observed in the resistant line following the test on immunosuppressed lambs.

Table 3

Establishment of infective larvae

Susceptible Resistant Significance


Expt. selection 1 37a NA

Expt. selection 2 45a 21a *

Test immunosuppressed lambs 35a 44b *

85

Establishment was calculated by dividing the number of worms observed at necropsy

divided by the infective dose (experimental selection 1, 2: susceptible n = 2, resistant n = 3;

test on lambs: susceptible n = 4, resistant n = 6). The experimental selection of the resistant

and susceptible lines took place in resistant and susceptible sheep respectively. The test

evaluated both the resistant and susceptible lines in immunosuppressed lambs. In certain

instances, the sheep hosts were not necropsied and thus no data is available i.e. NA.

Significant differences between the resistant and susceptible line are denoted with * where

ANOVA p ≤ 0.05. Significant differences between the passages of the same line are indicated

with different letters in superscript where ANOVA p ≤ 0.05.

Egg to larval development

The egg to larval development was significantly (ANOVA p ≤ 0.05) reduced in the

resistant line compared to the susceptible line during the experimental selection stage

(Table 4). When the lines were tested in the immunosuppressed lambs, the resistant line had

a significantly (ANOVA p ≤ 0.05) increased larval development ratio under winter conditions.

This was a probable contributing factor to the observed increase in relative fitness of the

resistant line following the test on immunosuppressed lambs.

Table 4

Egg to larval development ratios

Susceptible Resistant Significance:


Winter

Expt. selection 1 0.39a 0.24a

Expt. selection 2 0.95b 0.71b *

Test immunosuppressed lambs 1.23c 1.35c *

Spring

Expt. selection 1 0.72a 0.42a

Expt. selection 2 0.99a 0.71b

Test immunosuppressed lambs 1.02a 1.39c

86

Summer

Expt. Selection 1 0.15a 0.39a

Expt. selection 2 1.02b 1.39b

Test immunosuppressed lambs 0.45a 0.39a

Larval development ratios were calculated by the number of L3 larvae stages attained

divided by the number of eggs present. The experimental selection of the resistant and

susceptible lines took place in resistant and susceptible sheep respectively. The test

evaluated both the resistant and susceptible lines in immunosuppressed lambs. Significant

differences between the resistant and susceptible line are denoted with * where ANOVA p ≤

0.05. Significant differences between the passages of the same line are indicated with

different letters in superscript where ANOVA p ≤ 0.05.

Faecal egg counts

The FEC of the resistant line was significantly reduced compared to that of the

susceptible line for the first two passages yet following the test in immunosuppressed lambs,

this difference was negligible (Table 5).

Table 5

Faecal egg counts

Susceptible Resistant Significance:


FEC

Expt. selection 1 448 ± 364a 51 ± 60a *

Expt. selection 2 396 ± 180a 146 ± 118a *

Test immunosuppressed lambs 200 ± 70a 181 ± 72a

The FEC shows the mean number of eggs per gram (EPG) of host faeces ± the confidence

interval (experimental selection 1, 2: susceptible n = 2, resistant n =3; test on lambs:

susceptible n = 4, resistant n = 6). The experimental selection of the resistant and susceptible

lines took place in resistant and susceptible sheep respectively. The test evaluated both the

87

resistant and susceptible lines in immunosuppressed lambs. Significant differences between

the resistant and susceptible line are denoted with * where ANOVA p ≤ 0.05. Significant

differences between the passages of the same line are indicated with different letters in

superscript where ANOVA p ≤0.05.

Discussion

The results of this study provide the first known example of gastrointestinal

nematodes adapting to the challenge of resistant sheep hosts. The intensive selective

pressure of the resistant sheep provoked adaptive responses in the resistant line in as few as

three generations. The increased relative fitness of the resistant line following the test on

immunosuppressed lambs may be attributed to the augmented survival traits at both the

parasitic stage (establishment) and free living stage (egg to larval development) suggesting

the simultaneous divergence of not one, but a cluster of phenotypically correlated traits was

occurring. There appeared to be no benefits, or costs, to the reproductive fitness of the

resistant line perhaps suggesting these two fitness components, reproduction and survival,

evolved independently. Alternatively, other authors have suggested that survival and

reproduction should logically be negatively correlated on the grounds that one always

necessitates the redirection of vital resources away from the other (Partridge and Harvey,

1985; Bell and Koufapanou, 1986; Reznick, 1992; Chehresa et al., 1997).

These results contrast with the previous studies which did not detect any adaptation

of GINs to resistant sheep for up to 10 generations of serial passage using FEC as a sole

measure (Adams, 1988; Albers and Burgess, 1988; Kemper et al., 2009; Saulai et al., 2010).

This highlights that FEC alone do not accurately reflect the entire adaptive capacity of the

GINs. Indeed, even the results of the present study show highly variable FEC that do not

exhibit any significant differences between the resistant and susceptible lines. Thus, had

these studies incorporated more life-traits measures, it is possible an adaptive response

would have been observed.

Our results have important implications on the dynamics of GIN infection should they

be applied to a field situations. Firstly, they show the presence of resistant sheep will drive

88

evolution in the GIN populations. Secondly, while our study demonstrated increased survival

capacities in T. circumcincta that is not to say all GIN species, or even all populations of the

same species, will respond to the challenge in the same way. Instead, it is likely there are

equally adaptive alternative directions in which to respond (Poulin, 1998) which would be

hard to predict in advance. Finally, if a greater number of larvae were able to survive over

winter with an increased capacity to infect, this would constitute a much greater force of

infection for the young susceptible immune-naïve lambs and their immune-compromised

lactating mothers during the periparturient rise (Boag and Thomas, 1971) when they’re

turned out onto pasture in spring.

Conclusions

The observed adaptations occurred rapidly in only three generations. Should the GINs

experience similar conditions over several years, it is not impossible to anticipate that

greater adaptations would be made to their life traits which could reduce resistant sheep to

levels of infection currently equated with susceptible sheep. A possible solution to extend

the shelf-life of this method is to maintain a portion of susceptible sheep in an otherwise

resistant flock. These sheep would harbor a portion of the GIN population and thus maintain

alleles for susceptibility in the GIN population to dilute the spread of resistant alleles. This is

known as a refugia concept and the same principle is applied to reduce the spread of

anthelmintic resistance in GIN. In conclusion, the results of this study should caution the

application of control measures such as selectively breeding sheep for resistance without

consideration for the adaptive evolution it will drive in the GIN populations and which may

ultimately shape our control challenges of tomorrow.

Conflict of interest

None of the authors of this paper has a financial or personal relationship with other people

or organisations that could inappropriately influence or bias the content of this paper.

Acknowledgments

The technical help of J. Cortet, C. Sauvé and the staff from Langlade INRA genetic

experimental unit during the pre-selection of T. circumcincta in the field is gratefully

acknowledged. E.S. was funded by General Pico Faculty of Veterinary Science in Argentina

89

and Fonamer project. C.C. is a grateful recipient of Marie Curie programme

“NematodeSystemHealth” grant. Results were presented as an oral communication at the

British Society of Parasitology Spring Meeting, Bristol, 9th – 11th April 2013.

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Gaba, S., Chadoeuf, J., Monestiez, P., Sauve, C., Cortet, J., Cabaret, J., 2006. Estimation of

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abomasum strongyle nematode infections in sheep at necropsy: Tentative proposals

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a simplified technique. Veterinary Parasitology 140, 105-113.

Gilleard, J.S., Beech, R.N., 2007. Population genetics of anthelmintic resistance in parasitic

nematodes. Parasitology 134, 1133-1147.

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Limouzin, C., 2004. A short-term divergent selection for resistance to Teladorsagia

circumcincta in Romanov sheep using natural or artificial challenge. Genetics

Selection Evolution 36, 217-242.

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Trends in Parasitology 20, 477-481.

Kemper, K.E., Elwin, R.L., Bishop, S.C., Goddard, M.E., Woolaston, R.R., 2009.

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term

exposure to sheep that were genetically resistant or susceptible to nematode

infections.

International Journal for Parasitology 39, 607-614.

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diagnostic de routine et le controle des infestations parasitaires des bovins, ovins,

equins et porcins. Annals of Parasitology 45, 321-342.

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Reznick, D., 1992. Measuring the costs of reproduction. Trends in Ecology & Evolution 7,

42-45.

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Nématodes parasites du tractus digestif des ruminants. PhD thesis, Université

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92

Desiccation tolerance of gastro-intestinal nematode third stage larvae:

exploring the effects on survival and fitness

Chylinski C., Lherminé E., Coquille M., Cabaret J.

INRA, UMR 1282, ISP 213, 37380 Nouzilly and F. Rabelais University, 37000 Tours France.

Abstract

The free-living third stage larvae (L3) of gastrointestinal nematodes are able to tolerate extreme

weather conditions such as desiccation, but little is known about the consequent effects this has on

their fitness. This study explored how the desiccation of Haemonchus contortus L3 larvae affected

their absolute fitness by examining their success at consequent life-cycle stages for a complete

generation, and comparing them against a control. The stages examined include establishment,

fecundity, larval development and pathogenicity. The results show that while desiccation greatly

reduced the survival of the L3 prior to infection in sheep, their absolute fitness was not negatively

impacted. Instead it appears desiccation slightly augmented H. contortus fitness by triggering

increases in fecundity. The study further explored what influence different GIN species (H. contortus,

Trichostrongylus colubriformis, Teladorsagia circumcincta), isolates and age of L3 had on their

capacity to revive following various periods of desiccation. The results showed desiccation tolerance

varied as a function of each of these variables. The greatest L3 survival was found in T. circumcincta

followed by T. colubriformis and finally H. contortus. Significant variation was observed between

individual species isolates and as a function of age. The results of this study carry important practical

implications for the epidemiological understanding of gastrointestinal nematode species of economic

importance.

93

Introduction

In arid and semi-arid areas, the free-living third stage larvae (L3) of gastro-intestinal nematodes (GIN)

are often subjected to extreme weather conditions. The presence of an external cuticle provides

them with greater tolerance against adverse climatic conditions that the proceeding free-living

stages lack. There is however a trade-off for this protective sheath. The barrier also prevents the L3

from feeding meaning they have finite energy reserves with which to find and infect a sheep host

(Selvan et al. 1993; Medica and Sukhedo 1997). Adverse environmental conditions have been shown

to accelerate the depletion of these reserves (Vlassoff et al. 2001). Studies by Siamba et al. (2011)

demonstrated the lipid contents of the GIN Haemonchus contortus L3 decreased as a function of

increased heat stress. However, the same study also found lipid metabolism to be negligible in L3

exposed to desiccation conditions. This suggests that under desiccation conditions H. contortus L3

enter a non-metabolic state known as anhydrobiosis, to conserve energy while preserving life

(Siamba et al. 2011). Anhydrobiotic organisms have been found in many taxa, including nematodes

(Clegg 2001; Watanabe 2006) and the state is considered to be fully reversible when more hospitable

moisture conditions are met (Persson et al. 2011). However, survival of these conditions does not

necessarily mean the L3 remain unaffected by the experience. Studies have shown that desiccated H.

contortus L3 have a significantly reduced capacity to ex-sheath and infect their sheep hosts (Siamba

et al. 2011). We do not know if these negative effects continue into the consequent life stages by

affecting fecundity or egg quality for example. Nematodes are well known to have remarkable

plasticity in their life cycle (Poulin 1998). Challenges that may negate their fitness at one life stage

may be compensated by altering aspects of survival or reproduction at consequent life stages. The

aim of this study was to explore how the desiccation of L3 impacts consequent life stages and overall

fitness of the GIN using H. contortus as a model species due to its capacity to survive in very arid

regions. The study further explored what influence different GIN species, isolates and age had on the

capacity of L3 to revive following various periods of desiccation and whether their survival was

affected by repeated revival cycles.


Evaluating the impact of desiccation on H. contortus fitness

Sheep

The study used nine Ile-de-France male lambs, three months of age. The breed is considered to be

susceptible to infection by H. contortus. All lambs were maintained in worm free conditions prior to

94

the experiment and their worm-free status verified with negative faecal egg counts (FEC) prior to the

experiment. The lambs were randomly allotted into one of two groups; one receiving the control H.

contortus infection (n = 4) the other receiving the desiccated infection (n = 5).

Sheep measures

The sheep were weighed at 0 and 33 days post infection (dpi) using walk-on electronic scales. Blood

samples were collected in heparin tubes from the jugular vein of the sheep at 0, 15 and 29 dpi using

a 10 ml gauge syringe. Blood haematocrit levels were established manually using a Hawksley micro-

haematocrit reader.

Infective doses

A 6 month old H. contortus Weybridge isolate was used for the infections. All the sheep received an

infective dose of 10,000 L3 larvae per os which had been left at room temperature for 24 hours prior

to infection, or manipulation. The doses for the desiccated group were deposited on a glass slides

and exposed to 24 hours of desiccation at room temperature before infection. The desiccated L3

were then revived by submerging the glass slide in water for three hours prior to the infection.

Faecal egg counts

Faecal samples were collected from all sheep on 15, 20, 21, 22, 23, 26, 28, 29 and 33 dpi to carry out

FEC using a modified McMaster technique (Raynaud 1970) in a sodium chloride flotation solution,

accurate to 50 eggs per gram (EPG) of faeces.

Egg to L3 larvae development ratio


into infective L3. This was done by culturing 5 x 5 gram faecal samples for each sheep in conditions

favourable to H. contortus development i.e. 23°C, 70%H, 10 days (Rossangio 1992). The L3 were then

extracted from each 5g sample of faecal matter using the Baermann funnel technique over a 24 h

period at room temperature (Baermann 1917), and counted under a microscope to obtain the

number of L3 developed per 5 g of faeces. Only living L3 were included in the count. The number of

L3 counted for each 5 g sample was then divided by 5 to obtain the number of L3 per 1g of faeces,

then divided again by the sheep’s respective FEC for that day. This provided the ratio of larvae

developing and surviving from the H. contortus eggs excreted.

Establishment in lambs

95

The capacity of the L3 to establish in their sheep host was determined by first counting the number

of adult worms found in the abomasum at necropsy (33 dpi) following the procedure described by

Gaba et al. (2006). The fourth stage larvae (L4) were also counted after having extracted them from

the abomasal mucosa by incubating them in water at 37°C for four hours, washing them thoroughly

in water which was saved, and then put through a 30µm mesh sieve to collect any L4. The total

number of adults and L4 counted for each sheep was divided by the number of L3 in the infective

dose (i.e. 10,000) to see how many were able to establish and the percentage was calculated.

Fecundity

The fecundity of the adult female worms was evaluated as described in Cabaret and Ouhelli (1984).

In brief, the total daily egg output per sheep was determined. Given the FEC only provide a measure

for 1 gram of faeces, the FEC on 33 dpi (just prior to necropsy) was multiplied by the total quantity of

faecal matter (QFM) produced in a day. The QFM was calculated using the following formula (back-

transformed from logarithm) developed specifically to the conditions of this study, based on a linear

regression between the logarithm of the weight of faecal excretion collected over a 24h period and

the logarithm of the metabolic weight (W0.75) of Ile-de-France male lambs:

QFM= 0.041W0.75 p=0.00; r=0.95

Where W is the weight of the individual lamb (kg).


necropsy to give the number of eggs produced per female, per day.

Absolute Fitness

Absolute fitness (W) for the control and desiccated H. contortus groups was determined to reflect

their capacity to survive and reproduce. This was done by multiplying the egg to L3 larvae

development ratios (i.e. number of L3 per gram of faeces) by the QFM to provide the total number of

living L3 produced in a day. This was done for two different days for each sheep i.e. 28 and 29 dpi.

The number of L3 produced in a day was then divided by the number of L3 in the infective dose i.e.

10,000 to provide the daily absolute fitness value (W) for the H. contortus in each sheep. A mean

absolute fitness value was calculated for the control and desiccated group based on the daily

absolute fitness value for each sheep in the group based on the three different days.

96

Accounting for the reduced survival of the desiccated infective doses in establishment and survival

Proceeding experiments within this study (described below) l ooked at the capacity of desiccated

Weybridge H. contortus L3 to revive in water following various periods of desiccation. The results

show that after 24 hours of desiccation only 35% of these L3 revived. Twenty-four hours was the

same length of time the infective doses for the desiccated group were exposed prior to infection. We

may therefore assume that from the original infective dose of 10,000 L3, the desiccated group rather

received an infective dose of 3,500 L3. Given the infective dose is incorporated into calculations for

both the establishment and absolute fitness, additional values for these have been included to

account for the differences in infective doses.

Female morphology

Adult female H. contortus vary in the morphology of their vulvar flaps. Three main morphs exist

including linguiform, smooth and button. Within the linguiform morph, there are four sub-morphs

referred to as A, B, C or I (LeJambre 1977). To determine if there was a correlation between a specific

morphology type and fitness, 100 female H. contortus worms were retrieved from the abomasum of

each sheep at necropsy and their specific morph-type determined.

Desiccation tolerance variables: GIN species, isolate, age and repeated revival cycles

The capacity of GIN L3 to revive following various periods of desiccation (i.e. 1 hour, 24 hours, 1

week, 1 month) was explored relative to the characteristics of species, isolate and age.

GIN species

The three most economically important GIN species of sheep were compared including H. contortus,

Trichostrongylus colubriformis and Teladorsagia circumcincta.

GIN isolates

The choice and number of isolates compared was simply a result of those available in the laboratory

at the time. All isolates compared were 3-12 months old at the time of study.

Four lines were compared in H. contortus including i) ISEs: an isolate susceptible to all anthelmintics

(AH) with no previous exposure to resistant hosts (see Roos et al. 2004 for isolate history); ii) ISER:

this isolate derived from ISES but had been selected in resistant Blackbelly sheep for three

generations prior to the present study; iii) Weybridge: an AH susceptible isolate; iv) KOK: an isolate

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resistant against the three main AH classes i.e. levamisole, benzimizadole and the

tetrahydropyrimidins and macrocyclic lactones.

Two isolates were compared in T. colubriformis including i) Weybridge: a levamisole resistant isolate;

ii) Pomroy: a levamisole and pyrantel resistant isolate.

Three isolates were compared in T. circumcincta including i) Sorin: a benzimidizole resistant isolate;

ii) Niort: a levamisole resistant isolate; iii) Pomroy a levamisole and pyrantel resistant isolate.

Age of L3 larvae

Age was compared by separating the GIN isolates into two groups: young i.e. those which had been

extracted less than six months prior to the study; and old i.e. those isolates which had been extracted

6-12 months prior to study. All isolates had been maintained in Roux flasks with water at 4°C at a

concentration of approximately 2000 L3 per 1 ml. The young isolates included: H. contortus ISES, ISER

and Weybridge; T. colubriformis Pomroy; T. circumcincta Pomroy. The old isolates included: H.

contortus ISES, Kok and Weybridge; T. colubriformis Pomroy and Weybridge; T. circumcincta Sorin and

Niort.

Desiccation process

The L3 were left at room temperature for 24 hours prior to any manipulations to allow them to

regain full mobility following storage at 4°C. The desiccation tests were performed by depositing 5 x

10 µl drops of the L3 stock onto a standard glass slide to obtain approximately 20 L3 per drop i.e. 100

L3 tested for each condition. The exact number of living L3 per drop were counted under a

microscope and recorded. The glass slides were then left in a plastic box with surrounding moisture

from damp tissue paper for either 1 hour, 24 hours, 1 week or 1 month. Following the appointed

time for each slide, the original drops were refreshed by adding another 10 µl of water on top of the

drops containing the L3. After 30 minutes, the number of surviving L3 from the original drop were

counted under a microscope and recorded. Living L3 were separated from the dead or moribund L3

based on movement and the integrity of their internal structures. For each sample, the survival

percentage was calculated as a function of the number of L3 at time zero.

Repeated desiccation and revival of H. contortus

The capacity of the Weybridge H. contortus isolate to withstand repeated cycles of desiccation and

revival was tested. This was done using a sponge substrate instead of a glass slide to better imitate

natural morning dew conditions that can occur in the field, even in very dry areas. A two inch

squared piece of Spontex® sponge was dampened with water and placed into a petri dish. Two-

98

hundred H. contortus Weybridge L3 were deposited directly on top of each sponge and maintained

outside in under shelter for 1, 2, 3 and 4 weeks. This was conducted during May in central France

where average daily temperatures range from 13-20°C. Three different conditions were examined for

each desiccation period: i) control: continually supplied with water; ii) desiccated: received no

moisture; iii) revived: very light mist of water sprayed on to the sponge daily. There were replicates

for each condition tested. Following the allotted desiccation period, the L3 were extracted from the

sponge by placing the sponge and petri dish into a Baermann for 24 hours. The surviving extracted L3

were counted and recorded.


The comparisons of fitness and life-history traits between the control and desiccated sheep groups

were made using Mann-Whitney tests. Comparisons of the tolerance variables i.e. GIN species,

isolates, age and repeated desiccation/revival cycles were carried out using a general linear model

(GLM). Logarithm transformations were used prior analysis where needed to stabilize the variance.

Correlations were estimated with nonparametric Spearman rho (rs).

Results

Evaluating the impact of desiccation on H. contortus fitness and life-history traits

Fitness

The mean absolute fitness index (from two days) for the control and desiccated H. contortus groups

were 205 (± 51 SE) and 75 (± 45 SE) respectively. The absolute fitness values were very similar

between the two days sampled. Despite the near three fold difference, this was not found to be

significantly different (Mann-Whitney p = 0.1). However, when the fitness of the desiccated group

was adjusted for the 35% survival rate of the infective dose, the absolute fitness index was slightly

greater than the control at 214 (± 107 SE)

Establishment H. contortus

The capacity of the desiccated H. contortus L3 to establish in the sheep was significantly reduced

(Mann-Whitney p = 0.02) compared to the control. After 33 dpi, the mean percentage of worms

established from the infective dose was 14.6 % in the control group and only 3.6 % in the desiccated

group. If however, the establishment of the desiccated group is adjusted to account for the 35%

99

survival rate of the infective dose, the percentage increases to 10.2% removing the significant

difference against the control group.

The sex ratio of established worms was slightly more balanced in the desiccated group which had

52% females and 48% males, whereas the control group had 59% females and 41% males.

Fecundity H. contortus

The fecundity of the desiccated H. contortus increased to almost double that of the control group

with 58.6 and 30.3 eggs per female respectively (Mann-Whitney p = 0.02). A significant negative

Spearman correlation (rs= -0.77; p=0.02) was observed between worm number in the lambs and

fecundity.

H. contortus faecal egg counts

The mean FEC was significantly greater (Mann-Whitney, p = 0.05) in the control group compared to

the desiccated group at all time points except 15 and 26 dpi (Fig. 1). While the FEC of the control

group continues to increase after this point, the desiccated group remains relatively stable.

Fig. 1 Mean fecal egg count (FEC) of H. contortus throughout infection for the control and desiccated

larval groups

Egg to L3 larvae development rate H. contortus (Table 1)

100

There was no significant difference between the control and desiccated group in the egg to L3

development capacity. The mean development ratio of eggs that successfully developed and survived

as L3 was 67% in the control group and 61% in the desiccated group.

Table 1. Percentage of female vulvar morphotypes ± standard error from the control and desiccated

H. contortus groups.

Female morphology

Vulvar type

Control

(mean % ± SE)

Desiccated

(mean % ± SE)

Smooth 27 ± 1.7 25 ± 3.4

Knobbed 6 ± 0.1 8 ± 0.1

Linguiform 67 ± 1.7 70 ± 2.6

A 51 ± 0.7 45 ± 0.6

B 9 ± 0.5 14 ± 0.5

C 23 ± 0.8 17 ± 0.5

I 17 ± 0.4 24 ± 0.9

Vulvar morphology of female H. contortus

The occurrence of the three vulvar morphs was similar between the control and desiccated groups.

By far the most common morph was the linguiform. The knob morph was the least common.

Correlation analysis using Spearman’s rank coefficient demonstrated a significant positive

relationship between fecundity and the percentage of linguiform morphs. The linguiform sub-morph

A was predominant in both the control and desiccated groups. The two groups did not differ

significantly in the proportion that each linguiform sub-morph was observed.

Pathogenicity H. contortus

The desiccated H. contortus were responsible for significantly greater reductions (Mann-Whitney p =

0.00) in haematocrit compared to the control group, with mean reductions per worm observed to be

0.11% and 0.02% respectively.

