Tesi sul tema "Teladorsagia circumcincta"

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.

The main aims of this study were to identify and characterise proteins from T. circumcincta that may induce a protective immune response in the host and to learn more about the biology of the worm. In order to identify possible protective antigens, a complementary DNA (cDNA) library prepared from adult worms was screened with serum from an animal that was protected against a single challenge infection after vaccination with a T. circumcincta protein fraction (S3 TSBP). Forty five immunopositive cDNA clones were identified, of which sixteen had homology to galectin. Of the remaining clones, the majority shared homology with two metabolic enzymes, methylmalonate semialdehyde dehydrogenase and 10-formyltetrahydrofolate dehydrogenase, that have not been characterised in nematodes. A single clone with homology to the antioxidant enzyme, catalase, was also identified. These three enzymes were selected for further investigation on the basis of their roles in nematode metabolism and therefore, their potential as vaccine candidates. Characterisation of T. circumcincta excretory/secretory material (ES) was also performed. L4 and adult worms were cultured in vitro and the proteins released were separated by 1D electrophoresis and analysed by Tandem Liquid Chromatography Mass Spectrometry and N-terminal sequencing. This identified proteins showing similarity to, amongst others, metabolic enzymes, structural components, antioxidants, globin-like proteins and cysteine proteases, present in online databases but not previously characterised in T. circumcincta. This study has identified several novel T. circumcincta proteins that may have potential as future vaccine or drug targets. It has also provided further information regarding the biology of the worm.

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.

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The mechanisms whereby parasitic nematodes become resistant to anthelmintics, particularly ivermectin, remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of gene expression changes. The present study focused on Teladorsagia circumcincta; the predominant parasitic gastrointestinal nematode species in the UK and the predominant resistant species. The role of changes in gene expression were investigated in an ivermectin-susceptible isolate (CVL) and a multidrug resistant isolate (MOTRI), utilising a range of molecular biological techniques. In the first experiment, a panel of novel putative ivermectin resistance genes were identified from T. circumcincta, comprising 11 partial P-glycoprotein (Pgp) and 3 partial Cytochrome P450 (CYP) sequences. Both Pgps and CYPs have been implicated in the handling and metabolism of xenobiotics in other biological systems, but have not been investigated in T. circumcincta to date. Initial results, using semi-quantitative PCR identified changes in expression of this panel of genes between the CVL and MOTRI isolates. Constitutive differences in expression of the Pgps and CYPs between CVL and MOTRI were determined using the ΔΔCt TaqMan® real-time PCR method. A statistically significant increase in expression was observed for TeciPgp-9 NBD2 across all life-cycle stages but most notably in eggs (55-fold increase). A statistically significant reduction in expression of TeciPgp-2 NBD2 was observed in all but the adult stages of MOTRI compared to CVL. Analysis of a 208 base pair sequence of TeciPgp-9 NBD2 identified high levels of polymorphism, with at least four non-coding SNPs evident in the MOTRI isolate. These results merit further investigation. Inducible changes in the expression of the Pgps and CYPs were investigated in MOTRI before and after ivermectin treatment, using real-time PCR. Statistically significant fold changes in expression in most of the genes occurred in at least one life-cycle stage. Inducible expression of TeciPgp-2 NBD2 and TeciPgp-9 NBD2 was investigated further by comparing adult MOTRI parasites with those recovered three days after in vivo ivermectin exposure, and by exposing pools of MOTRI xL3 to ivermectin in the larval migration inhibition test. The survivors of ivermectin exposure exhibited a statistically significant reduced 13.68-fold expression of TeciPgp-2 NBD2 compared to MOTRI. Similarly, the MOTRI xL3 able to migrate in the presence of ivermectin in the LMIT had a 1.88-fold reduction in TeciPgp-2 NBD2 expression compared to MOTRI xL3 unexposed to ivermectin. These results indicate that inducible changes in TeciPgp-2 NBD2 and TeciPgp-9 NBD2 expression can occur, but the experimental design is critical to being able to identify the changes. In a more global approach, the transcriptomic response of MOTRI adults to in vitro ivermectin exposure was investigated using Roche 454 sequencing, generating 98,685 novel EST sequences, providing an important resource for a genome resource-poor organism. Objective bioinformatic analysis of the two datasets revealed statistically significant differences in the mean expression levels of the KEGG orthologous groups for ‘translation’, ‘amino acid metabolism’ ‘carbohydrate metabolism’ and ‘xenobiotic degradation and metabolism’. On combining the two datasets, and through application of a novel statistical method, 16 clusters of ESTs were identified as containing statistically significant differences in the mean proportion of exposed reads compared to unexposed reads under the conservative model, whilst a further 355 clusters were found to have statistically significant differences under the liberal model. One-way suppression subtractive hybridisation (SSH) was used to identify genes exhibiting increased expression in MOTRI adults compared to CVL adults. 28 contiguous sequences were identified from the SSH experiment; 6 contiguous sequences were selected for validation; 5 of these results were confirmed using semi-quantitative PCR. Each contig was BLAST searched against the Roche 454 dataset; contig SSH14 aligned most closely to one of the statistically significant clusters in the conservative model, SSHs 5, 6, 10 and 23 aligned most closely to statistically significant clusters in the liberal model. This suggests that changes in expression in these sequences occur both constitutively, between CVL and MOTRI isolates, and inducibly, following ivermectin exposure. This work has shown that changes in gene expression, particularly the constitutively reduced expression in TeciPgp-2 NBD2 and the constitutively increased expression in TeciPgp-9 NBD2 (coupled with the presence of SNPs) could play a role in allowing multidrug resistant T. circumcincta to survive ivermectin exposure. Roche 454 sequencing and SSH approaches identified gene expression changes associated with in vitro ivermectin exposure and ivermectin resistance. These could form the basis of a novel panel of candidate resistance genes whose altered expression profiles may allow multidrug resistant T. circumcincta to survive ivermectin exposure by some, as yet identified, mechanism. Finally, we have also shown that a multidrug T. circumcincta isolate is affected by ivermectin exposure and that changes in gene expression could have a role to play in the ivermectin resistance phenotype in T. circumcincta. The genetic changes underpinning these changes in gene expression remain to be elucidated, and need to be investigated in other isolates. These changes could form the basis of an ivermectin resistance molecular marker, to monitor the spread of resistance, and to evaluate management practices aimed at delaying its spread.

