Final version Thesis 17_05_2016

OLLSCOIL na hÉIREANN
National university of Ireland, Dublin
UNIVERSITY COLLEGE DUBLIN
Gastrointestinal nematode control in sheep:
Immunological and nutritional strategies
DVM Rocco Sebastiano
PhD 2015

Gastrointestinal nematodes control in sheep:
Immunological and nutritional strategies
DVM Rocco Sebastiano
A thesis submitted to the National University of Ireland, Dublin in
fulfilment of the requirements for the degree of Doctor of
Philosophy
July 2015
Supervisors of research:
Prof Torres Sweeney, School of Veterinary Medicine, University College
Dublin, Belfield
Dr Barbara Good, Teagasc, Animal Production Research Centre, Athenry,
Co. Galway
Dr Tim Keady, Teagasc, Animal Production Research Centre, Athenry, Co.
Galway

I
Declaration
I declare that this thesis has never been submitted for a degree at the National University of
Ireland, Dublin or at any other university. I declare that the work contained within this thesis
is my own
--------------------------------------
Rocco Sebastiano

II
Table of Contents
Declaration I
Summary XV
CHAPTER 1 1
Literature review 1
1.1 Overall introduction 2
1.2 Sheep industry in Ireland 3
1.2.1 Financial overview of sheep farm system 2012 4
1.4 Gastrointestinal nematodes 6
1.4.1 Epidemiology of GINs in Ireland and in cool temperate climates 7
14.2 GIN lyfe cycle 10
1.4.3 Nematodirus battus and pathological effect on the host 11
1.4.4 Teladorsagia circumcincta and its pathological effect on the host 13
1.5 Nematode control strategies 15
1.5.1 Anthelmintic and anthelmintic resistance: potential tools that can prolong the life span
of available drugs 15
1.6 Alternative solutions to anthelmintics 17
1.6.1 Grazing management 17
1.6.2 Biological control 18
1.6.3 Role of nutrition on resistance to GINs 19
1.6.4 Vaccines 23
1.6.5 Genetic selection (phenotypic and genetic markers) 24
1.7 Immune response to GIN infection 27
1.7.1 Innate immunity 27
1.7.2 Adaptive immunity 29
1.7.3 Principal immunological mechanism controlling gastrointestinal nematodes 35

III
1.8 Dissemination of information to farmers in order to contain the development of
anthelminthic resistance in sheep 41
1.9 Thesis objectives 43
CHAPTER 2 44
Breed differences in humoral and cellular responses to experimental infection of lambs with
the gastrointestinal nematode Teladorsagia circumcincta 44
2.1.Abstract 45
2.2 Introduction 46
2.3 Materials and Methods: 47
2.3.1 Ethical approval 47
2.3.2 Animals 47
2.3.3 Experimental infection 48
2.3.4 Worm counts 49
2.3.5 Abomasal mast cell and eosinophil counts 49
2.3.6 Mucosal antibody recovery 50
2.3.7 IgA ELISA 50
2.3.8 Pepsinogen and haematology 51
2.3.9 Statistical analysis 51
2.4 Results 52
2.4.1 Worm burden and Faecal Egg Count 52
2.4.2 Pepsinogen 53
2.4.3 Serum and mucosal IgA 54
2.4.4 Mast cell and eosinophil counts in abomasal tissue 55
2.4.5 Haematology 56
2.5 Discussion 58

IV
CHAPTER 3 62
Evidence for differences in the timing of expression of genes influencing immune cell
trafficking in Suffolk and Texel lambs in response to an experimental infection with the
gastrointestinal nematode Teladorsagia circumcincta 62
3.1 Abstract 63
3.2 Introduction 64
3.3 Material and methods 66
3.3.2 Animals 67
3.3.3 RNA extraction 67
3.3.4 Reverse transcription of mRNA into cDNA 68
3.3.5 Quantitative Real-Time PCR assay 68
3.3.6 Enzyme-linked immunosorbent assay (ELISA) 71
3.4 Results 71
3.4.1 Mucosal barrier integrity: relative expression of extracellular matrix and mucosal
integrity genes 71
3.4.2 Relative gene expression of mediators and networks involved in lymph nodes
trafficking 71
3.4.3 Relative gene expression of Th1, Th2 and Treg mediators 73
3.4.4 Sphingosine phosphate-1 concentration in the serum 73
3.5 Discussion 77
CHAPTER 4 81
Can digestible undegradable protein in late pregnancy affect periparturient parasite egg count
and performance of ewes? 81
4.1 Abstract 82
4.2 Introduction 83
4.3 Materials and method 85

V
4.3.2 Forage 85
4.3.3 Concentrates 86
4.3.4 Animals and experimental design 86
4.3.5 Measurements 88
4.4 Results 91
4.4.1 Feed intake 91
4.4.2 Ewe performance 94
4.4.3 Faecal egg counts 95
4.4.4 Sward heights 96
4.4. 5 Herbage Larvae (L3) 97
4.6 Discussion 98
CHAPTER 5 101
Can digestible undegradable protein offered to ewes during late pregnancy affect performance
and immune competence of the offspring to gastrointestinal nematodes? 101
5.1 Abstract 102
5.2 Introduction 103
5.3 Materials and methods 105
5.3.2 Animal and experimental 105
5.3.3 Measurement 106
5.4 Results 110
5.4.1 Lamb performance 110
5.4.2 Sward heights 111
5.4.3 Larvae (L3) pasture contamination 112
5.4.4 Faecal egg count of Nematodirus spp. and ‘other thricostrongyles’ 113

VI
5.4.5 Worm burden 115
5.4.6 Evaluation of serum IgA and IgE specific to T. circumcincta 115
5.5 Discussion 117
CHAPTER 6 121
Discussion and Conclusion 121
Future research 125
REFERENCES 128

VII
List of Figures
Chapter 1
Figure 1.1 Consumption of meat per capita in Ireland (2011)……………………………......3
Figure 1.2 Family Farm Income-Sheep Farms 2010-2012…………………………………...4
Figure 1.3 Lamb mid-season production………………………………………………….. …5
Figure 1.4 Phylogenetic tree of the major sheep gastrointestinal nematodes in Ireland…...…6
Figure 1.5 Effect of protein deficiency in periparturient relaxation of immunity………… …9
Figure 1.6 Schematic diagram of Nematodirus battus life cycle……………………………12
Figure 1.7 Schematic diagram of Teladorsagia circumcincta life cycle……………………14
Figure 1.8 Entry of lymphocytes through HEVs-the multistep adhesion cascade…………..31
Figure 1.9 Sphingosine-1-phosphate’s metabolism………………………………………....32
Figure 1.10 The journey of lymphocytes into lymph nodes and their egress through the S1P
gradient……………………………………………………………………………………….33
Figure 1.11 Differential activation of CD4+ T cells and cytokines involved in the immune
response to T. circumcincta………………………………………………………………... ..35
Figure 1.12 Mast cell chemical mediators………………………………………….………..38
Figure 1.13 IgE receptors structure……………………………………………………….....39
Chapter 2
Figure 2.1 Adult worm burdens in the abomasum of Suffolk and Texel lambs following
infection with 3x 104
T. circumcincta ………………………………………………………52
Figure 2.2 Serum pepsinogen of Suffolk and Texel lambs following infection with 3x 104
T.
circumcincta …………………………………………………………………………………53
Figure 2.3 Serum and mucosal IgA of Suffolk and Texel lambs following infection with 3x
104 T. circumcincta……………………………………………………………………….. ..54

VIII
Figure 2.4 Abomasal mast cells and eosinophils of Suffolk and Texel lambs following
infection with 3x 104
T. circumcincta…………………………………………………………… ..55
Figure 2.5 Haematology variables of Suffolk and Texel lambs following infection with 3x
104
T. circumcincta………………………………………………………………………......56
Chapter 3
Figure 3.1 Relative gene expression of Sphingosine phosphate-1(S1P) metabolic enzymes in
the lymph node of Suffolk and Texel lambs following infection with 3x 104
T.
circumcincincta………………………………………………………………………………………...72
Figure 3.2 Sphingosine phosphate-1 (S1P) concentration in the serum of Suffolk and Texel
lambs following infection with 3x 104
T. circumcincta…………………………………... ..74
Chapter 4
Figure 4.1 The effect of concentrate DUP concentration and feed level on the silage dry
matter intake (kg/d) during the final 6 weeks of pregnancy……………………………….. ..93
Figure 4.2 The effect of concentrate DUP concentration and feed level on total dry matter
intake (kg/) during the final 6 weeks of pregnancy………………………………………... ..93
Figure 4.3 FEC of “other trichostrongyles”………………………………………………....95
Figure 4.4 Pre and post-grazing sward heights……………………………………………...96
Figure 4.5 Number of L3 larvae of Nematodirus spp. and “other trichostrongyles”………..97
Chapter 5
Figure 5.1 Pre and post-grazing sward heights from April to early October………………111
Figure 5.2 Number of L3 larvae of Nematodirus spp. and “other trichostrongyles”……...112

IX
Figure 5.3 Effect of concentrate DUP concentration and concentrate feed level offered to
ewes on late pregnancy on ‘Nematodirus battus’ egg count in the faeces of offspring…… 113
Figure 5.4 Effect of concentrate DUP concentration and concentrate feed level offered to
ewes on late pregnancy on ‘’other trichostrongyles’ egg count in the faeces of offspring... 114
Figure 5.5 Least squares means (± s,e,) for effects of concentrate DUP concentration and
concentrate feed level offered to ewes on late pregnancy on OD values for IgA and IgE
antibody in the serum of their offspring…………………………………………………… 116
List of Tables
Chapter 1
Table 1.1 Mid-season lamb production: gross output from 2011 to 2012……………………4
Table 1.2 GINs infection and correlated symptoms of lambs in Ireland from 3 weeks of age
onwards (Hynes Frank Teagasc booklet 2012)……………………………………………..…7
Table 1.3 Characterization of nutrients content in ruminant foods………………………... ..21
Chapter 2
No tables at this section.
Chapter 3
Table 3.1 Ovine specific primers used for real-time PCR………………………………… ..69
Table 3.2 Ovine specific primers used for real-time PCR………………………………… ..70
Table 3.3 Relative expression of genes involved in abomasal mucosa barrier integrity in
Suffolk and Texel lambs on days 0, 3, 7, 14, 21 relative to an experimental infection with 3x
104
T. circumcincta…………………………………….. ………………………………………..74

X
Table 3.4 Relative gene expression of lymphocytes trafficking mediators in abomasal lymph
nodes of Suffolk and Texel lambs on days 0, 3, 7, 14, 21 relative to an experimental infection
with 3x 104
T. circumcincta………………………………………………………………..………..75
Table 3.5 Relative gene expression of Th1, Th2, Treg mediators in abomasal mucosal tissue
in Suffolk and Texel lambs on days 0, 3, 7, 14, 21 relative to an experimental infection with
3x 104
T. circumcincta……………………………………………………………………....76
Chapter 4
Table 4.1 Ingredient compositions of the concentrates (kg/t)…………………………….....87
Table 4.2 Chemical compositions of the silage and concentrates…………………………...91
Table 4.3 Effects of DUP concentrate on silage dry matter intake (kg/day)……………… ..92
Table 4.4 Effect of treatment on ewe live weight and condition………………………….. ..94
Chapter 5
Table 5.1 Effect of concentrate DUP concentration and feed level offered to ewes in late
pregnancy on the performance of their progeny……………………………………………110
Table 5.2 Effect of concentrate DUP concentration and feed level offered to ewes in late
pregnancy on total abomasal worm burden and total T. circumcincta and Trichostrongylus
axei burden………………………………………………………………………………….115
Table 5.3 Comparison between DUP supplied by the 4 treatments and requirements during
late pregnancy………………………………………………………………………………117

