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&lt0.001) 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&gt0.05). 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&lt0.01). 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&lt0.05) 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&lt0.01 for both). In serial biopsy tissue, immuno-suppression did prevent a rise in tissue IgA that was apparent in CIF animals (p&lt0.01) 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.

Orientador: Luis Octavio Regasini / Resumo: Um dos maiores problemas sanitários encontrados pela pecuária são os nematoides gastrintestinais (NGIs), os quais causam perdas significativas aos pecuaristas de pequenos ruminantes. O controle de NGIs é realizado com base na administração repetida e imprópria dos anti-helmínticos (AHs) convencionais, havendo uma crescente presença de resíduos no meio ambiente ou em produtos de consumo. Acima de tudo, o desenvolvimento de resistência em populações de NGIs tornou-se um problema mundial. A fim de preservar a saúde dos hospedeiros, bem como retardar a resistência, torna-se necessário identificar métodos alternativos ao uso de AHs, tais como o uso de plantas, seus extratos e substâncias. Pterogyne nitens Tul. (Fabaceae), árvore nativa brasileira, apresenta indicações de uso popular como vermífugo, bem como de diversas substâncias bioativas, tais como: ácidos fenólicos, flavonoides, alcaloides guanidínicos, terpenos e esteróis. O presente estudo teve como objetivo investigar os efeitos de extratos e substâncias de P. nitens em diferentes estágios do ciclo de vida de três isolados susceptíveis originários do Brasil e da França de duas espécies de NGIs, Haemonchus contortus e Trichostrongylus colubriformis. Foram preparados três extratos etanólicos a partir das folhas (EEL), frutos (EEFR) e flores (EEFL); e oito substâncias fenólicas isoladas [duas flavonas (sorbifolina e pedalitina), dois flavonóis (quercetina e rutina), um flavan-3-ol (ourateacatequina) e três ácidos fenólicos (ác... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: One of the considerable health problems encountered by livestock is gastrointestinal nematodes (GINs), as they cause significant losses to small ruminant breeders. The control of GINs is based on the repeated and improper administration of synthetic anthelmintics (AHs), causing an increase of environmental residues and in food. Furthermore, the development of resistance in GIN populations has become a worldwide problem. In order to preserve the health of the hosts, as well as reduce resistance, it became necessary to identify AH alternative methods, such as the use of medicinal plants and their extracts and compounds. Pterogyne nitens Tul. (Fabaceae), brazilian native tree, presents popular use as a vermifuge and several bioactive compounds, such as phenolic acids, flavonoids, guanidine alkaloids, terpenes, and sterols. The aim of the current study was to investigate the effects of extracts and compounds from P. nitens against the different stages of the life cycle of three susceptible isolates from Brazil and France of two GIN species, Haemonchus contortus, and Trichostrongylus colubriformis. Three ethanolic extracts were prepared from leaves (EEL), fruits (EEFR), and flowers (EEFL). Eight phenolic compounds were isolated [two flavones (sorbifolin and pedalitin), two flavonols (quercetin and rutin), one flavan-3-ol (ouratecatechin) and three phenolic acids (caffeic acid, ferulic acid, and gallic acid)]. In addition, two flavonoids (chrysin and morin) were obtained commercial... (Complete abstract click electronic access below) / Doutor

This study was stimulated by a publication of Mr Mike Bosch (Bosch 2007) that Cereus jamacaru DC (Cactaceae) used by him on his farm is effective for gastrointestinal nematode control in livestock. This plant, widely known as Queen of the night, is a serious invasive weed. We evaluated this claim in in vitro assays and in vivo experiments in sheep experimentally infected with Haemonchus contortus and Trichostrongylus colubriformis. This is the first report of such an anthelmintic trial in livestock involving C. jamacaru. The first study was to repeat the farmer’s work under strictly controlled conditions. In vivo studies were conducted to determine the possible direct anthelmintic effects of C. Jamacaru on ovine gastrointestinal nematodes. Eighteen sheep were allocated to three groups and were infected with 4000 H. Contortus and 6000 T. Colubriformis infective larvae given in 3 divided doses over a period of three days. From day 0 until day 49 of the experiment, sheep were