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.

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.

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.

Mes travaux de these s'inserent dans le cadre general des recherches developpees sur la resistance aux benzimidazoles (bzs) chez teladorsagia circumcincta, trichostrongle parasite de petits ruminants. Dans une premiere partie, une etude sur la diversite genetique a tout d'abord montre que t. Circumcincta est en fait un complexe d'especes qui regroupe au moins deux especes distinctes t. Circumcincta et t. Goat. Elle a aussi mis en evidence chez t. Circumcincta (espece la plus frequente du complexe), une absence de structuration des populations a l'echelle regionale (< 200 km) et une faible structuration a plus large echelle. Compte tenu des caracteristiques des elevages caprins etudies et des parametres demographiques de t. Circumcincta, ce resultat traduit l'existence d'une faible derive genetique qui permet le maintien d'alleles rares (dont les alleles de resistance) dans les populations parasites. Dans une seconde partie, ces travaux se sont interesses a l'influence de differents types de traitement anthelminthiques sur la selection des genotypes resistants et aux consequences de l'acquisition de la resistance aux bzs sur certains traits de vie des parasites. En absence de traitement, la proportion des genotypes reste constante ce qui indique que le succes reproductif des genotypes resistant et sensibles est semblable. La resistance aux bzs augmente proportionnellement avec le nombre de traitements aux bzs. Il est donc tres difficile de combattre la resistance, lorsque les premiers genotypes resistants sont apparus dans la population parasite.

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.

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 is a common abomasal parasite of sheep in temperate regions and is one of the major causes of parasitic gastroenteritis (PGE) in growing lambs. Control of infection is achieved using anthelmintic drugs; however, this practice is rapidly becoming unsustainable due to widespread anthelmintic resistance within the T. circumcincta population. Sheep can acquire protective immunity against this parasite; immunity involves local and systemic antibodies and immune cells which can impair worm growth and fecundity and lead to expulsion of the parasites from the abomasum. Vaccination against this parasite is therefore a feasible option of control. A recent study showed that a recombinant vaccine cocktail containing 8 T. circumcincta antigens significantly reduced the faecal egg count and worm burdens of immunised sheep, compared to an adjuvant-only control group. However, the recombinant antigens induced a suboptimal antibody response to the recombinant antigens. This suggests that differencies between the native antigens and their recombinant versions may exist, possibly due to variations in structure and/or post-translational modifications (PTMs). The main aim of this work was to use a novel expression system, the free living nematode Caenorhabditis elegans, to generate alternative recombinant versions of two of the T. circumcincta antigens used in the 8-antigen vaccine, cathepsin F (Tci-CF-1) and monocyte Migration Inhibitory Factor (Tci-MIF-1). This was achieved by micro-injection of C. elegans worms with plasmids containing the cDNA sequences of Tci-cf-1 and Tci-mif-1 followed by purification of recombinant Tci-CF-1 and Tci-MIF-1 from the transformed worms. Immune recognition, enzyme activity and biological effects on sheep cells of the recombinant antigens were characterised. The results show that immunisation-induced antibodies bind to native Tci-CF-1 purified from T. circumcincta L4 ES, whereas infection-induced antibodies were unable to bind the recombinant Tci-CF-1 versions. Further characterisation of recombinant Tci-CF-1 versions expressed in C. elegans or Pichia pastoris showed that in order to be enzymically active, these proteins require cleavage of the pro-peptide by an exogenous enzyme and that some differences were present in the glycosylation of the recombinant versions and native Tci-CF-1. Characterisation of both recombinant Tci-MIF-1 versions showed that although both are enzymically active, neither showed a significant inhibitory effect on the migration of sheep monocytes or on the activation of sheep macrophages in vitro compared to unstimulated controls. It is speculated that Tci-MIF-1 may be involved in T. circumcincta larval development rather than host immunosuppression.

