Parmi les nématodes gastro-intestinaux ayant un impact majeur sur la santé des ruminants Haemonchus contortus est l’espèce la plus pathogène. Actuellement, le contrôle des populations parasitaires repose sur les traitements anthelminthiques dont l’efficacité est limitée par l’émergence de parasites résistants. L'établissement de nouvelles stratégies de lutte (thérapeutiques ou vaccinales) repose actuellement sur l'identification de nouvelles cibles moléculaires. Ainsi, les gènes spécifiquement régulés lors de la phase précoce d’interaction du parasite H. contortus avec son hôte sont des cibles de choix. Afin d’identifier ces gènes, 4 banques d’H. contortus enrichies en ADNc spécifiquement exprimés au stade L4 (5 jours après infestation) ont été obtenues par la technique d’Hybridation Suppressive et Soustractive (SSH). Sur 400 clones criblés (dot-blot, RT-PCR), 51 clones d’intérêts ont été regroupés en 10 contigs. Les candidats possédant un peptide signal de sécrétion ont fait l’objet d’une étude plus approfondie. Les homologues de ces candidats ont été recherchés chez les deux autres principales espèces de strongles gastro-intestinaux (T. circumcincta et T. colubriformis) dans le but d’évaluer leur polymorphisme. La production et la purification des protéines recombinantes correspondant à ces candidats a également été réalisée afin de tester leur éventuel pouvoir immunogène. / Gastro-intestinal nematodes such as Haemonchus contortus have a major impact on health of small ruminants world-wide. The control of infections remains largely based on anthelminthic treatments, but spreading of resistance has reduced their efficiency. An attractive solution would be the development of anti-nematode vaccines. Genes expressed during the early parasitic stage of H. contortus constituted our main targets. We have developed an approach based on SSH (Suppressive Subtractive Hybridization) technique and generated 4 subtracted cDNA libraries of H. contortus enriched in cDNA specifically expressed during L4 stage (five days post infection). 400 clones were analyzed by dot-blotand 51 clones regrouped in 10 contigs. All contigs were validated by RT-PCR. Homologues of candidates possessing a signal peptide were searched in T. colubriformis and T. circumcincta to evaluate their polymorphism. Recombinant proteins of theses candidates were produced and purified in order to know if they have a good vaccine potential with cross protection against two major gastro-intestinal nematodes.

The studies submitted herein have contributed to our understanding of ruminant immunology, host-parasite interactions during ruminant infection with nematode parasites, and potential vaccine strategies to combat parasitic gastroenteritis (PGE). PGE of sheep and cattle, caused by T. circumcincta and O. ostertagia respectively, is a major problem for the global farming industry both in terms of productivity and animal welfare. To date control of these parasites has relied on the use of anthelmintic drugs however the emergence of widespread anthelmintic resistance is driving the search for alternative methods of control. As ruminants do acquire immunity in the field, vaccination is one such alternative under investigation. The first three papers contributing to this thesis used modern immunological tools alongside a locally developed surgical technique to revisit a model of nematode infection in sheep, investigating the composition and kinetics of the ovine local immune response to infection with Teladorsagia circumcincta via cannulation of the efferent gastric lymph duct. A protective local secondary immune response was observed in sheep which had previously experienced infection with T. circumcincta, but was absent from naive sheep. This immune response consisted initially of a rise in TE and BE cell activity peaking at 3 and 5 days post challenge respectively, followed by a secondary parasiteEspecific IgA response from 5 days post challenge which correlated with stunting of parasite growth. Significant parasite loss occurred by 2 days post challenge, prior to detection of the secondary immune response, suggesting critical early events in the host-parasite interaction and the potential importance of larval antigens in these interactions. No difference was observed in either the manifestations of immunity, or the magnitude and quality of the immune response, between adult sheep and lambs. The fourth and fifth papers describe vaccine trials carried out in bovine and ovine hosts using detergent soluble proteins derived from 4th larval stage Ostertagia ostertagi and Teladorsagia circumcincta respectively as antigens. Substantial reduction in total faecal egg output of up to 85% was observed in the calf trials, but not in the sheep trials which attained a maximum reduction of 29% in total faecal egg output. The sixth paper is a transcriptomic study carried out using the Roche 454 sequencing platform to investigate the immediate responses of Teladorsagia circumcincta upon encountering ovine host tissue of either immune or naive status. Following larval exsheathing and 4 hours of exposure to either immune or naive abomasal environments the transcript level of several genes was observed to differ. Genes which were most upregulated in response to encountering the immune environment included a peptidyl-glycine alpha-amidating mono-oxygenase homologue and a small heat shock protein. The studies described herein represent a body of work carried out using up-to-date tools and technologies. The first three papers confirmed the existence of critical early events in the host-parasite interaction, pointing to the potential use of larval antigens as vaccine candidates described in the trials in papers 4 and 5, and leading to the in-depth transcriptomic analysis described in paper 6. Papers 4 and 5 demonstrated that while Teladorsagia circumcincta and Ostertagia ostertagi have similar life cycles and host-site predilection, and both the ovine and bovine host can develop immunity to incoming parasitic larvae in the field, important differences may exist in either the proteome of the fourth stage larvae and/or the nature of the host response. Paper 6 revealed that changes in T. circumcincta transcript levels in response to ovine-host immune status can be detected early in the host-parasite interaction.