XI
Acknowledgements
There are so many people I need to thank for their assistance, support and advice throughout
my PhD journey. I would like to thank my supervisor, Professor Torres Sweeney for her
guidance and encouragement, throughout the course of this study. I would also like to express
my gratitude to Dr. Barbara Good (Ph.D), Dr. Tim Keady (Ph.D) and Dr. J.P Hanrahan (Ph.D)
for their important contributions as associate supervisors. I would like to extend my gratitude
to Marie Curie actions for funding this project through the FP7 scholarship program. My
gratitude also goes to Professor Michael Stear coordinator of this project and Denise Ritchie
from the University of Glasgow for their assistance and organization of very interesting
meetings throughout this PhD.
My deepest gratitude goes to the technical staff of UCD School of Veterinary Science Centre
Dr. Marion Ryan (Ph D) for her assistance with the gene expression data analysis and proof-
reading this thesis and to Mr. Kevin Thornton for his assistance with the ELISA and
histological analyses and especially for his encouragement and support during these years.
I wish to thank all staff of Animal and Grassland Research and Innovation Centre Teagasc
Athenry, Co. Galway for technical assistance during my trial. Particularly, I express my
deepest gratitude to Henry Walsh, Anne Donovan and Noel McNamara. Special thanks to
Padraig O’Boyle for his parasitological expertise and for helping me settle in Athenry. I
would be remiss if I did not express my special thanks to all undergraduate French and Irish
students and my Italian friend Riccardo who help out during sampling and lab analysis.
My heartfelt thanks to my fellow old and new labmates, Albin, Anindya, Bahar, CarrieAnn,
Cormac, Giseli, Meike, Stafford and Vanessa for always being there and bearing with me the
good and bad times during my wonderful days of PhD. I also would like to thank particularly
Anindya and Stafford for proof-reading of this thesis and suggestions.
My deepest gratitude goes to my parents, my wife Natalia, and to my little daughter Eva.
Without their support this dissertation would have not been possible. I consider myself the
luckiest in the world to have such a supportive family, standing behind me with their love and
support.

XII
Summary
The principal aims of this thesis were to identify genetic differences between
susceptible and resistant breeds in order to identify potential markers for selection and
to evaluate the effect of nutritional strategies on GIN epidemiology and on the
development of immune response in lambs.
The first objective of this study was to identify the physiological and immunological
parameters linked to resistance/susceptibility of sheep breeds to gastrointestinal
nematodes. The second aim was to evaluate the effects of digestible undegradable
protein (DUP) on the control of periparturient relaxation of immunity (PPR) when
supplemented during late pregnancy, in addition to the immune response of the progeny
to gastrointestinal nematodes.
In the first experiment, Texel and Suffolk lambs were experimentally infected with 3 x
104
third stage (L3) larvae of GIN species Teladorsagia circumcincta. Subsequently,
parameters related to the parasitological, humoral and cellular immune responses were
measured. Texel lambs had significantly lower worm burdens, plasma pepsinogen and
higher numbers of mast cells in the abomasal tissue compared to the Suffolk. Mucosal
IgA concentrations were established at an increased rate in the Texel compared to the
Suffolk breed. It has been concluded previously that an early and localised IgA response
is associated with greater abomasal infiltration by mast cells and that eosinophils
contribute to the more resistant characteristics observed in the Texel breed. The
increased level of plasma pepsinogen, evident in the Suffolk, implies that there is
greater level of abomasal tissue damage associated with GIN infection in this breed
(Chapter 2).
The expression of genes associated with barrier function in the abomasum including;
the mucin layer tight junctions and targets implicit in tissue re-modelling and repair
were explored in Texel and Suffolk lambs infected with 3 x 104
third stage (L3) larvae
of GIN species T. circumcincta. In addition, the relative expression of cytokines,
involved in innate and adaptive immunity, was determined in the mucosal tissue and
lymph nodes. Whilst a significant day effect was observed for most of the genes

XIII
associated with mucosal integrity (down regulation from day 0 to 3 post infection)
significant breed differences were observed for the expression of metalloproteinase
genes. The Texel breed presented up regulation of these genes at 14 days post infection;
suggesting that this breed is potentially more capable of restoring mucosal integrity
after the infection with T. circumcincta. The expression of the CCL19/CCL21/CCR7
axis, which is known to play an important role in the lymphocyte entry of secondary
lymph nodes, did differ between the two breeds. In addition, the expression of the
Sphingosine-1 phosphate (S1P) enzymes involved in the metabolism of S1P was
differently expressed overtime in the two breeds. This suggests that further
consideration of the mechanisms involved in lymphocyte trafficking could progress our
understanding of the genetic differences of immunity to GIN (Chapter 3).
In the second trial, eighty-five naturally infected pregnant ewes Belclare and Belclare x
Scottish Blackface ewes were housed and received four different dietary treatments
consisted of 2 concentrates differing in digestible undegradable protein (DUP)
concentration and in feed levels for the last 6 weeks of pregnancy. The effect of DUP on
the control of the PPR was evaluated in addition to the immune response of their
progeny.
The live weights and body condition scores of the ewes were recorded at week -6 and -5
pre-lambing, within 24 hours of parturition, and at weeks 5 and 14 (weaning) post-
lambing. Strongyle faecal egg counts (FEC) were determined for each ewe at 6 weeks
and then 3 weeks pre-lambing, at lambing and then post-partum at weeks 1, 3, 4, 5, 6, 7,
8, 9 and 10. It was concluded from the results that the different levels of DUP
supplementation used in this study, had no effect on the control of magnitude of PPR
(Chapter 4).
Lambs from the eighty-five ewes were weighed at birth and subsequently at weeks 5,
10, 14, 21 and 29 weeks. Individual faecal samples were collected from all lambs at 10,
12, 14, 16, 18, 20, 21 and 29 weeks of age, for FEC measurement. Composite samples
were taken for each treatment group at weeks 5, 6, 8, and then at weekly intervals from
weeks 18 to 29 inclusive. In addition, blood samples were collected at 10, 12, 14, 16,
20, 24 and 26 weeks to determine serum IgA and IgE antibodies specific for T.

XIV
circumcincta. At slaughter, the abomasum was recovered from 12 lambs per dietary
treatment. It was concluded that changing the level of maternal DUP supplementation
during late pregnancy did not influence the immune response in lambs in response to
nematode infection (Chapter 5).

XV
This work is dedicated to my parents, my wife Natalia
and my little girl Eva

2
1.1 Overall introduction
Gastrointestinal nematodes (GIN) are arguably the most pervasive problem facing
worldwide sheep production. Endoparasitic infections cost the livestock industry in excess of
$ 3 billion per year globally (Jackson et al. 2009). It is been estimated that in Ireland € 24
million are spent on controlling internal parasites in cattle and sheep and the cost of
subclinical nematode infestations in the UK is £ 84 million (Nieuwhof and Bishop, 2005).
Lambs are susceptible to infection with several different species of nematodes. However,
climate conditions and age influence the predominant species present in the gastrointestinal
tract at any particular time point (Stear et al. 2009). In Ireland, lambs at the age of 5 to 10
weeks (April/May) are parasitised predominately by Nematodirus battus whereas, after 10
weeks of age (May/early June onwards) Teladorsagia circumcincta and Trichostrongylus
spp are the prevalent species (Hynes, 2012).
Economic losses derived from gastrointestinal parasitism are characterized predominately by
subclinical infections resulting in weight loss, reduced weight gain and reduced reproductive
efficiency. At present, the control of nematodes relies on grazing management, anthelmintic
treatment or both (Miller and Horohov, 2006). Nevertheless, studies on the incidence and
prevalence of anthelmintic resistance (Papadopoulos et al. 2012) have led to greater interest
in alternative parasite control strategies (Sayers and Sweeney, 2005; Stear et al. 2007). One
of these alternative solutions to anthelmintics is to take advantage of the host’s immune
response, which might be used in selection programs to increase the level of resistance in the
population (Miller and Horohov, 2006). Research of genetic variability (Keane et al. 2006;
Goddard et al. 2010; Periasamy et al. 2014) and environmental factors such as diet (Coop
and Kyriazakis, 1999; Houdijk et al. 2001; Houdijk, 2012; Torres-Acosta et al. 2012)
influencing the expression of immune genes have been part of most recent studies as control
of GIN.

3
1.2 Sheep industry in Ireland
Sheep farming in Ireland is a significant contributor to the national economy. Hence, in order
to maintain and develop good standards of production and to be competitive on the market,
the major long-term challenges are improving ewe prolificacy to improve stocking rate and to
manage the challenge of increase resistance to anthelmintics (Diskin and McHugh, 2012a).
In terms of the overall number of ewes, national population increased dramatically in the
1980s, growing from 1.5 million in 1980 to 4.75 million in 1992. Since 1992 however,
numbers have gradually declined, with 2.5 million ewes recorded currently (Diskin and
McHugh, 2012a).
Ireland is 340% self-sufficient in lamb meat, resulting in over 71% of the total production
valued at €180m being exported in 2011 (60% of Irish lamb meat have been exported to
France). However, even though Ireland is an efficient producer in sheep meat, lamb domestic
consumption compared to other meat has been estimated to be only 2% per capita (Fig.1.1).
Figure 1.1 Consumption of meat per capita in Ireland (2011).
Beef and veal
27%
Pork
36%
Lamb
2%
Poultry
33%
Other
2%

4
1.2.1 Financial overview of sheep farm system 2012
The Teagasc National Farm Survey estimated that there were 12,580 sheep farms in Ireland in
2012. The average Family Farm Income (FFI) in 2012 was €16,898 compared to 2011
average FFI of €19,050 (Fig. 1.2). This represents an 11% decline on average 2011 farms
income.
Gross output value declined by 6% due to lower lamb prices, while the total production costs
increased by 8%. This increase in input expenditure was almost entirely due to feed cost
increases, with concentrate feed expenditure increasing by 20% in 2012 (Tab1.1) (Kinsella,
2013).
Table 1.1 Mid-season lamb production: gross output from 2011 to 2012.
Average 2011 Average 2012 % change
Gross output (€/hectare) 1077 1013 -6%
Concentrates (€/hectare) 148 174 +18%
Weaning rate 1.29 1.22 -5%
Lamb carcass (kg) per hectare 190 175 -8%
18055
19050
16898
5000
7000
9000
11000
13000
15000
17000
19000
2010 2011 2012
FFI€/Farm
Figure 1.2 Family Farm Income-Sheep Farms 2010-2012.