Les études épidémiologiques sont nombreuses sur ce parasite. Les travaux relatifs à l'écologie sont assez rares, ceux concernant la morphométrie ou la génétique, très rares. Des études antérieures ont montré l'isolement des fermes caprines en ce qui concerne les infestations par les helminthes. Les méthodes d'élevage sont très différentes selon les fermes, et les populations parasitaires pourraient être diversifiées. L'objet du présent travail est d'estimer la variabilité morphologique, écologique et génétique du nématode teladorsagia circumcincta. Quatorze populations de t. Circumcincta provenant de chèvres issues de fermes caprines situées dans le centre-ouest de la France ont été étudiées. Les informations concernant l'historique des fermes (ancienneté de l'élevage, origine et nombre de chèvres achetées pour constituer le troupeau lors de la création de la ferme, etc) et le mode d'élevage (taille du troupeau, utilisation des surfaces de paturages, traitements anthelminthiques, etc) ont été recueillies. La morphologie des ufs et des larves infestantes ainsi que l'écologie des stades libres ont été étudiées dans un sous-echantillon de huit populations. La variabilité génétique a été appréciée au moyen d'isoenzymes (malate déshydrogénase -mdh, lactate déshydrogénase -ldh, phosphoglucomutase -pgm, mannose-phosphate isomérase -mpi et glucose-phosphate isomérase -gpi), pour les 14 populations naturelles et pour trois générations d'un sous-échantillon de cinq isolats maintenus dans des conditions de laboratoire. Les populations naturelles de t. Circumcincta se différencient selon la présence d'un électromorphe supplémentaire, à migration très rapide au locus mdh-1 qui a été mis en évidence dans plusieurs fermes caprines. Nous distinguons une souche vraisemblablement caprine (avec une fréquence élevée de cet allèle) et une souche ovicaprine (avec une fréquence très faible ou l'absence de cet allèle). Nous supposons que celles-ci sont reliées à l'historique des chèvres introduites dans chacune des fermes et dépendant de l'utilisation de paturages contaminés par des chèvres ou des moutons. Un déficit en hétérozygotes a été observé pour toutes les fermes et pour tous les systèmes enzymatiques. Des variations très faibles des moyennes (valeurs absolues) ont été observées pour la morphologie des stades libres, ufs et larves infestantes. Les distributions de quelques caractères morphologiques des larves, bien que plus informatives, restent insuffisantes pour caractériser les populations. Les différences entre fermes restent modestes et la variabilité morphologique dépend plus des conditions environnementales que de l'origine des isolats. La méthode que nous avons établie pour étudier la variabilité écologique du développement des ufs en larves infestantes semble valide. Certaines populations sont adaptées à la sécheresse. D'autres se developpent mieux dans des conditions caracterisées par une humidité élevée associée à une température froide (4c) et d'autres encore par une humidité élevée combinée à une température chaude (supérieure a 23c). Deux stratégies indépendantes apparaissent parmi les populations étudiées. La première correspond à un succès élevé de l'infestation par la première génération de larves reliée à une faible survie des larves produites dans les générations suivantes. La seconde stratégie se caractérise par une fertilité élevée des femelles avec une aptitude médiocre des ufs produits à évoluer en larves. Les trois estimations de la variabilité ne convergent pas complètement. Cela tient sans doute au fait que pour chaque estimation de variabilité, seules quelques mesures sont réalisées. Il semble qu'une convergence apparaisse pour les isolats maintenus depuis quelques générations dans les conditions de laboratoire : les isolats se ressemblent plus entre eux qu'avec leurs populations d'origine. De ces résultats, nous pouvons présumer que les données obtenues à partir de populations de nématodes maintenues en conditions expérimentales peuvent se montrer trompeuses et devraient être validées dans les conditions naturelles

Throughout the world, control of parasitic nematodes in livestock has been compromised by the emergence and spread of anthelmintic resistance. Teladorsagia circumcincta is the most important gastrointestinal nematode parasite of small ruminants in temperate regions and the major resistant species in the United Kingdom (UK). In most cases the genetic factors which underpin resistance to broad-spectrum anthelmintics are still poorly understood. Recent work conducted independently in New Zealand (NZ) and Scotland has implicated the involvement of a particular P-glycoprotein (Pgp) gene, Tci-pgp-9, in multiple-anthelmintic resistance in T. circumcincta. The focus of this study is to further characterise Tci-pgp-9 and its possible role in ivermectin (and multi-drug) resistance using two UK field isolates of T. circumcincta, one which is anthelmintic susceptible (MTci2) and another that is multiple-anthelmintic resistant (MTci5). The generation of full-length cDNA sequence data from these isolates allowed genetic comparisons which identified the presence of nine non-synonymous SNPs in the Tci-pgp-9 coding sequence of the MTci5 isolate. The 3.8 kb, Tci-pgp-9 transcript from the MTci2 and MTci5 isolates shared 95.5 % identity at the nucleotide level and 99.5 % identity at the protein level. Twelve sequence variants were identified in the first internucleotide binding domain, designated Tci-pgp-9-IBDA, some of which shared a high level of identity with sequence variants identified in near-isogenic NZ strains. Multiple allelic variants were present in the majority of individuals, but a reduction in the number of allelic variants present in individuals of MTci5 relative to the MTci2 isolate was evident. A further reduction in the number of alleles present in individuals was also observed in individuals derived from an IVM treated population of MTci5, suggesting that IVM treatment applied purifying selection pressure. Quantitative real time PCR analysis showed a 3.7-fold increase in Tci-pgp-9 gene copy number in the MTci5 isolate relative to the MTci2 isolate, which was consistent with a 3.4-fold increase observed in the NZ study. None of the common haplotypes identified were unique to any given isolate, and the relationship between haplotype and copy number was not straightforward. This study provides evidence that Tci-pgp-9 is under anthelmintic selection, but the precise role of this specific P-glycoprotein gene, and its alleles, in the phenotypic expression of anthelmintic resistance in T. circumcincta remains to be determined.