The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites to affect the farming of sheep and goats. T.circumcincta infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all currently available classes of anthelmintic drugs, it is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Infection of sheep with T.circumcincta triggers local changes in the abomasum characteristic of a T helper type-2 (Th2) driven immune response, including local eosinophilia, mastocytosis and increased mucus production, which leads to expulsion of the parasite. However, this protective immunity develops slowly during repeated exposure, wanes rapidly, and does not appear to be evident in young lambs. Vaccination to provoke early onset of protective immunity has therefore been suggested as an alternative means of control in the face of spreading anthelmintic resistance. Greater understanding of the development of immunity to T.circumcincta, and why this is delayed in lambs, would be useful in vaccine development. This thesis focuses on cytokine transcription profiling of the ovine abomasal mucosa and local lymphatic tissues. Changes in cytokine transcription over the course of a challenge infection with T.circumcincta were defined in helminth naïve sheep, and in previously infected sheep which have developed a degree of immunity during an eight week trickle infection, to clarify the mechanisms by which this immunity is orchestrated. This work demonstrated a clear Th2 cytokine response in the abomasal mucosa over the course of infection, which developed earlier and was more pronounced in the previously infected sheep; possibly owing to a population of polarised Th2-type cells built up during the previous infection. Suppression of Th1 cytokine transcription was also a prominent finding in the draining lymph node, which likewise occurred earlier in the previously infected sheep. Repetition of this experiment using younger lambs provided a possible explanation for the reduced resistance to T.circumcincta in this age group. While Th2 and proinflammatory cytokine responses in the abomasal mucosa demonstrated similar trends to those found in the older sheep, little suppression of Th1 cytokine transcription was observed in the draining lymph node. It is therefore suggested that the increased susceptibility of young lambs to T.circumcincta is not due to an inability to generate adequate Th2 responses, but an inability to suppress transcription of antagonistic Th1 cytokines.

Teladorsagia circumcincta is a parasitic nematode which is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The parasite has developed resistance to the major anthelmintic drug classes and this challenges its future control. Vaccination is a potential alternative control method since sheep are able to develop protective immunity against this parasite. Although potential vaccine candidates have been revealed, the increasing gene datasets suggest that vaccinetarget selection may be aided by screening methods such as RNAi. This is a reverse genetic mechanism that causes highly specific gene silencing which was initially described and applied to defining gene function in Caenorhabditis elegans. Nevertheless, its application was more difficult than anticipated in parasitic nematodes because of the inconsistency of the silencing effect. In the unsuccessful cases, did the dsRNA penetrate the parasite and activate the RNAi pathway? Thus far, there are no internal controls that indicate the activation of the pathway. Are the RNAi pathway genes constantly transcribed or are they ‘switched on’ in response to the dsRNA exposure? The initial aim of the study was to determine potential marker genes in the RNAi pathway that could indicate the activation of the pathway in C. elegans. After the exposure to dsRNA from two target genes, the transcript levels of three candidate marker genes (Ce-dcr-1, Ce-ego-1 and Ce-rsd-3) were examined and showed that exposure to dsRNA has no effect on the transcript levels of these genes making them inappropriate markers for the activation of the RNAi pathway. The two target-genes were Ce-cpr-4 and Ce-sod-4 which had been proven to be consistently susceptible and refractory to RNAi, respectively. Another aim of the project was to develop an RNAi platform in T. circumcincta for use as a screening method for potential vaccine candidates. The targets selected for the in vitro RNAi included: five members of the Activation-associated Secreted Proteins (ASPs); a Macrophage migration Inhibitory Factor-like (Tci-mif-1) and a Surface Associated Antigen gene (Tci-saa-1), all of which have been associated with vaccine-induced protective immunity. The selection of the ASPs was based on a bioinformatic and transcriptomic analysis of the ASPs in T. circumcincta. The results showed successful knock-down only for three out of five ASP targets after 1 hour of soaking in gene-specific double stranded RNA (dsRNA) which illustrates the inconsistency and the target specificity of RNAi in T. circumcincta which has been observed in the past with other parasitic nematodes. Inconsistencies were also observed within the successful ASP targets with the results not being reproducible after several successful experiments. Potential reasons for the