5
1.3 Lamb production in cool temperate climate
The lowland sheep flock is the major source of lamb output, accounting for 85% of carcass
output. The hill flock and mountain flocks account for the remainder (Diskin and McHugh,
2012b). Lamb production is divided as mid-season production (Fig 1.3) and early-season
production where lamb mid-season production is characterized by the mating in autumn and
lambing in spring (March/April), whereas the lamb early-season production is outlined with
breeding season initiated by oestrus induction in summer and lambing in winter (December).
Nevertheless, prime lamb production in Ireland is mid-seasonal and grass-based, with
lambing normally targeted to coincide with the start of the grass growth in spring (Keady and
Murphy, 2013). Therefore, lambs are born in late March to early April weaned and separated
from their mothers on other pastures at 3-4 months of age. Lambs continue grazing until 6-7
months of age (period when the grass stops growing). At this stage lambs are sorted and they
can be used as replacement for old breeding stock or sold for meat.
Figure 1.3 Lamb mid-season production.

6
1.4 Gastrointestinal nematodes
Nematodes belong to their own unique phylum Nematoda (Hodda, 2007). Two major classes
of nematodes are recognised: the Secernentea and the Adenophorea (Anderson and Anderson,
2000). However, the major parasites causing gastrointestinal disease in ruminants belong to
the class Secernentea order Strongylida (Sutherland and Scott, 2009). The order Strongylida
includes five superfamilies: the Diaphanocephaloidea, Ancylostomatoidea, Strongyloidea,
Trichostrongyloidea and Metastrongyloidea. The superfamily Trichostrongyloidea includes
the three most relevant species present in sheep in Ireland (Nematodirus battus, Teladorsagia
circumcincta and Trichostrongylus spp. (Fig. 1.4). The Strongylidae are characterized by the
presence of a copulatory bursa in the male and are thus called bursate nematodes (Anderson,
2000).
Figure 1.4 Phylogenetic tree of the major sheep gastrointestinal nematodes in Ireland (green
boxes).
Nematoda
Adenophorea
Enoplida
Trichuris
Trichinella
Secernentea
Strongylida
Trichostrongyloidea
Teladorsagia
circumcincta
Trichostrongylus spp.
Nematodirus spp.
Stroingyloidea
Ancylostomatoidea
Diaphanocephaloidea
Metastrongyloidea
Ascaridida
Oxyurida
Rhabditida
Spiruda
PHYLA CLASSES ORDERS SUPERFAMILIES SPECIES

7
1.4.1 Epidemiology of GINs in Ireland and in cool temperate climates
In Ireland the main parasites infecting young animals in spring and later on in the season are
internal parasites (round worms and coccidia). Until 3 weeks of age lambs are protected by
passive immunity provided by antibodies in the colostrum. From 3 to 10 weeks of age
Nematodirus battus and Eimeria are the predominant species and later in the season the most
important parasites affecting lambs are Teladorsagia circumcincta and Trichostrongylus spp
(Table 1.2) (Hynes, 2012).
Table 1.2 GIN infection and correlated symptoms of lambs in Ireland from 3 weeks of age.
MONTHS MARCH APRIL/MAY JUNE ONWARDS LATE SUMMER/AUTUMN
LAMBS
AGE
0-3 weeks 3 -8 weeks 5-10 weeks 10 weeks onwards
PARASITE
Lambs are
not heavily
infected due
to the
passive
immunity
provided by
the
colostrum
Eimeria Nematodirus battus
Teladorsagia
circumcincta
Trichostrongylus
spp
SYMPTOMS
 Acute
diarrhea
 Blood in
scour
(maybe)
 Poor thrift
 Diarrhea
 Wasting
 Dehydration
 Mortality
 Diarrhea
 Weight loss
 Dehydration

8
While adult sheep become immune competent to GIN, a temporary decline in resistance is
recognised in periparturient ewes (Taylor, 1935). This transitory phenomenon known as
periparturient relaxation of immunity (PPR) enables arrested parasitic larvae to resume their
development in the host and/or allow ingested L3 to establish (Beasley et al. 2010). Therefore,
PPR and the consequent increase in worm burden and faecal egg excretion plays an important
role in the epidemiology of GIN infections as it represents a major source of infection for
naïve-offspring (Beasley et al.2010). Various hypothesis for the occurrence and magnitude of
PPR have been postulated (Gruner et al. 1992; Houdijk, 2008). However, the reason of the
periparturient breakdown in resistance is unclear but was summarised by Barger (1993) as
being variously attributed to, lack of antigenic stimulation, hormonal suppression and poor
nutrition. Several studies have been confirmed that nutrition plays an important role in the
modulation of the magnitude of PPR (Bown et al. 1991; Donaldson et al. 1998; Houdijk et al.
2001; Sakkas et al. 2009). The PPR was associated with a waning of resistance during the
winter months due to an absence of antigenic stimulation (Soulsby, 1957). This now seems
unlikely as larvae, particularly Teladorsagia circumcincta, have been reported to remain
present on pasture, in significant numbers, throughout the winter (Waller et al. 2004a).
The role of the lactogenic hormone, prolactin, has been the subject of much research attention
due mainly to its high concentration at times of PPR. In a study which looked specifically at
the role of prolactin levels and their relationship with the PPR, Coop et al. (1990) found that
the rise in egg output and therefore the presumed relaxation of resistance occurred prior to the
peak of prolactin secretion. These authors observed absence of the relaxation in immunity to
T. circumcincta in barren ewes with an induced milk production through artificially raised
plasma prolactin.
Numerous studies have suggested that the resistance of sheep to GIN can be influenced by
differences between the requirement for, and supply of, metabolizable protein (MP) (Bown et
al. 1991; Donaldson et al. 1998; Houdijk et al. 2001; Sakkas et al. 2009). The MP requirement
increases by 35% in twin bearing ewes during late pregnancy, due to foetal growth and
mammary development (reproductive efforts) (Robinson et al. 1977) (Figure 1.5).
Gastrointestinal nematodes affect the availability of protein to the host through reductions in
feed intake and/or reductions in the efficiency of absorption of nutrients (Coop and
Kyriazakis, 1999; Zaralis et al. 2009). In addition, protein losses due to leakage of plasma
protein and increases of mucoprotein production should be considered (Stear et al. 2009).

9
Hence, nematode egg output might be reduced by MP supplementation at a time of higher
requirement of nutrients (Donaldson et al. 1998; Coop and Kyriazakis, 1999; Kahn et al.
2003a; Houdijk et al. 2005). Conversely, even though MP supplementation per se reduces the
magnitude of PPR, the evaluation of protein quality is an important factor to be considered in
GIN control (Donaldson et al. 2001; Houdijk, 2012).
Figure 1. 5 Effect of protein deficiency in periparturient relaxation of immunity (Coop et
al.1999).

10
14.2 GIN life cycle
Adult strongylid nematodes exist as females and males; the females produce different
numbers (depending on the species) of typically ovoid, strongylid eggs (70–150 μm), which
are excreted in the faeces into the external environment. In T. circumcincta and
Trichostrongylus spp. the first-stage larva (L1) develops inside the egg then hatches (within
1–2 days, depending on environmental conditions) and develops through to the second-stage
larva (L2). Both the L1 and L2 feed on bacteria and other microorganisms in the external
environment (faeces). After moulting, the ensheathed third-stage larva (L3) develops (usually
within 1–2 weeks, depending on species, temperature, humidity, pH and/or other factors) (Fig
1.7). However, a significant difference has been displayed in N. battus, in which the
development from L1 to L3 occurs within the eggs and the hatching of eggs needs a period of
chilling (winter) to trigger hatching (Fig. 1.8).
The surface of nematodes is covered by a collagen rich cuticle secreted by the underlying
epidermis (hypodermis). The composition of this cuticle is variable according to the life-cycle
stage. For example, the free-living stage L3 displays a specific epicuticular glycan (CarLa)
(Harrison et al. 2003) that it is not present in L4 and in the adult stages. Interestingly, this
glycan has been used for CarLa test, a new tool to identify GIN resistance in sheep (Shaw et
al. 2012) (for more details see chapter 6).The cuticular sheath around the L3 prevents it from
feeding but protects it from relatively harsh environmental conditions. After the L3 is ingested
by the animal and passes through the stomach(s), it exsheaths and then it develops through to
the fourth-stage larva (L4) and subsequently the adult at the predilection site in the alimentary
tract. The time from the L3 to the production of eggs by the adult female is usually 3–4 weeks
(Roeber et al. 2013).

11
1.4.3 Nematodirus battus and pathological effect on the host
Nematodirus battus, a parasite of the small intestine, is one of the most pathogenic organisms
in sheep in cool temperate climates (Denwood et al. 2008). It was first isolated in the UK
(Crofton and Thomas, 1951). Nevertheless, over the years, N. battus has been subsequently
isolated in Norway (Helle, 1969), Netherlands (Borgsteede et al. 1978), in the USA (Hoberg
et al. 1986), Germany (Bauer, 1989), Denmark (Thamsborg et al. 1996), Poland (Fudalewicz-
Niemczyk et al. 1996), Canada (Lichtenfels et al. 1997) and Sweden (Lindqvist et al. 2000).
This nematode presents a different life cycle compared to the other GIN; larvae develop to the
infective third stage within the egg and these eggs only hatch when the temperature exceeds
10°C following a cold spell. N. battus outbreaks occur usually in late spring in those years
with suitable weather (Fig 1.6). However, more recently, warmer and more variable weather
suggests that outbreaks could also occur in late autumn. Infected lambs develop acute enteritis
with watery diarrhoea accompanied by inappetence and weight loss (Denwood et al. 2008). N.
battus presents a very interesting feature, it induces a rapid and protective immune response in
infected young lambs (< 3months of age) which is not observed in other nematode infections
where the immunity develops over 10- 12 months of age (Winter, 2002). Therefore, an
alternative approach to identify resistant animals might be based on the evaluation of intensity
and time of occurrence in developing immune response against N. battus within 10 weeks of
age with the CarLA test (before appearance of T. circumcincta and Trichostrongylus spp
infections). Hence, once resistant animals are identified (through the saliva IgA level against
CarLA) selective treatment and genetic selection can be implemented in the flock.

12
Figure 1.6 Schematic diagram of Nematodirus battus life cycle.

13
1.4.4 Teladorsagia circumcincta and its pathological effect on the host
Teladorsagia circumcincta a predominant abomasal parasite in sheep in cool temperate
climate such as Scotland and Ireland from summer to autumn (Good et al. 2006; Stear et al.
2009). It causes hyperplastic gastritis which might lead to protein deficiency (Scott et al.
2000). During T. circumcincta infection important changes occur in the normal architecture
of the fundus of the abomasal mucosa. The numbers of chief and parietal cells are generally
reduced and the number of cells with mucous phenotype is increased. Moreover, the epithelial
barrier is breached as the tight junctions of the epithelial cells are destroyed. These changes
result in increased mucus production, abomasal fluid pH, gastrin and pepsinogen plasma
concentration and a concurrent decrease in the amount of albumin and fructosamine (Stear et
al. 2009). The destruction of the tight junctions allows epithelial growth factor, produced in
the salivary gland, to bind to its receptors on the inner surface of the epithelial cells (Playford
et al. 1995). This initiates increased mucus production, decreased acid production, increased
cell division and migration. The pH rise inhibits the autocatalytic conversion of pepsinogen to
pepsin and causes pepsinogenaemia. The decreased acid production leads to
hypergastrinaemia which might cause inappetance (Fox et al.1989; Bado et al. 1998).
However, causes of inappetance in sheep are still unknown (Zaralis et al. 2008). The loss of
proteins, following the damage of the mucosa, is responsible for the decrease of circulating
albumin and fructosamine (Stear et al. 2001).