Desiccation tolerance variables: GIN species, isolate, age and repeated revival cycles

GIN species

101

The three different GIN species tested had significantly different capacities to survive desiccation

conditions at all the time points tested i.e. 1 hour, 24 hours, 1 week (Table 2). None of the GIN

species survived 1 month of desiccation. All of them encountered a decrease in survival within the

first hour of desiccation and continued to decrease as a function of time. After one week of

desiccation conditions, T. colubriformis had the greatest number of surviving L3 (40%) followed by T.

circumcincta (22%) and finally H. contortus (2%) (Fig. 2).

Table 2. Influence of GIN species,

isolate and age on the L3

capacity to withstand desiccation

for 1 hour, 1 day and 1 week

using General Linear Models

with interaction analyses where

significance p ≤ 0.05. NA: not

applicableDesiccation period

Source of variation Significance

1 hour Model (R2 = 0.766) 0.000

Species 0.000

Isolate 0.001

Age 0.202

Species * Isolate 0.000

Species * age 0.006

Isolate * age 0.019

Age * Isolate * Species NA

24 hours Model (R2 = 0.848) 0.000

Species 0.000

Isolate 0.000

Age 0.803


Species * age 0.001

Isolate * age 0.000


1 week Model (R2 0.912) 0.000

Species 0.000

102

Isolate 0.000

Age 0.001


Species * age 0.517

Isolate * age 0.005


Fig. 2 Mean percentage of L3 surviving ± standard error for three GIN species following various

periods of desiccation. Significant differences between the species denoted with *

GIN isolate

The GIN isolates tested varied significantly both within their respective species, and between GIN

species at all time interval (Table 2).

GIN L3 age

0

10

20

30

40

50

60

70

80

90

100

I hr 24 hr 1 week 1 month

Mea

n su

rviv

al L

3 %

Desiccation time

GIN species

H. contortus

T. colubriformis

T. circumcincta

*

*

*

103

The age of the L3 did not significantly influence the survival of the L3 until after 1 week of desiccation

conditions (Fig. 3). Prior to that, the influence of age was highly variable between GIN species and

isolates (Table 2).

Fig. 3 Mean percentage of L3 surviving ± standard error for young (≤ 6months) and old (≥6months) L3

following various periods of desiccation. Both young and old L3 tested included isolates from H.

contortus, T. colubriformis and T. circumcincta. Significant difference between the young and old

denoted with *

Repeated desiccation and revival of H. contortus

The survival rate of the desiccated H. contortus L3 dropped to 65% after one week and 44% after two

weeks (Fig. 4). None of the desiccated L3 survived past this point. The survival rate of revived L3 was

significantly higher than the desiccated L3 (p=0.00). However, the rehydration conditions did not

extend life span with no L3 surviving past two weeks of desiccation/revival cycles. The control L3 had

the greatest survival rate at all time intervals; losses were minimal at week 1 at 5% which increased

to 30% by week 4. The survival rate of the desiccated group was significantly (p=0.00) better when

desiccated on the sponge substrate compared to the glass slide.

0

10

20

30

40

50

60

70

80

90

1hr 24 hr 1 week 1 month

Mea

n su

rviv

al L

3 %

Desiccation time

Age of L3

Young

Old

*

*

104

Fig. 4 Mean percentage of L3 surviving ± standard error for H. contortus following various periods of

desiccation under three different conditions: i) control ii) desiccated iii) daily repeated revival with

water

Discussion

The results of this study show that the desiccation of H. contortus L3 larval stages reduced the

absolute fitness of the infecting population by nearly 3-fold, although this was not found to be

significant. Closer examination of the life-history stages revealed this decrease in fitness to result

exclusively from a significantly reduced capacity to establish in their host compared to the control

group. This was the only life stage that performed significantly worse than the control. Similar

observations were made by Siamba et al. (2012) who owed the reduction in establishment of

desiccated H. contortus L3 to a delayed capacity to ex-sheath following uptake by their host. It is

important to note that our calculation of establishment percentage here assumed that all the L3 in

the infective dose survived the desiccation conditions prior to infection. However, our consequent

studies demonstrated that only about 35% of H. contortus were able to survive the same conditions

the desiccated infective doses experienced (i.e. 24 hours on a glass slide) (Fig. 2). Re-calculations of

establishment based on this reduced infective dose of the desiccated group actually suggest that the

establishment of the surviving desiccated L3 does not differ that much from the control, with 10.2%

and 13.9% establishment respectively. Accordingly, the absolute fitness of the desiccated and control

group were nearly identical (214 vs. 205). Thus, the initial reduction in the absolute fitness of the

desiccated group we observed was rather a result of fewer L3 surviving desiccation to go on and

infect the sheep, and not an outcome of reduced performance at consequent life-stages. Indeed, it

0

20

40

60

80

100

120

1 2 3 4

Mea

n su

rviv

al L

3 %

Desiccation time (weeks)

H. contortus desiccation and repeated revival

Control

Desiccated

Repeated revival

105

rather appears that the desiccated group experiences a boost to their absolute fitness with the

females producing nearly double the number of eggs compared to the control group. This was not

reflected in the FEC owing to the reduced number of established females in the desiccated group,

but it does appear that the experience of desiccation conditions triggered augmentations in the

reproductive output of the population. This did not extend to increased development of eggs to L3

larvae which remained very similar between the control and desiccated groups. It remains possible

the L3 were impacted on a finer scale. Studies by Rossanigo and Gruner (1996) found a liner

correlation between faecal moisture content and length of L3 in various GIN species ). While there

was no repercussion on the survival of shorter worms, they were found to have slower migration

speeds which in turn could negatively impact the GIN fitness via reduced transmission.

No differences were observed in the ratio of female vulvar morphs in H. contortus between the two

groups either. To date, differences in morph ratios have only been observed by comparing H.

contortus from distinct geographic regions (LeJambre 1977). Although a positive correlation was

found between the lingiform morph and fecundity, this was not unexpected given the

disproportionate representation of the linguiform (around 70%). To truly understand if this is the

case it would be necessary to count the number of eggs in utero of each morph. Further studies need

to be done to understand the selective advantages that led to the evolution of the three morphs.

There were no significant differences in haematocrit levels between the control and desiccated

groups (i.e. 15 dpi: 37 and 36%, 29 dpi: 30 and 31% respectively - data not shown). Yet given there

were significantly fewer worms in the desiccated group, this suggests the worms in this group

provoked greater losses in haematocrit per worm compared to the control group, and were thereby

more pathogenic. It is possible that their increased fecundity induced greater feeding to cope with

the heavy energetic demands of reproduction (Viney and Cable 2011). Or alternatively, it may be a

false relationship whereby high haematocrit levels controlled for low infection intensities, which in

turn, released any potential density-dependent effects on fecundity. Regardless of the explanation,

the results suggest that desiccation may increase the pathogenicity of the infection to the sheep

host.

The capacity of the L3 to survive desiccation conditions appears to be dependent on the GIN species,

the respective isolate and their age. Of the three GIN species examined, H. contortus (2%) had the

lowest capacity to survive desiccation, followed by T. colubriformis (22%) and then T. circumcincta

(40%). This was rather surprising given H. contortus is the most prevalent of the three species in arid

environments, we predicted they would survive desiccation conditions the best. Studies into the

desiccation resistance of the non-infective egg to second stage larvae also found H. contortus had the

lowest tolerance of the three species (Rossanigo and Gruner 1994). The capacity to survive

106

desiccation varied significantly within a single species. This begs the question as to whether

desiccation tolerance is something that may be selected for with repeated experience. The age the

L3 in a population also influenced their survival capacity, decreasing as a function of age. This was

somewhat expected given the older L3 would have less energy reserves to withstand the desiccation

stress. Regardless of GIN species, isolate or age, none of the L3 survived past two weeks of

desiccation conditions. This time was not extended in the presence of small amounts of water in the

revival cycles with H. contortus, although it did increase the number of L3 which survived at week

two. We may thereby infer that desiccation tolerance of the free-living H. contortus stages does not

contribute to their capacity to survive in arid environments. Instead, it rather appears to be a

mechanism to help them persist short periods of desiccation. A more likely explanation for the

survival of H. contortus in areas which have dry seasons spanning 9 months or more may be found in

the work of Jacquiet et al. (1995). Their research found the female H. contortus survived in the sheep

but stopped their reproductive output completely during the dry season, only to be re-started in time

with the wet season to maximize the free-living stages chances of survival. These findings

complement previous studies investigating the capacity of various sheep GIN species to survive over

wintering conditions (Makovcava et al. 2009). In this case, it was T. colubriformis that was found to

have the greatest capacity to survive unaffected by cold conditions, via greater tolerance of L3 on

pasture and by entering an arrested developmental phase inside the host. The study found T.

circumcincta to be less tolerant of cold conditions with increased numbers found in milder climates,

and H. contortus was barely present at all. With increasing information available on the climatic

effects and GIN survival, we can improve our epidemiological understanding of the disease, a central

element to the formulation of successful control programmes to determine when and for how long

pastures remain dangerous (Makavcava et al. 2009).

In conclusion, desiccation conditions clearly appear to regulate the survival of L3, but this does not

negatively impact their consequent fitness. Given L3 survival varies between isolates of a given

species, we question whether this trait may be selected for with repeated exposure to extend both

the number of L3 surviving and the length of time they are able to tolerate it. While this study looked

at the consequent effects of desiccation on H. contortus fitness and life-traits within a single

complete generation, it would be interesting to see if the observed increases in fecundity of the

desiccated group, and their influence on host haematocrit, persisted into consequent generations.

These results carry practical implications in the understanding of the epidemiology of GIN infections.

Quantifying the direct and synergistic effects of climatic variables is necessary to improving

predictive capacities of disease modeling (Harvell et al. 2002).

107

Acknowledgements

C. Chylinski is a grateful recipient of a PhD grant from the EU Marie Curie Project

“NematodeSystemHealth”. We thank W. Pomroy, Massey University, New Zealand, for providing

anthelmintic resistant isolates.

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110

Storage of gastrointestinal nematode infective larvae for species preservation

and experimental infections

C.Chylinski1, J. Cortet1, G. Sallé1, P. Jacquiet2, J. Cabaret1

1 INRA, UMR 1282, ISP 213, 37380 Nouzilly and F. Rabelais University, 37000 Tours France

2 Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31300 Toulouse, France

Abstract

Techniques to preserve the infective third stage larvae of gastrointestinal nematodes are of

considerable interest to preserve rare species and to maintain a stable source for routine

experimental infections. This study compares the relative pros and cons of the two most common

techniques, cryopreservation and refrigeration by comparing how they influence consequent

infection outcome parameters in terms of life-history traits and fitness as a function of time using the

gastrointestinal nematode of sheep Haemonchus contortus as a study species. Establishment

capacity was found to be significantly reduced in cryopreserved stocks of L3 compared to

refrigerated stocks, but this was followed by significant increases in their fecundity. Refrigeration did

not affect L3 stocks consequent fitness by 16 months (the maximum examined) although they did

incur a significant reduction in establishment, followed once again by an augmentation in fecundity.

The study highlights potential areas for bias in comparing studies using L3 larvae maintained for

different periods of time under different techniques.

111

Introduction

Techniques to preserve the infective third stage larvae (L3) of gastrointestinal nematodes (GIN) are

of great experimental interest, both to preserve rare species selected for particular aptitudes i.e.

anthelmintic resistance, morphology etc. and as a source for routine experimental infections.

Effective preservation techniques reduce larbour and financial costs associated with maintaining

differing isolates in constant cycle through their obligate hosts and further minimize the potential for

genetic drift or unexpected selection that may result from repeated passage (Chehresa et al. 1997;

Gasnier et al. 1992). Two techniques are commonly used in the preservation of GIN L3 stages. The

first is cryopreservation whereby the L3 are ex-sheathed and maintained in vials of physiological

serum in liquid nitrogen (Van Wyk et al. 1977). Studies using the sheep GIN Haemonchus contortus

have found that not only are the worms recoverable after 10 years of storage (Rew and Campbell

1983), they also remain infective to sheep (Campbell et al. 1973). The second technique is to

maintain stocks of L3 refrigerated at 4 °C (MAAF 1986). This technique takes advantage of GIN L3

physiology. The L3 possess an external protective sheath to provide greater resistance against

environmental variables however it also prevents the L3 from feeding (O’Conner et al. 2006). The L3

therefore exist on a limited energy budget. While elevated temperatures can cause a rapid decline in

their energy reserves by increasing their metabolic rate (Vlassoff et al. 2001), maintain L3 stocks

cooler temperatures can lowers their metabolism to sustain their energy reserves. While it is possible

to refrigerate most GIN species for up to 12 months (MAFF 1986), use of the stocks should be

avoided between four-16 weeks during which time development by may inhibited.

Cryopreservation has a clear duration advantage over refrigeration. Studies by Van Wyk et al. (2000)

found that H. contortus, among other nematode species, could survive and establish in their host

following more than 15 years in cryopreservation (Van Wyk and Gerber 2000). However, this did

produce highly variable results for establishment ranging from only a few percent up to 40% and this

was based on infections introduced directly into the abomasum or rumen by means of laparotomy

(Van Wyk et al. 2000) rather than traditional infections per os. On the other end of the spectrum,

stocks of GIN L3 maintained at room temperature are thought to loose their infectivity in a matter of

weeks (Kerboeuf 1978a; Mallet and Kerboeuf 1984; 1985). No studies could be found exploring the

effect of refrigeration on GIN L3 establishment.

The relative pros and cons of cryopreservation and refrigeration techniques on consequent GIN

infection outcome have never been compared. This study thereby compares the effects of

cryopreservation and refrigeration on the establishment capacity and fecundity of GIN using H.

controtus as a model species. Furthermore, the effect of various periods of refrigeration (three-16

112

months) on the viability of the L3 and consequent infection is explored by comparing their

performance at different life-history traits and determining their absolute fitness. Given GIN are well

documented to alter their life-history traits to compensate for any challenges (Chehresa et al. 1997;

Poulin 1998) these measures provide a more complete picture on the effects of refrigeration age and

enable direct comparisons between the stocks. To ensure there was no H. contortus isolate related

effects on these measures, two different isolates were compared. Similar studies were carried out on

the effect of cryopreserved stocks of GIN maintained for up to 15 years, but the results are limited to

information on establishment and fecundity (Van Wyk et al. 2000).


Sheep

The trial comparing cryopreserved vs. refrigerated stocks was tested on Ile-de-France male lambs, 3

months of age. The study comparing refrigerated stocks at various time intervals tested on 15

Romane rams, 9 months of age. Both breeds are considered susceptible to infection by H. contortus.

All sheep were maintained in worm free conditions prior to the experiment and their worm-free

status verified with negative faecal egg counts (FEC) prior to the experiment. This experiment was

approved (no 2012-06-10) by the Val de Loire ethical committee (no 19).

Cryopreservation Technique

This techniques was derived from that described by Van Wyk et al. (1977) with some small

alterations made. The main differences used here include: 1) following ex-sheathment of infective

larvae, they were washed four times with 10x Phosphate Buffered Saline (PBS) solution at pH 7.2; 2)

the larvae were cryogenized in 1x PBS; 3) freezing was done gradually decreasing by 1.4°C every

minute for 30 minutes in gas nitrogen before being introduced to liquid nitrogen where they

remained; 4) prior to use, the stocks were thawed at 30°C and 1x PBS was subsequently added. The

number of mobile L3 were counted under a microscope to determine the percentage surviving and

calculate the infective dose. Stocks were left at room temperature for 24h prior to infection in sheep.

Refrigeration Technique

Following extraction of the L3 larvae from faecal sample using the Baerman funnel technique

(Baermann 1917) the stocks were placed in Roux flasks in water and maintained at 4°C. Generally,

stocks are not used within the first 30 days following observations of better establishment after this

time point since a delay of maturation seems needed for attaining full infective capacity (Kerboeuf et

al. 1978a,b). Prior to infection, the stocks are removed from the fridge and left for 24 hours at room

113

temperature to revive fully. The living L3 were separated from the dead larvae by placing them once

again through a Baermann funnel. The stock densities were then calculated and then diluted to

obtain the desired infective dose.

Infective dose

All sheep received an infective dose of 10,000 L3 from their respective isolate per os.

Haemonchus contortus isolates

The study comparing the establishment and fecundity of cryopreserved vs. refrigerated stocks of H.

contortus compared two different isolates; ISE, an isolate that is susceptible to the three main

anthelmintics’ groups (see Roos et al. 2004 for line history) and Kokstad (KOK) an isolate obtained

from a farm in South Africa with resistance against the three main anthelmintic drugs i.e. Levamisole,

Benzimizadole, Macrocyclic lactones, kindly donated by J. Van Wyk. The length of time the isolates

had been preserved and the number of sheep the respective isolate was tested on are presented in

Table 1.

Table 1. Cryopreserved vs. refrigerated H. contortus infective larvae stocks. Details of isolates,

duration of preservation and number of sheep tested.

Isolate Method preservation Duration of preservation Number of sheep

ISE Refrigeration 4°C 15 months 1

10 months 2

9 months 2

3 months 2

Cryopreservation 2 years 2

KOK Refrigeration 4°C 4 months 1

6 months 1

Cryopreservation 2 years 5

The study comparing the fitness of refrigerated H. contortus stocks after various periods of time

compared two isolates. The ISE (described above) and ISE-BB, an isolate which originated from ISE

but had undergone a strong divergent selection process in Martinik Blackbelly sheep, a breed known

for its resistance against H. contortus infection (Terefe et al. 2007). This resulted in a strong

bottleneck for the ISE-BB isolate. The length of time the isolates had been preserved and the number

of sheep the respective isolate was tested on are presented in Table 2.

114

Table 2. Refrigerated H. contortus stock fitness after various periods of time: isolates, length of

preservation and number of sheep tested.

Isolate Duration of preservation

(months)

Number of sheep

ISE 4 4

7 3

ISE-BB 4 4

16 4

Faecal egg counts

Faecal samples were collected to carry out FEC using a modified McMaster technique (Raynaud

1970) in a sodium chloride flotation solution, accurate to 50 eggs per gram (EPG) of faeces. For the

cryopreservation vs. refrigeration trial, FEC were carried out on 28 to 35days post infection (dpi). For

the trial comparing fitness of refrigerated stocks after various periods of time, FEC were carried out

on 0, 21, 24, 28, 32 and 35 dpi.

Establishment

The capacity of the L3 to establish in their sheep host was determined by counting the number of

adult worms found in the abomasum at necropsy following the procedure described by Gaba et al.

(2006). The fourth stage larvae (L4) were also counted after having extracted them from the

abomasal mucosa by incubating them in water at 37°C for four hours, washing them thoroughly in

water which was collected and put through a 30µm mesh sieve to collect any L4. The total number of

adults and L4 counted for each sheep was divided by the number of L3 in the infective dose (i.e.

10,000) to see how many were able to establish and the percentage was calculated.

For the trial comparing cryopreserved vs. refrigerated stocks, necropsy took place at 35-40 dpi. For

the trial comparing fitness of refrigerated stocks after various periods of time, necropsy took place at

35 dpi.

Fecundity

For the cryopreservation vs. refrigeration trial, fecundity was determined by dividing the last FEC

On 28 to 35 dpi by the number of female worms counted.

For the trial comparing refrigerated stocks after various periods of time, the fecundity of the adult

female worms was evaluated as described in Cabaret and Ouhelli (1984). In brief, the total daily egg

115

output per sheep was determined. Given the FEC only provide a measure for 1 gram of faeces, the

FEC on 35 dpi (just prior to necropsy) was multiplied by the total quantity of faecal matter (QFM)

produced in a day. The QFM was calculated using the following formula (back-transformed from

logarithm) developed specifically to the conditions of this study, based on a linear regression

between the logarithm of the weight of faecal excretion collected over a 24h period and the

logarithm of the metabolic weight (W0.75) of Romane rams:

QFM= 0.041W0.75 p=0.00; r=0.95

Where W is the weight of the individual sheep (kg).


necropsy to give the number of eggs produced per female, per day.

Egg to L3 larvae development ratio


into infective L3. This was done by culturing 5 x 5 gram faecal samples for each sheep in conditions

favourable to H. contortus development i.e. 23°C, 70%H, 10 days (Rossanigo 1992). The L3 were then

extracted from each 5g sample of faecal matter using the Baermann funnel technique over a 24 h

period at room temperature (Baermann 1917), and counted under a microscope to obtain the

number of L3 developed per 5 g of faeces. Only living L3 were included in the count. The number of

L3 counted for each 5 g sample was then divided by 5 to obtain the number of L3 per 1g of faeces,

then divided again by the individual sheep’s respective FEC for that day. This provided the ratio of

larvae developing and surviving from the H. contortus eggs excreted.

Absolute Fitness

Absolute fitness (W) for the four isolate/age groups was determined to reflect their capacity to

survive and reproduce. This was done by multiplying the egg to L3 larvae development ratios (i.e.

number of L3 per gram of faeces) by the QFM to provide the total number of living L3 produced in a

day. This was done for three different days for each sheep i.e. 28, 32 and 35.The number of L3

produced in a day was then divided by the number of L3 in the infective dose i.e. 10,000 to provide

the daily absolute fitness value (W) for the H. contortus in each sheep. The mean absolute fitness

value was then calculated for each of the four isolate/age groups to provide the mean daily fitness.


A general linear model was used on raw or transformed data when gaussiann distribution was not

present using SPSS software Version11.5.

116

Results

Cryopreservation vs. refrigeration

The refrigerated stocks established significantly (p = 0.02) better than the cryopreserved stocks.

Neither the isolate nor the age of the refrigerated stock interacted with the establishment (Table 3).

The mean establishment rate for the cryopreserved stocks was 20 % ± 13.4 SD (both isolates

included) and 32% ± 8.2 SD for the refrigerated stocks (both isolates and all ages included).

Using GLM analyses we found that both the isolate (p = 0.026) and the age of the refrigerated stock

(p = 0.048) interacted significantly with the fecundity. Fecundity was greater in the KOK (6.2 eggs per

female ± 0.9 SD) than in ISE (4.4 eggs per female ± 0.9 SD). The mean fecundity for all cryopreserved

stocks was greater (6.2 eggs per female ± 0.9 SD) than for the refrigerated stocks (4.4 eggs per

female ± 1.1 SD).

Table 3. General Linear Model analyses comparing effect of cryopreserved vs. refrigerated stocks on

establishment and fecundity including effect of isolate and length of refrigeration.

Source F Significance

Establishment

(log transformed)

Corrected model 2.511 0.108

Intercept 615.016 0.000

Cryopreserved vs.

Refrigerated stocks

7.230 0.020

Isolate 0.736 0.408

Age of refrigerated stock 0.101 0.756

Fecundity Corrected model 7.350 0.005

Intercept 457.449 0.000

Cryopreserved vs.

Refrigerated stocks

4.844 0.048

Isolate 6.413 0.026

Age of refrigerated stock 0.014 0.907

Fitness of refrigerated stocks compared after various periods of time in storage

The results show there was no significant differences in fitness between the isolates, irrespective of

length of time the L3 were stored (Table 4). There was however significant differences between the

life history traits of the ISE-BB old compared to the others. This group had a significantly diminished

117

capacity to establish in their host. This was not reflected in the fitness as the same group also had a

significantly increased fecundity.

These results suggest that L3 stocks can be maintained for a minimum of 7 months without any

effect on their fitness and life-history traits. Somewhere between 8-16 months of storage, there is a

reduction in infectivity, but not overall fitness.

Table 4. The daily absolute fitness value and mean life-history trait ± standard deviation of different

ages of H. contortus isolates stored at 4°C. Significant differences between isolates denoted with

different letters in superscript. Significant differences (S) p = 0.05, not significant (NS). comparison

ISE

4 mths

(N = 4)

ISE

7 mths

(N = 3)

ISE BB

4 mths

(N = 4)

ISE BB

16 mths

(N = 4)

Significance

Fitness 46.39 ± 9.6 35.7 ± 8.97 38.53 ± 7.83 55.89 ± 0.72 NS

Establishment

(No. adults)

4341 ± 1826a

4341 ± 1826a

5351 ± 603a

2398 ± 500b

S

FEC (EPG) 6918 ± 169 5527 ± 1262 5535 ± 1004 4523 ± 1024 NS

Fecundity

(eggs/female)

3599 ± 2429 3148 ± 1585 2836 ± 783 4572 ± 1400 NS

Egg-larvae

development

0.06 ± 0.01a 0.06 ± 0.02

a 0.06 ± 0.02

a 0.1 ± 0.02

b S

Discussion

The cryopreserved stocks (20%) demonstrated a significantly reduced capacity to establish compared

to the refrigerated stocks (32%). In this case the cryopreserved stock was only two years old but it

attained a lower establishment capacity than that of the 13.8 year old cryopreserved stock (28.5%)

observed by Van Wyk et al. (2000). Two procedural differences may account for the differences

between these studies; i) The present study froze the L3 in increments of gradually decreasing

temperatures whereas Van Wyk et al. (2000) froze the L3 directly in liquid nitrogen; ii) the present

study infected the sheep per os whereas Van Wyk et al. (2000) infected the sheep parentally.