inconsistencies were examined with the duration of larval storage being a critical factor. Larvae stored for a short or long period of time were susceptible and refractory to RNAi, respectively. Experiments were also conducted to investigate how the ASPs relate to extracellular microvesicles (EMVs). These vesicles are considered to play an important role in the intercellular communication between parasites and their hosts, and thus represent potentially useful vaccine and/or drug targets. Transmission electron microscopy (TEM) confirmed that EMVs are excreted / secreted by the parasite and the proteomic analysis revealed several types of proteins within the vesicles such as: ASPs, Actins, Metallopeptidases, and RAB proteins. A comparative analysis of EMVs, EMV-free ES (Excretory / Secretory) and total ES products showed that approximately 35% of the proteins found in the vesicles could also be identified in EMV-free ES and in total ES products, whilst the remaining 65% were present only in EMVs.

Control strategies against the parasitic nematode Teladorsagia circumcincta are problematic under current sheep management systems. Infection with the parasite, particularly in young lambs, results in significant production losses therefore sustainable worm control is being sought. It has been established that variation in resistance to T. circumcincta is under genetic control and the development of resistance is an acquired characteristic and has an immunological basis. This project investigated the immunological response to infection, of lambs with predicted resistance or susceptibility to T. circumcincta. Specifically, the study aimed to identify immune response-associated genes that were differentially-expressed in resistant and susceptible lambs and attempted to identify mutations in these genes. This study was part of a long term project that aims to identify genetic marker/s to aid in marker-assisted selection (MAS) for resistance to T. circumcincta. Real time reverse transcription-quantitative polymerase chain reaction (real time RTqPCR) was performed on abomasal mucosa and lymph nodes from 55 lambs used in a previous experiment. The lambs had been either trickle-infected with 2,300 infective larvae every two days over three months (infected resistant/susceptible, n=45) or sham-dosed (non-infected control, n=10). Lambs were ranked in relation to faecal egg count (FEC) and adult worm count (AWC) at post mortem; zero or low FEC (resistant) to high FEC (susceptible). Histopathology showed only mild pathological changes in the abomasal mucosa of resistant lambs but heavy lymphocytic inflammatory infiltration in the mucosa and submucosa of infected susceptible animals. Measurements of a range of cytokine transcripts and cell markers associated with the four major CD4+ T cell subsets identified IL6, IL21, and IL23A as significantly increased by at least two-fold in abomasal lymph nodes and abomasal mucosa of susceptible lambs in comparison to resistant animals. Highly significant (P<0.02) positive correlations were found between IL6 (ρ=0.35), IL21 (ρ=0.54) and IL23A (ρ=0.38) transcript levels and AWC. Similarly, there were highly significant (P<0.01) positive correlations between FEC and IL6 (ρ=0.41), IL21 (ρ=0.65) and IL23A (ρ=0.31). In contrast, significant negative correlation (P<0.04) between IL23A with IgA antibody levels (ρ=-0.31) was found. There was also a significant positive correlation (P<0.03) of TGFB1 levels with AWC (ρ=0.42) and FEC (ρ=0.32) in the abomasal mucosa. These data suggests that susceptibility to T. circumcincta is linked to the activation of the inflammatory TH17 T cell subset and that this chronic inflammatory response was inappropriate to clear worm infection. High resolution melt analysis failed to identify single nucleotide polymorphisms in the coding regions of IL21 and IL21R. This is the first report of the involvement of TH17 response in GI worm infection in sheep. Similar gene expression studies involving the known upstream and downstream players of the TH17 response could be done.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

Parasitic nematodes of grazing livestock represent an increasing economic and welfare problem for British agriculture. By investigating specific life-cycle stages of these parasites, it may be possible to identify key molecules or pathways that are required for the survival of the worms, and thus exploit these for future control strategies. It has been shown previously that the third larval stages (L3) of the ovine parasitic nematode Teladorsagia circumcincta produce high levels of transcript for the enzyme GTP-Cyclohydrolase relative to later developmental stages. As the ratelimiting factor in the production of tetrahydrobiopterin, GTP-Cyclohydrolase is required for a number of different biochemical pathways, including those involved in the production of serotonin and melanin. As the L3 do not feed, it can be hypothesised that, if finite resources are being used in the production of transcript encoding this enzyme, then it may be important for survival. In this thesis, a number of approaches were taken to explore the function of GTPCyclohydrolase in the life-cycle development of T. circumcincta. The closely related