14
Figure 1.7 Schematic diagram of Teladorsagia circumcincta life cycle.

15
1.5 Nematode control strategies
1.5.1 Anthelmintic and anthelmintic resistance: potential tools that can prolong the life
span of available drugs
Anthelmintics are drugs used to remove existing burdens or to prevent establishment of
ingested L3. Treatment of gastrointestinal helminthiasis mainly involves commercially
available anthelmintics such as: benzimidazoles and probenzimidazoles (albendazole,
fenbendazole, oxfendazole, mebendazole); nicotinic agonist (such as levamisole);
macrocyclic lactones (including abamectin, doramectin, ivermectin, and moxidectin);
aminoacetonitrile derivate groups (monepantel) (Good, 2012); spiroidoles (derquantel) (Little
et al. 2010). However, since the first suspicion of resistance to phenothiazine (Drudge et al.
1958), anthelmintic resistance became an evident threat in sheep production (Sargison et al.
2007, Papadopoulos et al. 2012).
Anthelmintic resistance (AR) is considered as the ability of a worm population to survive
anthelmintic doses which would be lethal for susceptible populations (Torres-Acosta and
Hoste, 2008). Cases of anthelmintic resistance have been reported for benzimidazole or
levamisole. Moreover, increased number of occurrences of resistance to macrocyclic lactones
(especially ivermectin) and monepantel (Bartley et al. 2015; Van den Brom et al. 2015); has
been noticed among GIN population. The most common genera, which have been displayed
AR so far are T. circumcincta, H. contortus, and Trichostrongylus spp (Papadopoulos et al.
2012).
At present, different classes of anthelmintics are available for farm animals. Nevertheless,
since the introduction of ivermectin in 1981, no new anthelmintics were developed and
introduced for use in livestock until the recent launch of monepantel (Kaminsky et al. 2008)
and derquantel combined with abamectin in New Zealand (Little et al. 2010). Novel
anthelmintics progress for farm animals might be a theoretical solution to contain AR,
conversely the extreme cost associated with the development of new drugs make this
hypothesis extremely unlikely (Kaplan and Vidyashankar, 2012). Therefore, an accurate use
of the available anthelmintics has to be followed in order to contain the development of
resistance and prolong their lifespan. Several measures have been suggested to contain and
slow the development of anthelmintic resistance such as : (a) administration of proper dose of

16
AH; (b) usage of AH only when necessary; (c) usage of appropriate AH; (c) reducing
dependence to AH; (d) avoiding the introduction of resistance onto a farm by treating
purchased stock on arrival followed by a quarantine period; (e) maintaining anthelmintic
susceptibility in worms population through targeted selective treatment; (f) testing for
anthelmintic resistance (Good et al. 2012); (g) employing of combination of different classes
of anthelmintics (Leathwick, 2012).
Two key methods to contain development of anthelmintic resistance are the prevention of the
introduction of resistant nematodes onto farms and slow down the development of
anthelmintic resistance in flocks (Kenyon et al. 2009). One of these approaches proposed to
prolong the lifespan of available anthelmintics it is to maintain a population of parasites in
refugia (unexposed to drug) which will preserve the genes for susceptibility within parasite
population (Martin et al. 1981; Van Wyk, 2001; Soulsby, 2007). The term “refugia”, in
according to the parasitological community, refers to those subpopulations from within either
the infrapopulation (the parasitic stages of host) or suprapopulation (free-living stages on
pasture which are not exposed to anthelmintic treatment (Van Wyk et al. 2002). The obvious
major pre requisite in the refugia-based method to contain onset of resistance is to allow
parasites in refugia to complete their life cycle and pass on the susceptible alleles to the next
parasite generations. Hence, pre-parasitic stages derived from those worms in refugia can
dilute the resistant genotype on pasture and later on reduce the probability that resistant
worms might mate with other resistant adults (Van Wyk et al.2006). Strategies that involve
refugia-based approaches to contain anthelmintic resistance include targeted or strategically
timed whole flock treatments and targeted selective treatments (TST). TST strategies require
the ability to identify and then address the treatment to those animals within a flock that are
parasite susceptible (Laurenson et al. 2013). Hence, animals intended to be treated might be
identified using pathophysiological markers such anaemia and dag score, parasite-based
markers such as FEC and production indices such as milk production and live weight gain
(Bisset et al. 2001; Van Wyk and Bath, 2002; Riley and Van Wyk, 2009). Laurenson et al.
(2013) have compared various markers as determinant criteria for TST and concluded that
compared to live weight or weight gain, the faecal egg counts (FEC) led to higher levels of
flock performance for a given percentage of animals drenched. Conversely, FEC presents
some limits. For example, difference in the genetic growth attributes of the host population
(Stear et al. 1996a) sampling errors (Stear et al. 2009), variation in the fecundity of individual

17
GIN species (Stear et al. 1999b) are limitations in using FEC as a parameter identifying
resistance in the flock.
The combination of different classes of anthelmintics as solution to extent the useful life of
anthelmintics has been already proposed around 30 years ago (Smith, 1990; Barnes et al.
1995). However, based on this hypothesis, (Leathwick, 2012) designed a mathematical model
to investigate the effectiveness of a anthelmintic combination using one of the new drugs
such as monepantel and derquantel combined with a member of macrocyclic lactone (ML)
such as abamectin. In this study, it has been highlighted that in all simulations parasites
developed resistance to new actives more slowly when used in combination with other
anthelmintics. Therefore, the combination of different anthelmintics might be considered as a
potential tool to contain development of resistance to the new drugs and at the same time
exploiting old drugs that don’t have any effect whether administrated singularly.
1.6 Alternative solutions to anthelmintics
Scientists in the last decades have been investigating alternative solutions to overcome
anthelmintic resistance. These methods fall into 5 categories: grazing management, biological
control, nutrition, vaccination and genetic approaches (Stear et al. 2006; Sayers & Sweeney
2005).
1.6.1 Grazing management
Methods based on strategies of grazing management have been described since the end of the
1960s. Grazing management strategies can be classified as: (a) preventive, (b) evasive and (c)
diluting (Michel, 1985). Preventive strategies rely on putting worm-free animals onto a clean
pasture, or by suppressing worm egg output by anthelmintic treatment in the early part of the
grazing season until the initial population of infective larvae on pasture has declined to safe
levels. However, the practice of dosing and moving animals to clean pasture is actively
discouraged (Molento et al. 2004). Leathwick et al. (2008) suggested that animals should be
moved prior to drenching and treatment delayed until the desired levels of refugia (part of the
worm populations which is not exposed to anthelmintic treatments) have built up on the new
pasture to ensure that unselected parasites were transferred to clean pasture.

18
Evasive strategies do not attempt to limit contamination of the pasture but rely on movement
of livestock before the number of larvae becomes a significant source of infection.
Diluting strategies refer to concurrent grazing of susceptible animals with resistant animal
such as adults from the same species or with different livestock species in order to reduce the
herbage infestation. In temperate regions of the world considerable benefits have been
achieved in worm control for both sheep and cattle parasites by interchange grazing between
these two species of livestock (Waller, 2006). However, sheep/cattle combination in parasite
control has to be used with caution in the longer term. There is some evidence that parasites
primarily of cattle may show increased ability to infect sheep and cause clinical disease
(Barger, 1997).
The combination of anthelmintics and grazing management might be highly recommended as
effective solution to control GIN. Conversely, this combination in Australia and New Zealand
has proved to select anthelmintic resistance (Besier, 1999; Leathwick et al. 2008) due to the
fact that any parasites that survive anthelmintic treatment carry resistance genes.
1.6.2 Biological control
The use of nematode-predatory fungi such as Duddingtonia flagrans have shown the ability
to reduce the number of infective larvae on the pasture and consequently moderate the
intensity of infection (Waller et al. 2004b). This species (formerly Trichothecium flagrans)
belongs to the Deuteromycetes, members of the class Fungi Imperfecti, which are well known
as nematode destroying fungi (Cooke and Godfrey, 1964; Barron, 1977).
D. flagrans spores present the advantage of being able to pass through a gastrointestinal tract
without losing its predatory capabilities (Larsen et al. 1992, 1998; Gronvold et al. 1993a).
However, other potential fungi in association with D. flagrans might be used as alternative
solutions to anthelmintics. In Ireland, twenty-nine nematophagous fungi were observed, of
which 12 were predatory and 17 were endoparasitic. M. cionopaga, D.coniospora and H.
rhoissilensis aside from D. ﬂagrans were detected in fresh faecal samples indicating that they
may have survived the gastrointestinal tract and therefore they might be a viable option as a
biological control agent (Kelly et al. 2009). However, to obtain optimal results, daily diet
supplementations with fungal spore (Waller and Thamsborg, 2005) are necessary. Therefore,

19
a dependence upon daily administration of fungal spores represents the main disadvantage of
this strategy (Stear et al. 2006).
1.6.3 Role of nutrition on resistance to GINs
Several studies have shown that nutrition plays a fundamental role in gastrointestinal
nematodes control (Bown et al. 1991; Coop and Kyriazakis, 1999; Houdijk et al. 2001,
Sakkas et al. 2009, Sakkas et al. 2012; Torres-Acosta et al. 2012). The nutritional anti-
parasitic activity might be considered arising both from indirect and/or direct effects of
nutrients on parasites, in which the first modulates the host immune response (Houdijk et al.
2005) and the latter exploits anti-parasitic property of metabolites contained in some plants
(Werne et al. 2013).
Amongst nutritionists, it has been recognized that nutrient scarcity in reproducing and
growing animals promotes the establishment, survival and fecundity of gastrointestinal
nematodes due to penalization of immune functions (Houdijk et al.2001, Houdijk, 2012;
Sakkas et al. 2012). However, a question, on nutrient scarcity and its effect on the host
immunity has been addressed whether the resistance to GINs is sensitive to metabolizable
energy (ME) o metabolizable protein (MP) scarcity (Bown et al.1991, Sakkas et al.2009).
Convincing evidence from ruminant and monogastric studies have shown that host resistance
to GINs is sensitive to metabolizable protein (MP) scarcity and not to (moderate) metabolic
energy (ME) scarcity (Houdijk et al.2001, Bown et al.1991; Sakkas et al. 2009). Related to
this fact, two hypothesis have been postulated as to why immune response to GINs is not
sensitive to ME scarcity: (a) the energy required to mount an immune response is low and the
organism can access their own reserves, or (b) the immune system relies on protein, or
specific amino acids as a source of energy. The latter has been demonstrated by the fact that
alanine and glutamine, two non-essential amino acids are used as source of energy in
preference to glucose to many immune cells in-vitro (Newsholme et al. 2003).
Body functions, such as immune response, reproduction, lactation, growth, need nutrients.
Nutrients as constituent part of foods include carbohydrates (sugar, fibres, and starches)
nitrogen compounds (amino acids, non-protein nitrogen compounds); lipids (fatty acids,
glycerides) minerals and vitamins (Tab 1.3). The energy used for metabolism is called