Although studies have suggested these different routes of administration should result in little

difference in GIN success (Van Wyk and Gerber 1980), the possibility cannot be excluded entirely.

The two studies did however share one thing in common, a high variability in the establishment

capacity of cryopreserved stocks. Why differences exist within the same protocol is hard to explain. It

118

seems probable that the capacity to survive and revive from cryopreservation conditions is not

homogenous across all individuals of a population. Studies exploring H. contortus in the extreme

conditions of desiccation found that only about 35% of the population were able to survive exposure

and revive following hydration thereafter (Chylinski et al. under review). The present study verified

that the infective doses of cryopreserved stocks consisted of only living L3, yet it is possible that the

physiological and metabolic demands of reviving from a frozen state negatively impacted the lipid

reserves of the L3 and their consequent capacity to establish. A reduced establishment capacity

owing to reduced lipid reserves was observed in desiccated H. contortus L3 (Siamba et al. 2011;

2012).

Given the refrigerated H. contortus stocks were not subjected to such extreme conditions, this likely

contributed to their better establishment capacity observed in this study. The refrigerated stocks had

another advantage over the cryopreserved stocks in that they still maintained their external

protective sheaths which were otherwise removed prior to cryopreservation. In natural infections,

the L3 do not ex-sheath until arrival at the rumen, this likely affords them some degree of protection

against the hostile host environment en route. The lack of sheath in the cryopreserved L3 would have

made survival to establishment for the cryopreserved stocks more challenging when infected per os.

Interestingly, the cryopreserved stocks appeared to respond to the challenge of their preservation

conditions by augmenting their reproductive output, an effect which was not triggered in the less

extreme conditions of refrigeration. Fecundity also varied significantly as a function of H. contortus

isolate in cryopreserved stocks. Previous studies have noted that the performance of H. contortus

isolates at different life-history traits can vary substantially, but sometimes with little overall effect

on fitness (Chylinski et al. under review).

The fitness results from refrigerated stocks suggest that stocks of H. contortus L3 may be maintained

at 4°C for up to 7 months without any change to their respective fitness and life-history traits. After

16 months of storage however, there appears to be a significant reduction in their capacity to

establish as seen in the 16 month old ISE-BB. This is in sharp contrast to what was observed for the

the GIN Trichostrongylus retortaeformis maintained at 24°C which showed decreases in

establishment as early as 9 weeks (Kerboeuf 1978a; Mallet and Kerboeuf 1985). This highlights the

important role a cooler temperature has to play in extending the viability of the L3.

Despite the significant reduction in establishment of the 16 month old ISE-BB isolate, they did not

incur any costs to their absolute fitness. Instead, the isolate appeared to compensate for this

119

reduced establishment by significantly augmenting their fecundity. This echo’s what was observed

for the cryopreserved stocks of L3 above. Similar patterns of decreased establishment followed by

increased fecundities as in older compared to younger stocks of GIN have previously been observed

in the Humeau isolate of H. contortus (3 vs. 11 months old maintained at 4°C) (G. Sallé personal

communication 2014); T. colubriformis (fresh vs. 9 weeks old maintained at 24°C) (Mallet and

Kerboeuf 1985); Heligimosoides polygyrus (fresh vs. 9 weeks old maintained at 22°C) (Kerboeuf

1978b). The later study also showed that H. polygyrus infectivity was also reduced within the first

three weeks of age suggesting a maturation period was required before maximal establishment could

be achieved (Kerboeuf 1978b).

The experimental implications of these results suggest H. contortus can be maintained for a

minimum of 7 months without any negative impacts on infectivity, other life-traits or fitness.

Although age does eventually impact establishment and other life-traits we are not able to specify

exactly when this happens except to say somewhere between 7 – 16 months. As reductions in

establishment capacity as a function of L3 age have been observed across species i.e. H. contortus, T.

colubriformis (Mallet and Kerboeuf, 1985) and H. polygyrus (Kerboeuf 1978a), it may occur in other

GIN species as well. However, the rapidity the infectivity is affected may be specific to the species,

and certainly, as a function of the temperature in which they are maintained (Mallet and Kerboeuf

1985). Similar studies for other common GIN species maintained experimentally may be useful. It

does not appear that L3 ageing is an isolate related issue, at least within the first four months.

While the cryopreserved stocks experienced a reduced capacity to establish relative to the

refrigerated stocks, we cannot conclude that their establishment was altogether poor. Indeed, we

maintain that there is a place for cryopreservation, especially for the long-term maintenance of the

isolates or rare GIN species. The relative aim of the experiment at hand will also influence whether

refrigerated or cryopreserved stocks should be used. For example, where H. contortus establishment

is of prime relevance to a study, refrigerated stocks less than 7 months of age would clearly be more

useful. However, where offspring production is of interest such as for multiplication and culturing of

stocks, cryopreserved stocks would be perfectly adequate. Importantly, in the interest of comparing

and contrasting different experimental studies in the literature, we suggest it would substantially

reduce bias if refrigerated stocks less than 7 months of age were used in laboratory experiments

using H. contortus.

Acknowledgments

120

C. Chylinski is a grateful recipient of Marie Curie ITN funding “NematodeSystemHealth”. Thanks to T.

Chaumeil and team for their care of the sheep.

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NEMATODE DISUCSSION

All of the selective pressures studied here had a negative impact in one way or another on H.

contortus. The duration of these costs however were largely limited to single life-history traits and

the losses consequently recuperated to maintain fitness. These results strongly underline the

importance of looking at fitness as a whole to understand the effects of a selective pressure.

Focusing on the effect to a single life-trait can otherwise result in very misleading conclusions being

drawn. The survival and reproduction components of fitness often acted as counterbalances for each

other. There were several instances throughout these studies whereby reductions to H. contortus

survival were redressed with alterations in reproduction. For example, the study on anthelmintic

resistance (AHR) found the susceptible ISE isolate to incur substantial decreases in establishment in

resistant sheep compared to susceptible sheep, but this was followed by greatly augmented

fecundity and egg to L3 development to maintain fitness. The cryopreserved stocks of H. contortus L3

had a significantly reduced capacity to establish in their host compared to refrigerated stocks, but

they also had significant increases in fecundity to compensate. Similarly the 16-month old

refrigerated stock of H. contortus L3 experienced significant reductions in their establishment

compared to stocks 4- or 7-month old stocks, but no differences in fitness were observed owing to

their increased fecundity. Finally, exposure of L3 to desiccation conditions killed 65% of the H.

contortus infective dose, but those survivors had such an increased fecundity that they achieved

similar levels of fitness to those infections which received 100% living L3 in their infective dose. This

capacity to rectify the costs of challenges localized to one life-history trait by altering their efforts at

others has clearly emerged as one of the driving forces behind H. contortus success.

Trying to determine how selective pressures are shaping the long-term success of H. contortus is

rather more complicated. It requires an indication of how their adaptation potential will be affected.

Demonstrating any kind of adaptation or evolution in in action is generally not amenable to the time

frame of PhD research. Yet we were able to side-step this slightly in two ways. Firstly, in comparing

the different H. contortus isolates with varying AHR capacities, the adaptation had already taken

place. Access to the anthelmintic susceptible ISE isolate provided a benchmark with which to draw

success comparisons against. Secondly, by serial passaging the GIN populations through sheep

experimentally, adaptation could in essence be accelerated by increasing the number of successive

generations exposed to a single selective pressure. Generally on the field, GIN populations are

thought to complete two full life cycles. With serial passage, we were able to complete six life-cycles

in about the same time to explore the influence of resistant sheep on H. contortus success. We shall

discuss how AHR and resistant sheep are influencing H. contortus populations of the future in turn.

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Of all the selective pressures studied, being AHR had the most extensive negative effects on H.

contortus success. The discussion shall largely refer to the costs of AHR in reference to the LEV

resistant BOR isolate as recent tests make us confident in their resistance status. Following the

introgression of the multi-AHR KOK isolate with the susceptible ISE isolate to produce KOKISE, we

cannot be sure of their continued resistance status except to know their susceptibility to LEV had

increased significantly. The BOR isolate experienced numerous costs compared to ISE including:

reduced fitness, reduced gene expression, reduced responsiveness/plasticity to challenge of resistant

sheep, increased vulnerability to heat and cold stress and increased pathogenicity. We can thereby

say with a degree of confidence that in the absence of exposure to the drug, LEV resistance reduced

the success of H. contortus. It was very interesting to note that there was a positive correlation

between reduced fitness and reduced gene expression in the BOR isolate and increased fitness and

increased gene expression in the ISE isolate. It is somewhat logical to assume that the greater the

number of genes being expressed would enable a greater choice of fitness responses to draw upon in

the face of selective pressures. Indeed, genetic variation has been predicted to correlate with fitness

and in the case of heritability’s, with evolutionary potential (Franklin 1980; Ralls and Ballou 1986;

Soulé 1986; Frankham 1995; Lande 1995). Given H. contortus success is often correlated back to its

comparatively higher levels of genetic diversity compared to the other common GIN, the BOR isolate

provides a nice inverse example of this within a species. There was however an inconsistency in this

argument whereby the intorgressed KOKISE isolate had the greatest number of genes expressed but

this did not earn them the greatest level of fitness.

The study exploring the effect of anthelmintic resistance contrasts with those of the other selective

pressures studied in one obvious way, it was measuring how a phenotype affected fitness rather than

an external factor. Each of the external selective pressures largely target specific life history traits:

sheep resistance targeted parasitic L3 to adult stages; density dependence limited adults and

fecundity; hot and cold temperature stresses affected free-living egg to L3 stages, while desiccation

and storage (cryopreservation and refrigeration) affected L3 stages. And more often than not, any

negative costs were recovered at later in the life-history. The AHR however negated every life-history

trait examined. This highlights that H. contortus transcriptome to be another contributing factor to

their success, and limiting it can thereby be a weakness. It opens the question as to the potential

value of the continued use of LEV in the field to maintain LEV-resistant populations that are, in effect,

more vulnerable to control.

The effect of resistant sheep on GIN success was examined in three different studies: i) to explore

whether exposure to resistant Martinik Blackbelly sheep increased H. contortus fitness; ii) to explore

whether exposure to Romanov sheep selectively breed for resistance increased fitness in T.

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circumcincta; iii) to test for an interaction with AHR isolates. Only one of the studies obtained results

which favour the efficacy of resistant sheep. The exposure of H. contortus to Martinik Blackbelly

(MBB) sheep observed the sheep strongly regulated H. contortus fitness via reduced establishment

and FEC. Furthermore, six generations of passage did not drive evolution of the H. contortus fitness

when consequently tested in susceptible sheep. In effect, this supports the main arguments in favour

of selectively breeding resistant sheep for control; reduced infection intensity, reduced

contamination and minimal likelihood for H. contortus adaption to overcome the control.

Direct contradictions for these results were observed in T. circumcincta passaged for three

generations in resistant Romanov. While the resistant sheep continued to regulate fitness

significantly, they simultaneously selected for increased fitness in T. circumcincta when consequently

tested on susceptible sheep, with significantly increased establishment and egg to L3 development.

This would indicate that the T. circumcincta were ‘learning’ from their experience to become more

aggressive in their infectivity and transmission strategy. The results of the AHR study also show

disappointing results following a single infection in resistant MBB whereby none of the H. contortus

isolates irrespective of AHR status experienced a reduction in fitness compared to their performance

in susceptible sheep.

The fact that T. circumcincta appeared to have a greater adaptation potential to sheep resistance in

fewer generations compared to H. contortus was surprising. It may be that we over-estimated the

greater genetic variability in H. contortus compared to T. circumcincta to result in adaptation, or it

may also result from differences in the intensity and type of host pressure they were exposed too.

Given MBB’s resistant status is largely attributed to their capacity to reduce the H. contortus success

from their first primary infection we may assume that their protective responses maximize on the

early innate responses. The Romanov breed however is not renowned for their resistance, and

resistance was likely achieved through different mechanisms. Where the Romanov sheep were

selected for their resistance status, the MBB were not. Furthermore, where the resistant status of

the Romanov sheep stayed stable throughout the three passages, the MBB resistance fluctuated

considerably between passages. Thus it maybe that both the consistency of exposure and the

mechanism of resistance T. circumcincta were exposed to were a greater driver of GIN fitness

compared to the inconsistent performance of MBB. However, what we saw in the experiment using

MBB is perhaps a better representation of what we would see in a field scenario of resistant sheep.

Even among selectively breed sheep, the intensity of the host response will likely vary between the

flock meaning that successive generations of GIN will not face a consistent selective pressure that is

needed to drive adaptation. Our interpretation of these results thereby support the potential

sustainability of selectively breeding sheep for resistance, but they do however cast some doubt over

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their efficacy in GIN control. The relative evidence found in this thesis for and against selectively

breeding sheep is considered further in the general discussion.

Climatic stress presented a reliable barrier to H. contortus free-living stages. The exposure of H.

contortus to one day of desiccation reduced the surviving population by 65%. It is interesting to

question whether the remaining 35% had a genetic predisposition to survival or whether the result

was rather more random. If a genetic basis was involved, chances for adapting the population would

appear somewhat unlikely for the same reason overcoming resistant sheep seems improbable, the

lack of consistent exposure to the selective pressure. It seems unlikely desiccation alone could block

H. contortus completely given the surviving 35% of the population consequently attained similar

levels of fitness to the non-desiccated group owing to massive increases in fecundity. The heat and

cold stress the egg to L3 stages were exposed to in the AHR study were fairly crushing to H. contortus

success. However, had we followed these stages into the second generation it is possible these costs

could have been recuperated. In light of the studies examining the effects of refrigeration and

cryopreservation on H. contortus fitness, we may speculate that dry hot climates would be more

detrimental to their success than the cold. Where the cooler temperatures seem to preserve the life

of the L3, heat and desiccation stress likely accelerates it. Examples from the field however do not

support this completely, where H. contortus have continued success in the deserts of Mauritania

where rainfall is restricted to only several days a year.

In conclusion, the results of these studies consistently highlight the H. contortus capacity to

“respond” to challenges by altering other life-history traits is one of the central factors in their

success. It seems that both the free-living and the parasitic stages possess the necessary sensory

machinery to induce an appropriate response. This puts forth a strong argument for the use of

integrated parasite management (IPM) approach to GIN control, whereby pressure should be applied

to each of the life-history traits to minimize the chance for consequent recuperation of costs by

augmenting other life-history traits.

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Table 1. The fitness results of the H. contortus in response to different selective pressures. The breed

and age of the sheep must be considered in comparing fitness between studies

Selective pressure H. contortus fitness

ISE susceptible sheep 91.2 ± 62.6

ISE resistant sheep 90.8 ± 136.6

BOR susceptible sheep 19.5 ± 27.8

BOR resistant sheep 26.5 ± 20.7

H. contortus isolate exposed to susceptible sheep

H. contortus isolate exposed to resistant sheep

Not desiccated L3 205 ± 51 SE

Desiccated L3 214 (± 107 SE

L3 stocks refrigerated 4° for 4 months 38.53 ± 7.83

L3 stocks refrigerated 4°C for 16 months 55.89 ± 0.72

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SHEEP INTRODUCTION

Over the last 40 years, sheep resistance biology has come a along way. Since it first became clear FEC

could be used as a fairly reliable indicator of GIN parasite burdens (LeJambre 1971) the hypothesis

that sheep could be selected for GIN resistance was put into play. During the 1980s, a number of

genetic selection studies showed that a concentrated selected pressures could produce sheep with

consistently low FEC which were resistant to GIN (Widon 1991; Morris et al. 1993; Woolaston and

Piper 1996). Our understanding about the genetic basis for resistance has continued to advance

further still with the aid of genetic mapping studies (Beh et al. 2002; Beraldi et al. 2007; Davies et al.

2006; Dominik et al. 2010; Kemper et al. 2011; Matika et al. 2011; Sallé et al. 2012, 2014). Selective

breeding is now in a position where it can successfully incorporate resistance traits into a selection

index which takes into account other production trait goals such as finer wool or faster growth rate

(Hunt et al. 2013).

Of all the factors known to influence sheep resistance i.e. breed, age, nutrition, reproductive status

etc., sex has received comparatively little attention. According to the literature, males of many

species are more susceptible than females to infections caused by parasites, fungi, bacteria and

viruses (Klein 2000). Despite the importance of this finding, no work could be found exploring the

mechanisms behind the sex effect of sheep with GIN. There are several studies which document

males being more prone to parasitic infections compared to their female counterparts (Poulin, 1996;

Moore and Wilson, 2002; Morand et al., 2004; Krasnov et al., 2005,; 2012; Perez-Orella and Schulte-

Hostedde, 2005; Hoby et al., 2006; Cowan et al., 2007). However, this pattern does not seem to be a

universal rule, as many counter examples exist (Behnke et al., 1999; Farrari et al., 2004; Fuentes et

al., 2004; Ferrari et al., 2004; Milazzo et al., 2010; Hillegass et al., 2008; Sanchez et al., 2011). These

contradictory examples stress that the processes that may explain sex biased parasitism are still

poorly understood. Determining whether there is a sex effect in resistance to H. contortus thereby

provides one of the focuses of this work.

For any selection programme to be successful, it is essential that the superior individuals can be

identified accurately and economically from among the candidate breeding stock. There are direct

and indirect means to help make those selections. The only real direct measure by which nematode

burden can be determined is by killing the sheep and counting the worms in the abomasum. Clearly,

this does not lend itself nicely to selective breeding programs. The next best thing is faecal egg

counts (FEC) of the number of GIN eggs passed in the host faeces. The FEC have been shown to have

moderate (0.61) to high (0.91) correlation to nematode burdens (Baker et al. 1991; Stear et al;

1995a; Bisset et al. 1996) and in essence, may be considered a direct measure. The measure of FEC

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bring the added advantage of being the most practical way to estimate potential pasture

contamination from transmission stages, which is in one sense, also a direct measure (Axford et al.

2000).

Indirect traits include physiological, immunological, genetic or hormonal parameters. Parasite

infections induce pathology at levels which can range from mild through to severe. Even mild

symptoms can still have a significant economic impact in commercial livestock production so the

measurement of these is important. Although the site of infection and many of the sites generating

immune responses inside the animal are not accessible for measuring the animal’s phenotype, many

sample types can be accessed readily, and most have been used to measure parasite responses in

sheep. For GIN, some specific pathologies are measurable and quantitative. Haemonchus contortus

feed on large quantities of blood, and this effect can be measured by monitoring blood haemoglobin

concentration or haematorit levels. These measurements are correlated with FEC and also the total

worm burden as measured by the mass of worms collected post mortem (Le Jambre et al. 1971).

Another measurable response to parasite infection is a rise in blood eosinophils. The phenomenon

has been linked to parasite resistance (Dawkins et al., 1989; Buddle et al., 1992; Rothwell et al.,

1993), but its use to evaluate animals kept under field conditions was found to be limiting

(Woolaston et al., 1996). Again, however, the main criterion used to evaluate the measure was

correlation with FWEC; perhaps this trait is worth re-evaluation (Hunt et al. 2013).

Antibody responses to parasite infection are measurable and can be qualitatively and quantitatively

indicative of the type of immune challenge and the resulting response. GIN typically induces IgA, IgE

and IgG1 responses in sheep, and these have been associated with genetic resistance (Gill et al.,

1993; Bendixsen et al., 2004). A more recent immunological parameter is the use of salivary

antibodies for carbohydrate larval surface antigen (termed CarLA) specifically against the GIN

Trichostrongylus colubriformis is suggested to be a useful correlate with nematode resistance in

sheep (Harrison et al. 2008).

Hormones have also recently emerged as a potential indicator with studies showing the

concentration of ghrelin, an appetite controlling hormone, circulating in blood correlated with

nematode resistance (Ingham et al. 2011). Significantly, the ghrelin concentrations were predicative

in uninfected sheep with basal concentrations of ghrelin shown to be more than two fold higher in

genetically susceptible sheep.

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For genetic markers, two strategies can be used to identify genes influencing resistance. The first

does not require an existing genetic map and uses the analysis of candidate genes, which are

expressed genes that may be expected to play a role in regulating resistance (i.e. genes encoding

immunoglobulins, MHC antigens, T cell receptor molecules etc) (Axford et al. 2000). The second

method relies upon linkage maps and genome wide analyses for quantitative trait loci (QTL)

detection. This method is based upon the use of polymorphic DNA markers to tag specific genes or

regions of the genome carrying resistant genes.

With the exception of the hormone marker ghrelin, all of these markers require the sheep are

infected to determine their resistance status. We question whether it is possible to look at biological

markers that will give a predictive indication of resistance outcome.

We selected to monitor host body temperature in relation to infection outcome. The regulation of

body temperature is a fundamental homeostatic function governed by the central nervous system

(CNS) of homeothermic animals (Nakamura, 2011). Any external or internal threats to the

homeostatic balance, such as an infection, can produce fluctuations in body temperature. Monitoring

body temperature is thereby considered to be one of the most relevant physiological parameters

available to gain insight into the pathophysiological response of an infection in animal models

(Williamson et al. 2007).

Fever has been found to be particularly advantageous in surviving bacterial infections by enhancing

immune cell activities (Rosenspire et al. 2002) and making the internal thermal environment out of

the temperature range optimal for pathogen growth (Miyamoto et al. 1995). This has resulted in host

temperature being monitored as a predictor for some bacterial diseases. We questioned whether the

same could be done for H. contortus infection in sheep.

In natural populations of sheep or other animals, resistance to GIN is the exception rather than the

norm. It suggests that this degree of resistance is rarely favoured by natural selection. It begs the

question as to whether there is a cost for GIN resistance to the host? At first sight this may seem

surprising since we might expect the hosts survivorship and reproductive potential to increase in

relation to the degree of elimination of a parasite. This is likely to be a naïve expectation however as

it ignores both the cost of elimination to the host and the fact that the cost of infection and thus the

benefit of attacking the parasite may not be simple linear function of parasite burden (Behnke et al.

1992). The cost of infection by H. contortus is substantial (Simpson et al. 2000 etc) with the

developmental worms causing substantial pathophysiological damage (Bueno et al. 1983) and the

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adults causing massive losses in blood. The concept that the immune response may be a source of

pathology to the host has been noted before (Simpson 2000; Greer 2008; Williams 2011). Pathology

due to immune responses can be grouped into two broad areas, first loss of bodyweight due to a

competition for nutrients between growth and second, the immune system and the direct effects of

immune mechanisms on tissues and or metabolic systems (Williams et al. 2011). Indeed, with the

effects of these combined, the can very easily start to mimic the effects of infection previously

attributed to the parasites alone. It is not that certain who is responsible for which symptom. To

disentangle these possible effects of the parasite and host, we explore this further in this chapter.

We further consider whether the pathophysiological damage caused by the early developmental

stages is painful for the sheep host.

Key findings

A sex effect exists in the capacity of Martinik Blackbelly to resist infection to H.

contortus

Mean FEC for females > 1 EPG, males = 745 EPG

Females greater immune gene expression for mucosal defense, males greater

immune gene expression for Th2 response

Females greater local gene expression, males greater systemic gene expression

Higher sheep body temperature before H. contortus infection correlated to

lower FEC

Resistant sheep experience pathophysiological costs of infection

No pain was detected within these specific experimental conditions

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Exploring immune gene expression relative to sheep resistance against

Haemonchus contortus: A story of sex?

Caroline Chylinski1, Jacques Cortet1, Christelle Grisez2, Françoise Prevot2, Philippe Jacquiet2, Jacques

Cabaret1

1 INRA et Université de Tours, UMR 1282, ISP 213, 37380 Nouzilly, France

2 Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31300 Toulouse, France

Abstract

Mounting evidence suggests that a sex effect exists in the capacity of hosts to resist parasitic

infections, whereby the males are consistently found to be the ‘weaker sex’. Using sheep infected

with the gastrointestinal nematode Haemonchus contortus as a model, this study explored whether

the sex effect could be attributed to differential expression of genes associated with the protective

response. An experiment using 49 H. contortus infected adult Martinik Blackbelly sheep (24 ewes, 25

rams) found low levels of infection in the males (mean 745 EPG), but near complete resistance in the

females (mean <1 EPG). Eight of the most extreme sheep were selected for further analyses using

quantitative RT-PCR i.e. the most resistant and susceptible males and females. Ten immune genes

previously implicated in the resistance of Blackbelly lambs were compared in the abomasal lymph

nodes and mucosa tissues. Data analyses focused on qualitative trends and networks. The results

show the males had a stronger expression of the Th2 cytokines (IL-4, IL-5, IL-13) typically associated

with resistance. The females had a greater expression of genes associated with the mucosal

defenses, including the secretion of lectins (Intelectin 2, Galectin 15) and epithelial repair (Trefoil

factor 3). Furthermore, up-regulation occurred most frequently in the abomasal lymph nodes for the

males but the abomasal mucosa in females. This is the first known attempt to explore the sex effect

in adult sheep resistance against gastrointestinal nematodes. The results provide interesting insights

and potential new avenues for future research into this phenomenon.