The parasitic nematode, Teladorsagia (Ostertagia) circumcincta is the primary cause of Parasitic Gastro-Enteritis (PGE) in lambs in Britain. Control of this parasite has largely depended on the use of broad spectrum anthelmintic drugs since their inception three decades ago. Widespread and unconstrained use of anthelmintics has resulted in selection for resistant strains of nematode, particularly within the T. circumcincta species. Control of PGE now involves optimizing parasite control whilst preserving the susceptibility of the parasites to the anti-parasitic drugs. Two aspects of the epidemiology of T. circumcincta are investigated in this thesis. First, the effect of temperature on the development and survival of the free-living stages is investigated. The conventional nematode development models are replaced by more sophisticated and biologically meaningful methods of describing temperature-dependent development rate phenomena in nematodes. The effect of geographical, temporal and developmental variation on the population dynamics of T. circumcincta are explored to determine possible sources of observed variability in infection levels in the field. Next, a suite of models generic to most direct life cycle parasites undergoing intensive drug therapy, is constructed and analysed. Provision is made within these models to explore the impact of important life history events such as refugia and immigration on the evolution of resistance. A novel technique in resistance control involving overwhelming a resistant strain of nematode with a susceptible strain is modelled and suggestions made for the practical implementation of such a method.

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.

The kinetics of the host's immune responses to challenge infection were studied and identified clear patterns in plasma IgA activity, peripheral eosinophil counts, faecal egg counts and plasma pepsinogen concentrations but not in plasma IgG activity. It was determined that when used in parallel and when tested at multiple time points, these parameters have much greater potential as markers of resistance than when used individually or more importantly if only assessed on a single occasion. Further work investigated the recognition of stage specific parasite antigens by host plasma IgA by Western blotting. After adjusting for differences in the activity of IgA in each plasma sample the work in this thesis identified that preferential recognition of a different set of antigens was associated with resistance in the group of experimentally challenged animals compared to previous publications. Additionally, and for the first time this investigation was also carried out on naturally infected animals. There was little correlation in the patterns of antigen recognition between the experimentally challenged and naturally infected animals. Finally, the role of MHC was investigated and it was determined that MHC heterozygotes produced significantly more plasma IgA then MHC homozygotes but did not harbour significantly shorter worms. The analysis also confirmed in naturally infected sheep that there was no obvious relationship between MHC polymorphism and antigen recognition. The results suggested that resistance was due to the recognition of several molecules rather than a single molecule. The work detailed in this thesis has further increased our understanding of the complex host/parasite relationship and has confirmed that selective breeding using the various phenotypic and genetic markers studied is possible. However, this will only be viable if the tests involved in assessing these traits become cheaper and easier to perform, especially if they are to be carried out by the farmer, on the farm.

La resistance aux benzimidazoles (bzs) chez t. Circumcincta, un nematode parasite de petits ruminants peut si aucune mesure limitant son developpement n'est preconisee, devenir un probleme majeur chez les eleveurs caprins et ovins. Les objectifs du travail realise furent donc d'etudier d'une part le determinisme genetique des mecanismes qui conferent une resistance aux bzs chez ce nematode et d'autre part d'effectuer une approche populationnelle de ce phenomene. Nous avons ainsi pu montrer que la resistance aux bzs chez t. Circumcincta semble principalement liee a une mutation sur le gene cible de l'anthelminthique, la beta-tubuline. Cette mutation a l'origine du changement en position 200 de la proteine d'une phenylalanine par une tyrosine est non seulement recessive puisque seuls les individus homozygotes resistants survivent aux traitements, mais elle semble aussi presente dans la plupart des populations naturelles etudiees de ce nematode. Il existe cependant dans ces populations une relation positive entre la proportion d'individus homozygotes mutants et le niveau de resistance evalue par la dose letale 50. La poursuite des travaux a montre qu'il n'existe aucune difference en conditions experimentales entre les individus des trois genotypes (rr, rs, ss) au niveau de leur fitness. Les premiers resultats obtenus sur le terrain et certaines donnees epidemiologiques qui revelent qu'aucune reversion de la resistance n'est observee meme lorsque les bzs ne sont plus utilises semblent confirmer ce resultat. Concernant l'origine des alleles mutants, il fut montre que cette mutation pouvait etre portee par differents alleles du gene de la beta-tubuline, ce qui suggere que la resistance a pu parfois apparaitre de facon independante dans les elevages. Mais en revanche certaines populations de nematodes partagent les memes alleles mutants pouvant s'expliquer soit par la presence de ces alleles mutants avant la constitution des troupeaux, soit par leur dispersion en raison de processus migratoires. Enfin les populations resistantes de nematodes se caracterisent par un faible polymorphisme du gene de la beta-tubuline, qui semble resulter d'un effet d'auto-stop genere par l'envahissement de la mutation avantageuse dans une population entrainant avec elle d'autres mutations voisines et neutres. Les perspectives a envisager a la suite de ces travaux sont nombreuses et consisteront dans un premier temps a completer les connaissances acquises sur le determinisme genetique de la resistance aux bzs chez ce nematode, notamment dans le cas de fortes resistance a ses composes. Les aspects populationnels seront aussi poursuivis par une etude sur le terrain de la fitness et par un travail plus approfondie sur l'origine des alleles mutants. Enfin d'autres protocoles sont envisages dans un but applique de gestion de la resistance soit en limitant la selection de mutants soit en identifiant les techniques d'elevages favorables a la selection des individus resistants.