20
metabolic energy (ME) and it is provided predominately by digestion of carbohydrates and
fats contained in foods and in the microbial fatty acids of microorganisms present in the
rumen. However, whilst, carbohydrates ingested by ruminants are fermented by
microorganisms and then absorbed as volatile fatty acid (VFA) (acetic acid, propionic acid
and butyric acid) in the rumen, the nitrogen fraction also known as crude protein (CP), is
absorbed in the intestine (McDonald, 2011). CP in relation to its degradability in the rumen is
divided into two types: the undegradable protein (UDP) and the effectively rumen degradable
protein (ERDP) (Tab 1.3). UDP once bypassed the rumen goes directly in the abomasum and
intestine where is digested and absorbed, whilst ERDP degraded in the rumen is font of
energy for ruminal microorganisms by its carbon chain and font of nitrogen that is reused to
synthetize microbial protein that will be secondly digested and absorbed by ruminants. All
protein absorbed resulting of the sum of ERDP and UDP is called metabolizable protein
(MP). The concept of metabolizable protein (MP) and quality (ratio between the two
constituents DUP/ERDP) as factor influencing the efficiency of immune response against
GIN will be analysed later on in the chapters 4 and 5 of this thesis.
Most of the current studies on the influence of protein supplementation on immune response
to gastrointestinal nematodes have been performed individually either in ewes (Houdijk et al.
2002; Houdijk et al. 2004; Zaralis et al. 2009; Rocha et al. 2011; Sakkas et al. 2012); or in
lambs after birth (Bown et al. 1991; Coop et al. 1995; Kahn et al. 2000; Strain and Stear 2001;
Greer et al. 2009). However, to date very little is reported in the literature on the influence
that quality and availability of protein during foetus growth which might have effect on the
development of immune response to gastrointestinal parasites later on in life. Based on this
hypothesis, an Australian study performed by Francoise et al (2012) showed that protein
supplementation during the first 100 days of pregnancy failed to enhance resistance of weaned
Merino lambs against H. contortus. In this study pregnant ewes were either fed with normal
protein diet (12% CP) or with high protein diet (21% CP) in their first 100 days of pregnancy,
a period in which fetal ovine thymus and spleen are supposed to be developing (Mackay et al.
1986; Jeong et al. 2001; Press et al. 1993).

21
However, protein supplemented had: (a) an identical ratio in digestible undegradable protein
(DUP) to effectively rumen degradable protein (ERDP); (b) poor quality, since cotton seed
and sunflower meal present 0.45 and 0.23 DUP/ERDP ratio respectively (Houdijk, 2011;
Keady, 2012) and moreover protein supplementation per se didn’t consider the amino acid
profile that might influence the efficiency of development of immune response (Houdjik
2011).
Table 1.3 Characterization of nutrients content in ruminant foods.
Food contents Rumen parameters Absorbed nutrients
Metabolizable
sources
NITROGEN/PROTEIN
NF (nitrogen
fraction)
UDN (undegradable nitrogen)
DUP AA (digestible
undegradable protein)
Metabolizable
protein (MP)
ERDN (effectively rumen
degradable nitrogen such as
urea) ERDP
Microbial amino acids
CARBOHYDRATES,LIPIDS
PFF (potentially
fermentable
fraction)
Starch, cell walls
Starch, cell walls
 VFA
 Microbial fatty acids
 Long chain fatty acid
 VFA
 Glucose
Metabolizable
energy (ME)
NFF (non-
fermentable fraction
 Volatile fatty acids (VFA)
 Lactate
 Lipid

22
Direct effect of nutrition on GIN has been taken into consideration. Plants or their extract
from the beginning have been used as phytotherapeutic in human and veterinary medicine
(Hoste H. et al. 2011). Even though, in medicine, preparations of plants and/or plant extracts
were usually administered as therapeutic for a short period when animals/humans were
infected a new approach has been considered in using plants in medicine as nutraceuticals
(Min et al. 2003; Hoste et al. 2006). A nutraceutical is any substance that may be considered
as a food or part of a food which provides health benefits, including the prevention and
treatment of disease (Andlauer and Furst, 2002). Most of the results of nutraceutical use in
parasitised ruminants were correlated with the consumption of tannin-rich (TR) legume
forages. Tannin is a secondary compound of plants characterized by free phenolic groups and
can be divided into two groups, hydrolysable and condensed (proanthcyanidins). However,
the latter is the most studied compound and the most common type of tannin found in forage
legumes (Reed, 1995). Their anthelmintic effect has been reported in sheep, goats and deer
(Hoste et al. 2006) and this is demonstrated by their ability to radically reduce nematode
burden (Niezen et al. 1998b). The variation of anthelmintic properties of plants has been
attributed to plant secondary metabolites (PSM). A dose-dependent relationship between the
anthelmintic efficacy and the concentration of plant extracts and/or PSMs (tannins or
flavonoids) has been widely confirmed by in-vitro assays (Barrau et al. 2005; Molan et al.
2003; Paolini et al. 2004). Studies in goats and sheep showed divergent anthelmintic effect
after administration of (TR) legume forages.
For instance, Paolini et al. (2003a, b) found that in goats fed with sainfon (Onobrychis
viciifolia) the reduction in larval establishment for T. circumcincta, T. colubriformis and H.
contortus was 70, 66 and 33% respectively, suggesting that the effect of this plant depends on
the species. Conversely, some authors suggested that the anatomical location of nematodes
might represent a major factor to consider since divergent effect were found between the
abomasal vs the intestinal species (Athanasiadau et al. 2001).

23
1.6.4 Vaccines
Although vaccination might be considered as an ideal alternative to anthelmintic treatments
the multitude of antigens present on the different larval stages and on the different genera
make this hypothesis far from viable in the future. An ideal vaccine has to provide an efficient
immune response towards a variety of different nematodes and larval stages, it has to be cost-
effective and it must not require repeated administration during the season (Knox, 2000; Knox
et al. 2003). Recently, vaccine adjuvants have received increased attention since they are the
main drivers of both the magnitude and type of adaptive response generated after vaccination
(De Veer and Meeusen, 2011, Pulendran and Ahmed, 2011). In two recent studies in sheep,
adjuvants such as DEAE-dextran and the recombinant part of the catalytic serine/threonine
phosphatase 2A (PP2Ar), showed their potentiality by stimulation of immune response
against H. contortus and T. circumcincta (Piedrafita et al. 2013; Fawzi et al. 2013).
Despite this, the common routes of vaccination are either intramuscular or subcutaneous; a
new approach through the mucosa has been considered. Mucosal administration offers more
advantages such as ease administration, reduction in adverse effect and moreover mucosa
represents the place where most pathogens invade the host tissue and where the immune
stimulation occurs. Mucosal administration such as intranasal immunization have been
investigated in mouse models (McGuire et al. 2002) and in pigs (Tsuji et al. 2004) and
recently a similar study has been performed in sheep. Fawzi et al. (2013) demonstrated that
the intranasal administration of a recombinant part of the catalytic serine/threonine
phosphatase 2A (PP2Ar) with E. coli walls can elicit a partially protective response against H.
contortus and T. circumcincta in lambs.
In addition, in a recent study of H. contortus L3s (free-living and activated third-stage larvae)
was observed that some proteins related to energy metabolism and parasite invasion were up-
regulated during L3 exsheathment. These proteins involved in these biological processes
might be interesting candidates for vaccine development or targets for drug discovery (Wang
et al. 2016).

24
1.6.5 Genetic selections (phenotypic and genetic markers)
Genetic selection for resistance and resilience to GIN have been considered as an alternative
solution to chemotherapy in several countries (Sayers et al. 2005; Keane et al. 2006; Torres-
Acosta and Hoste, 2008; Saddiqi et al. 2010; Hassan et al. 2011; Riggio et al. 2013; Periasamy
et al. 2014; Ahmed et al. 2015a). Selection for resistance has been traditionally based on the
quantitative measurement of phenotypic traits such as faecal egg count (FEC) (Nieuwoudt et
al. 2002; Gruner et al. 2004; Shaw et al. 2012; Venturina et al. 2013), a trait that presents
heritability for a single measurement in the range from 0.2 to 0.4, the same magnitude of
heritability presented in milk production in dairy cattle (Nicholas, 1987) and it is highly
correlated (r=0.61–0.91) with nematode burdens (Stear et al. 1995a). However, the
relationship between FEC and worm burden in lambs of 6-7 months of age predominantly
infected by T. circumcincta was found to be convex (Bishop and Stear, 2000); i.e. lambs with
higher number of adults produced fewer eggs compared to their counterparts with lower
number of adults where the FEC was higher. This fact demonstrated that FEC in T.
circumcincta is not an ideal indicator of resistance (Stear et al. 2009).
Besides faecal egg counts, other phenotypic markers may be measured such as packed red cell
volume (Gray et al. 1987) eosinophilia (Stear et al. 2002), parasite-specific IgA (Stear et al.
2004; Sayers et al. 2008; Hassan et al. 2011; Ahmed et al. 2015b), IgG serum levels (Sayers
et al. 2008; Hassan et al. 2011), gastrin, fructosamine and albumins plasma levels (Stear et al.
2009). Conversely, even if, phenotypical markers might appear as potential tools to identify
resistant animals, sometimes they might not be entirely reliable. For instance, parasite-specific
IgA and eosinophilia indicate resistance in older animals (Doligaska et al. 1999), packed red
cells is a useful marker for blood sucking nematodes such as H. contortus, pepsinogen is
suitable for abomasal nematodes such as T. circumcincta (Stear et al. 2009; Ahmed et al.
2015b). It has been assessed that animals with ‘high levels’ of anti-CarLA (carbohydrate
larval surface antigen present on the infective-stage larvae L3 of all trichostrongylid
nematodes) IgA saliva presented 20-30% lower FEC, improved growth rate post weaning, and
no tendency for increased breech-soiling (Shaw et al. 2012). Hence, this measurement might
offer a practical, rapid and easy method for identify resistant animals in the flock.

25
Phenotypic markers such as FEC present more limitations compared to genetic markers. In
fact, phenotypic markers are only expressed after infection, whereas genetic markers don’t
require infection to be detected and they are able to identify resistance in animals of any age
(Beh and Maddox, 1996). Additionally, phenotypic records are labour-intensive and farmers
are reluctant to record these traits.
Many of the economically important traits in sheep such as disease resistance, growth rate,
body composition and wool characteristics are the result of multigene expression (Crawford et
al. 1995). The identification of individual genes linked to resistance to parasites is
problematic. Therefore, broader areas of the genome called quantitative trait loci (QTLs) have
been identified (Sutherland and Scott, 2010). QTLs, stretches of DNA containing or linked to
the genes connected with resistance to sheep strongyles, have been identified on sheep
chromosome 3 (OAR 3) (Periasamy et al. 2014) in loci on OAR4, OAR6 (Matika, 2011;
Beraldi et al. 2011) and OAR20 (Dukkipati et al. 2006b). Moreover, novel QTLs associated
with resistance to H. contortus and T. colubriformis have been recognized within OAR6,
OAR 14 and OAR22 in Africa red maasai sheep (Silva et al. 2012). Recent studies conducted
in sheep populations from three countries (France, Italy, Scotland) identified a large number
of genomic regions and single nucleotide polymorphism (SNP) markers associated with
nematode resistance (Sallé et al. 2012; Riggio et al. 2013). In one of these two studies an
extensive phenotyping was achieved through measurement of FEC, packed cell volume,
worm burden, worm female length, abomasal pH and serum /mucosal specific IgG in 1.275
Romane x Martinik Black Belly backcross lambs after infection with H. contortus. The QTLs
regions identified were on chromosome (OAR) 5, 12, 13 and 21. A locus of OAR 21 (PGA5)
was associated to pepsinogen concentration and a 10-Mbp region affecting FEC was found on
OAR12 (Sallé et al. 2012).
A number of genes have been linked with the ability of sheep to resist infection to GIN. The
three most studied molecular markers linked to FEC are genes of the MHC II (molecular
histocompatibility complex) (Schwaiger et al. 1995; Sayers et al. 2005; Valilou et al. 2015),
IFNG (IFNγ) (Coltman et al. 2001; Sayers et al. 2005b) and IL-4 (Benavides et al. 2009).
The most studied gene included in the MHC complex is DRB1 (Schwaiger et al. 1995; Sayers
et al. 2005; Hassan et al. 2011; Atlija et al. 2015). Conversely, the highly polymorphic nature
of this region which has over 80 alleles makes it unlikely that these genes will be suitable
markers for GIN resistance (Sayers and Sweeney, 2005).