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Introduction

Sheep resistance to gastrointestinal nematode (GIN) infection has received substantial attention in

the past. In wake of the rapid development of GIN resistance against the most commonly used

anthelmintic drugs, selectively breeding sheep for resistance against GINs is increasingly seen as a

key tool for the sustainable control of parasites (Waller and Thamsbourg, 2004). Numerous factors

have been identified to influence the relative resistance status of sheep including breed (Terefe et

al., 2007), individual susceptibility as reflected in aggregated distribution of nematodes (Gaba et al.,

2005 ), age (Kosi and Scott, 2001), nutrition (Wallace et al., 1996; Coop and Kyriazakis, 2001) and

reproductive status i.e. the peri-parturient rise (PPR) (Sykes, 1994). There is mounting evidence to

suggest that another variable may be in play, that of sex-biased parasitism. Studies in other host-

parasite systems have consistently found males to be the ‘weaker sex’ when it comes to resistance

(Poulin, 1996; Moore and Wilson, 2002; Morand et al., 2004; Krasnov et al., 2005, 2012; Hoby et al.,

2006; Cowan et al., 2007). Sex differences in sheep against GIN infection have been observed

(Barger, 1993). Yet despite the importance of this finding, no work could be found exploring the

mechanisms behind the sex effect of sheep with GIN. The aim of this study was to thereby explore

whether the sex effect was evident in sheep infected with Haemonchus contortus, and if so, whether

these differences could be attributed to the differential expression of genes implicated in the

protective response.

Several studies have been done contrasting the gene expression of resistant sheep breeds against

susceptible breeds (Lacroux et al., 2006; Terefe et al., 2007). The results of these studies found that

both breeds elicited similar immune genes, but their level of expression was greater in resistant

sheep (Terefe et al., 2007). We hypothesized differential gene expression could also explain the sex-

biased parasitism against GIN whereby the females elicited stronger levels of immune-gene

expression compared to the males. To explore this sex effect, the study selected an H. contortus

resistant breed of sheep, Martinik Blackbelly (MBB). While the majority of previous studies have

explored differential gene expression on a more extreme scale i.e. resistant vs. susceptible breeds,

this study looks at resistance on a finer scale i.e. differentiating between succumbing to low levels of

infection and rejecting the infection completely. This was done by first infecting a group of 24 female

and 25 male MBB sheep with H. contortus. After establishing the female sheep were significantly

more resistant than the males based on faecal egg counts (FEC) and worm counts, four female and

four male MBB were selected for further study of immune gene expression using quantitative RT –

PCR. The results of this study find distinct patterns of immune gene expression between the sexes as

well as differences in the local vs. systemic expression of the genes.

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Sheep and parasitological data

The study used 24 female and 25 male MBB sheep. All sheep were 18 months old at the start of the

experiment and had never been mated. None of the sheep had previous experience of GIN infection,

as verified by negative faecal egg counts prior to the study. The sheep were maintained indoors

throughout the study, with the males separated from the females in different pens. They were fed

hay and feed concentrate ad libitum and had open access to water. All sheep were administered an

infective dose of 10,000 H. contortus L3 infective larvae per os.

Faecal samples were collected from all sheep on 0, 14, 19, 21, 25, 28, 32, 39, 42 and 53 dpi to carry

out FEC using a modified McMaster technique (Raynaud, 1970) in a sodium chloride flotation

solution, accurate to 50 eggs per gram (EPG) of faeces.

Blood samples were collected from all sheep on 0, 6, 19, 25, 32, 42, 53 dpi. The samples were drawn

from the jugular vein of the sheep into two heparin and two dry tubes using a 10 ml gauge syringe.

These samples were used to determine the pepsinogen levels using a colorimetric assay described by

Kerboeuf (1975) and haematocrit percentage using a manual Hawksely micro-haematocrit reader.

The sheep were weighted using walk-on scales on 0 and 63 dpi.

Sheep were necropsied at 63 dpi by electronarcoses and ensanguination. The number of worms

established in the sheep was determined by counting the number of adult worms found in the

abomasum following the procedure described by Gaba et al. (2006). Tissue samples were taken from

the sheep including 2 x 2 cm piece of the pyloric (P) and fundic (F) regions of the abomasal mucosa

and a 3g sample of abomasal lymph nodes (ALN) from various nodes. The tissue samples were

immediately put in RNAlater and stored at -20°C.

RNA extractions and quantitative RT-PCR

Based on the parasitological data of FEC and worm counts, four female and four male Blackbelly

sheep were selected for further analyses using quantitative (q)RT-PCR. The two sheep with the

highest and lowest FEC were selected from within each sex to represent the most extreme

susceptible and resistant individuals respectively.

Total RNA from abomasal fundic mucosa and draining lymph nodes of the sampled animals

were extracted following the commercial RNeasy Mini Kit (Qiagen). The RNA quality of the

recovered RNA was monitored by A260/A280 spectrophotometry. RNA were subsequently

135

reverse transcripted to cDNA with a Reverse Transcriptase commercial kit (Invitrogen). For more

details, these procedures have been extensively described in Lacroux et al. (2006).

The genes selected for study using qRT-PCR are outlined in Table 1. These were selected based on

results from previous studies which observed these genes to be upregulated in Martinik Blackbelly or

resistant Martinik Black Belly – Romane back cross sheep following infection with H. contortus

(Terefe et al., 2007; Sallé et al., 2014). The relative expression of these genes was conducted on the

sheep outlined in Table 2.

Table 1. Genes selected for study using qRT-PCR, their implied function and the tissue in which their

expression was studied; ALN (abomasal lymph node) or abomasal mucosa P (pyloric), F (fundic)

regions.

Gene Implied function Tissue examined

TNF-α Pro-inflammatory ALN

TFF3 Heal epithelium Mucosa (P, F)

OX40 T-cell activation / co-stimulation ALN

IFN-ƴ Th1 cytokine ALN

CXCL14 Neutrophil chemokine ALN

CCL26 Eosinophil chemokine ALN

Galectin 15 Lectin ALN and Mucosa (P,F)

Intelectin 2 Lectin ALN and Mucosa (P, F)

IL-4 Th2 cytokine ALN and Mucosa (P, F)

IL-5 Th2 cytokine ALN and Mucosa (P, F)

IL-13 Th2 cytokine ALN

Primers were designed for these particular genes using the "primer 3" NCBI website (Salle et al.

2014). The qPCR was performed using a SYBR green PCR kit (Qiagen) with three replicate per sample.

Gene expression data was normalized against five housekeeping genes (s26q, HPRT, ACTB, SDH, TyQ)

which acted as endogenous controls for each individual sheep based on their gene-wise stability

value as reported in Vandesompele et al. (2002). Differential expression was tested following the

136

DDCt method of Livak and Schmittgen (2001). The cycle time (Ct) value of the gene of interest was

corrected by the average level of reference genes expression. A Wilcoxon test was applied to

determine any significant difference between the compared groups, i.e. p < 0:05. The complete data

processing was performed using a homemade R script (R software, http://CRAN.R-

project.org/doc/FAQ/R-FAQ.html). No significant differences were observed here and consequently,

a more sensitive approach to gene expression analyses was adapted and described below.

Analyses

Determining what differences in gene expression constitute levels importance is not straightforward.

While statistics can of course be applied, these tests ignore the possibility that even small changes in

gene expression may have big impacts on the phenotypic outcome, either acting individually or in

synergy with other expressed genes and their proteins. We thereby placed greater emphasis on the

looking at the ‘trend’ of gene expression. This was done using a binary matrix approach which simply

attributed increases or decreases in gene expression using binary code to highlight patterns in gene

expression. More specifically, for each gene examined the Ct expression values for the 8 sheep were

ranked in order and the median value selected. Individual sheep with Ct values below the median

were coded 0, those above were coded 1. Differences between the males and females were

highlighted if ¾ of individuals from one sex had codes that opposed ¾ of the individuals from the

opposing sex. Principal Component Analyses were then carried out to understand the relationship

between individual sheep and their respective gene expression.

Results

Table 2 presents the parasitological data for the both female and male MBB. Significant differences

between the sexes were observed for the mean EPG, worm burden and pepsinogen.

http://cran.r-project.org/doc/FAQ/R-FAQ.html

http://cran.r-project.org/doc/FAQ/R-FAQ.html

137

Table 2. Parasitological data for the female (n = 24) and male (n = 25) MBB. Means based on samples

taken on various days throughout experiment: EPG n = 7 i.e. 21, 25, 28, 32, 39, 42, 53 dpi ;

pepsinogen n = 6 i.e. 6, 19, 25, 32, 42, 53 dpi. PCV loss difference between 63-0 dpi. * denotes the

sex with significantly greater result (ANOVA p = 0.05).

EPG

range

EPG

mean

Worm burden

range

Worm burden

mean

Mean

PCV loss

Mean

pepsinogen

Mean

weight gain

(kg)

Females 0 – 120 < 1 0 – 217 11 10.75 966* 1.7

Males 0-11149 745* 0 – 1685 435* 7.4 607* 1.8

The parasitological data for the eight sheep selected for qRT-PCR analyse are presented in Table 3.

The two sheep with the highest and lowest EPG, implying susceptibility and resistance respectively,

were selected from each sex.

Table 3. Parasitological data for the female (4) and male (n = 4) MBB selected for qRT-PCR analyses.

Means based on samples taken on various days throughout experiment: EPG n = 7 i.e. 21, 25, 28, 32,

39, 42, 53 dpi ; pepsinogen n = 6 i.e. 6, 19, 25, 32, 42, 53 dpi. PCV loss difference between 63-0 dpi.

No. Sex Resistance

status

Mean

EPG

Total

worms

PCV loss Mean

pepsinogen

Weight

gains (Kg)

1 F S 5 43 13 1311 1.3

2 F S 15 0 16 1009 1.4

3 F R 0 0 5 597 4.9

4 F R 0 0 14 904 3.9

5 M S 4081 13 13 859 - 2.4

6 M S 2106 1672 8 777 3.0

7 M R 25 25 1 889 2.9

8 M R 25 25 4 1036 2.3

Sex differences observed in gene expression are presented in Table 4. The females had a

comparatively greater expression than the males of Intel2 (ALN), Gal15 (P), TFF3 (P), IL-4 (F) and OX-

40 (ALN). The males had comparatively greater expression of IL-4 (ALN), IL-5 (P) IL-13 (ALN) and

CCL26 (ALN).

138

Table 4. Genes observed to be differentially expressed between the sexes where ¾ of the sheep from

one sex consistently had greater (+) expression than the median and ¾ of the opposing sex

consistently had less than the median (-).

Group

(n=4)

Intel2

(ALN)

Gal15

(P)

TFF3

(P)

IL-4

(F)

IL-4

(ALN)

IL-5

(P)

IL-13

(AL N)

CCL26

(ALN)

OX40

(ALN)

Female + + + + - - - - +

Male - - - - + + + + -

PCA analyses show that gene expression in females is better described by changes in the abomasal

mucosa (P and F regions combined) (Fig. 1) whereas the male gene expression was better described

by changes in the ALN (Fig. 2) (variance 60%). In the females, the two susceptible sheep (1 and 2)

are positioned close together away from the two resistant sheep (3 and 4). The position of the

susceptible and resistant males is mixed.

139

Fig. 1: Female PCA analysis showing relationship between individual sheep (circle) and specific gene

(triangle) expression. Only those genes which well describe the individuals are shown. The

significance of the relationship increases as you proceed along the axes from the intersection.

Susceptible sheep = 1, 2; resistant sheep = 3, 4.

Fig. 2: Male PCA analysis showing relationship between individual sheep (circle) and specific gene

(triangle) expression. Only those genes which well describe the individuals are shown. The

significance of the relationship increases as you proceed along the axes from the intersection.

Susceptible sheep = 5, 6; resistant sheep = 7, 8.

Discussion

The parasitological results from this study clearly demonstrated that female MBB are significantly

better at resisting H. contortus infection than their male counterparts. This supports previous

findings of a female biased sex effect in other host parasite systems (Poulin, 1996; Moore and

Wilson, 2002; Morand et al., 2004; Krasnov et al., 2005, 2012; Hoby et al., 2006; Cowan et al., 2007).

140

A striking difference was identified in the gene expression patterns between the two sexes, as well as

the tissues in which they were expressed. The female MBB were observed to have greater expression

of genes associated with mucosal barrier defense compared to the males including Intel2 (G), Gal15

(P) and TFF3 (P). The mucus layer in the gastrointestinal tract forms the first line of defense to the

external environment (Rinaldi et al., 2011) and it is known that a vigorous and effective mucosal

immune response is essential for the development of resistance to GIN in sheep (Ingham et al.,

2008). Mucins or mucus glycoproteins are considered to be essential components of the mucus

barrier (Rinaldi et al., 2011). Lectins specifically have been implicated in the immune rejection

response (Meeusen et al., 2005; French et al., 2008) which is supported here by the up-regulation of

Galectin 15 and Intelectin 2 in the female MBB. It is thought these proteins bind both to mucins and

to the parasite surface increasing both mucus viscosity and its adherence to the parasite (de Veer et

al., 2007; Vasta et al., 2009) to consequently expel them. Galectin 15 expression has previously been

shown to be greatly up-regulated in helminth larval infections of the gastrointestinal tract (Dunphy et

al., 2000). Likewise, up-regulated levels of Intelectins have been observed in the mucosa of sheep

infected with the GIN Teladorsagia circumcincta during parasite expulsion (French et al., 2008). We

may deduce that the up-regulation of these innate barrier defenses in the female MBB kill or expel

the early larval stages of H. contortus before establishment could take place. However, given the pre-

patent period of H. contortus is 21 days (Veglia, 1915) we as yet lack sufficient data to pinpoint at

which developmental stage the larvae are expelled. As the larval stages are known to provoke

substantial pathophysiological damage to the host (Bueno et al., 1982), early expulsion of the arriving

L3 would likely be a more optimal strategy.

The female MBB further exhibited an up-regulation of the TFF3 gene. Trefoil factors are also major

secretory products of normal mucus secreting cells in the epithelium of the GI tract (Ingham et al.,

2008) and are thought to play a major role in wound responses to maintain mucosal surface integrity

as well as pathological processes (Hoffmann et al., 2005; Thim et al., 2001). The up-regulation of this

gene would suggest the females experienced a degree of epithelial damage. This is supported in part

by the significantly increased levels of pepsinogen observed in the females compared to the males,

both on a group level (n = 24) and for the eight sheep analysed with qRT-PCR (n = 4). The increased

pepsinogen levels likely resulted either from direct damage of the developing larvae (Paynter, 1992)

or from increased inflammation of the mucosal tissue owing to the infiltration of leucocytes (Serrano

et al., 1997).

Our results suggest the females may have achieved higher levels of H. contortus resistance as a result

of greater innate mucosal defenses targeting larval stages compared to the males. Targeting larval

stages is also considered a preferable strategy as adult stages are thought to modulate host

141

immunity to promote their own survival, after which, potential effector mechanisms become less

effective (Else et al., 2009). Thus, the rate at which an effector response develops in relation to the

parasite growth rate is at least, if not more important than the level of response (Datta et al., 2005).

Terefe et al., (2007) made similar observations in comparing MBB against the susceptible Romane

breed of sheep following H. contortus infection whereby the MBB up-regulated Th2 cytokines faster.

This may also apply to the observed differences in resistance between the female and male MBB in

this study. Indeed, the female MBB were observed to have up-regulated OX40 expression. OX40 is a

member of the TNF superfamily and has been shown to play a role in the initiation and progression

of antigen-specific T cell activation and co-stimulation (Ekkens et al., 2003). It was perhaps somewhat

surprising to find that the expression levels of genes associated with the innate immune response

were still up-regulated 63 dpi and largely in the absence of any adult worms. It must be emphasized

that these results represent the gene expression in a moment in time. It is entirely possible

substantial differences would have been observed in gene expression earlier on in infection. It would

be interesting to compare the same genes between the males and females at more regular intervals

earlier on in an H. contortus infection. But in this case, it should be kept in mind that under natural

conditions of infections in the field, animals are continuously exposed to infection while grazing and

increases in mucous viscosity may reduce the successful establishment of further incoming larvae

(Robinson et al., 2011).

The immune expulsion of GIN is typically associated with T helper type 2 (Th2) responses (Balic et al.,

2000; Pena et al., 2006; Miller and Horohov, 2006). The associated cytokine profile of a Th2 response

includes interleukin (IL) -4, IL – 5 and IL – 13 (Meeusens et al., 2005). Interestingly, a greater Th2

response was associated with the male MBB rather than the females, including up-regulated

expression of IL-4 (ALN), IL-5 (P), IL-13 (ALN). Thus, either the females expelled the infection prior to

the proper development of an adaptive Th2 response, or, it is possible that an increase in the Th2

environment beyond a certain threshold may bring no additional benefit to the host (Livak and

Schmitthen, 2001).

Following PCA analyses, the results showed a strong divide between the males and females regarding

the tissues with which their gene expression was more closely associated. Where the females were

more closely associated with local expression in the abomasal mucosa, the males were more closely

associated with expression in ALN. There is conflicting views in the literature on whether the local

abomasal response is more important than systemic ALN. Munoz-Guzman et al. (2006) argue that the

local response is more important for protection as it is in closer proximity with the parasite.

Alternatively, Gossner et al. (2013) suggested that given the immune response to the GIN takes place

in the ALN, the events within the node determine the quality and quantity of the immune response

142

and consequent clinical outcome of infection. The results of this study would rather agree with the

view of Munoz-Guzman et al. (2006) that the local response is more important for resistance. It

should further be noted that expression throughout the abomasal mucosa was not found to be

homogenous between the pyloric and fundic regions. These regions vary histologically and

physiologically (Banks, 1986) with H. contortus largely demonstrating a preference for the fundic

region (Dash et al., 1985; Terefe et al., 2009). These findings support previous studies which also

found the immune response to differ between the regions based on the number of CD4+ and WC1(+)

T-cells, tissue eosinophils and IgA+ plasmas cells (Munoz-Guzman et al., 2012). It is also interesting to

note that neither all the females nor all the males had identical patterns of expression. The PCA

analyses graphs show two members of each sex to be associated with different gene expression than

the other two. This may suggest that the protective response is largely individual and cannot be

categorized on a breed, or even a sex level. While this requires further research, if true it could have

far reaching effects on our current approach to understanding the genetic and immunological basis

of sheep resistance to GIN, as well as our strategy to selective breeding.

In summary, the results of this study suggest that female MBB are better able to control H. contortus

infection compared to males as a result of greater expression of mucosal barrier defense genes as

well as a more localized expression of immune genes in the abomasal mucosa rather than the ALN.

This is the first known study that has correlated parasitological differences in sheep sex resistance to

differing patterns of immune gene expression. It would be of immediate interest to further test

whether the differential levels of gene expression are directly correlated to the consequent levels of

protein production. It is possible that there are other contributing factors to the sex effect observed

here such as sex steroids. Males for example are hypothesized to be more susceptible to infection

than females not only because androgens can modulate immune-competence, but because sex

steroid hormones affect disease resistance genes and behaviors that make males more susceptible to

infection (Klein, 2000). This would provide another interesting avenue to explore in the future.

Understanding sex-based differences in disease pathogenesis likely has an important role for optimal

GIN disease management in both sexes.

Acknowledgements

Caroline Chylinski is a grateful recipient of Marie Curie ‘Nematode Systems Health’ ITN funding. We

gratefully acknowledge the assistance of Thierry Chaumel and team for their assistance with animal

care.

143

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Disentangling the relative contribution of the parasite and the host to

consequences of Haemonchus contortus infection in sheep

Chylinski, C., Cortet, J., Neveu, C., Cabaret, J*.

Introduction

Infection with the gastrointestinal nematode Haemonchus contortus is marked by severe damage to

the pathophysiological well-being of their sheep host and substantial production losses for the

farmer (Sadiqqi et al. 2011). Traditionally, symptoms of infection have been attributed exclusively to

the parasite alone (Sykes 2010) with direct correlations thought to exist between infection intensity

and symptom severity (Kenyon et al. 2009). Accordingly, this has led to the exploitation of common

H. contortus symptoms such as anaemia and reduced live weight gains to be exploited in the field for

the identification of heavily infected sheep (i.e. anaemia via FAMACHA©: Malan et al., 2001; Kaplan

et al., 2004; Mahieu et al., 2007; Kenyon et al., 2009; live weight gains: Coop et al., 1977; 1988;

Hubert et al., 1979; Kenyon et al., 2009). However, it has long been known that resistant sheep under

challenge from GIN do not necessarily remain symptom free, despite the absence of infection

(Clunies-Ross 1932). This suggests a potential role for host-induced influence on the symptom

manifestation. This study thereby aimed to disentangle the relative contribution of the sheep host

and the H. contortus parasite to the symptoms of infection. The results of this question have

immediate importance for our understanding of the infection biology and consequently, how much

faith we can place in certain symptoms to act as indicators of infection intensity.

This was done by comparing infection symptoms in three different groups of sheep with established

H. contortus resistant phenotypes to reflect high, medium and low resistance to infection. The study

monitored three different pathophysiological parameters in the sheep including haematocrit

(anaemia), reduced live weight gains and pepsinogen. While the first two indicators are more

commonly used in field scenarios, pepsinogen is largely used experimentally as an indicator of

abomasal damage from GIN infection (Kerboeuf et al. 2002). By and large, anaemia is thought to

result from the haematophagous activities of the adult H. contortus (Baker et al. 2003; Saddiqi et al.

2010a; b), while the pathophysiological destruction cause by the migrating GIN larval stages is

thought to reduce the functional and absorption capacity of the abomasum affecting pepsinogen

production and nutrient absorption (i.e. live weight gains) respectively (Simpson 2000). The potential

for host-induced symptoms may lie in the high energetic demands of eliciting an effective protective

response (Sykes 2010) against H. contortus infection along with possible immune mediated

149

pathology (Williams et al. 2011). The early non-specific protective responses against infection are

thought to be especially energetically demanding (Sykes 2010), and indeed, pivotal in determining

respective immune-competencies to control H. contortus infection (Terefe et al. 2007). This study

thereby focuses specifically on the symptoms of early H. contortus infection spanning the first 32

days. If the parasites are responsible for the symptoms, the severity of the symptoms should be

worse in the low resistance (Low R) group of sheep. If the host-induced effects contribute more to

the symptoms then the high resistance (High R) group should experience the worst symptoms. If

however the symptoms result from an interaction between the parasites and host protection against

infection then the medium resistance (Med R) group will demonstrate the most severe symptoms.


Sheep

This experiment was approved by the regional ethical committee of Loire Valley (Comité d’Ethique

du Val de Loire, national number 19) under the reference 2011-07-03.

All the sheep used had been reared and penned indoors and had no previous experience of

gastrointestinal nematode infection, as verified by negative faecal egg counts (FEC) prior to the

study. They were fed a diet of hay, feed concentrate and cereals with open access to water and

grouped according to their levels of resistance to H. contortus infection.

The high resistance group consisted of twelve Martinik Blackbelly ewes, 18 months of age. The

medium resistance group consisted of twelve Martinik Blackbelly rams, 18 months of age. The low

resistance group consisted of fifteen Romane rams, 7 months of age. The respective resistant or

susceptible statuses of these sheep breeds have been well documented, but predominantly in lambs

(Terefe et al. 2008).

H. contortus infection

All sheep received a single infective dose of 10,000 H. contortus L3 larvae using the ISE isolate (see

Roos et al. 2004 for full isolate history).The presence and intensity of the H. contortus infection in the

sheep host was carried out using faecal egg counts (FEC). These were performed using a modified

McMaster technique (Raynaud 1970) in a sodium chloride flotation solution, accurate to 50 eggs per

gram (EPG) of faeces. These were carried out at three time points throughout infection: 21, 25, 32

days post infection (dpi). FEC are considered as a reliable phenotypic parameter available to reflect

150

the intensity of a GIN infection on a group basis (Cabaret et al. 1998) and the hosts ability to control

it (Woolaston, 1992; Kemper et al., 2009; Axford et al., 2000).