parasite, Dictyocaulus viviparus, was used as a model organism to explore the role of GTP-Cyclohydrolase and serotonin production with regards to larval arrest, or hypobiosis. This process occurs readily under experimental conditions in D. viviparus, which is not possible with T. circumcincta. Quantitative PCR was used to examine GTP-Cyclohydrolase transcript levels in two different strains of D. viviparus, one that enters larval arrest when exposed to cold conditions and one that does not. No differences were observed between the two strains suggesting that GTP-Cyclohydrolase was unlikely to be involved in hypobiosis. The model nematode, Caenorhabditis elegans, was used to perform functional complementation experiments to assess the role of GTP-Cyclohydrolase in the cuticle, as it has been shown previously that C. elegans GTP-Cyclohydrolase mutants have a ‘leaky cuticle’ and are killed by lower doses of anthelmintics and bleach than the wild-type worms. The T. circumcincta gene for GTP-Cyclohydrolase was able to restore cuticular integrity of C. elegans GTP-Cyclohydrolase-deletion mutants, suggesting that the role played by the protein in both species is similar. In vitro inhibition experiments using a chemical inhibitor of GTP-Cyclohydrolase showed that T. circumcincta larval development was disrupted in the presence of the inhibitor. It was also shown that T. circumcincta L3 that were exposed to sunlight produced melanin, suggesting that the levels of GTP-Cyclohydrolase observed in the preparasitic stages of T. circumcincta may be required for the synthesis of melanin. Together, these data suggest that GTP-Cyclohydrolase is required by the preparasitic stages to survive on pasture. Ultraviolet radiation has been shown previously to be harmful to T. circumcincta L3, so if the melanin production provides protection from this, then it would be crucial for the survival of the pre-parasitic stages.

Teladorsagia circumcincta, an economically-important abomasal nematode of small ruminants in temperate regions worldwide, is currently controlled with a combination of anthelmintics and pasture management. Anthelmintic resistance has emerged and vaccination is a potential alternative control strategy, as protective immunity in sheep can be acquired after repeated exposure to the parasite. Abomasal mucosal IgA responses in immune sheep have been correlated with delayed worm development and reduced faecal egg counts. However, recombinant vaccine development against parasitic nematodes has had limited success, and one of the reasons may be unsuitable expression systems for antigen production leading to incomplete or inadequate post-translational modifications such as glycosylation and tertiary protein folding, resulting in incorrect epitope structures for antibody binding. In this thesis, to address this issue, “native” infective larval (L3) antigen targets of protective immune responses and synthetic peptide sequences which mimic structural epitopes on these antigens were identified. Abomasal mucosal IgA was used as a probe to identify native immunogenic antigens from T. circumcincta L3. IgA was purified from abomasal mucus of animals rendered immune by repeated experimental infection and a custom antibody-affinity column was created and used to purify antigens from an L3 somatic PBS-soluble extract. Affinity purified L3-antigen-specific IgA levels in sheep with varying levels of immunity to T. circumcincta were positively correlated (rs = 0.853, P < 0.001) with both the total IgA concentration in efferent gastric lymph after parasite challenge, and with the percentage of inhibited fourth-stage (L4) larvae present in the gastric glands of the immune hosts (rs = 0.534, P = 0.007). In contrast, a negative correlation between the levels of affinity-purified L3 antigen-specific IgA and total T. circumcincta burden was observed (rs = -0.565, P = 0.004). Proteomic analysis of the IgA-affinity purified L3 extract identified a number of proteins which represent potential vaccine candidate molecules in other helminth species, including paramyosin, superoxide dismutase, galectin, activation-associated secreted proteins and fatty-acid retinol-binding proteins. As a first step towards the development of a novel vaccine based on IgA-binding peptide mimics of native structural epitopes, phage display libraries were used to screen antibodies, from sheep rendered immune to T. circumcincta by experimental infection. These antibodies were affinity-purified before use and specifically bound T. circumcincta L3 glycans or, alternatively, surface antigens on exsheathed T. circumcincta L3. Five peptide sequences which mimic L3 antigenic epitopes were identified and positive correlations existed between peptide-specific IgA levels and both the total IgA concentration in efferent gastric lymph after parasite challenge and the percentage of inhibited L4 present (rs > 0.621, P < 0.001 to P < 0.05). In contrast, negative correlations between the levels of peptide-specific IgA and the total nematode burden were observed (rs > -0.528, P < 0.01 to P < 0.05). In conclusion, the selected phage clones may therefore represent vaccine candidates if they could be presented to the ovine immune system in an appropriate fashion.