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

This thesis describes a series of three experiments designed to estimate the nutritional cost of the immune response to the gastrointestinal nematodes Trichostrongylus colubriformis and Teladorsagia circumcincta in sheep. For each experiment, animals were allocated hierarchically by liveweight into one of four groups that were either infected (group IF), similarly infected and concurrently immuno-suppressed with weekly intramuscular injections of 1.3mg kg liveweight (LW)-1 of methylprednisolone acetate (group ISIF), immuno-suppressed only (group IS) or remained as controls (group C). Body composition of all animals was estimated using x-ray computer tomography prior to infection and at the conclusion of each study with bodyweight and faecal nematode egg counts (FEC; eggs gram-1 of fresh faeces (epg)) measured along with blood samples taken for the determination of levels of serum proteins, phosphate and antibodies. In the first trial (Chapter 3), the nutritional cost of both the acquisition and maintenance of immunity to gastro-intestinal nematodes was investigated using immunologically naive 5-month-old lambs and immunologically competent 17-month-old ewes during infection with 2,000 and 4,000 L3 infective T. colubriformis larvae d-1, respectively (80 L3 T. colubriformis larvae kgLW-1 d-1). Profiles of FEC and comparative worm burdens at slaughter indicated an effective immune response was maintained in IF ewes and developed in IF lambs while successfully suppressed in both ISIF lambs and ISIF ewes and was confirmed by serum antibody titres. The typical reduction in voluntary feed intake as a consequence of infection was observed in IF lambs (0.30, p<0.001) but not in IF ewes, ISIF lambs or ISIF ewes, and appeared to be associated with L3 IgA. Gross efficiency of use of metabolizable energy (ME) for net energy (NE) deposition was reduced by 0.20 in lambs during acquisition of immunity and by 0.16 in ewes maintaining an established immunity. Infection in immuno-suppressed animals reduced efficiency by 0.05 and 0.15 for lambs and ewes. These findings allowed the hypothesis that the reduction in feed intake and nutrient utilization in young parasitized sheep is caused by physiological signalling associated with the acquisition phase of the host immune response to infection, rather than simply the damage caused by the parasite per se. The second trial (Chapter 4) investigated the influence of metabolizable protein (MP) supply on the metabolic disturbances associated with the acquisition phase of the immune response during infection with 2,000 L3 T. colubriformis d-1. Groups of lambs were offered either a low protein (L; 62g MP kgDM-1) or high protein diet (H; 95g MP kgDM-1). Patterns of total daily egg excretion indicated that an effective immune response was developed in HIF, but not LIF, HISIF nor LISF and was confirmed by comparative worm burdens. The proportionate reduction in feed intake in immunologically normal animals was reduced through the provision of additional protein, being 0.12 in HIF and 0.23 in LIF. Regardless of diet, infection did not cause a reduction in feed intake in immuno-suppressed animals (p>0.05). Infection proportionately reduced the gross efficiency of ME utilization in immunologically normal animals by 0.23 in HIF (p=0.09) and by 0.51 in LIF (p=0.01), but not in immuno-suppressed animals. Immuno-suppression did not suppress serum L3 IgA levels in seven of the eight HISIF and four of the eight LISIF animals. Furthermore, only four out of the eight immunologically normal animals from both the HIF and LIF groups displayed an L3 IgA response. Consequently, regardless of immuno-suppression treatment, animals were termed as IgA responders (HR or LR) or non-responders (HN or LN). Feed intake was proportionately reduced from day 22 by 0.15 in HR (p=0.03) and by 0.32 in LR (p=0.01), but was not significantly reduced in HN or LN. Gross efficiency of ME utilization was significantly reduced for LN animals only, being proportionately 0.59 (p<0.01). These findings allowed the conclusion that additional MP reduced the consequence of immunological signalling that was displayed in reduced feed intake and in nutrient utilization, both of which appeared to be associated with an IgA response. It is hypothesized that the lessening of nutritional disturbance observed in high protein and immuno-suppressed animals could be a consequence of altered physiological signalling during the immunological cascade. The third trial (Chapter 5) utilized lambs infected with the abomasal parasite T. circumcincta to explore the possibility that the reduction in feed intake and nutrient utilization is a universal phenomenon of the acquisition phase of the immune response to nematode parasites inhabiting different organs along the gastrointestinal tract. In addition, immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from a further twelve animals that were surgically fitted with an abomasal cannula and either infected (CIF) or concurrently infected and immuno-suppressed as described previously (CISIF). The development of immunity in IF animals was accompanied by a 0.17 proportional decrease in feed intake between days 15 to 28 of infection (p<0.05) and a 0.20 proportional reduction in nutrient utilization (p=0.07), none of which were observed in ISIF animals. While FEC and worm burdens indicated successful immuno-suppression in ISIF animals, both serum IgA and total antibody production were not reduced. The development of immunity in CIF was reflected in an increase in both mast cells and globule leukocytes in serial abomasal tissue biopsies, both of which were reduced in CISIF (p<0.01 for both). In serial biopsy tissue, immuno-suppression did prevent a rise in tissue IgA that was apparent in CIF animals (p<0.01) although these changes were not reflected in serum IgA levels. It appears that the alleviation of the reduction in feed intake and nutrient utilization in young lambs through the use of corticosteroid induced immuno-suppression may be a universal phenomenon for both intestinal and abomasal parasites, but the association with and/or role of IgA during infection with T. circumcincta is unclear. In summary, the reduction in feed intake and nutrient utilization in sheep during infection with both the abomasal nematode T. circumcincta and the small intestine nematode T. colubriformis appears to be associated with a component(s) of the acquisition phase of the host immune response, rather than, as conventionally assumed, the direct mechanical damage of the parasite per se. It is hypothesised that the nutritional disturbance as a consequence of infection in young lambs may be the result of pro-inflammatory cytokines involved in immunological signalling that may also be associated with the production of IgA, the effects of which can be reduced through the provision of adequate MP. These studies provide evidence that the immune response to gastrointestinal parasites is nutritionally costly to the animal and have implications for application of manipulations that are intended to promote the development of a strong immune reaction in high producing animals.