Haematocrit

Blood samples were drawn from the jugular vein of the sheep using a 10 gauge syringe into heparin

tubes. The haematocrit was calculated manually using a Hawksely micro-haematocrit reader and

carried out on 0, 21, 25, 32 dpi. To determine the overall loss in haematocrit, the value at 32 dpi was

subtracted from the value at 0 dpi.

Pepsinogen

Blood samples were drawn from the jugular vein of the sheep using a 10 gauge syringe into dry

tubes. The samples were centrifuged to separate the serum which was then used to determine

pepsinogen levels following the colormeteric assay described by Kerboeuf et al. (2002). This was

carried out at 0, 14, 21, 25, 32 dpi. To determine the overall increased in pepsinogen, the value at 32

dpi was subtracted from the value at 0 dpi.

Live weight gain

The sheep were weighted using walk-on scales on days 0 and 32 dpi. The value at 32 dpi was

subtracted from the value at 0 dpi to obtain the overall weight gain.

Faecal egg counts

The faecal egg counts (FEC) were carried out to validate the presences and intensity of H. contortus

infection. These were performed using a modified McMaster technique (Raynaud, 1970) in a sodium

chloride flotation solution, accurate to 50 nematode eggs per gram (EPG) of faeces.

Statistical analysis

Statistical analyses were carried out using the SPSS version 11 software package. Calculations were

based on general linearized models (GLM) and estimated marginal means (EMM) were derived from

this. The analysis of variance (ANOVA) was used to determine significant differences and post-hoc

Student Newman-Keuls analysis were used to identify between which groups they existed. The FEC

data was log transformed prior to analysis as it did not follow a normal distribution.

151

Correlation analysis was carried out using Spearman’s Rank coefficient to determine any

relationships between the measures taken. These incorporated all data, irrespective of resistance

status. Correlations to temperature were made on 7, 9, 11, 13, 20, 25, 32 dpi.

Results

The FEC and the worm counts verify the significant differences between the three sheep groups in

their capacity to resist infection (Table 1). The High R group remained virtually uninfected, the Med R

group had low levels of infection and the Low R group had high levels of infection.

The weight gain was inversely related to the level of infection where the greatest weight gain was

seen in the most heavily infected Low R group, followed by the Med R and finally the High R gained

the least weight (Table 1). This difference was significant (ANOVA p = 0.001). When weight gains are

divided by the corresponding FEC, the Med R group gained slightly more weight than the Low R per

worm, although this was not significant (Table 2).

The High R and Low R group showed similar losses in haematocrit, the Med R group lost significantly

less (ANOVA p = 0.001) (Table 1). However, when haematocrit losses are divided by the FEC it shows

the Med R group experienced greater losses per worm compared to the Low R (Table 2). In

comparing the haematocrit losses of the group at 0, 21 and 32 dpi, both the High R and Med R

experienced their losses before 21 dpi and stayed stable thereafter. The Low R group experienced

their haematocrit losses after 21 dpi.

The changes in pepsinogen did not vary significantly between the High R and Low R group, however

the Med R group experienced significantly greater (ANOVA p = 0.007) increases (Table 1). When

pepsinogen increases were divided by the FEC, the Med R group experienced greater pepsinogen

increases per worm than Low R.

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Table 1. Estimated Marginal Means value for examined parameters for each sheep resistance group.

Includes significant differences (ANOVA p <0.05) where different sub-groups (Student-Newman

Keuls) are denoted with different letters in subscript.

High R Med R Low R Significance

FEC 0a 1620b 5066c

0.000

Worms 0.6a 493a 4110b 0.000

Weight gain 0.43a 3.1b 4,9b 0.001

Haematocrit loss 12a 8b 14a 0.001

Pepsinogen increase 112a 500b 48a 0.007

Table 2. Weight and pathophysiological indicator value per worm ± SE. Calculated by dividing the

mean pathophysiological parameters by the corresponding mean FEC of the period for each sheep

resistance group. Includes significant differences (ANOVA p <0.05) where different sub-groups

(Student-Newman Keuls) are denoted with different letters in subscript.

High R Med R Low R Significance

Weight/FEC 0.433 ± 0.268 0.007 ± 0.268 0.001 ± 0.001 0.42

Haematocrit loss /FEC 12.417 ± 0.695 0.013 ± 0.695 0.003 ± 0.622 0.00

Pepsinogen/FEC 112 ± 40 0.494 ± 40 0.01 ± 36 0.08

The differnces seem enormous, probably because we divided the variable by FEC which is near

naught for High R

Discussion

The results of the FEC and worms counts confirm the significantly different resistance status’ of the

different sheep groups. Given the High R group remained virtually parasite free, we may largely

attribute any changes in pathophysiological parameters to result from the host protective response.

The high FEC and worm burden of the Low R group would suggest they exerted comparatively little

153

effort in a protective response and any changes in pathophysiological parameters would be

representative of worm-induced costs. Finally, the Med R group represent the continuum between

host-induced and worm-induced pathophysiological costs. No single group experienced the greatest

expression of all symptoms suggesting that the parasites alone were not solely responsible for

inducing the symptoms evaluated.

Indeed, weight gains were inversely correlated to the H. contortus infection intensity (FEC and worm

burden) whereby the greatest weight gains were seen in the most heavily infected Low R group. This

goes against findings made by Bishop et al. (1996) who observed negative correlations between FEC

and live weight gain in Scottish Blackface lambs. However, numerous other studies have also found

very small differences in weight gains following comparisons of GIN resistant and susceptible sheep

(Amarante et al. 2004; Mugambi et al. 2005; Saddiqi et al. 2010a, b, Vanimisetti et al al. 2004;

Bricarello et al. 2007). There is some evidence to suggest that these reductions in weight gain are

driven by the sheep protective response. Coop and Kyriazakis (2001) attributed reduced sheep

production to the partitioning of nutrients towards immune responses. A study by Greer et al. (2005)

suggested that live weight gains of lambs infected with the GIN Trichostrongylus colubriformis may

result from reduced appetites during the development of immunity, a symptom which was reversed

in lambs that were administered corticosteroids. The study also found that appetite reduction did

not occur in sheep with acquired immunity (Greer et al. 2005) which rather isolates this symptom to

early primary infections. However, we cannot confirm in the present study whether there was a

reduction in appetite in any of the groups as food intake was not monitored. Additionally, caution

should be exercised in drawing weight comparisons across sheep of different breeds, sex and ages

such as those used in this study, as these factors may influence the relative genetic performance

potential and size of the sheep (Saddiqi et al. 2012).

Examining the losses in haematocrit highlighted an example whereby the host protective response

may strongly influence the severity and timing of the anaemia symptom. In this parameter, both the

High R and Low R groups experienced similar losses in haematocrit. Interestingly, the groups

experienced their losses at different moments; the High R and Med R groups before 21 dpi and the

Low R group after 21 dpi. This is important as the pre-patent period of H. contortus is 21 dpi (ref).

This highlights that haematocrit losses are occurring even before the commencement of blood

feeding activities which are traditionally blamed for haematocrit losses. Potential explanations as to

the host-induced losses in haematocrit may result from the stimulated host protective response

redirecting energy away from non-immune tissues (Sykes 2010) such as the production of red blood

cells (RBC). Studies have demonstrated that pro-inflammatory cytokines, which are part of the early

protective response, may negatively affect RBC production by reducing their stimulatory activity

154

(LeGrand and Alcock, 2012) and life-span (Adamson 2008). This however requires further study to

confirm in the case of H. contortus infected sheep.

Increases in pepsinogen were most clearly seen in the Med R group, whereas the Low R and High R

experienced significantly smaller changes. This is somewhat surprising given good correlations have

been found between GIN infection intensity and the level of circulating serum pepsinogen (Kerboeuf

et al. 2002) owing to the damage of the developing GIN (Paynter 1992). Here it appears that the

interaction between both host and parasite factors make the greatest contribution to this symptom

i.e. Med R, and neither the H. contortus (Low R) or host response (High R) alone can be blamed.

Potential host factors contributing to increased pepsinogen are thought to result from the protective

inflammatory response of the mucosal tissue (Serrano et al. 1997) increasing the permeability of the

abomasal mucosa and the diffusion of luminal pepsinogen into the circulating blood stream (Simpson

2000).

The results of this study demonstrate that the H. contortus parasites alone are not responsible for

the symptoms of reduced live weight gains, anaemia or pepsinogen. Instead, it appears the host

protective response may have a contributing influence on the symptom outcome. It appears that the

Med R group had arguably the greatest expression of symptoms, suggesting that being either highly

resistant or susceptible may be a better host strategy to minimize symptoms than being in between

the two. It is important to note however that these results only represent the symptoms up until 32

past a primary infection. It is likely that in a field situation, a Low R group would continue to

experience a decline in pathophysiological parameters as the infection progresses. Thus, both the

Med R and certainly the High R strategy would payoff more in the long-run. These results carry

implications for the use of pathophysiological indicators during early H. contortus infection based on

these parameters where false positive may occur.

Acknowledgements

C. Chylinski is a grateful recipient of Marie Curie ITN “Nematode Systems Health” PhD funding. Many

thanks to T. Chaumeil and team at the animal platform INRA Nouzilly.

155

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Do gastrointestinal nematode infections induce pain in their sheep host?

Caroline Chylinski 1, Jacques Cortet 1, Didier Crochet 2, Juliette Cognié 3, Jacques Cabaret 1

1 INRA, ISP, UMR 1282 Nouzilly 37380 and University of Tours, Tours 37000-F,

2,INRA, PFIE, Nouzilly, 37380-F, 3 CIRE Nouzilly 37380-F

Abstract

Gastrointestinal nematodes are well documented to provoke extensive pathophysiological damage in

their sheep host which we hypothesized to be painful. The presence of pain would have immediate

relevance to production associated costs as well as for livestock welfare. This study explored whether

gastrointestinal nematode infections induce pain in their ruminant hosts using sheep infected with

Haemonchus contortus as a model species. Numerous parameters were incorporated to detect pain

including those relating to sheep host physiology (haematocrit, eosinophils, cortisol, body

temperature), clinical examinations (escape response or vocalization following percussion and

palpation of infected region), lesions (pepsinogen) and performance (weight). The use of analgesic

pain relief was also used to verify results. Despite evidence of tissue damage and indications of

general illness in the H. contortus infected sheep, no other indicators suggested pain ensued within

this model system.

160

1. Introduction

Gastrointestinal nematodes (GIN) of livestock cause disease of major socioeconomic importance

worldwide (Reober et al., 2013). These parasites are well documented to provoke extensive

pathophysiological damage in their sheep host (Simpson 2000) which is considered painful (McClure,

2000). Yet pain as a consequence of GIN infection has never been examined in livestock animals. The

question could have serious implications for the welfare of livestock given the wide-scale prevalence

of the GIN, as well as for farm economics given previous studies have suggested the presence of pain

may reduce production performance (Fourichon et al., 1999; Earley and Crowe, 2002). The aim of

this study was to thereby explore whether GIN infections induce pain in their ruminant hosts using

sheep infected with Haemonchus contortus as a model species.

Previous studies have reported that H. contortus provoke the greatest pathophysiological damage to

their host during the first couple of weeks after initial infection, during which time the

developmental larvae stages penetrate the host abomasal mucosa twice (Christie et al., 1975; Bueno

et al., 1982; Simpson, 2000). This results in lesions of the mucosal tissue and HCl glands which in turn

may alter local gastric secretions (Simpson, 2000). It is these specific events we hypothesized would

induce pain in the sheep. This is the first known study that has attempted to assess sheep pain in

response to GIN infection. The only other relevant document that could be found came from the

Official Veterinary Office in Switzerland (1992) which tried to ascribe a threat level of pain for

commonly researched infections in animals ranging from 0 (no pain) to 3 (maximum threat of pain).

Trichostronglyids were rather vaguely assigned a category 1-3 depending on the dose. However, this

was based on expert opinion and it is not clear which biological or pathophysiological parameters or

data were used to determine this pain threat.

Evaluating pain in sheep is not a straightforward task. They have evolved to demonstrate minimal

indications of weakness lest they appear weak in the face of predators (Fitzpatrick et al., 2006).

Prunier et al. (2012) proposed combining various indicators would improve the efficiency of pain

assessment. This study thereby incorporated several indicators relating to sheep host physiology

(haematocrit, eosinophils, cortisol, body temperature), clinical examinations (escape response or

vocalization following percussion and palpation of infected region), lesions (pepsinogen) and

performance (weight). To ensure the parameters were H. contortus pain specific, the study measured

them in various conditions including in the presence of analgesic pain relief during which time any

pain should have been reversed. The aims and results of this study present the study of veterinary

parasitic diseases from a novel and often overlooked point of view.

161

2. Materials and methods

2.1 Sheep

A total of 16 Romane rams, 9 months of age were used. This breed of sheep is susceptible to H.

contortus infection (Terefe et al., 2007). The sheep were reared indoors and had no prior experience

of GIN infection before the experiment as validated by negative faecal egg counts (FEC). Throughout

the experiment the sheep were allotted into groups of four and maintained in pens indoors. The

sheep had open access to water and were fed hay ad lib and feed concentrate.

2.2 Experimental design

The sheep were divided into four groups of four sheep. The groups consisted of a non-infected

control group, an analgesic without infection group, an H. contortus infection group and an H.

contortus infection group that also received analgesics. The experimentation was approved (no 2012-

06-10) by the Val de Loire ethical committee (no 19).

The eight sheep which received the H. contortus infection were administered 2,500 L3 infective

larvae over four consecutive days per os. It was thought that by spreading the infection over

sequential days it would extend the window in which pain may be experienced and detected. At 25

days post infection (dpi) the infected sheep were treated orally with Ivermectin (Ivomec® as per

manufacturers instructions : 200µg/ kg bodyweight) and left for 10 days. Faecal egg counts were

carried out during this time to ensure the infection had cleared. The same sheep were then re-

infected a second time in the same way described before. Previous studies have suggested that after

initial tissue damage, consequent damage may result in hypersensitivity of the area (Benson et al.,

2012) which may augment the pain experienced in a second infection. The sheep were sacrificed by

means of electronarcoses and exsanguination and necropsied 25 days after the second infection (60

days post first infection).The worm counts were then performed by examining a 1/10 aliquot of the

abomasal contents as described in Gaba et al. (2006). The mucosa was also examined for worms by

immerging them in water at 40°C for four hours, then sieving the water to collect any worms,

particularly looking for the fourth stage larvae (L4).

The eight sheep which received the analgesic did so over the moments when the developing larvae

are thought to penetrate the abomasal mucosa and pain was hypothesized i.e. 1, 2, 3, 4, and 9, 10,

11, 12 dpi. This was repeated for the second infection. The analgesia consisted of a combination of

butorphenol Torbugesic® 1% (0.5 ml per sheep) and microdoses of ketamine Imalgène® 1000mg

(0.15 ml/sheep) given intramuscularly on day 0 to 10 dpi on prime- and re-infection.

162

2.3 Faecal egg counts

The faecal egg counts (FEC) were carried out to validate the presences and intensity of H. contortus

infection. These were performed using a modified McMaster technique (Raynaud, 1970) in a sodium

chloride flotation solution, accurate to 50 nematode eggs per gram (EPG) of faeces.

2.4 Blood samples

Blood samples were collected from all 16 sheep on the following days over the course of the

experiment (covering both infections) -3, 3, 24, 35, 51, 60. The samples were drawn from the jugular

vein of the sheep into two heparin and two dry tubes using a 10 ml gauge syringe. These samples

were used to determine the pepsinogen, haematocrit, eosinophil and cortisol levels of the sheep.

Pepsinogen levels were determined from blood serum samples following the colorimetric assay

described by Kerboeuf et al. (2002). The haematocrit percentage was determined manually using a

Hawksely micro-haematocrit reader. Eosinophils were counted using an automatic counter (MS9-5

Counter, Melet Schloesing Laboratories) calibrated specifically for sheep. Cortisol levels were

established following Orgeur et al. (1998).

2.5 Weight

The 16 sheep were weighted using walk-on scales on days -3, 28, 32, 51, 60 dpi.

2.6 Percussion/palpation

The sheep were examined clinically by trained personnel for indications of pain by percussing and

palpating the abomasal region externally. Following stimulation of the potentially painful area, the

sheep behavior and posture was monitored. The specific responses looked for include attempts to

escape or avoid abomasal stimulation, focused direction looking towards the area or vocalization.

2.7 Core body temperature

Body temperature was selected as a pain indicator based on previous studies which recorded

decreases in sheep (Stubsjøen et al., 2009) and cattle (Stewart et al., 2008) eye temperature

following external pain treatments. The body temperature was recorded in all 16 sheep using

prototype MEDRIA® thermobolus transponders. These were swallowed by the sheep six days before

the first infection and were maintained in the rumen throughout the study. From the rumen, the

transponder transmitted a temperature signal to a corresponding MEDRIA ® GSM radio base and

records the data to an internet based file at five-minute time intervals.

163

To validate whether sheep body temperature decreased as a function of pain, it was necessary to

stimulate the abomasal region with an established pain source. The administration of capsaicin-like

substances has previously been observed to stimulate pain behaviours (Craft et al., 1993; 1995;

Pandita et al., 1997). We thereby selected Tabasco® to be used as the pain stimulant. A single

uninfected two year old Berrichon du Cher ewe was used and fitted with a Medria® transponder

three days prior to pain stimulation. The sheep abomasum was then located using a SonixTouch

ultrasonography device (Ultrasonix Medical Corporation130-4311 Viking Way Richmond, BC V6V

2K9 Canada) with a convex 2D 2-5 Mhz probe, and injected with 5ml of Tabasco® at 10 am. The

sheep was necropsied the following day and the abomasum examined.

2.8 Data and statistical analyses

Between groups comparisons were performed using the Kruskall and Wallis non-parametric test as

the data did not follow a normal distribution. A general linear model was used to analyse sheep body

core temperature. A cluster analysis was undertaken in order to simultaneously regroup the different

response parameters to infection. It was performed using an UPGMA method based on the

Spearman coefficient of correlation.

3. Results

3.1 Infection based on faecal egg counts

Only the infected groups excreted EPG. The Infection + analgesic group had a significantly higher

(Kruskal-Wallis p = 0.04) EPG than the Infection group. The anthelmintic treatment after the first

infection effectively removed the infections with 0 EPG. The second infection did not appear to

establish in either of the infected groups as the EPG remained negative.

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Fig. 1: Mean FEC ± standard error for each sheep group throughout the experiment.

3.2 Lesions as based on pepsinogen

Pepsinogen levels were not significantly different between the groups during the infection. A

difference at p=0.10 could be evidenced between the infection and non-infected groups. For both

infected groups, the pepsinogen levels decreased during the anthelmintic treatment and did not rise

again during the second infection. This suggests the L3 larvae did not survive to penetrate the

abomasal mucosa to continue their development.

0

1000

2000

3000

4000

5000

6000

7000

8000

Before infection 1st infection AHT 2nd infection

EPG

Time during experiment

FEC

Control

Analgesic

Infection

Infection + analgesic

165

Fig. 2: Mean pepsinogen (mU TYR) ± standard error for each sheep group throughout the

experiment.

3.3 Weight

The mean weight for each of the groups was very similar at the beginning and end of the experiment

and no significant difference could be evidenced. From three days before infection to 16 days post

the second infection (total 59 days) the groups gained approximately 15 kg.

200

300

400

500

600

700

800

900

1000

Beforeinfection

1st infection AHT 2nd infection

mU

TY

R

Days post infection

Pepsinogen

Control

Analgesic

Infection

Infection +analgesic

166

Fig. 3: Mean weight (kg) ± standard error for each sheep group throughout the experiment.

3.4 Anaemia based on haematocrit

Both H. contortus infected groups (Infection and Infection + analgesic) incurred a significant (p=0.01)

decrease in haematocrit during the first infection (Table 1). The decrease was reversed following the

AHT and continued to increase through the second infection suggesting there were no adult worms

present to feed. The non-infected groups (Control and Analgesic) maintained relatively stable

haematocrit levels throughout the experiment.

3.5 Response to infection: Eosinophils

Eosinophil levels did not vary significantly during the experiment. The only significant difference

between groups (p=0.04) was recorded at the end of the experiment, and this was mostly due to the

infected group alone.

3.6 Response to infection: Cortisol

Cortisol levels did not vary significantly between the groups at any of the times examined.

60

65

70

75

80

85

90

95

Before infection 1st infection AHT 2nd infection

Kilo

gram

s

Days post infection

Weight

Control

Analgesic

Infection

Infection + analgesic

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Control Analgesic Infection Infection + analgesic

Haematocrit Before infection 37 ± 0.7 37 ±0.7 36 ± 1.2 36 ± 0.4

(%) 1st

infection 34 ± 0.6 34 ± 0.5 27 ± 0.7 23 ± 0.4

AHT 33 ± 0.9 34 ± 0.4 29 ± 0.5 29 ± 0.3

2nd

infection 32 ± 0.9 33 ± 0.8 33 ± 0.3 32 ± 0.5

Eosinophils Before infection 2.41 ± 0.53 3.14 ± 0.54 1.85 ± 0.15 2.23 ± 0.36

(1000/mm3) 1st

infection 5.39 ± 0.62 3.18 ± 0.28 2.7 ± 0.49 3.10 ± 0.25

AHT 5.59 ± 0.78 6.01 ± 0.89 4.24 ± 0.86 4.88 ± 0.49

2nd

infection 4.35 ± 0.57 3.93 ± 0.5 1.44 ± 0.17 2.55 ± 0.21

Cortisol Before infection 9.47 ± 2.52 7.7 ± 1.03 4.82 ± 1.69 12.34 ± 4.21

(ng/ml) 1st

infection 10.46 ± 3.31 11.7 ± 2.59 13.97 ± 5.19 4.67 ± 2.58

AHT 10.29 ± 2.26 8.26 ± 3.31 8.43 ± 3.89 9.48 ± 3.97

2nd

infection 7.54 ± 2.14 7.79 ± 2.99 10.56 ± 3.36 6.87 ± 3.02

Table 1. Infection parameters for each group throughout the four periods of experiment including

mean haematocrit, eosinophils and cortisol ± standard error.

3.7 Pain based on palpation / percussion

None of the 16 sheep responded to the palpation / percussion examinations throughout the

experiment.

3.8 Core body temperature

The results show that each group had varying temperature norms even before infection, ranging as

much as 0.8°C in a single day (Table 2). Analyses using General Linearized Models (GLM) revealed

temperature varied according to the hour, day and sheep group. The GLM analyses further revealed

the temperature changed in all the groups (even the non-infected) following the first infection.

However, even when the individual sheep temperatures were examined on an hourly basis, no

consistent patterns of temperature increases and decreases were found within any of the groups.

Thus, it appears there were no short or long term responses to infection or analgesic treatments.

168

Time during experiment (days) Control Analgesic Infection Infection +

analgesic

Before 1st infection

Day-3 39.6a 39.4

b 39.3

b 39.6

a

Day-2 39.2a 39.6

a 39.6

b 40.0

c

Day-1 39.6a 39.3

b 39.2

b 39.5

c

1st infection

Day 0 39.7a 39.4

b 39.4

b 39.4

b

Day 1 39.6a 39.5

a 39.3

b 39.5

a

Day 2 39.5a 39.6

a 39.4

b 39.6

a

Before 2nd

infection (AHT)

Day-3 39.4a 39.4

a 39.6

b 39.8

c

Day-2 39.3a 39.5

b 39.5

b 39.6

c

Day-1 39.6a 39.3

b 39.5

b 39.7

c

2nd

infection

Day 0 39.8a 39.6

b 39.8

a 40.0

a

Day 1 39.8a 39.6

a 39.7

a 39.6

a

Day 2 39.7a 39.6

a 39.8

a 39.9

b

Table 2: Mean daily (07:00 – 00:00) temperature based on hourly means (temperatures documented

every five minutes) for each group at several time points throughout the experiment. Letters in

superscript denote the groups that significantly differed from each other on the day examined.

3.9 Cluster analysis (UPGMA)

UPGMA analysis showed the variables of pepsinogen, weight, eosinophils, cortisol, haematocrit and

body temperature (before infection, 1st infection, AHT, 2nd infection) combined well to separate

those sheep which were infected with H. contortus, and those that weren’t (Fig. 7).

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Fig. 8: UPGMA analysis separating individual sheep based on their values for the variables of

pepsinogen, weight, eosinophils, cortisol, haematocrit and body temperature (before infection, 1st

infection, AHT, 2nd infection). Sheep 1 - 4 = Control; 5 - 8 = Analgesic ; 9 – 12 = Infection; 13 – 16 =

Infection + Analgesic).