26
Comparison between transcriptome analyses with genome-wide association studies in
identifying QTLs affecting host resistance represent a promising molecular technology to
identify mechanisms involved in immune response against GIN in ruminants (Li et al. 2012).
The transcriptome is a complete set of transcripts in a cell, and their quantity, for a specific
developmental stage or physiological condition (Wang et al. 2008). Several technologies have
been developed to determine the transcriptome such as microarrays, sequencing and tag-based
sequencing. However, a new molecular biology tool identified as RNAseq, has shown more
precise measurement of levels of transcripts and their isoforms than the aforementioned
methods (Wang et al. 2008).
A recent study has determined the importance of the whole transcriptome study applied in
sheep (Ahmed et al. 2015a). Firstly, the entire transcriptome of lymph node tissue of Suffolk
and Texel breed was profiled and then differentially expressed genes, pathways and networks
characterizing resistance and susceptibility were determined. The four most significant
differentially expressed pathways were all related to “antiviral and antibacterial immunity”.
Noteworthy was the fact that all these pathways, before infection, were more expressed in
Suffolk breed compared to Texel breed suggesting that Suffolk presents a more active
immune response to virus and bacteria. Moreover, Texel (resistant breed) presented balanced
Th1/Th2/Treg gene expression after 7 days post infection onwards compared to Suffolk
(susceptible breed) where response was unbalanced after 7 days post infection. The
importance of analysing all genome expression over the time represents an innovative
approach to identify the differential genes expression and the mutual interactions that confer
resistance or susceptibility to gastrointestinal nematodes.

27
1.7 Immune response to GIN infection
The immune response is divided into innate and adaptive immunity. Whilst the first is
aspecific and provides an early line of defence, the adaptive immunity is specific and
develops within several days. The innate immunity refers to various defences associated with
initial infection of the gastrointestinal tract. These include epithelial wall and mucus,
inflammatory responses, gut motility and phagocytosis. The acquired immunity involves the
ability of the host to recognise specific invasive organisms (including parasites) and to act
selectively to eliminate these. This encompasses both humoral (antibody production) and
cellular (specific cell production) immunity and lymphocytes are the main cells involved in
this response (Abbas et al. 2012). The function of the immune system in animals is to provide
defence against infections, in order to maximize fitness (Viney et al. 2005). Nonetheless, an
excessive and ineffectual activity of the immune system against parasitic infection can result
in damage to host tissues (immunopathology) (Graham et al. 2005).
1.7.1 Innate immunity
The innate immune response to GIN infections has received less attention than the acquired
immunity. However, the innate immunity has been recognized as a critical component in the
development of the adaptive response and as a driver of vaccine induced immunity (De Veer
et al. 2007). Epithelia play an important role in innate immunity by constituting a physical
barrier to invading microorganisms and by synthesizing an array of defence effector
molecules (Tjabringa et al. 2005). The surface of the gastrointestinal tract is covered with a
layer of mucus, mainly produced by epithelial goblet cells and comprised mostly of highly
glycosylated mucin molecules (Deplancke and Gaskins, 2001). Mucin covering the luminal
surface of the gastrointestinal tract provides defence as physical barrier to GIN establishment
(Shea-Donohue et al. 2015b) and promote worm expulsion (Hasnain et al. 2011). These
effector molecules present in the mucus, may regulate innate and adaptive immunity (Zanetti,
2004) and also promote wound healing (Aarbiou et al. 2003; Heilborn et al. 2003).

28
Lectins, for example are carbohydrate-binding-proteins released into mucus from epithelial
goblet cells in which it has been proposed that they can recognize and bind antigen on the
parasite surface and promote the expulsion by forming a bridge between the parasite and the
mucins (Sutherland and Scott, 2009). In recent years studies showed that a novel intelectin 2,
normally not expressed in no-challenged animals, is responsible of up regulation of IL-4,
sheep mast cell protease-1 (sMCP-1) and ovine galectin-14 (OvGal-14) in the abomasal
mucosa. Considering the involvement of IL-4 in Th2 response to nematode infection, it is
possible that this lectin plays an important role in the recognition of GINs (French et al.2007,
2008).
The initial sensing of infection or tissue damage is mediated by the interaction between
pathogen-associated molecular patterns or damaged associated molecular patterns
(PAMPs/DAMPs) with pattern recognition receptors (PRRs) (Mc Rae et al. 2015). These are
present on the plasma membrane and on the endosomal membranes of antigen presenting cells
(APCs) such as Toll-like receptors (TLRs), C-type lectin-like receptors, or in the cytoplasm
such as NOD-like receptors (NLRs) and RIG-like receptors (RLRS) receptors. With the
exception of some NLRs, the sensing of PAMPs or DAMPs by PRRs up regulate the
transcription of genes involved in inflammatory responses (Osamu & Shizuo, 2010).
However, in parasitic infections, it has been hypothesized that nematodes-associated
molecular patterns (NAMPs) such as glycan moieties, or escretory/secretory products might
be the factors eliciting the immune response (De Veer et al. 2007).
The nervous system, is also considered part of the innate immune response. In fact, nerve
fibres are particularly dense at intestinal and skin surfaces, where they trigger perceptions of
pain, touch and temperature; the first two are likely to be induced by the mechanical and
proteolytic actions of invading helminth parasites (De Veer et al. 2007). Most of the
neuropeptides are pro-inflammatory mediators involved in the recruitment and activation of
the innate immune response. One of the most studied neuropeptides is substance P, released
by peripheral nerves that mediate pain perception, increase the production of pro-
inflammatory cytokines and chemokines by leukocytes and can directly induce release of
vasoactive mediators from mast cells (Wang et al. 1995).

29
1.7.2 Adaptive immunity
The generation of the adaptive immune response occurs in the lymph nodes and requires
interactions between naïve lymphocytes and dendritic cells (DC) (von Andrian and Mempel,
2003; Lian and Luster, 2015). While dendritic cells coming from tissues are transported to
secondary lymph nodes through afferent lymphatic vessels, naïve B and T cells enter these
organs through the high endothelial venules (HEVs) via a multistep adhesion cascade
orchestrated by chemotactic mediators (Gowans and Knight, 1964; Girard et al. 2012). High
endothelial venules are blood vessels present in physiological condition only in lymphoid
organs in the paracortex (Gowans and Knight, 1964). Two families of G protein-coupled
receptors play essential roles in lymphocytes migration: chemokines receptors (CXCR5,
CCR7, and CXCR4) and sphingosine-1-phosphate receptor (S1PR1). The initiation of effector
T cells by cells interaction and cytokines environment have been widely investigated in the
immune response to GIN, however, there is lack of information on the mechanisms involved
lymphocytes trafficking. The evaluation of lymphocyte trafficking might represent a new
approach to advance our understanding on the immunological mechanisms associated with
resistance/susceptibility to GIN in sheep.
1.7.2.1 Dendritic cell: antigen presentation and regulation of HEVs phenotype
Immature DC, in the site of infection, engulf antigens and break them down into peptides that
are loaded onto molecular of histocompatibility complexes (MHC) (Cyster, 2005). After
exposure to inflammatory stimuli, DC undergo maturation, upregulate the chemokine receptor
CCR7 that allow DC to enter lymph vessels and to access the T-cell area of draining lymph
nodes under CCL19 and CCL21 chemotaxis (Dieu et al. 1998; Yanagihara et al. 1998;
Sallusto et al. 1998; Iwasaki and Medzhitov, 2015). While in transit, DC begin to generate
chemokines that allow to attract T cells in the lymph node (Sallusto et al. 1998).
.

30
1.7.2.2 Entry of immune cells through HEVs in the lymph nodes
Naïve B and T lymphocytes traffic to lymph nodes via HEVs is regulated through a multistep
adhesion cascade: rolling, sticking, crawling and transmigration (Fig 1.8). Lymphocytes
circulating in the blood tether and roll on HEV walls through the binding of L-selectin
(CD62L) to GlyCAM or MADCAM-1 (HEVs of mesenteric lymph nodes and Payer’s
patches) proteoglycans expressed on the surface of HEV endothelial cells. Subsequently,
rolling lymphocytes are activated by chemokines that are either produced by HEVs CCL21 or
transcytosed through HEVs and that are immobilized on the luminal surface. Signalling
through the G protein-coupled receptor CC-chemokine receptor 7 (CCR7), together with the
shear force of blood flow, induces conformational changes in the lymphocyte integrin
lymphocyte function-associated antigen 1 (LFA1), which mediates firm binding (sticking) to
intercellular adhesion molecule 1 (ICAM1) and ICAM2 on the endothelium. After arresting
the incoming leukocytes are able to breanch tight junctions between blood endothelial cells
and migrate deep into the lymph node parenchyma following gradient of CCR7 ligands
(CCL19, CCL21 and CXCL13) synthetized by resident dendritic and fibroblastic reticular
cells (von Andrian and Mempel, 2003; Girard et al. 2012).
After crossing the HEVs, lymphocytes migrate to different areas of the lymph node following
a network made by fibroblastic reticular cells (Fig. 1.10). Naïve T cells express CC-
chemokine receptor (CCR7), the receptor for CCL21, and CXC-chemokine receptor
(CXCR4), the receptor for for CXCL12, whilst naïve B cells (but not naïve T cells) express
CXCR5, the receptor for CXCL13, in addition to CCR7 and CXCR4 (Girard et al. 2012). T
cells migrate to T cells area in the lymph node paracortex, whereas B cells enter the B cell
follicles in the cortex. The migration of lymphocytes is driven by CCL19, CCL21 and
CXCL13 synthetized by dendritic cells and fibroblastic reticular cells (Girard et al. 2012).
However, if lymphocytes fail to recognize specific antigens within a few hours to days, they
return to the circulation through efferent lymph vessels and the thoracic duct (Gowans and
Knight, 1964). It has been estimated that B and T cells spend ‘exploring a mouse lymph node
around (24 hours) and (8-12 hours), respectively (Tomura et al. 2008). The egress of naïve
lymphocytes depends on the interaction between a bioactive lipid mediator called
sphingosine-1-phosphate and one of its complementary receptors S1PR1.