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

The purpose of this study was to undertake detailed molecular and phenotypic characterisation of a MDR isolate of T. circumcincta (MTci5) with particular focus upon the mechanisms underlying benzimidazole (BZ) resistance.  MTci5 was isolated from a farm in Central Scotland, which employed a suppressive anthelmintic dosing regime and was closed in 2002 when control of the parasite population became unsustainable.  Underpinning all of the experiments in this study was an anthelmintic selection process whereby the MTci5 isolate was pressured individually with three broad-spectrum anthelmintics (benzimidazole, ivermectin and levamisole). There are three main areas of investigation in this study, the first being an investigation of the population genetic structure or a MDR isolate.  A central question was whether the MDR phenotype of MTci5 is conferred by the inheritance of genes present in a single interbreeding population or whether there is genetic sub-structuring, whereby discrete sub-populations of the isolate each show resistance to different anthelmintics.  Microsatellite analysis was employed to investigate the population genetic structure of the MTci5 isolate.  The results suggest that the MTci5 isolate is a single, freely interbreeding population with triple resistance, showing no evidence of genetic sub-structuring. The second area of investigation was the role of the F200Y isotype I ß-tubulin mutation in the determination of BZ resistance and the potential involvement of this mutation in resistance to ivermectin (IVM) and levamisole (LEV). There was no evidence of an effect of IVM or LEV selection upon the F200Y isotype I ß-tubulin mutation. The third area of investigation was the origin and diversity of BZ resistance alleles in the MTci5 isolate.  Single strand conformation polymorphism (SSCP) analysis of small region extending through exons 1 and 2 and intron 1 of the isotype I ß-tubulin gene was used to assess the genetic diversity of this locus in the MTci5 isolate and of five other UK T. circumcincta populations. Results are consistent with the theory of multiple independent, spontaneous mutations at the P200 locus of the isotype I ß-tubulin gene.