This UPGMA analyses further clearly separated several of the variables into two main groups. It

shows the sheep temperature before infection, regardless of group, is not related to the temperature

post-infection (including the first or second infection or during the AHT). The body temperature post-

infection is shown to be most closely related to weight and pepsinogen. The baseline body

temperature was more closely associated with eosinophils, cortisol and haematocrit.

170

Fig. 9: UPGMA analysis of the relationship between the following variables pepsinogen (Pepsi),

weight, eosinophils, cortisol, haematocrit (Haem) and body temperature (before infection (BaseT), 1st

infection (1st_inft), AHT (BreakT), 2nd infection (2nd_infT)).

3. 10 Response to inflammation of abomasum: Tabasco® injection

No significant difference could be evidenced in comparing the sheep temperature before (39.45°C) ,

during (39.10°C) or after (39.23°C) the Tabasco®injection. This was due to high variability between

days before Tabasco injection. The sheep was necropsied the day after infection and upon

examination of the abomasum, the injection site was easily located by the presence of red inflamed

mucosae. This suggests that sheep body temperature does not react to abomasal inflammation and

likely pain stimulus.

171

Hours

before

Tabasco®

injection

Temperature (°C) 3

days before

Tabasco® injection

Temperature (°C) 2

days before

Tabasco® injection

Temperature (°C) 1

days before

Tabasco® injection

Day of tabasco

ingestion

H-3 (07:00) 38.8 38.8 38.5 38.9

H-2 (08:00) 38.8 40.0 38.6 39.1

H-1 (09:00) 39.0 39.1 39.2 39.1

H0

(10:00)

39.3 39.4 39.5 39.2

H+1

(11:00)

39.3 39.4 39.5 39.2

H+2

(12:00)

39.0 39.0 39.9 39.0

H+3

(13:00)

38.7 38.6 40.0 39.0

H+4

(14:00)

38.6 38.9 39.7 39.1

H+5

(15:00)

38.6 38.6 39.4 38.9

H+6

(16:00)

38.9 38.6 39.3 39.1

Table 3: Mean hourly temperature for the sheep which received an injection of Tabasco® into the

abomasum. Hours in day before or after Tabasco® injection denoted by H.

4. Discussion

The elevated levels of pepsinogen in the H. contortus infected sheep confirm that tissue damage had

taken place from the developing larvae. Furthermore, a measure of general illness could be assumed

in the infected sheep as documented by a significant drop in haematocrit. Despite these indications

of damage and disease, none of the other indicators suggested pain ensued. The results for each of

the parameters shall be discussed in sequence.

It was slightly surprising to see that the second H. contortus infection did not establish in any of the

eight sheep tested, especially given the Romane breed is susceptible to infection (Terefe et al., 2007).

It is possible the trickle infection approach with low numbers of L3 enabled the sheep to build a more

effective adaptive immune response than a single large infective dose would have done. Although all

172

the pain indicators were examined in the second infection, discussion will largely refer to the primary

infection which established. It is interesting to note that the presence of analgesics increased the H.

contortus infection intensity with significantly elevated FEC compared to the Infection group, yet the

group simultaneously had reduced lesions as seen by lower pepsinogen levels. Studies with the

analgesic drug morphine found the drug to be associated with a degraded host innate defence

barrier in mice (Frenklah et al., 2006), inhibited proliferation of T cells (Cheng et al., 2006; Wang et

al., 2001; Chang et al., 2011) and the suppression of T cell mediated immunity by means of inhibiting

TCR signalling (Lysle et al., 1993; Borner et al., 2009). This may account for both the increased

susceptibility to infection and reduced lesions resulting from controlled inflammatory response.

Minimizing the local inflammatory reactions by limiting cellular recruitment and other effector

mechanism may reduce the disturbance in vascular permeability for the pepsinogen to escape into

the blood stream (Simpson, 2000; Terefe et al., 2007).

None of the sheep demonstrated any reaction to the percussion and palpation of the abomasum. In

the presence of pain, we may have expected a nociceptive withdrawal reflex, an involuntary and

rapid movement mediated by a reflex arc synapsing in the spinal cord that protects the animal from

potentially damaging stimuli (Prunier et al., 2012). Such movements have been observed in recently

castrated lambs following palpation of the scrotum (Thornton and Waterman-Pearson, 1999).

Similarly sheep, among other animals, have frequently been recorded to increase the number and

features of vocalisations during painful events (reviewed by Watts and Stookey 2000; Manteuffel et

al. 2004), yet here, they remained silent throughout the clinical exam. Likewise, monitoring the

sheep weight revealed nothing more than an assurance that the sheep were kept in very comfortable

conditions, all the sheep having gained on average 15 kg in 60 days.

Body temperature revealed itself to be a highly individualistic parameter, with variations observed

between and within the groups even before the first infection. Regardless of their initial body

temperature, we expected to see sudden temperature decreases in the infected group if pain was

present. But this was not observed. This was based on findings that external pain treatments caused

reductions in eye temperature of sheep (Stubsjøen et al., 2009) and cattle (Stewart et al., 2008)

resulting from sympathetically mediated vasoconstriction from a stimulated fight or flight response

(Stubsjøen et al. 2009). It may be the recorded drop in eye temperature was specific to the external

acute pain treatments experienced in these studies, whereas the potential visceral pain stimulated

by H. contortus may be more chronic. Pain studies in post-laparotomy mice actually found increased

temperature as a result of pain (Arras et al., 2007), which we could assume would be a more chronic

than acute form of pain. Alternatively, it may be that changes to the eye temperature are not

mimicked by changes in body temperature. Yet, a study investigating pig body temperature at six

173

different subcutaneous locations found direct linear correlations between these sites with rectal

temperature (Lohse et al., 2010).But other studies have noted that the temperature of sheep ear-

pinna decreased while core body temperature increased in response to different stressors (Ingram et

al., 2002; Lowe et al., 2005). Given body temperature is a fundamental homeostatic function

governed by the control of the central nervous system of homeothermic animals (Nakamura, 2011),

numerous external or internal threats may disturb the homeostatic balance to produce fluctuations

in body temperature. Indeed, some of these may even have conflicting effects. For example, while it

remains possible that pain causes a decrease in body temperature, it is well established that tissue

destruction is closely followed by inflammation (Coop et al., 1976), a known trigger for increasing

temperature (Gallin, 1989). Thus, any decreases in temperature resulting from pain could be masked

by the increases resulting from inflammation. No temperature increase was evidenced in the sheep

administrated Tabasco® despite inflammation being clearly present, thus, the inflammation in

digestive tract may not result in elevated core body temperature in sheep.

Based on the results of this study, we conclude that H. contortus infections under the conditions

described in this study do not provoke any detectable level of pain. It is entirely possible that the

presence of pain may alter as a result of greater infection intensity or even as a result of the sheep

breed infected. Greater inflammation for example has been recorded in sheep breeds more resistant

to infection (Terefe et al., 2007) and it is possibly different in susceptible breeds such as Romane. We

remain confident that the numerous indicators in this study should be sensitive enough to detect

pain. The UPGMA analyses confirm their sensitivity to discriminate H. contortus infected and non-

infected sheep. While the lack of evidence for pain in this study for H. contortus infections is

ultimately good news for the welfare of the sheep, it became evident within the research of this

study that pain, as a consequence of infection, is an area massively overlooked in veterinary

medicine and warrants further investigation.

Acknowledgements

Caroline Chylinski is a grateful recipient of Marie Curie ‘Nematode Systems Health’ ITN funding. We

gratefully acknowledge the assistance of the INRA PRC Hormone laboratory for their assistance with

cortisol analyses and Thierry Chaumel and team for their assistance with animal care and Mikael

Rioux PFIE INRA for their assistance counting the eosinophils.

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178

SHEEP HOST DISCUSSSION

In the entirety of this thesis research, only one barrier was identified that could block the success of

H. contortus so unequivocally, the protective response of MBB ewes. Their virtual zero tolerance of

the H. contortus establishment, as indicated by the negative FEC, would suggest the compatibility

filter between themselves and H. contortus was completely closed, akin to being out-with the GINs

host-specificity range. This level of resistance is exceptional. The finding that greater resistance was

conferred as a result of differential immune gene expression is not necessarily a new discovery

(Lacroux et al. 2006; Terefe et al. 2008) however, the fact that it resulted as a consequence of sex

very much is.

Considering the extent of our current knowledge on resistant sheep, our ability to identify them with

phenotypic, genetic or even immunological parameters, and our capacity to harness so effectively

their genetic traits in selective breeding, it seems unmatched that we still know comparatively little

about the immunological mechanisms by which they operate. Clearly there is a strong place for the

numerous studies which have helped identify the genes involved in resistance, but there is some

distance between the identification of genes and their translation into what is happening on an

immunological level. But eliciting the mechanisms of sheep resistance is a bit like trying to hit a

moving target. There is substantial inter-individual and intra-individual diversity in the sheep host

response, so that two sheep of the same breed are unlikely to elicit identical immunological

reactions. The elusiveness of the immune mechanisms involved in sheep resistance is confounded

further still by the numerous interacting factors such as age, exposure, nutrition and reproductive

status. If immunological studies are to advance, these factor need to be controlled experimentally. If

females are consistently more resistant than males, or are able to maintain lower levels of infection

even in more susceptible cases, this may provide more interesting comparative models for

immunological studies.

This finding can stimulate many other interesting questions; Is the sex effect in sheep resistance

limited to H. contortus, or can it extend to other nematodes and pathogens? Is it common across

sheep breeds? Even in terms of reduced susceptibility? But first we must consider whether it is

consistent even within MBB females. Other results in this research would suggest not. The study

presented in the previous chapter exploring how sheep resistance would influence H. contortus

fitness used nine MBB females, all of which presented positive FEC on their first infection. For four of

these sheep, it was their third experimental infection with H. contortus so they are not strictly

comparable, but we may have even expected to see their resistance increase in this case. Perhaps

179

the origin and/or environment of the sheep played a role. All the female MBB used in this study

originated from Bourges and experiments took place in Tours. The five primary infected female MBB

in the other study originated from Guadeloupe where they remained throughout the experiment.

We may also consider this sex-biased parasitism in terms of the natural life history of sheep. There is

a hypothesis that males maximize their fitness via mating rates, while females invest more in their

progeny. In polygynous systems, such as for wild sheep, competition for females should favour bigger

males that, under energy restraints, may result in a trade-off between growth and immunity (Moore

and Wilson, 2002; Zuk, 2009). On the contrary, females are expected to invest in longevity and to

then select for higher investment in immunity (Rolff, 2002). Previous studies have attempted to

measure immune investment in wild animals based on spleen size on the basis enlargement may

reflect host immune activation due to the expansion of the splenic B cell pool(Bordes et al. 2012). We

briefly tried to extrapolate this measure to use in experimental sheep based on the spleen weight

and FEC of 19 sheep consisting of male and female MBB (not the ones used in the sex effect study)

and male and female Romane sheep. No correlations were made (data not shown). And it is further

difficult to speculate on how much natural life history traits continue to impact a species that has

been domesticated for thousands of years.

One final, slightly more abstract consideration on why females demonstrated greater resistance than

males could relate to their social environment. A study in rats infected with Escherichia coli found

that females kept in groups experienced exacerbated inflammatory responses and sickness

behaviours than if they were kept alone. Conversely, males kept in groups had attenuated

inflammatory responses compared to solo animals. The sex effect study maintained the males and

females separately in groups. While we have no evidence that this was an influencing factor on our

results, it is nonetheless interesting to know that it has the potential to be.

If we were to imagine that this sex-biased influence on sheep resistance were to be established as a

certainty in the future, we could then theorize as to the implications this may have on sheep farming

and selective breeding. Regardless of the production target, be it meat, wool or milk, at least 90-95%

of the flock is likely to be represented by females anyway, so the only change it may bring is to

suggest more attentive GIN management strategies focused on the remaining males. The lambs

produced in these systems are largely sold for meat which, on French farms, occurs at about 6 – 7

months of age. The females are not necessarily conferred an advantage over their male counterparts

as their immature sexual development makes them equally susceptible at this time. A study

monitoring the FEC of male and female MBB and Romane lambs over three successive H. contortus

infections demonstrate that the sex effect develops in both breeds, to varying degrees, but only after

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the second infection coinciding with the age of sexual maturity (Biovipar project personal

communication). What the sex effect could mean in terms for selective breeding is substantially

limited by the obvious fact the males are the fertilizers. They are even used for selection of female

milk production traits based on the amount of milk their offspring produced. So in practical terms, it

seems the applicative value of the sex effect does not carry as much impact as it biological interest

for sheep farming, although it may be of interest applied to wild populations of sheep.

For selective breeding purposes, numerous biomarkers exist to identify the resistance status within

sheep including FEC, genetic and molecular markers (e.g. single nucleotide polymorphisms), parasite

induced pathology or immune responses such as haematocrit or eosinophil levels respectively. All of

these biomarkers require that the sheep is infected before a measure can be taken. There is only one

known exception to this and that is the prospect for the use of ghrelin hormone as a predictive

indicator. The results of the temperature data that show that higher temperature before infection is

correlated with lower FEC after infection. Thus, sheep temperature potentially offers a predictive

parameter to determine the sheep response prior to an H. contortus infection. The value of

monitoring temperature in a sheep flock could be coupled with several other flock health and

welfare factors. Some very preliminary investigations with the sheep temperature in this research

suggest that it may also be a useful marker for when sheep under some form of stress. Observations

of the sheep temperature during periods of transport or at the abattoirs show sharp inclines in their

temperature (data not presented) with similar results found in other studies (Vinkers et al. 2009).

Conceivably, a similar response would occur if sheep were under threat from a predator or thief,

both of which are regular occurrences in some regions. Additionally, several previous studies have

documented an increase in body temperature is common in response to viral and bacterial diseases

(Bouwknect et al. 2007). Although the Medria© equipment used to monitor the sheep temperature

in this study was limited to work within a 400m radius, there are very likely prospects to extend this

signal. With increasing emphasis put on the food security, that is ‘the sustainable production of

sufficient amounts of high quality affordable and safe foods to maintain the well-being of human

populations worldwide’ (Fitzpatrick 2013) farmers are increasingly moving towards extensive farming

systems with greater flock sizes. In such cases, the use of remote sensing technologies for alert

systems is a particularly compelling to achieve higher levels of animal welfare at sustained levels of

profitability (Goddard et al. 2006).

The study of pain showed a greater consideration for sheep welfare than is normally applied in GIN

research. Although our results showed no indication of pain in the confines of the experimental

conditions and parameters tested, ultimately a positive note for the sheep, it did draw attention to

the lack of comparative work done in this area. Increased livestock welfare has been shown to be

181

beneficial for production in numerous studies. For example, by reducing negative emotional states

such as fear and stress, this can reduced the frustration that animals may experience when they are

unable to express their behavioral needs (Huges and Duncan 1988; Nicol 1992; Wood-Gush and

Vestergaard 1989). In turn, reduced stress in livestock animals has been equated with enhanced

productivity with increased resistance to disease and better immunity (Siegel and Honaker 2014). No

studies could be found exploring the connection between sheep welfare and GIN resistance directly.

Given the potential production benefits increased welfare could include, I suggest this could be

worthy to explore further in the future, especially pertaining to sheep housed indoors over winter

conditions. During the research of this work, it became strikingly apparent how much production

interests are prioritized above animal welfare interests. Establishing a link between the two could go

a long way to improving welfare standards in increasingly intensified production systems.

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FARMER INTRODUCTION

Farmers represent the final piece of the GIN success puzzle. While they clearly do not share the same

biological intricacies as the GIN do with their sheep host, they can be viewed as a type of

orchestrator of these interactions. It is their decisions that can prevent, or facilitate, the encounter

between the parasite and host via their husbandry decisions. And it is their decisions that can to a

point, control the infection outcome. But the decisions a farmer makes are entirely dependent on the

knowledge and tools he has available to apply to the situation. In both instances, the most advanced

knowledge and tools are developed by scientific research. But there is a decided distance between

what happens in the laboratory and what is applied in the field. Farmers don’t read scientific journals

any more often than we go to the farms to discuss our results with them. So for all the money and

effort that is being poured into veterinary research, how can we be sure it is getting put to use in the

field?

By and large we may assume that it is the role of the veterinarian or animal health providers to

provide the best counsel for GIN control strategies. But research has shown that they can often have

differing awareness and knowledge of parasitic infection resulting in contrasting proposals for

control (Saddiqi et al. 2012). Mixed messages evidently bring farmers mistrust in the animal health

providers resulting in farmers acting in accordance with their own views (Saddiqi et al. 2012). The

problem of GIN control has increased in complexity substantially since the development of

anthelmintic resistance. Prior to this, GIN control instructions for the farmer largely consisted of

using anthelmintics at will. Given how cheap, effective and easily administered this solution was it

was readily applied, eventually to its own detriment. But a single generation later we’re asking

farmers to forget prior counsel and trust new strategies of integrated parasite management (IPM).

These include components such as the use of selectively bred sheep, pasture management

strategies, optimized nutrition, nematophagous fungi, and the targeted selective treatment (TST) of

anthelmintics only to those sheep ‘most in need’. To be carried out fully, many of these controls

require a certain understanding of GIN disease biology, epidemiology and biological tools to target

treatment as well as a substantial investment in time and labour. It could perhaps be understood if

the farmers are not eager to comply. Especially given GINs are only one of many potential health

concerns the farmer must try and control, with the bacteria causing foot rot being of particular

concern for sheep farmers. This drove us to question the relationship between farmers and GIN

control further.

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Given the importance of farmers in GIN control, it is astonishing how under-studied it has been. We

target some of the more sociological aspects of farmers GIN control strategies in trying to

understand their understanding of the infection and their motivation to control it. First we

questioned to what extent GIN control counsel is needed. Farmers do after all have more knowledge

on their flock than a scientist in a laboratory does. Perhaps having greater freedom over his decisions

is pertinent to enable an element of farm-specific control. For example, the optimal husbandry

practices for sheep in Guadeloupe will vary substantially from those needed in French flocks.

Secondly, we explored whether farmers have the appropriate motivation to apply GIN control

strategy by evaluating their understanding of the disease. Motivation will largely be a trade-off

between the perceived threats of a disease, and the perceived benefits of enacting a control. Finally,

we evaluate the efficacy of some of the biological tools available to farmers that are supposed to

enable them to make the best GIN control decisions.

Key findings

Farmers do not have an accurate perception as to the threat GIN present

This should be addressed before any corrective GIN control measures can be effected

Greater farmer autonomy in husbandry decisions results in greater GIN infection intensities

Rules rather than recommendations for GIN control may be needed

The efficacy of pathophysiological indicators (FAMACHA©, DISCO, weight gains) to detect

sheep in need of treatment varies depending on the moment their used in an infection, the

breed of sheep and the infecting H. contortus population

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Trade-off between farmers’ autonomy and control of sheep parasitic gastro-intestinal

nematodes in conventional and organic farms

Jacques Cabaret 1,4*, Caroline Chylinski 1, Salah Meradi 2, Gabriel Laignel 3, Christian Nicourt 4,

Bourhane Bentounsi 5, Marc Benoit 3.

1 INRA and François Rabelais University, UMR 1282, Nouzilly, FRANCE

2 Biological Sciences, Batna University, Batna ALGERIA

3 INRA, URH, Theix, FRANCE

4 INRA, RiTME, Ivry/Seine, FRANCE

5 Veterinary Department, Parasitology, Mentouri University, Constantine ALGERIA

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Introduction

Craftsmen generally place high value in their work. They organize their own learning and

development in their craft, normally by means of apprenticeships or learning from peers

rather than in a school environment. Their work often extends into the family environment

with other members participating in the enterprise to varying degrees (Dubar et al. 2011).

Autonomy is thereby a central part to a craftsman’s trade. Farmers demonstrate a similar

profile in their approach to work, although their job is often part of a much wider

hierarchical system of contractors which appears to be in opposition with autonomy.

Integration in farming systems can be applied at two different levels: 1) general integration

of a production system such as in poultry and pig industries where a private integrator

provides young animals, food and sometimes housing and therapeutics; 2) local integration

at farm level were the farmer decides on the complementarity of agriculture and husbandry,

the animals to be bred, food management, housing and therapeutics. Local integrated

farming systems refer to agricultural systems that integrate livestock and crop production

(Chan, 1985) and can be seen to various degrees in organic and conventional semi-extensive

meat sheep farms. Autonomy is particularly prized in organic farming (Barres et al., 1985).

This paper takes the view that a farmers autonomy is in direct opposition to general

integration but corresponds to the greater flexibility of local integration systems.

Gastrointestinal nematode (GIN) are a frequent problem for the husbandry practices in

grazing meat sheep farms according to the views of the veterinarians (Perry et al., 2002), the

farmers (Cabaret et al., 2009; Ouzir et al., 2011), or both (Sadiqqi et al., 2012). The life cycle

of GIN entails both a free-living life phase whereby the egg to larvae stages are exposed to

186

the environmental elements, and a free-living phase when the infective larvae are consumed

and the developed adults shed future generations of eggs back onto pasture with the host

faeces. Importantly, none of the stages, free-living or otherwise are easily seen by the

farmer. Instead, the farmer must rely on indications from the sheep flock as to the presence

and severity of the infection, but this is not an easy task for the farmers (Cabaret 2003;

Saddiqui et al. 2012). While the farmers have a variety of systems for the direct or indirect

control of GIN at their disposal (Barger 1997, Thamsbourg et al. 1999; Cabaret 2003), the

decree in which their applied is likely influenced by the farmers personal belief system rather

than on the technical knowledge alone. An example of this can be seen in organic sheep

farmers who reduce the authorized number of anthelmintic treatments depending on their

assessment of the need for them (Cabaret et al. 2009).

The threat of human disease can be understood in the context of the health belief model

(Abraham and Sheeran 2009), and was adapted in this study to understand the problem of

GIN by farmers. The model is based on the treat perception and the behavioural evaluation.

Threat perception constitutes what the perceived susceptibility to health problem is, in this

case GIN, and the perceived consequences of the health problem. The behavioural

evaluation is a balance between the benefits of a recommended health behavior, for

example if lambs gain more weight after an anthelmintic treatment, and the barriers to

enacting the behaviour such as the cost of the anthelmintics, the time spent treating the

sheep or managing pastures. Certain GIN control practices will thereby be more or less

attractive to the farmer depending on his beliefs relating to the importance of GIN and the

expected returns from these practices. Other beliefs, not directly related to GIN, may also

impact the intensity of a GIN infection. For instance, it is widely believed among organic

meat sheep farmers that increased autonomy in their practices can improve the success of

187

the farm, and possibly extend to the control of GIN (Cabaret et al. 2009). The exact definition

of autonomy as they relate to specific farming practices i.e. diversity of productions, feed

origin, therapeutics, commercialization of farm production and agricultural learning, will be

detailed in full. This research aimed to compare the level of general or specialized autonomy,

specifically relating to GIN treatment and pasture management, to the levels of GIN infection

to farms operating under different belief and knowledge situations in similar climates.


The farms

The study focused on private farms which bred sheep for meat. All of them relied primarily

on pasture to feed the flocks. Twenty conventional farms were studied in the Batna region of

eastern Algeria (coded A) which experienced a steppic climate. The coldest month in the

area was January (average 5°C) and the hottest July (average 25°C). The drought period

extended from June to September. Sixteen farms in the Centre of France were studied,

located in the semi-mountainous region of Auvergne and exposed to a fresh temperate

climate. The coldest month was January (5°C) and the hottest was July (21°C). Nine of these

farms reared the sheep following conventional methods (coded FC), the other 7 farms

followed organic regulation (Coded FO). Information regarding the autonomy variables on

the farms was collected via questionnaire led by one of the investigators or farm technicians.

Parasitological data

Faecal egg counts (FEC) of gastrointestinal nematodes (GIN) were obtained from the faeces

of adult animals on two/three composite faecal adult samples at the end of Spring. These

were used as a proxy to determine the infection intensity on a farm. The technique was

188

standard (MAFF, 1986, modified McMaster technique) and accurate to 50 or 15 eggs per

gram (EPG) of faeces respectively for FO/FC or A.

The autonomy variables

Variables were coded from 0 (absence of autonomy) to 3 (highest autonomy).

Table 1. Definition of autonomy variables coded from 0 (absence of autonomy) to 3 (highest

level of autonomy).

Variables Description

Agriculture Absence (0) of agriculture to approximately

50% of activity (3), thus farmer not depending

only on agriculture or animal breeding

exclusively.