31
Figure 1.8 Entry of lymphocytes through HEVs-the multistep adhesion cascade (Girard et al.
2012).
1.7.2.3 Sphingosine-1-phosphate and its role in lymphocyte egress
The exit of lymphocytes from lymph nodes is dependent on sphingosine 1-phosphate receptor
(S1PR1) expression and on the S1P gradient between the lymphnodal parenchima and the
blood stream (Cyster, 2005). The concentration of S1P is regulated by three enzymes S1P-
kinase, phosphatase, and lyase (Schwab et al. 2005; Pham et al. 2010; Proia and Hla, 2015)
(Fig 1.9). S1P is present at relatively high concentrations in the blood and lymph compared
with tissues. This concentration gradient is maintained because an S1P-degrading enzyme,
S1P lyase, is ubiquitously present in tissues, so the tissue concentration of the lipid is less than
in the lymph and blood (Schwab et al. 2005). The understanding of the role of S1P and
S1PR1 in lymphocyte cell trafficking is based in large part on studies of the effects of a drug
called fingolimod (FTY720), which binds to S1P1R and causes its down-modulation from the
cell surface with consequent sequestration of lymphocytes in lymph nodes.

32
Figure 1.9 Sphingosine-1-phosphate’s metabolism.
The expression of S1PR1 is very low when naïve cells are circulating in the blood stream due
to the high concentration of S1P. However, once a naïve lymphocyte enter a lymph node, the
low S1P concentration gradually induce (it may take several hours) the re-expression of
S1PR1 allowing lymphocytes to respond to the S1P gradient and then egress the lymph node.
It should be considered that, the expression of S1PR1 is suppressed for several days after
antigen-mediated activation of naive T cells, and therefore the ability of the cells to leave the
lymphoid tissue in response to an S1P gradient is impaired.

33
Figure 1.10 The journey of lymphocytes into lymph nodes and their egress through the S1P
gradient (Girard et al. 2012).
1.7.2.4 Effector T cells and immune response to gastrointestinal nematodes
The cellular and humoral immune response to GIN are regulated by cytokine pathways
generated through antigen activated T cells (Patel et al. 2009). In the lymph nodes, naïve T
cells differentiate into distinct subset (Th1, Th2, Th17 and Treg) of effector cells in response
to antigens and the cytokines (Amsen et al. 2007).
Sheep infected with T.circumcincta have showed a predominant Th2 response characterized
of local eosinophilia, mastocytosis and increased mucus production (Craig, 2010). It is
already established that the ability to mount a Th2 response is associated with parasite control
whilst an up-regulation of Th1 genes is linked to susceptibility to GIN infections (Andronicos
et al. 2010). Nevertheless, a balancing of the immune response is essential to elude

34
uncontrolled Th2 activation that might be result in severe inflammatory reactions and
immunopathology (Mulcahy et al. 2004).
The maturation into the Th2 cells phenotype is promoted by IL-4 cytokine (Reynolds and
Maizels, 2012; Reynolds and Dong, 2013). This cytokine activates the transcription factors
(STAT) 6 and GATA3 which stimulates the differentiation of CD4+ T cells to the Th2 subset
(Amsen et al. 2009; Zhu et al. 2010). IL-4 produced by Th2 cells amplifies this response and
inhibits the development of Th1 and Th17 (Abul et al. 2012). Th2 cells drive B cells in IgE
and IgA synthesis as well as mast cells maturation and proliferation (Ansel et al. 2006). Two
additional cytokines are synthetized by Th2 cells: IL-5 and IL-13. The first stimulates the
maturation of eosinophils whilst the second, in concert with IL-4 stimulates IgE synthesis,
promotes tissue healing fibrosis and enhances worm expulsion by increased mucosal
permeability, mucus production and muscle contraction (Madden et al. 2002; Wynn, 2003;
Meeusen et al. 2005).
The differentiation into Th1 cells phenotype is promoted by IL-12. This cytokine activates
STAT4 and triggers the production of the Th1 transcription factors T-bet (TBX21) and HLX.
Th1 cells then synthesis interferon gamma (IFNγ) (Venturina et al. 2013) which has been
thought to be related to Th2 inhibition and IL-4 down regulation (Pulendran, 2004).
The activation of CD4+
T cells into Treg is triggered by IL-2 cytokine. The importance of
Treg activation is to prevent the pathological consequences of prolonged immune activation
(Belkaid & Tarbel, 2009). CD4+
Treg cells are identified by their receptors IL-2RA (CD25)
and the transcription factor FOXP3 (Hori et al. 2003; Fontenot et al.2005). TGFβ and IL-10
are the two cytokines involved in controlling T cell response (Ouyang et al. 2011).
A recent study characterized the immune response in Texel and Suffolk lambs challenged
with T. Circumcincta (Ahmed et al. 2015a). Texel lambs (resistant) showed a synchronized
Th1/Th2/Treg immune response compared to Suffolk lambs. This equilibrium in sheep
demonstrates the important role of it played in the GIN resistance, as previously showed in
murine and human models (Maizels and Yazdanbakhsh 2003; Belkaid Tarbel 2009).
Moreover, it has been hypothesized that the activation of Th17 T cell subset might be related
to the inability to control L3 larval colonization, adult worm infection and egg production
(Gossner et al. 2012 a, b).

35
Figure 1.11 Differential activation of CD4+ T cells and cytokines involved in the immune
response to T. circumcincta (Venturina et al. 2013).
1.7.3 Principal immunological mechanism controlling gastrointestinal nematodes
In sheep the immune response towards GIN develops by 10-12 months of age with some
variability amongst parasite species (Vlassoff et al. 2001). Parasitic diseases are the result of
interactions amongst the host, the environment and the parasite. Variation of one of these
variables can result in a modification in the course and outcome of the infection. There is a
general consensus on the idea that immune response requires metabolic resources. It has been
proposed that immune response to gastrointestinal parasite has 6 potential costs to the host.
Five phenotypic costs arise from: a) increased metabolic activity; b) reduced nutrient
availability due to anorexia; c) altered priorities for nutrient utilization; d) change in size and
turnover of pools of immune cells and properties; e) immunopathology from inappropriate or
excessive immune activation. A sixth cost is the genetic cost which originates from a change
in the capacity of offspring to express production and life-history traits following selection for
pasture resistance (Colditz, 2008).

36
Resistant sheep present one or more of the following features: fewer adult nematodes, more
inhibited larvae, shorter adult nematodes and decreased production of nematode eggs (Stear et
al. 2007a). In lambs at the end of the first grazing season, the heritability of adult worm length
is very strong whereas the heritability of egg production is moderate. The heritability of worm
number is low while there is no detectable genetic variation in the number of inhibited larvae
(Stear et al. 2007a). Quantitative immune genetic analysis suggests that there are two major
mechanisms of resistance: IgA-mediated response which controls worm growth and fecundity
and type I Hypersensitivity reaction (section 1.7.3.1) that regulate worm number. However,
suppression of worm growth develops before regulation of worm number (Venturina et al.
2013). The mechanism of IgA action is not completely clear, there are different theories. It
has been indicated that IgA antibodies can control larval colonization, development and egg
production (Stear et al. 2004; Lacroux et al. 2006; Halliday et al. 2007) by specific binding to
both larvae and adults or to nematode secretions. Other mechanisms of protection have been
identified including inactivation of metabolic enzymes (Gill et al. 1993) and feed-suppression
that would result to reduce adult worm length and fecundity (Stear et al. 2004; Craig et al.
2007). IgE antibodies play an important role in parasite expulsion as high levels of IgE are
negatively correlated with FEC (Murphy et al. 2010). However, IgE response is associated
unfavourably with live-weight gain and, therefore is not a suitable parameter to be considered
as measure of selection for resistance (Shaw et al. 1999). The major mechanism controlling
the number of adult T. circumcincta appears to be mast cells degranulation (Seaton DS et
al.1989; Stear et al.1995). The action of IgE is thought to be through a classical Type I
hypersensitive reaction mediated by mast cell proliferation (Stear et al. 1995; Miller 1996;
Greer et al. 2008).
1.7.3.1 Hypersensitivity reaction type I
Type I hypersensitivity reaction assumes an important meaning in the resistance to parasitic
infection. It has already been demonstrated that mast cells are involved in the control of adult
worm number in Teladorsagia circumcincta infection. (Stear et al, 2009). IgE, mast cells and
eosinophils play a fundamental role in allergic inflammation as well as in innate and adaptive
immunity (Stone et al.2010). It is documented that during a parasitic infection or during an
allergy, antigens are taken up and processed by antigen-presenting cells (APCs), subsequently

37
APCs present antigens in the context of MHCII to the CD4+ naïve cells which will
differentiate into TH2 cells in the satellite lymph nodes. TH2 cells activated stimulate IgE
isotype switching and mast cells activation (Vercelli, 2009).
1.7.3.1.2 Mast cells
Mast cells are large, round cells (15 to 20 µm in diameter) distributed throughout the body in
connective tissue. Their cytoplasm is packed with large granules that stain with dyes such as
Giemsa Stain and Toluidine blue. Mast cells are bone marrow-derived cells that contribute to
a variety of allergic and other immune responses (Bochner and Schleimer, 2001). They are
important effector cells in nematode infections. Conversely, their involvement in parasitic
expulsion is dependent upon the nematode species (De Veer et al. 2007). Mast cells contain
many granules filled with histamine, heparin and proteases and can secrete IL-4 and IL-5 as
well as leukotrienes and chemokines following activation (De Veer et al. 2007). Mucosa mast
cell activation is mediated by cross-linking between IgE antibodies and foreign
antigens/allergen (Schubert, 1997). It has been shown that the major mechanism controlling
the number of adult worm in sheep infected by T. Circumcincta is the mast cells
degranulation (Seaton et al. 1984, Stear et al. 1995).

38
Figure 1.12 Mast cell chemical mediators (lipid mediators, chemokines and granules content)
1.7.3.3 IgE
Level of IgE in the serum is the lowest of the 5 immunoglobulin subtypes. It has the shortest
half-life (approximately 2 days) and it shows no transplacental transfer in human (Smith et
al.,, 2009). IgE is secreted by B cells; isotype switching in general requires transcription
through switch regions upstream of the new constant region. Isotype switching requires two
signals. Signal 1 mediated by IL-4 and IL-13 cytokines and a signal 2 mediated by CD40
ligand (CD40L) on T cells acting through CD40 on B cells (Vercelli, 2009). There are two
receptors for IgE: the low affinity IgE receptor (Fcϵ RII; CD23) expressed on the surface of B
cells, as well as other hematopoietic cells, and the high affinity receptor (Fcϵ RI) expressed on
mast cells and basophils.

39
Figure 1.13 IgE receptors structure.
The α-chain of FcϵRI binds to the Fc portion (C3 domain) of IgE and consists of an
extracellular domain, a transmembrane domain, and a short cytoplasmic tail with no signaling
motifs. The β subunit consists of 4 transmembrane domains with a single immunoreceptor
tyrosine–based activation motif (ITAM) and is associated with Lyn kinase. The γ subunits
form a disulphide linked dimer, and each subunit contains an ITAM. After aggregation of
FceRI by multivalent antigen recognized by bound IgE, Lyn phosphorylates tyrosine residues
in the ITAMs of the β and γ subunits. The tyrosine-phosphorylated γ subunit then recruits Syk
kinase. Syk activates a number of downstream signaling events associated with mast cell or
basophil activation (Rivera et al. 2008, Gilfillan AM et al, 2009; Mulsant et al. 2001).