This thesis describes a series of three experiments designed to estimate the nutritional cost of the immune response to the gastrointestinal nematodes Trichostrongylus colubriformis and Teladorsagia circumcincta in sheep. For each experiment, animals were allocated hierarchically by liveweight into one of four groups that were either infected (group IF), similarly infected and concurrently immuno-suppressed with weekly intramuscular injections of 1.3mg kg liveweight (LW)⁻¹ of methylprednisolone acetate (group ISIF), immunosuppressed only (group IS) or remained as controls (group C). Body composition of all animals was estimated using x-ray computer tomography prior to infection and at the conclusion of each study with bodyweight and faecal nematode egg counts (FEC; eggs gram⁻¹ of fresh faeces (epg)) measured along with blood samples taken for the determination of levels of serum proteins, phosphate and antibodies. In the first trial (Chapter 3), the nutritional cost of both the acquisition and maintenance of immunity to gastro-intestinal nematodes was investigated using immunologically naive 5-month-old lambs and immunologically competent 17-month-old ewes during infection with 2,000 and 4,000 L3 infective T. colubriformis larvae d⁻¹, respectively (80 L3 T. colubriformis larvae kgLW⁻¹ d⁻¹). Profiles of FEC and comparative worm burdens at slaughter indicated an effective immune response was maintained in IF ewes and developed in IF lambs while successfully suppressed in both ISIF lambs and ISIF ewes and was confirmed by serum antibody titres. The typical reduction in voluntary feed intake as a consequence of infection was observed in IF lambs (0.30, p<0.001) but not in IF ewes, ISIF lambs or ISIF ewes, and appeared to be associated with L3 IgA. Gross efficiency of use of metabolizable energy (ME) for net energy (NE) deposition was reduced by 0.20 in lambs during acquisition of immunity and by 0.16 in ewes maintaining an established immunity. Infection in immuno-suppressed animals reduced efficiency by 0.05 and 0.15 for lambs and ewes. These findings allowed the hypothesis that the reduction in feed intake and nutrient utilization in young parasitized sheep is caused by physiological signalling associated with the acquisition phase of the host immune response to infection, rather than simply the damage caused by the parasite per se. The second trial (Chapter 4) investigated the influence of metabolizable protein (MP) supply on the metabolic disturbances associated with the acquisition phase of the immune response during infection with 2,000 L3 T. colubriformis d⁻¹. Groups of lambs were offered either a low protein (L; 62g MP kgDM⁻¹) or high protein diet (H; 95g MP kgDM⁻¹). Patterns of total daily egg excretion indicated that an effective immune response was developed in HIF, but not LIF, HISIF nor LISF and was confirmed by comparative worm burdens. The proportionate reduction in feed intake in immunologically normal animals was reduced through the provision of additional protein, being 0.12 in HIF and 0.23 in LIF. Regardless of diet, infection did not cause a reduction in feed intake in immuno-suppressed animals (p>0.05). Infection proportionately reduced the gross efficiency of ME utilization in immunologically normal animals by 0.23 in HIF (p=0.09) and by 0.51 in LIF (p=0.01), but not in immuno-suppressed animals. Immuno-suppression did not suppress serum L3 IgA levels in seven of the eight HISIF and four of the eight LISIF animals. Furthermore, only four out of the eight immunologically normal animals from both the HIF and LIF groups displayed an L3 IgA response. Consequently, regardless of immunosuppression treatment, animals were termed as IgA responders (HR or LR) or non-responders (HN or LN). Feed intake was proportionately reduced from day 22 by 0.15 in HR (p=0.03) and by 0.32 in LR (p=0.01), but was not significantly reduced in HN or LN. Gross efficiency of ME utilization was significantly reduced for LN animals only, being proportionately 0.59 (p<0.01). These findings allowed the conclusion that additional MP reduced the consequence of immunological signalling that was displayed in reduced feed intake and in nutrient utilization, both of which appeared to be associated with an IgA response. It is hypothesized that the lessening of nutritional disturbance observed in high protein and immuno-suppressed animals could be a consequence of altered physiological signalling during the immunological cascade. The third trial (Chapter 5) utilized lambs infected with the abomasal parasite T. circumcincta to explore the possibility that the reduction in feed intake and nutrient utilization is a universal phenomenon of the acquisition phase of the immune response to nematode parasites inhabiting different organs along the gastrointestinal tract. In addition, immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from a further twelve animals that were surgically fitted with an abomasal cannula and either infected (CIF) or concurrently infected and immuno-suppressed as described previously (CISIF). The development of immunity in IF animals was accompanied by a 0.17 proportional decrease in feed intake between days 15 to 28 of infection (p<0.05) and a 0.20 proportional reduction in nutrient utilization (p=0.07), none of which were observed in ISIF animals. While FEC and worm burdens indicated successful immunosuppression in ISIF animals, both serum IgA and total antibody production were not reduced. The development of immunity in CIF was reflected in an increase in both mast cells and globule leukocytes in serial abomasal tissue biopsies, both of which were reduced in CISIF (p<0.01 for both). In serial biopsy tissue, immuno-suppression did prevent a rise in tissue IgA that was apparent in CIF animals (p<0.01) although these changes were not reflected in serum IgA levels. It appears that the alleviation of the reduction in feed intake and nutrient utilization in young lambs through the use of corticosteroid induced immuno-suppression may be a universal phenomenon for both intestinal and abomasal parasites, but the association with and/or role of IgA during infection with T. circumcincta is unclear. In summary, the reduction in feed intake and nutrient utilization in sheep during infection with both the abomasal nematode T. circumcincta and the small intestine nematode T. colubriformis appears to be associated with a component(s) of the acquisition phase of the host immune response, rather than, as conventionally assumed, the direct mechanical damage of the parasite per se. It is hypothesised that the nutritional disturbance as a consequence of infection in young lambs may be the result of pro-inflammatory cytokines involved in immunological signalling that may also be associated with the production of IgA, the effects of which can be reduced through the provision of adequate MP. These studies provide evidence that the immune response to gastrointestinal parasites is nutritionally costly to the animal and have implications for application of manipulations that are intended to promote the development of a strong immune reaction in high producing animals.