Husbandry Only sheep on the farm (0) to breeding of mixed

species (3) e.g. other ruminants, equids,

chickens etc. and thus not dependent on a single

production.

Feed Only seeded pasture and /or buying cereals from

external sources (0) to natural pasture, and/or

use of fallow field and/ or stubble fields available

(3). The autonomy increases with the possibility

to use a variety of pastures.

Therapeutics From exclusive and frequent use of synthetic

anthelmintic treatments decided by

189

veterinarians or technicians (0) to low number

of synthetic anthelminthic treatments and use of

alternative medicines decided by the farmer (3).

Commercialization One external buyer managing the

commercialization of the production (0) to the

farmer being involved in selling his animals and

products by himself at market or on the farm (3).

Knowledge/Learning The absence of schooling, the experience of

sheep for less than 20 years, no participation in

a breeders association (0), experience > 20

years, primary school education and

participation in a breeder association (2), high

experience of sheep breeding, secondary school

education or over and participation in a

breeders association (3).

Statistical procedures

The Kruskall-Wallis test was used to detect differences between A, FO and FC as the data did

not follow a Gaussian distribution. Spearman rho non-parametric correlation coefficient was

used to evaluate the relationships between variables. Cluster analysis was performed using

un-weighted arithmetic average clustering (UPGMA) and based on Gower similarity

coefficient (MVSP 3.1, 2001). These were constructed using Gower general similarity

coefficient (adapted to nominal and quantitative data) between characteristics. Gower

190

coefficient ranges from 0 to 1 (maximum similarity). The un-weighted clustering method

gives equal weight to each object (variable) in each cluster of variables. The UPGMA is

preferred to others (such as centroid) where primary connections among variables was the

aim of the analysis (Legendre and Legendre, 1998). The FEC were arranged into four

categories less than 50 EPG, 51-100 EPG, 101-200 EPG, >201 EPG.

Results

Description of GIN infection and autonomy variables (Table 2)

The infection as estimated from EPG was the lowest in Algeria. The autonomy was highest

for husbandry and commercialization in Algerian farms and for therapeutics in French

organic farms. No significant differences were found between organic and conventional

French farms regarding EPG, husbandry, feed and commercialization. The overall autonomy

was similar in FO and A, and was significantly higher than in FC.

Table 2. Level of GIN infection intensity (EPG) and corresponding level of autonomy across

farms.

Variables A: Algeria

conventional

(n=20)

FO: France

organic (n=7)

FG: France

conventional

(n=9)

Significance

(Kruskall and

Wallis test)

EPG 43* (40)** 186 (159) 414 (261) 0.00 FO,FC> A

Agriculture 0.45 (0.94) 0.57 (1.13) 1.22 (1.20) 0.09 A,FO>FC

Husbandry 1.75 (0.85) 0.86 (1.07) 0.67 (1.00) 0.02 A>FO,FC

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Feed 1.55 (0.60) 2.29 (0.49) 2.22 (0.97) 0.01 FO,FC>A

Therapeutics 1.60 (0.50) 3.00 (0.00) 0.78 (0.44) 0.00 FO>A>FC

Commercialization 3.00 (0.00) 2.14 (0.38) 2.00 (0.00) 0.00 A> FO,FC

Learning 1.90 (0.55) 1.71 (0.95) 1.00 (0.87) 0.03 A>,FO>FC

*Average and ** standard-deviation

Relationships between GIN infection and autonomy variables

The association between GIN infection and autonomy variables differed between the farms.

Gastrointestinal nematode infection was associated with food autonomy in A farms, to

therapeutic autonomy in FO farms and to none of the autonomy variables in FC farms.

Therapeutic autonomy is related to autonomy in learning and husbandry in both A and FC

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farms.

Fig 1. Algerian farms UPGMA analyses showing the relationship between autonomy variables

and GIN infection intensity. Abbreviations include com (commercialization), argic

(agriculture), therap (therapeutics), GIN (GIN infection intensity via EPG).

193

Fig 2. French organic farms UPGMA analyses showing the relationship between autonomy

variables and GIN infection intensity. Abbreviations include com (commercialization), argic

(agriculture), therap (therapeutics), GIN (GIN infection intensity via EPG).

194

Fig 3. French conventional farms UPGMA analyses showing the relationship between

autonomy variables and GIN infection intensity. Abbreviations include com

(commercialization), argic (agriculture), therap (therapeutics), GIN (GIN infection intensity

via EPG).

Discussion

The Algerian farms had the lowest GIN infection intensity based on FEC. This could be

influenced by the relative resistance status of the breeds used, pasture management or a

climatic environment that may favour the establishment of different GIN species with

different fecundities, hence the differences in FEC due to GIN communities. The helminth

fauna was different in the French Auvergne (Cabaret et al, 2002 Teladorsagia,

Trichostrongylus, Haemonchus in decreasing order of intensity) and Eastern Algeria (Meradi

2011: Teladorsagia, Marshallagia, Haemonchus and Trichostrongylus in decreasing order of

UPGMA

Gower General Similarity Coefficient

agric

husbandry

therap

learning

com

food

GIN

0,28 0,4 0,52 0,64 0,76 0,88 1

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intensity). Most of the species in the two regions had low fecundity especially Marshallagia

(Meradi, 2011). Thus the low FEC could be due in part to the GIN species. However the

proportion of Marshallagia was small and it could not explain entirely the low levels of EPG

observed. It may be related to the more extensive management of pastures in Algeria.

The belief that digestive-tract strongyles are playing an important role in sheep production is

common among farms from the regions we studied (Cabaret et al. 2009-France; Ouzir et al,

2011-Northern Africa). Autonomy in husbandry, including the use of other grazing species

with different susceptibilities to GIN, should reduce infection intensity given most GIN

species are host specific. This however was not observed and in organic farms, it was even

associated with higher infection. Feeding autonomy should also reduce GIN infection since

there is a wide range of pasture types and often highly extensive management of pastures,

but once again, no correlation was observed in the farms. The use of synthetic anthelmintics

is the major tool for control but other therapeutics such as phytotherapy or homeopathy are

also used (Benoit and Leignel, 2003; Saddiqui et al, 2012).The therapeutic autonomy

correspond to the limited use of synthetic anthelmintics and use of alternative drugs as

proposed in organic farming but also in developing countries like Algeria where access is

limited by their costs. Therapeutic autonomy was associated with higher infection by GIN.

This may be due to the use of alternative drugs with likely poor efficacy, the majority of

which remain to be evaluated.

Autonomy in farm practices was not associated with lower FEC. This means that farmers

cannot cope properly with GIN using only their knowledge (Bouilhol et al, 2011) and they

need other sources of knowledge. Although autonomy is highly valued among organic

breeders it cannot be considered as a major means for controlling GIN. Specific tools and

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well-trained health advisors (Cabaret et al. 2011) are needed for elaborating an efficient GIN

control.

Acknowlegements

C. Chylinski is a grateful recipient of a EU ITN grant Marie-Curie of the Nematode System

Health program. The research was funded in part by the ANR Dynrurabio project.

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Benoit M, Laignel G. Constraints under organic farming on French sheep meat productions: a

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role of the veterinarians. The Open Veterinary Science Journal, 2011; 5: 7-11.

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modelling 20. Elsevier. Amsterdam. 853 p.

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Laboratory Techniques. Reference Book 418. HSMO Books 1986, 159 p.

MVSP (Multivariate statistical package). User’ manual. Version 3.1. KCS, Pentraeth,

Wales, UK. 2001.

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Parasite, 2011; 18: 261-269.

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Vet. Parasitol. 180, 372-377.

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Perry BD, Randolph RF, McDermott JJ, Sones KR, Thornton PK (2002). Investigating in animal health

research to alleviate poverty. Kenya: International Livestock Research Institute 148.

Saddiqi H.A., Jabbar A., Babar W., Sarwar M., Iqbal Z., Cabaret J. Contrasting views of animal

healthcare providers on worm control practices for sheep and goats in an arid environment.

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186 DOI: 10.1016/S0304-4017(99)00035-7

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Exploring the limitations of pathophysiological indicators used for targeted

selective treatment in sheep experimentally infected with Haemonchus

contortus

Chylinski C., Cortet J., Neveu C., Cabaret J*.

INRA, UMR 1282 Infectiology and Public Health 37380 Nouzilly France

Abstract

Identifying which sheep to treat as part of a Targeted Selective Treatment approach to gastro-

intestinal nematode control relies entirely on the efficacy of the indicators. Indicators such as

FAMACHA© (anaemia), DISCO (diarrhea) and reductions in weight gains were designed specifically to

reflect those sheep experiencing symptomatic consequences of infection. Using the gastro-

intestinal nematode Haemonchus contortus as a model species, this study explored the utility and

sensitivity of these indicators under controlled experimental conditions on 63 adult sheep. The

potential effect of sheep with different H. contortus resistance phenotypes on indicator efficacy was

compared in three different phenotypes i.e. high (Blackbelly females) , medium (Blackbelly rams) and

low resistance (Romane rams). The potential effect of the H. contortus isolate on indicator efficacy

was also explored by using four different isolates, with varying anthelmintic resistance capacities, to

infect the sheep. We limited the study to the first month of infection to evaluate the interest of

these indicators as an early predictive means for controlling infection. The pathophysiological

indicators FAMACHA© and DISCO do not reflect infection intensity based on Faecal Egg Counts, nor

do reductions in weight gains. FAMACHA© was however a good indicator of anaemia with strong

correlations to haematocrit. There was little agreement among the three indicators to identify the

same animals in need of treatment and even combining them did not increase their predictive value

of infection intensity or relative host damage from infection. The indicator sensitivity was influenced

by the H. contortus isolate and sheep resistance phenotype in which they were tested. One isolate

200

was poorly infective but induced high levels of anaemia (FAMACHA©) and diarrhea (DISCO)

compared to the three others. The FAMACHA© and DISCO had higher values in the sheep group

with a medium resistance phenotype (Blackbelly rams) indicating higher levels of damage compared

to the high and low resistance phenotypes. We conclude that there is no ‘one size fits all’ approach

to the use of indicators for Targeted Selective Treatment and the indicators should be calibrated to

farm-specific conditions to increase their efficacy.

1. Introduction

Gastrointestinal nematode (GIN) infections are a severe limitation to ruminant production

systems worldwide (Kenyon et al., 2009). Control of these parasites has become increasingly

hampered by the indiscriminate use of anthelmintic (AH) drugs which has led to the spread of

anthelmintic resistance (AHR) (Kaplan, 2004). Targeted Selective Treatment offers a more

responsible approach to AH use whereby only those sheep most in need of therapeutics receive

treatment (Gomez and Georgi, 1991). TST greatly reduces the frequency of AH use compared to the

more traditional mass flock approach and it is also thought to reduce the spread of AHR by

maintaining populations of the worms in refugia (communities of worms that remain unexposed to

AH and thus maintenance of alleles for susceptibility: van Wyk, 2001; Besier and Love 2003; Jackson

and Waller, 2008; Leathwick et al., 2006; Kenyon et al., 2009). TST may be applied with the aim of

reducing pasture contamination by treating only the most heavily infected sheep or, more

commonly, it can be used to treat only those sheep which exhibit the greatest production-reducing

symptoms of infection such as anaemia, diarrhea or reductions in weight gain. Given the clinical signs

of infection are generally considered to be density dependent, increasing with greater GIN challenge

(Kenyon et al., 2009), it should theoretically be possible to achieve both aims at once; by identifying

and treating only the most heavily individuals, this in turn should also reduce production-related

losses. In practice however, this is less straight forward. The only available parasitological indicator

201

designed to reflect GIN infection intensities is that of faecal egg counts (FEC) to enumerate the

number of eggs excreted by the sheep. Yet FEC are both costly and time consuming making them ill

adapted for use in field scenarios. This leaves the option of using pathophysiological indicators which

instead were developed to identify specific GIN-induced symptoms of infection. The aim of this study

was to explore whether certain pathophysiological indicators can also be used to reflect infection

intensities as determined by FEC. The indicators selected for evaluation included FAMACHA©

(acronym of François Malan Chart) designed to reflect GIN-induced anaemia via changes in sheep eye

mucosa colour (Malan et al., 2001; Van Wyk and Bath, 2002); DISCO (acronym for diarrhea score)

designed to detect changes in faecal consistency resulting from GIN-induced gastric disturbances

(France: Cabaret et al., 2006; North Africa: Ouzir et al., 2011, Bentounsi et al. 2012); and reductions

in sheep weight gain based on the premise that GIN infections reduce productivity (Coop et al., 1977;

1988; Hubert et al., 1979; Stafford et al., 2009 : Malan et al., 2001; Kaplan et al., 2004; Mahieu et al.,

2007; Kenyon et al., 2009; Greer et al., 2009).

The study further questioned how effective the pathophysiological indicators were at

detecting sheep suffering symptomatically from GIN infection. While several studies have supported

their efficacy, the evaluations took place in field scenarios where sheep likely incur multi-infections

(i.e. bacterial, viral or parasitic) that could potentially misrepresent the severity of the symptoms

attributed to GIN infections. For example, in some studies lungworms have been present but not

accounted for in the evaluation (Ouzir et al. 2011, Bentounsi et al., 2012 among others). This study

thereby used controlled experimental conditions and a single GIN species of Haemonchus contortus

to explore the efficacy of the indicators while minimizing external sources of variation. Variation in

indicator efficacy was explored in two specific contexts. The first set of conditions explored the

potential effect of various populations of H. contortus. The isolates selected had differing capacities

to resist AH or survive in sheep with H. contortus-resistant phenotypes. Given different isolates may

differ in their life-history strategies i.e. establishment, fecundity, pathogenicity etc. we hypothesized

this in turn may influence the symptoms of the infection and the sensitivity of the indicators. The

202

second set of conditions explored whether sheep with different H. contortus resistant phenotypes,

including high, medium or low resistance based on significantly different FEC, influenced the

sensitivity of the indicators. Given the immunological responses of sheep are also thought to

contribute to the symptomatic manifestation of an H. contortus infection (Simpson 2000; Williams

2011) we hypothesized that not all symptoms would be expressed equally across the three resistance

phenotypes resulting in a skewed applicability value of the indicators across the resistance-

phenotype groups. Given adult sheep represent a large portion of the flock in he field and thereby

have an important role in GIN transmission, the experiments used young adult sheep. Finally, as the

indicators should ideally be sensitive enough to detect sheep in need of treatment as early as

possible to minimize GIN induced effects, the study focused specifically on the early infection by H.

contortus (i.e. 15-32 dpi).

2. Materials and methods

A total of 63 young adult sheep were used in the study: 24 Blackbelly ewes (18 months old),

24 Blackbelly rams (18 months old) and 15 Romane rams (9 months old). Several previous studies

have documented the H. contortus resistant and susceptible disposition of the Blackbelly and

Romane breeds respectively (Terefe et al., 2007; Salle et al., 2012). None of the sheep in the

experiment had previous experience of GIN infection as they were reared indoors. This was validated

by negative FEC carried out on the sheep prior to the study.

2.1 Parasitological indicator

Faecal egg counts were performed using a modified McMaster technique (Raynaud 1970) in a

sodium chloride flotation solution, accurate to 50 eggs per gram (EPG) of faeces. The oocysts of

coccidian were counted as they may interfere with DISCO and they remained low (448 ± 1448 SD

oocysts per gram of faeces as an average for all the period. The sheep were examined daily during

203

food distribution at their pens for other symptoms such as coughing, diarrhoea or abnormal

behavior. The sheep were further examined individually at the sampling dates to ensure they were

apparently free from other co-morbidity that may affect the indicators.

2.2 Pathophysiological Indicators

FAMACHA©: The system is based on a semi quantitative evaluation of the eye mucosal colour. This

colour was classified into one of five categories according to the FAMACHA© eye colour chart: 1 =

red, non-anaemic; 2 = red-pink, non-anaemic; 3 = pink, mildly anaemic; 4 = pink-white, anaemic; 5 =

white, severely anaemic (Van Wyk and Bath, 2002).

DISCO: This indicator uses the following values: 1 = normal sheep faeces in pellets; 2 = “soft” faeces

(similar to cow pat); 3 = diarrhoea (semi-liquid faeces). These scores correspond to 40, 25 and 15%

dry matter in faeces respectively as determined in grazing lambs (Cabaret et al., 2006).

2.3 Weight

The sheep were weighed using automatic walk on scales. Their weight gain ratio was

calculated by dividing the sheep weight at the end by their weight at the beginning of the

experiment.

2.4 Haematocrit

Blood samples were drawn from the jugular vein of the sheep using a 10 gauge syringe into

heparin tubes. The haematocrit values were determined by drawing blood from the heparin tubes

into capillary tubes, blocking them with gum and spinning them in a Hettich MIKRO 20 centrifuge at

1000 rpm for 10 minutes (G-force 672). The capillary tubes were then positioned in a Hawksley

micro-haematocrit reader to obtain the results manually.

2.5 Experiment 1: Influence of different H. contortus isolates on indicator efficacy

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This experiment used Blackbelly rams only. The sheep had open access to water and hay ad

lib and were provided with feed concentrate and cereals. They were penned in groups of 6 according

to the infecting H. contortus isolate used and maintained indoors throughout the experiment.

The four H. contortus isolates tested included: i) ISES: a line susceptible to all AH with no previous

exposure to resistant hosts (see Roos et al., 2004 for line history); ii) ISER: this line was derived from

ISES but had been selected in resistant Blackbelly sheep for three generations prior to the present

study; iii) RHS6: a line highly resistant to the AH levamisole (see Hoekstra et al., 1997 for line history);

iv) KOK: a line resistant against the three main AH classes i.e. levamisole, avermectin and

benzimidazole. This line was originally obtained from a farm in South Africa and kindly donated by J.

Van Wyk in 2000. It has since been maintained in the INRA laboratory. Each isolate was infected in six

sheep. All sheep were infected orally with 10,000 L3 larvae from their respective lines. The infection

period lasted 32 days during which time FEC, FAMACHA© and DISCO measures were taken at 21 and

32 dpi. The sheep were weighed and hematocrit determined at 0, 15, 21 and 32 dpi.

2.6 Experiment 2: Influence of different sheep H. contortus-resistant phenotypes on indicator efficacy

The second experiment used three groups of adult sheep that had no previous experience of

GIN infection, having been reared indoors and verified with FEC. These groups represented different

H. contortus-resistant phenotypes based on significantly different FEC relative to each other. The

high resistance group was comprised of 12 Blackbelly ewes (mean 2 EPG). The medium resistance

group consisted of 13 Blackbelly rams (mean 1664 EPG). The low resistance was made up of 15

Romane rams (mean 4684 EPG). Each of the sheep received a single infective dose of 10,000 L3 H.

contortus infective larvae from the ISES line. The infection lasted 32 days during which time FEC,

FAMACHA© and DISCO measurements were carried out at 21 and 32 dpi. The sheep weights were

taken at 0 and 32 dpi. Haematocrit was determined at 0, 15, 21 and 32 dpi.

2.7 Treatment cut-off limits

205

All the indicators were compared against FEC. Defining a FEC cut-off for treatment is a

controversial topic among researchers. The differing disposition of sheep breeds to either resistance

or resilience makes it unlikely that a single FEC cut-off could be applicable across breeds. Based on

previous experience using the Blackbelly and Romane breeds, we deemed ≥ 1000 EPG to be

considered a cut-off above which there would be negative symptomatic consequences for the

individual . A cut-off of ≥ 5000 EPG was also included in the results. Given haematocrit is considered

the “gold standard” of anaemia evaluation (Jain, 1986) and that FAMACHA© was expressly designed

to reflect it, comparisons were also drawn between haematocrit and FAMACHA©.

The cut-off limits used for the three indicators to identify individual sheep in need of

treatment have varied somewhat in previous studies (FAMACHA© and haematocrit: Reynecke et al.,

2011; Roberts and Swan, 1982; reductions in weight gain: Leathwick et al., 2006). The treatment cut-

offs used for FAMACHA© and DISCO were those typically recommended for sheep including

FAMACHA© value ≥ 3 (Bath et al., 2001; van Wyk and Bath, 2002; Burke et al., 2008; Vatta et

al.,2001) and DISCO value ≥ 2 although Cabaret et al. (2006) suggested this may need to be altered in

accordance with the sheep breed and GIN isolate involved. The weight gain indicator cut-off included

those sheep which gained ≤ 5% of their bodyweight at 0 dpi by 32 dpi. While previous studies used

higher cut-offs than this (Stafford et al., 2009) they used young lambs. Given the adult sheep used in

this study had comparatively less potential for growth compared to the lambs, the cut-off was

lowered accordingly.

To gain a general indication of how many sheep were suffering from one symptoms or

another as a result of the H. contortus infection, all the sheep which exceeded one or more of the

treatment indicators were pooled. The indicators were then compared against each other to explore

whether they identified the same sheep to be in need of treatment.

2.8 Statistical analyses

206

All data analyses were conducted using IBM SPSS software (Version 11.5). The efficacy of the

indicators and any significant differences between the groups were determined using One way

ANOVA and Student-Newman-Keuls tests. Where the data were not normally distributed (i.e. FEC)

logarithmic transformation was performed prior to performing ANOVA and non-parametric Kruskal-

Wallis (KW) tests. The comparison of indicators (FAMACHA© ≥ 3 DISCO ≥ 2) in relation to nematode

isolates was evaluated using Fisher exact test to retain bilateral significance. The Cohen’s Kappa

measure of agreement was used to calculate accordance among the different indicators to treat. A

negative value is indicative of disagreement between the indicators decision to treat, a value of

naught reflects independence between the indicators, a positive value over 0.40 indicated slight

concordance between the indicators whereas values over 0.60 are encountered when high

concordance exists between indicators.

3. Results

3.1 Influence of different H. contortus isolates on indicators efficacy (Table 1)

The FEC varied significantly between the different H. contortus isolates at 21 dpi (One way

ANOVA and KW p=0.019) as tested in 24 Blackbelly rams (Table 1). At 21 dpi, the ISEs isolate had

significantly greater FEC than the other three isolates. While these other three isolates increased

their FEC by 32 dpi, the ISES isolate (1570 EPG) maintained the greatest outputs which were nearly

double that of the ISER (941 EPG) and KOK (813 EPG) and four times that of RHS6 (373 EPG).

The FAMACHA © indicator values did not vary significantly between the isolates at 21 or 32

dpi (Table 1). Significant differences in the DISCO indicator values were observed at 32 dpi only

(Table 1). In this case, the RHS6 isolate approached significantly greater (ANOVA p = 0.007; KW

p=0.006) DISCO values than any of the other isolates, despite also having the lowest FEC. No

significant differences were observed in the weight indicator between the isolates (Table 1). These

207

results suggest that FAMACHA© and weight gain indicators remained unaffected by different H.

contortus isolates but the sensitivity of DISCO may be influenced by it.

Table 1

Mean indicator values per H. contortus isolate at 21 and 32 dpi

H. contortus isolate

Dpi Indicator* P ISES ISER RHS6 KOK

21 FEC 0.019 2376 ± 2939a 36 ± 84b 13 ± 20b 3 ± 6b

FAMACHA© ns 1.50 ± 0.84 1.14 ± 0.38 1.17 ± 0.41 1.33 ± 0.52

% FAMACHA© ≥ 3** ns 17 0 0 0

DISCO ns 1.67 ± 0.52 1.71 ± 0.49 1.33 ± 0.52 1.83 ± 0.41

% DISCO≥ 2* ns 50 67 33 50

Weight ratio ns 1.05 ± 0.38 1.06 ± 0.11 1.06 ± 0.02 1.03 ± 0.15

32 FEC ns 1570 ±1182 941 ± 921 373 ± 538 813 ± 862

FAMACHA© ns 2.17 ± 1.17 1.29 ± 0.49 2.00 ± 0.89 1.67 ± 0.52

%FAMACHA© ≥ 3** ns 33 0 33 0

DISCO 0.007 1.00 ± 0a 1.29 ± 0.49a 1.83 ± 0.41b 1.17 ± 0.41a

% DISCO≥ 2** p=0.030 17 67 67 33

Weight ratio ns 1.05 ± 0.05 1.06 ± 0.11 1.07 ± 0.06 1.05 ± 0.02

*Arithmetic mean indicator values ± standard-error (FEC (EPG), FAMACHA© (1-5),DISCO (1-3) at 21

and 32 dpi and final weight gain ratio (weight at 32 dpi divided by weight prior to infection) for each

of the H. contortus isolates. Significant differences (One-way ANOVA p ≤ 0.05) between H. contortus

isolates noted; ns indicates no significant difference. Significantly different (post-hoc Student-

Newman-Keuls p ≤ 0.05) subsets within H. contortus isolates denoted by letters superscript. **Fisher

exact test.