40
1.7.3.4 Eosinophils
Eosinophils are bone marrow-derived cells that contribute to a variety of allergic and other
immune responses. They present a cytoplasm containing granules that stain intensely with the
pink stain eosin. Two different types of granules are present: (a) small granules containing
arysulfatase, peroxidase, and acid phosphatase and, (b) large crystalloid granules with a core
made of major basic protein surrounded by a matrix containing eosinophil cationic protein,
eosinophil peroxidase, and eosinophil- derived neurotoxin (Rothenberg et al. 2006; Tizard,
2009). Eosinophils express Fc receptors for IgG, IgA, IgE, and are presumably able to
respond to cross-linking of these receptors by antigen binding the receptor-associated
antibodies (Stone et al. 2010).
Mast cell and Th2 cells produce IL-5 and chemokines known as eotaxin that stimulate the
release of eosinophils from the bone marrow. Eosinophils are then attracted to sites of cell
degranulation by molecules such as the eotaxin, histamine and its breakdown product
imidazolacetic acid, leukotriene B4, serotonin, and platlet activating factor (PAF) (Bochner
and Schleimer, 2001). Eosinophils can be primed for activation by a number of mediators
including IL-3, IL-5, GM-CSF, CC chemokines, and PAF. Eosinophils can be activated by
cross-linking of IgG or secretory IgA (most potent) (Stone et al. 2010). Eosinophils release
granule proteins that are toxic for parasite and may injure normal tissue.

41
1.8 Dissemination of information to farmers in order to
contain the development of anthelminthic resistance in sheep
Previous surveys evaluating strategies to control gastrointestinal nematodes (GIN) in sheep
concluded that most of sheep farmers in the UK were not adopting strategies designed to
control the development of resistance to anthelmintics in GIN (Coles, 1997; Sargison and
Scott, 2003). Hence, in order to reduce the development of AR, producers should be advised
on the correct strategies to be adopted to control GIN. However, the success is far from
simple due to a number of barriers most of which are associated with economic concerns, and
long term effectiveness (Woodgate and Love 2011; Moore et al. 2016). In a recent survey, a
questionnaire was distributed amongst English farmers to assess classes of anthelmintics type
of strategies used to control GIN and to determine the awareness of AR in sheep farming.
Conversely, even though awareness of AR was higher compared to previous surveys, a
disconnection between awareness and practice of nematode control was observed (Moore et
al. 2016). In fact, the highest preference of farmers using grazing management as a strategy to
control worms was the “dose and move strategy” well known to be a cause of increased rate
of AR (Sutherland et al. 2002; Moore et al. 2016).
The stages of an extension campaign typically include awareness, information seeking,
evaluation, trials and finally full adoption. Information to farmers on worm control can be
disseminated by several tools such as websites, farming magazines and interpersonal
communication. However, it should be considered that the level of literacy, age and
willingness to change are different in target people (Woodgate and Love, 2012). The
dissemination of information through the internet is often suggested a as a tool to encourage
practice change (Woodgate and Dook, 2002). In fact, farmers can receive monthly email
newsletter discussing worm issues and recommendations (Woodgate and Love, 2012). At
present several countries are providing on-line information through specific websites such as
WormBoss in Australia, WormWise in New Zealand EBLEX, NADIS, SCOPS in the UK and
Sheep Ireland in Ireland. In addition, newer technologies such as podcasts, webinars, weblogs
and wikis, widgets smartphone applications can represent new channels of communication for
new generations of farmers (Woodgate and Love 2012).

42
Producers can also be informed by using a variety of farming magazines and booklets such as
for example, Sheep farmer in the UK, Teagasc today’s farm, Technical Updates on Sheep
Production and Irish farmers Journals in Ireland. This tool represents a mean of
communication that can be addressed especially to the old generation of farmers reluctant to
the use of new technologies. Farmers can also be updated on worm control strategies by
interpersonal communications with vets, agricultural merchants and other farmers. The
interpersonal communication represents a direct method of information which does not
require particular efforts by farmers and therefore been easily adopted by different target
people. The proof that interpersonal communication is one of channels preferred by farmers
has been observed in a recent survey (Moore et al. 2016). In this survey farmers declared that
the first interlocutor would be the vet followed by agricultural merchants and other farmers
(Moore et al. 2016). In conclusion, better-informed farmers are recognized to make greater
use of information, advice and training, to participate more in government schemes and to be
more proactive in adjusting to change and planning for the future of the business (Gusson,
19998). Therefore, in order to reduce the development of AR in the GIN populations, farmers
should be periodically updated on worm control strategies; however the preferred channel to
be used must consider target people.

43
1.9 Thesis objectives
Even though breed differences in resistance/susceptibility to gastrointestinal nematodes in
sheep have been observed, the immunological mechanisms involved are still not well
understood. In addition, husbandry management, such as protein supplementation during the
last period of pregnancy might influence the impact on pasture contamination and affect the
development of immune response of lambs.
Based on these facts the objectives of this thesis were:
 To identify humoral and cellular markers associated with resistance/susceptibility to GIN
by comparison of the immune response between resistant and susceptible breeds and
evaluate the expression of a panel of genes associated with the innate and adaptive
immunity to GIN with qPCR and evaluate mechanisms involved in lymphocytes
trafficking.
 To evaluate the impact of maternal nutrition in late pregnancy on the magnitude of the
periparturient relaxation of immunity and subsequently on the development of
immunocompetence in their progeny to gastrointestinal nematodes.

44
Chapter 2
Breed differences in humoral and cellular responses to
experimental infection of lambs with the gastrointestinal
nematode Teladorsagia circumcincta
* A.M Ahmed, * R.S. Sebastiano, T. Sweeney, J.P Hanrahan, , A. Glynn, O.
Keane , A. Mukhopadhya, K. Thornton and B. Good
* Both authors have contributed equally to this work.
Received: 18 December 2013
Accepted: 11 December 2014
Published: 17 February 2015

45
2.1. Abstract
While Texel lambs have increased resistance to infection with the gastrointestinal
nematode Teladorsagia circumcincta compared to Suffolk lambs, the underlying
resistance mechanisms are still unknown. The aim of this study was to compare
parasitological, humoral and cellular responses of Texel and Suffolk lambs over time
following a single experimental infection with T. circumcincta. Gastrointestinal
nematode free (but not naïve) lambs received a single oral dose of 3×104
infective T.
circumcincta larvae. The variables examined included worm burden, mucosal and serum
IgA, abomasal mast cells and eosinophils, haematological parameters and plasma
pepsinogen. Texel lambs had significantly lower worm burden on day 14 and lower
plasma pepsinogen concentration from day 14 onwards than Suffolks, and their
response in mucosal IgA to infection occurred earlier. The results from the study
suggest that an earlier local IgA response in the Texel contributes to the resistant
characteristics of the breed, while the increased level of plasma pepsinogen in the
Suffolk lambs implies greater abomasal tissue damage arising from the nematode
infection.
Key words: Suffolk, Texel, Teladorsagia circumcincta, abomasal IgA, pepsinogen.

46
2.2 Introduction
Teladorsagia circumcincta is among the most important gastrointestinal nematode
(GIN) species affecting sheep production in temperate regions (Stear et al. 1998, Morgan
and Van Dijk, 2012). GIN infection has a negative effect on reproductive performance,
milk production, body weight, carcass quality and survival (Coop and Kyriazakis,
2001, Alberti et al. 2012). Extensive use of anthelmintics as a control strategy has
resulted in the evolution of anthelmintic resistance in various nematode species
(Papadopoulos et al. 2012). This together with consumer concern over drug residues
in animal products has promoted interest in the development of alternative methods of
GIN control, such as genetic selection for increased host resistance to GIN. A number of
studies have already identified established breeds that are relatively resistant to various
GIN species (Good et al. 2006, Amarante et al. 2004). In Ireland, the Texel breed is
more resistant to GIN infection than the Suffolk (Good et al. 2006). Identification of
physiological markers associated with resistance would facilitate the classification of
resistance status of individuals and thus contribute to the development of rapid reliable
markers for use in national sheep breeding programmes. Resistance to nematode
infection can be manifest as a combination of impaired larval establishment,
inhibition of larval development, reduced worm fecundity and/or accelerated worm
expulsion (Stear et al. 1999a, Li et al. 2012). Resistant animals may have more efficient
mechanisms for affecting some or all of these physiological processes. The only direct
method of identification of resistant animals is to measure worm burden; however, this is
not practical for use in breeding programmes, as it requires animals to be sacrificed
(Sayers and Sweeney, 2005). Faecal egg count (FEC) is positively correlated with worm
burden (Stear et al. 1995b) and has been proposed and used as a marker of resistance to
GIN (Cringoli et al. 2008). However, there are various limitations to FEC data including
variability due to host factors (age, gender, immune status, and stress), parasite
specific factors (variability in species composition, fecundity, developmental stage),
environmental factors (nutrition, climate), sampling accuracy and precision (Stear et al.
1996b, Villanúa et al. 2006).

47
It is hypothesised that physiological processes that determine host control of worm
burden will vary between resistant and susceptible animals following GIN challenge.
Previous data have suggested that resistance to GIN infection depends on the activation
of an effective Th2 immune response, which elicits a humoral immune response and
results in the recruitment of eosinophils and mast cells to the gastrointestinal
mucosa and the local production of IgA and IgE antibodies (Allen and Maizels, 2011).
Pepsinogen concentration in blood plasma reflects the extent of abomasal tissue
damage (Stear et al. 1999a) and is elevated in Suffolk lambs naturally infected
with GIN in comparison to Texel lambs (Sayers et al. 2008).
The objective of this study was to identify physiological markers in blood or abomasal
mucosa that that differ between Suffolk and Texel lambs following artificial challenge with
infective T. circumcincta larvae, and thus may be indicators of resistance/susceptibility to
T. circumcincta infection.
2.3 Materials and Methods:
2.3.1 Ethical approval
All procedures described in this study were conducted under experimental license from
the Irish Department of Health in accordance with the Cruelty to Animals Act 1876 and
the European Communities (Amendments of the Cruelty to Animals Act 1976)
Regulations, 2002 and 2005.
2.3.2 Animals
All lambs (32 Texel and 29 Suffolk) were sourced from the flock of purebred Suffolk
and Texel sheep maintained at Athenry Research Centre (Good et al. 2006). Lambs of
both the breeds were born indoors, from a synchronized mating programme, and then
all the lambs were moved to the same pasture for a 6-week period. Lambs were
weaned at about 6 weeks of age and moved indoors where they were maintained on a
concentrate-based diet with free access to water for the remainder of the experiment.
Upon housing, faecal sampling per rectum was attempted on all lambs, but sufficient
material was obtained from only 36 individuals (16 Texel and 20 Suffolk). All lambs
were then treated with ivermectin (Oramec, Merial Animal Health Limited) according

Final version Thesis 17_05_2016

Final version Thesis 17_05_2016

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