Le titre de la thèse concernant le succès des nématodes peut sembler extremement vaste. Notre projet était pourtant bien d’avoir une vue d’ensemble de l’ensemble des acteurs, à savoir les nématodes parasites du tube digestif mais également l’hôte et certains aspects de sa réponse, et enfin l’éleveur qui est celui qui décide du mode d’élevage et des traitements antiparasitaires. Nous avons essentiellement travaillé en conditions expérimentales et sur une espèce de ces strongles. En ce qui concerne les traits de vie des nématodes plusieurs grands phénomènes conditionnent leur succès : a) leur capacité assez variable selon les isolats à infester un hôte, b) l’interaction entre résistance aux antiparasitaires et cette capacité à infester, c) enfin leur aptitude à survivre aux stress climatiques au cours de leur phase non-parasitaire. / The success of gastrointestinal nematodes in their sheep hosts is so extensive that they present one of the leading threats to ruminant health and production throughout the globe. This thesis research identified three key factors which influence their success including the gastrointestinal nematode biology, the sheep host protective response and the farmers control decisions. Using Haemonchus contortus as a model species, we demonstrated that the success of GIN biology is aided by their capacity to overcome numerous selective pressures that target both parasitic and free-living stages in their life cycle. This was achieved by amplifying life-history traits following challenge to recoup any costs in survival and reproduction. In turn, high levels of fitness were maintained and they remained stable in the face of numerous selective pressures. Sheep have the capacity to exert almost perfect control over GIN success by blocking their life cycle through via protective responses.

Parasites have a major impact on host condition and fitness and thereby represent a strong selective force for individuals in wild populations. The main defence against parasite infection and associated morbidity is the host immune response, and consequently it is expected for there to be strong selection eroding genetic variation underlying immune responses in natural populations. However, studies in the wild have found considerable heritable variation underlying immune responses. Few studies have investigated the genetic variants underlying immunity in wild populations and are able to examine how genetic variation is maintained in the face of natural selection. The aim of this thesis is to investigate the selection on, and genetic variation underlying, immunity in a wild Soay sheep population by looking at antibody responses to the prevalent parasite Teladorsagia circumcincta. Anti- T. circumcincta antibody levels (IgA, IgE, IgG) were measured in neonatal plasma samples taken soon after birth, representing maternally-derived antibodies, and in samples from August yearly from four month old lambs and adults, representing endogenous antibodies. All three endogenously produced antibody measures in lambs and adults were repeatable and heritable. In addition, a genome wide association study run on the three antibody traits on August lamb and adult measures found associations between anti-T. circumcincta IgA levels and single nucleotide polymorphisms in a region on chromosome 24. There was evidence for age- and isotype- dependent negative associations between antibody isotypes and strongyle faecal egg counts (FEC). Further, there was evidence for age-dependent selection via positive associations between anti-T. circumcincta IgG and survival in females and annual fecundity in males. In comparison, there was no additive genetic variance underlying maternally-derived (neonatal) anti-T. circumcincta antibody levels in neonates, but maternal and maternal genetic effects explained a considerable proportion of the variance in these traits. There was evidence for associations between neonatal anti-T. circumcincta IgG and later offspring phenotype and fitness, independent of total antibody (IgG) transferred. We found that neonatal anti-T. circumcincta IgG levels positively predicted survival to four months old, as well as weight in August. In addition, neonatal anti-T. circumcincta IgG levels were associated with reduced strongyle FEC in August, and were associated with improved survival over the first winter. In early life, maternally-derived anti-helminth antibodies are important for early growth, survival, and parasite resistance, as well as first winter survival, while fitness benefits in adulthood were associated with higher endogenous anti-helminth antibody levels. This thesis illustrates that maternal effects and genetic variation can have strong effects on variation in immunity in the wild, and this variation in turn can have health and fitness consequences for individuals.