208

3.2 Influence of different sheep H. contortus-resistant phenotypes on indicator efficacy (Fig. 1a-e, )

The FEC varied significantly (ANOVA p = 0.00) between each of the three sheep resistance

phenotypes at both 21 and 32 dpi (Fig. 1a). While the high resistance phenotype sheep group

maintained near zero FEC throughout the infection, the low resistance group had almost double the

FEC of the medium resistance group at 21 dpi which increased to a 12 fold difference by 32 dpi (Fig.

1a).

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

21* 32*

EPG

DPI

a. FEC

High R

Medium R

Low R

c

b

a b

c

209

0

5

10

15

20

25

30

35

40

45

50

0* 21* 32*

Haem

atoc

rit %

DPI

b. Haematocrit

High R

Med R

Low R

a

a b b

b b

a

b b

0,8

1

1,2

1,4

1,6

1,8

2

21* 32*

Fam

acha

val

ue

DPI

c. Famacha

High R

Medium R

Low R

a a

a a

b

b

210

Figure 1 (a, b, c, d, e): Arithmetic mean indicator values ± the standard deviation (a: FEC (EPG), b:

Haematocrit( %); c: FAMACHA© (1-5), d: DISCO (1-3) at 21 and 32 dpi and d: final weight gain ratio

(weight at 32 dpi divided by weight prior to infection) for each of the sheep resistant phenotype

groups tested i.e. high, medium and low resistance (R). Significant differences* (One-way ANOVA p ≤

0.05) between sheep resistant phenotypes. Significantly different (post-hoc Student-Newman-Keuls p

≤ 0.05) subsets within H. contortus isolates denoted by letters above bars.

0,80,9

11,11,21,31,41,51,61,71,8

21 32*

Disc

o va

lue

DPI

d. Disco

High R

Medium R

Low Ra

a a a

b

b

0,8

0,9

1

1,1

1,2

High R Medium R Low R

Wei

ght r

atio

Sheep resistance group

e. Weight gain ratio

211

The haematocrit values differed significantly between the three sheep resistance phenotypes

(Fig.1b). FAMACHA© values also differed significantly between the three sheep resistance

phenotypes at 21 (ANOVA p = 0.02) and 32 (ANOVA p = 0.00) dpi (Fig.1c). Post-hoc Student-Newman-

Keuls comparisons revealed that the medium resistance group experienced significantly greater

FAMACHA© values than both the high and low resistance group. This did not correspond to the

suggested infection intensities of the groups based on FEC observations. While both the high and low

resistance groups maintained FAMACHA values of approximately 1 at both time points, the medium

resistance group had values around 1.3 at 21 dpi which increased by 38% at 32 dpi to 1.8.

The sheep resistance phenotype groups also differed significantly in their DISCO values, but

only at 32 dpi (ANOVA p = 0.01) (Fig.1d). Post-hoc Student-Newman-Keuls comparisons showed

similar results to the FAMACHA© where the medium resistance group experienced greater DISCO

values than either the high or low resistance groups. Once again, this does not correspond to the

groups’ infection intensities as reflected by the FEC.

The weight gain ratios also varied significantly (pairwise comparison p = 0.003) among the

sheep resistance phenotype groups with the high resistance group gaining significantly less (p=0.001)

than the medium and low resistance groups (Fig. 1e).

These results suggest that none of the three indicators performed consistently across

different sheep resistance phenotypes.

3.3 Number of sheep to exceed indicator treatment cut-offs (Table 2)

The efficacy of the indicators was examined in all 63 H. contortus infected sheep used in both

experiments i.e. H. contortus isolates (n = 24) and comparing sheep resistance phenotypes (n = 39).

Eighteen sheep exceeded FEC ≥ 1000 EPG and 11 sheep exceeded ≥ 5000 EPG at 32 dpi. For

haematocrit, seven sheep had haematocrits= 22% whereas none had haematocrits=19% at 32 dpi.

Only four sheep exceeded the FAMACHA© indicator ≥ 3 at 32 dpi, only one of these sheep was also

212

identified by haematocrit. Eight sheep exceeded the DISCO ≥ 2 at 32 dpi all of which belonged to the

medium resistance (Blackbelly ram) phenotype. Thirteen sheep gained ≤ 5% of their body weight by

32 dpi, all except one of these sheep belonged to the medium resistance (Blackbelly ram) phenotype.

Table 2

Number of sheep to exceed indicator cut-off

No. sheep to exceed cut-off

Indicator Cut-off 15 dpi 21 dpi 32 dpi

FEC ≥ 1000 0 16 18

≥ 5000 0 6 11

Haematocrit ≤ 22 0 1 4

≤ 19 0 0 0

FAMACHA© ≥ 3 1 1 4

≥ 4 0 0 2

DISCO ≥ 2 0 15 8

≥ 3 0 0 0

Weight gain ≤ 5 NA NA 13

≤ 1 NA NA 4

A total of 63 H. contortus infected sheep were analyzed. Indicators included FEC (EPG), Haematocrit

(%), FAMACHA© (1-5), DISCO (1-3) at 15, 21 and 32 dpi and final weight gain ratio (weight at 32 dpi

divided by weight prior to infection).

3.4 Correlations between indicators, FEC and haematocrit (Table 3 and 4)

213

The Kappa coefficient agreement found there to be no accordance between the FEC and any

of the three indicators including FAMACHA©, DISCO and reductions in weight gains. Accordance was

observed however between haematocrit and FAMACHA© (Table 3) ranging from 0.48 - 0.68 at 32

dpi. No other indicators were found to be related to each other based on Kappa analysis.

Table 3

FAMACHA© value and mean corresponding haematocrit value

Haematocrit %

FAM ACHA© 15 dpi 21 dpi 32 dpi

1 35.1 ± 5.3 (57) 33.7 ± 5.3 (56) 31.4 ± 5.7 (41)

2 30.4 ± 5.6 (5) 27 ± 5.7 (6) 29.9 ± 6.7 (18)

3 26 (1) 25 (1) 25 ± 1.73 (3)

4 21 (1)

FAMACHA© and haematocrit values compared in 63 sheep ± standard deviation at 15, 21 and 32 dpi.

The number of sheep within the FAMACHA© cut-off is noted in brackets.

Combined, the indicators cut-offs were exceeded a total of 25 times in 16 sheep at 32 dpi. This

included FAMACHA ≥ 3 = 4; DISCO ≥ 2 = 8; weight gains ≤ 5% of initial body weight = 13. Only one of

these sheep exceeded the cut-off for all three indicators (data not shown). From the 13 sheep which

gained ≤ 5% of their body weight at 32 dpi (Table 4) two of these also exceeded the FAMACHA© cut-

off and two of them exceeded the DISCO cut-off. From the eight sheep which exceed the DISCO cut-

off, only one of them also exceeded the FAMACHA© cut-off. It should be noted that 15 of the sheep

which had FEC ≥ 1000 EPG did not exceed the treatment cut-off for any of the other indicators.

Table 4

214

Number of cases of indicator agreement in 63 sheep on 32 dpi

No. of cases of indicator agreement between:

Total cases above cut-off Haematocrit FAMACHA© DISCO Weight gain

FEC 18 4 4 0 3

Haematocrit 4 0 0 0

Famacha© 4 2 1

DISCO 8 2

4. Discussion

Three main findings emerge from the results of this study which focused on the efficacy of

different pathophysiological indicators during the first month of an H. contortus infection. Firstly, the

pathophysiological indicators FAMACHA© and DISCO do not reflect infection intensity based on FEC,

nor do reductions in weight gains. Secondly, there is little agreement among the three indicators to

identify the same animals in need of treatment. Finally, the indicator sensitivity was influenced by

the H. contortus isolate and sheep resistance phenotype in which they were tested.

Given all three indicators FAMACHA©, DISCO and weight gains were designed to reflect sheep

suffering most from infection, irrespective of infection intensities, perhaps it should not be surprising

that no correlation was found between them and FEC. Indeed, several studies support that those

sheep with the greatest clinical symptoms of infection (less resilient) are not necessarily the most

heavily infected (Clunies-Ross, 1932; Williams 2011). Nonetheless, our study proceeded to examine

this possibility in the belief that these two factors, infection intensity and pathophysiological

symptoms, cannot remain completely unrelated, especially in heavily infected individuals. This is

somewhat supported by previous findings which found correlations between FEC and FAMACHA ,

DISCO, and body scores which are relative to reduced weight gains (Bentounsi et al., 2012;

215

Rosalinski-Moraes et al., 2012). In practical terms, based on the results of this study none of the

indicators appeared to be a useful tool for a TST approach aiming to reduce transmission stages

which require targeting and treating the most heavily infected individuals of a flock. It is important to

highlight that the sheep in the present study were maintained in comfortable conditions which may

have limited the stress of the infection. Furthermore, the sheep were only monitored during the first

month of infection, it possible symptoms may have changed more thereafter.

Even allowing for some degree of discord between the indicators and FEC, we at least expected a

considerable amount of overlap among the three indicators to identify the same individuals in need

of treatment. This was not the case. From the 63 infected sheep used in this study, 18 were identified

for treatment based on a cut-off of FEC ≥ 1000 EPG at 32 dpi. Pooling the positive treatment

identifications from all three indicators (FAMACHA ≥ 3 = 4; DISCO ≥ 2 = 8; weight gains ≤ 5% initial

body weight = 13 at 32 dpi) served to provide a general indication of which individuals were suffering

from the pathophysiological costs of infection. A total of 16 sheep exceeded the cut-off for one, or

more, of the indicators. However, from these 16 sheep, only one of the individuals was identified by

all three indicators, who incidentally had a FEC of only 15 EPG at 32 dpi. Indeed, from all 25 positive

treatment identifications made by the indicators, only 3 of the cases had FEC ≥ 1000 EPG. Thus,

based on the little overlap between the three indicators found in this study, it does not appear they

are individually sensitive at detecting general pathophysiological suffering in the sheep as a result of

H. contortus infection. With the very low number of sheep that exceeded the FAMACHA© and DISCO

cut-offs we question how sensitive they were in respect to detecting anaemia or diarrhea

respectively. It could be that not all the symptoms (anaemia, diarrhea and weight loss) are expressed

equally across different infecting GIN populations or different breeds of sheep. This is supported by

our findings that the infecting H. contortus isolate (i.e. DISCO) and sheep-resistance phenotype (i.e.

FAMACHA©, DISCO, weight gains) significantly altered the sensitivity of the indicators to detect

sheep in need of treatment. Sensitivity here refers solely to the positive identification of a sheep in

need of treatment by exceeding their pre-defined cut-off, this does not necessarily infer accuracy i.e.

216

potential for false positives or false negatives. All three indicators increased in sensitivity by 32 dpi

compared to 21 dpi suggesting they may increase further still as the infection progresses and greater

pathophysiological penalties are experienced. Additionally, it could be the indicators are better

suited to different stages of an H. contortus infection. For example, DISCO may be more applicable to

the pre-patent period of an infection during which time the pathophysiological damage (Simpson,

2000) and/or immune response (Meeusen 1999; Williams 2011) may provoke diarrheal responses.

FAMACHA© may be more useful after this to coincide with the maturation of adults and

commencement of the blood-letting activities (Baker et al., 2003; Saddiqi et al., 2010a, 2010b).

Along with several other studies (Van Wyk and Bath, 2002; Reynecke et al., 2011), a correlation

was found between FAMACHA© and haematocrit. However, given the few sheep which were found

to exceed the treatment cut-off of ≥ 3 (four sheep) and ≤ 22% (four sheep), for the indicators

respectively, this agreement is largely based on sheep which are seemingly healthy in that respect

and do not necessarily reflect a decrease in haematocrit with an increase in FAMACHA© score. All

the FAMACHA© measures were carried out by a single experienced evaluator which minimizes the

likelihood these low counts resulted from human error. Two possible explanations may account for

this. Firstly, it is possible the experimental setting of our study offset the general stress and anaemia

normally experienced by sheep in field scenarios where the majority of FAMACHA© evaluations have

taken place (Van Wyk and Bath, 2002; Reynecke et al., 2011). The sheep in our study were sheltered

from environmental stress and well supplied with high quality food, a factor thought to improve

sheep resilience to infection (Coop and Kyriazakis, 1999). For example, studies observed increased

protein uptake during an H. contortus infection reduced the extent of blood loss and

hypoalbuminaemia (Abbott et al., 1986a, 1986b, 1988; Wallace et al., 1995). Secondly, it is possible

that the arbitrary haematocrit cut-off of ≤ 22% or even ≤ 19% was too high for the sheep in this

study, even though other studies have gone as low as using ≤ 15% as a cut-off (Reynecke et al., 2011).

No precise haematocrit value has been determined to reflect anaemia of clinical importance across

breeds (Burke et al., 2007). It may instead be more relevant to consider the total overall reduction in

217

hematocrit for each individual, rather than if they exceed a specific value at a single time point. For

example, if we presume a total drop in haematocrit ≥ 15% by 32 dpi was important and indicative of

a sheep in need of treatment, in that case 15 of the 63 sheep in our study would be considered in

need of treatment, 13 of which sheep also had a FEC ≥ 1000. In this hypothetical case, the four sheep

which FAMACHA© identified for treatment at 32 dpi appears a weak and unreliable reflection of the

sheep in need of treatment. The FAMACHA© values varied significantly between the sheep resistant

phenotypes suggesting the indicator may require some form of calibration. FAMACHA© was

developed and verified predominantly in a single sheep breed, the Merino (Van Wyk and Bath, 2002)

although some studies have also evaluated it in indigenous cross-breeds (Gauly et al., 2004; Ouzir et

al., 2011). Nonetheless, this system has not accounted for potential inter-breed variations. This is

important given previous studies have demonstrated fundamental differences in eye colour

measurements between different breeds of sheep (Moors and Gauly, 2009) which may compounded

further by peripheral factors i.e. vasoconstriction due to the stress of capture, local inflammation and

physiological status (Di Loria et al., 2009).

All eight sheep identified by DISCO to be in need of treatment belonged to the medium

resistance phenotype (Blackbelly rams) suggesting it was more inclined towards a diarrhoeal

response than the high (Blackbelly ewes) and low (Romane rams) resistance phenotypes. Other

studies have also found certain sheep breeds to have greater dispositions to diarrhoeal responses

(Ouzir et al. 2011) and that DISCO had a greater efficacy when applied to young lambs than in ewes

(Cabaret et al., 2006). It is difficult in our experiment to relate DISCO only to a breed effect since one

group was constituted of ewes and there is likely a confounding effect between sex and breed.

Additionally, the diarrhoeal response was found to be significantly greater in those sheep infected

with RHS6 H. contortus isolate suggesting it could be more pathogenic than the other three isolates.

Based on these findings, the applicability of DISCO as a pathophysiological indicator seems to be

limited to sheep resistance phenotypes and infecting H. contortus populations.

218

Reductions in weight gain appeared to be the most sensitive of three indicators with 13 sheep

gaining ≤ 5% of their initial body weight by 32 dpi. This was significantly correlated to sheep-

resistance phenotype where surprisingly, the high resistance group (i.e. lowest FEC) experienced the

lowest weight gains, and the low resistance group (i.e. highest FEC) had the greatest weight gains.

These results should be interpreted cautiously given the differences in breed, sex and age of the

three sheep resistance phenotypes which will influence the genetic determinants of weight (Bisset

and Morris, 1996; Bisset et al., 2001). While all sheep were considered adults at the time of infection,

the low resistance group was not only several months younger but consisted of a breed (Romane)

selected for high weight gain performance which likely allowed for greater weight gain potential

than the other two resistant phenotype groups.

Based on the results of this study focused only on the first month of infection, none of the

indicators FAMACHA©, DISCO or reduced weight gains have the capacity to reflect H. contortus

infection intensity. Furthermore, no one indicator could be recommended over the others in terms of

identifying sheep generally suffering from the pathophysiological consequences of an early H.

contortus infection. While reductions in weight gains identified the most sheep in need of treatment,

the lack of correlation with FEC combined with the breed, sex and age determinants involved in the

comparison leave its accuracy still questionable, at least during the first month of infection. The

value of FAMACHA© and DISCO also appear to be nematode and sheep specific and consequently,

farm specific. Thus, there does not appear to be a ‘one size fits all’ approach to the use of indicators

for TST. We acknowledge that other studies found these indicators to be reliable in themselves under

different conditions (with several species of gastrointestinal infections and in sheep of various ages),

yet in light of our inconclusive results, we may question whether this was a direct result of the

indicator efficacy or partly a random effect. Work by Gaba et al. (2012) has demonstrated both with

models and a small field study that by simply treating 20% of a flock at random once a month, this

can be equally as effective as TST at counter-selecting for AHR with no additional cost to flock

production. Our results show that the indicators are not of much use at the early stage of infection.

219

We do not however preclude the interest of TST in farm conditions where infection is continual with

daily grazing. The results of the present study and others (Swan and Roberts, 1982; Reynecke et al.,

2011) have highlighted the difficulties in using single arbitrary cut-offs for the indicators. Additionally,

there is a sociological aspect to this too whereby the decision of which indicator and which cut-off to

use is ultimately made by the farmer, and not the researcher. His choice will be shaped largely by his

previous experience of the GIN problem, the interpreted severity of the problems and the perceived

production benefit of the indicator and treatment used (Abraham and Sheeran, 2009).

Acknowledgments

C. Chylinski is a grateful recipient of European Marie Curie ITN “Nematode Systems Health”

PhD funding. The animals were provided by the Bourges INRA Center and we are grateful to F.

Bouvier for information on the sheep. Many thanks to T. Chaumeil and team at the animal platform

INRA Nouzilly. We thank F. Borgsteedt and J. van Wyk for providing Haemonchus strains.

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FARMER DISCUSSION

As parasitologists, we understand the threat of GIN from many angles. We see their global

prevalence with epidemiological tools, we are fascinated by the genetic diversity their extensive

population sizes enable, we marvel at the flexibility of their life-history traits, we know the

pathophysiological damage they inflict and we grimace when we pick them out of a sheep’s

abomasum. This information lends itself very easily to understanding the need for appropriate GIN

control strategies. Farmers on the other hand are exposed to none of these points of view. In fact,

the GIN likely remain a rather abstract notion with Farmers likely never physically seeing any of their

life cycle stages. The only indicator of their existence to the farmer is symptomatic signs such as loss

of condition and anaemia. Flocks of 500 sheep or more are increasingly common and while it is by no

means acceptable, it is perhaps understandable if these symptoms, many of which likely crossover

with other co-infections, do not make themselves immediately apparent to the farmer. Thus, a

farmers understanding of the threat of disease is largely based on the information he receives about

it. The results in this chapter clearly highlight that whatever level of information they’re receiving, it

is not sufficient. Farmers are underestimating the threat that GIN pose. In absence of an accurate

threat perception, we may assume this is followed by a lack of motivation to apply recommended

(often labour intensive) control strategies. Thus, the first step in improved GIN control would appear

to be improved dissemination of knowledge. But who is responsible for this? And equally important,

who would be effective at this? Animal health advisors have not proved to be reliable to date

(Saddiqi et al. 2012). Given the continuing problem, nor do the farmer magazines often popular

among them. How much should researchers assume a role in this and if we did, would the farmers be

inclined to listen to us?

Once clear understanding of the problem is established in the farmer, and their appropriate

motivation addressed, GIN recommendations will likely be applied more effectively. But the results

herein also suggest that these recommendations may be better off as rules. Given greater farmer

autonomy resulted in greater GIN infection intensities perhaps farmers would react better to rules

rather than options. Yet we cannot assume that universal rules will be effective globally. The results

investigating the efficacy of the pathophysiological indicators showed their efficacy could be as

capricious as depending on the day post infection their applied. On a field scenario, where infection

is likely to be more continual than the single doses applied in these studies, this knowledge would

not be particularly useful. Many of the studies throughout this thesis support the many factors that

interact to influence the farm specific situation i.e. local H. contortus population, their relative AHR

status, the sheep in the flock and their relative GIN resistance status, climatic effects, husbandry

226

choices etc. This very much points to the conclusion that there is not a one size fits all approach to

GIN control.

The three main results of this chapter suggest there is a need for the farmers improved knowledge,

focused motivation, understanding of the right GIN control strategies and the tools to apply to farm

specific cases. It is thereby apparent that the gap needs to be bridged between Farmers and Science

to obtain the most efficient GIN control. Given the sums of money that are lost annually from GIN

infection i.e. direct production losses, cost of therapeutics and GIN controls, and their inevitable

ineffective application, more money should be invested in an effective knowledge dissemination

plan. The role of a parasite control strategist could act as the go between for farmers and advancing

research, keeping abreast of scientific developments while working alongside farmers to aid in

developing the best GIN control action plan for that farm.

227

GENERAL CONCLUSIONS

Our impetus to understand GIN success is largely with the view to control it. In that respect, the

definition of success is largely different to each of the players studied in this research; the GIN, the

sheep host and the farmer.

For the GIN the definition of success remains the same, to maximize fitness via altering their survival

and reproductive strategies. The repeated demonstration of H. contortus altering their life history

traits in response to the selective pressures presented in this thesis showed their success in action.

This flexibility is undoubtedly one of our key conclusions to their success. Haemonchus contortus

overcame, desiccation, ageing , cryopreservation and certain instances of resistant sheep by altering

their life-history traits. The only obstacle they were not able to overcome was the resistance of

Martinik Blackbelly females. But the multiple variations in life history traits showed there is not a

single road to success, but success can be achieved by many different routes. We cannot necessarily

expect that the same challenges will consistently provoke the same response within an H. contortus

isolate, and certainly not between isolates given the AHR study documented H. contortus specific

strategies. But we can certainly acknowledge that there are multiple, equally adaptive strategies to

overcome physical barriers for H. contortus.

In terms for the AHR result, it seemed interesting to note that a reduction in fitness via reduced

flexibility in their life history strategies seemed to be linked with reduced transcriptomic expressioin.

If we were to run with this idea we may question what possible targets that may present in GIN

control. Although techniques exist to “turn-off” certain gene expression via methods such as RNA

interference, and this could theoretically target transcriptomic regulator genes, this does not lend

itself nicely to control a grand scale. There is always a possibility that a virus exists which could target

transcriptomic expression, but this would need to be a very specific virus and the applicative value

would be complicated.

There is a theory that suggests parasites that want to optimize their success should limit their

pathogenicity as it is in their interest to preserve their “living environment” (Combes 1997). Indeed,

this would very likely be the sheep hosts view of success as well. However several authors (reviewed

by Toft & Aeschlimann 1991; Ewald 1995; Ebert and Herre 1996) have demonstrated that this

question is more complex and that natural selection does not necessarily favour peaceful coexistence

(Ewald 1995). Some parasites increase their fitness by increasing their pathogenic effect in order to

obtain a higher fecundity or a higher probability of transmission. Others decrease their fitness by

decreasing their pathogenicity if they can benefit from a longer survival in the host (Comber 1997).

To understand why the selective pressure can result in different parasites evolving in opposite

228

directions, probably the most influential factor to consider is whether there is a cost of virulence to

the parasite which corresponds to the cost of resistance for the host i.e. the virulence of the parasite

is the loss of fitness of the host due to pathogenic effects. For any parasite-host system, there is an

“ideal” pathogenicity (Lipsitch and Nowak 1994) which is a compromise between the benefits and

costs of virulence. For MBB females, that ideal pathogenicity was to encounter none at all. Event

though they encountered some degree of pathophysiological costs in exerting this resistance, it

appears this was the more tolerable option compared to infection.

The lessons learnt regarding the farmers are extremely valuable to the future progress of GIN

control. Currently husbandry practices, lack of knowledge, attitude and open communications are

enabling the success of GIN. Education is needed for the farmers to see the value in their efforts

before any corrective behaviours can be applied to their GIN control strategies.

As with all experimental studies, there are limitations to their application in the field. On the side of

the H. contortus, the use of single dose infections with 10,000 infective L3 was were largely used in

the present studies do not reflect the reality of the field. Instead, lower daily L3 intake on a daily

basis is more probable. This in turn limits what we saw in the sheep experiment. For example

perhaps continued trickle infections would eventually induce greater pain responses, or the resistant

MBB females would weary and succumb to infection. Nonetheless, these studies have been valuable

in enabling us to disentangle relative contributions of isolated factors.

This thesis research provides new insight in to the factors influencing GIN success and highlights a

new approach to see the problem of GIN in the bigger picture. We recommend that each of the three

factors should be considered in greater depth, especially from the point of view of the farmer, if

advances in GIN control are to be made.

229

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