The series of experiments described in this thesis were designed to investigate the role of suckling or late weaning in the response of young lambs to nematode infection. All experiments were conducted outdoors with grazing animals and no supplementation but for suckled groups of lambs whose counterparts were weaned to ryegrass – white clover swards. The parasite of interest was mainly Teladorsagia circumcincta solely but with mixed infection of Trichostrongylus colubriformis in one instance. In Chapter 3 (first experiment), the hypothesis that milk per se may have a direct effect on nematode development, rather than an indirect effect through enhancement of host immunity by superior nutrient supply was tested. Sixty, twinborn lambs were used, allocated to one of eight groups formed by either dosing lambs from 42 days of age or not with the equivalent of 1000 or 250 L₃ T. circumcincta larvae d⁻¹ until five days before necropsy, while a twin was either weaned at 39 days of age, suckled as single or twin until necropsy on day 84. The possibility that weaning one of a twin set onto pasture in close proximity to the ewe would cause abnormal ewe and lamb behaviour was tested by replicating the work in twins maintained as twins but in which one twin received equivalent of 250 and the other 1000 L₃ T. circumcincta larvae d⁻¹. This showed no abnormal ewe nursing or lamb suckling behaviour as a result of weaning a twin in a set. Relatively low faecal egg counts (FEC) and a two to three fold lower worm burdens suggest suckling could reduce larval establishment. Inability to detect peripheral titres of immunoglobulins supports this conclusion. An intra worm-population regulation of T. circumcincta, indicated by a pattern of greater egg-laying by a numerically smaller but physiologically better developed nematode population in suckled lambs measured in eggs 'in utero' and worm length made interpretation of FEC difficult. Suckling significantly improved weight gain and carcass weights, but early weaning did not reduce resilience to infection. In Chapter 4 (second experiment), 40 pairs of twin lambs, average age of 39 days, were either infected with the equivalent of 1000 L₃ T. circumcincta larvae d⁻¹ or not, while one twin was weaned and the other allowed to continue suckling. Necropsy was carried out on groups of five and six lambs from each of the uninfected and infected treatments, respectively, at mean age of 84, 112, and on six lambs from each group at 140 days of age. This serial slaughter allowed further confirmation of the hypothesis in Chapter 3 but also investigated the long-term effect of suckling on resistance or resilience of lambs at the trial when immune responses were anticipated to be developing. An in vitro direct larval challenge (IVDC) study, to monitor larval establishment, was carried out on tissue explants from necropsied lambs. Suckled lambs consistently showed lower FEC (P < 0.05) and worm burdens (P < 0.05) at every phase of the trial. Within the infected groups, % in vitro larval rejection suggested earlier immune responses in the weaned lambs by day 84, which was not consistent with lower worm burdens in suckled lambs but appeared similar in the subsequent necropsies. Lambs continued to show better growth due to suckling while weaning did not reduce the resilience of lambs confirming observations in Chapter 3. The immunoglobulin profile suggested the commencement of immune responses in lambs from the period after the 84th day necropsy, with significantly greater (P < 0.01) IgA titre in the infected groups, and the suckled lambs towards the end of the trial on day 140. A vaccinating effect of early exposure to parasites was coincidentally revealed as a result of unintentional pasture larval contamination, seen in suckled non-infected lambs shedding fewer eggs and harbouring fewer worms during the later necropsies compared with their weaned non-infected counterparts. In Chapter 5 (third trial), 93 pairs of twin lambs, 47 pairs of which received a vaccinating mixed infection of T. circumcincta and T. colubriformis larvae (60 L₃ / kg W / d) at ratio 40:60, respectively during the period 36 – 103 days of age, were either weaned early on day 51 or later on day 108. All lambs were drenched on day 108 and groups received challenge infections from day 116, at same rate with the vaccinating infection, or not, which ceased five days before respective necropsies. Necropsies were carried out on selected lambs on days 108, 184 and 218. The direct effect of milk on larval establishment appeared to feature only in the T. circumcincta populations on slaughter day 108. The long-term benefit of late weaning for development of resistance was conditional on lambs receiving the vaccinating infection, and appeared to be more pronounced in the small intestine, reflected by a greater reduction of T. colubriformis populations in that organ than of T. circumcincta populations in the abomasum. A negative consequence of enhanced immune response was the suggestion of an increased metabolic cost in reduced performance of lambs. In conclusion, the work provides support to the hypotheses that: (a.) suckling may reduce the establishment of nematode larvae through the direct effect of milk, (b.) may enhance rapid development of host immunity to infection, and (c.) it further suggests that lack of larval experience during suckling may have long term negative implications for host resistance. Finally, it suggests that milk may play little role in the enhancement of host resilience to infection and, on the contrary, that additional metabolic cost may be associated with a more rapid development of immunity resulting from larval challenge while suckling.

Two studies were conducted to investigate anthelmintic resistance in goat parasites in New Zealand. In Study 1 parasites from goats on a farm with a long history of problems with anthelmintic efficacy were used to infect sheep for a controlled slaughter study. Nineteen lambs were acquired, effectively drenched and housed. Each was infected with a mixture of larvae comprising Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis and Oesophagostomum venulosum. After 28 days lambs were restrictively randomised into 3 groups based on faecal egg counts. Group 1 was left untreated (n=6), Group 2 (n=6) was given a single dose of abamectin (0.2mg/kg) + levamisole HCL (8mg/kg) + oxfendazole (4.5mg/kg) (“Matrix Oral Drench for Sheep”®, Ancare, New Zealand) and Group 3 (n=7) was treated at twice the dose rate of Group 2. Fourteen days after treatment all animals were killed for total worm counts. The mean burdens of T. circumcincta in Group 1 was 337, in Group 2 was 68 (efficacy 80%) and in Group 3 was 10 (efficacy 97%). The mean burdens of T. colubriformis in Group 1 was 375, in Group 2 was 220 (efficacy 41%) and in Group 3 was 81 (efficacy 78%). Although the worm burdens in these lambs were low, all animals were infected with each of these two species except for T. circumcincta in Group 3 where only 3 lambs were infected. Efficacy against other species was 100%. These results clearly indicate that a single dose of a combination drench was ineffective against two species and even when a double dose was used the efficacy against T. colubriformis was only 78%. In Study 2 a survey of drench efficacy was conducted on 17 goat farms using the DrenchRite® larval development assay. Evidence of concurrent resistance to benzimidazoles, levamisole and ivermectin was detected in T. colubriformis and T. circumcincta on 11/17 and 3/14 respectively. Only 5 of 14 farms had previously undertaken some form of testing for drench resistance prior to this survey. Evidence from these two studies suggests that severe anthelmintic resistance is common on goat farms in New Zealand