drenched once a week with fresh blended C. jamacaru plants with the core removed at the same (32.33 g/sheep) or double the dose (64.66 g/sheep) used by Mr Bosch. No negative effects of the double dose were observed during the period of the experiment. Faeces were collected twice a week for faecal egg count. Based on the in vivo experiments, C. Jamacaru was not effective in reducing H. Contortus and T. Colubriformis in sheep to the 70% reduction levels of the control treatments considered to be a useful reduction in FEC. Nevertheless, its in vivo activity was substantial at the higher dose and reduced the FECs by 65%. To investigate the matter further some additional experiments were carried out. Specimens of C. Jamacaru were collected and dried in the shade. For the phytochemical analysis and in vitro experiments, the dried material was milled to a fine powder, it was extracted with acetone and five fractions (hexane, butanol, water, chloroform and 35% water in methanol) were obtained by solvent-solvent fractionation. The chemical composition of the fractions and crude extract was analysed by thin layer chromatography using three solvent systems of varying polarity and pH. To detect the separated compounds, vanillin-sulphuric acid-methanol was sprayed on the chromatograms and heated at 110°C for optimal colour development. The antioxidant activity in plant extracts may influence the immune systems of animals and have an indirect effect. The antioxidant activity was therefore determined. For qualitative analysis of antioxidant activity, the 2,2, diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates was used as a test for the radical scavenging ability of the compounds present in the different extracts. In the DPPH qualitative analysis of antioxidant activity there were a number of antioxidant compounds present in some of the extracts and fractions but the activity appeared to be low. This observation was confirmed in the TEAC quantitative analysis of antioxidant activity. Even the fraction with the highest activity was about 8 times less active than trolox or ascorbic acid. It therefore appears that stimulation of the immune system by antioxidant activity does not explain the results found on the farm. The antibacterial and antifungal activities were determined against 4 bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) and 3 fungi (Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans). The minimum inhibitory concentration (MIC) values for plant extracts varied from 0.04 to 2.5 mg/ml. E. coli was the most sensitive and the fungi were in general resistant to the extract and fractions. In general the activity was not very good, especially if the dosage in an aqueous system on the farm is considered. Antimicrobial activity also does not explain the results obtained. The nematocidal activity was evaluated by an egg hatch assay and larval development viability assay (LDVA) in vitro. The acetone extract inhibited egg hatchability and killed infective larvae of H. Contortus nematodes of sheep in a concentration-dependent manner. The percentage inhibition of egg hatching of the acetone extract and the butanol, chloroform, 35% water in methanol, hexane and water solvent-solvent fractions were 100%, 100%, 94%, 91%, 9% and 16%, respectively. The percentage inhibition of larval development of the acetone extract and chloroform, 35% water in methanol, hexane and water fractions were 93%, 84%, 49%, 85%, 31%, respectively. The chloroform fraction was significantly more active than all the other fractions (p<0.05). The extracts had in vitro activity against the nematodes. The activity was however much lower than the positive control, albendazole. It is disappointing that our results do not provide an explanation for the success obtained by Mr Bosch on his farm. Even if we do not understand how it works it may be useful to prepare suitable dosages (using low level technologies adaptable to rural conditions) for use by resource-poor rural communities where C. jamacaru occurs as an invasive weed. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Les nématodes gastro-intestinaux (NGI) représentent un problème sanitaire majeur dans le monde entier en systèmes de production à l’herbe des élevages de moutons et de chèvres. La maîtrise de ces parasites est désormais compliquée par l'apparition de résistances aux anthelminthiques (AH) chez les nématodes. L'utilisation de plantes riches en tanins (PRT) comme AH non conventionnels est une des alternatives possibles au contrôle chimique de ces maladies parasitaires. Cependant, le mécanisme d'action de ces plantes sur les vers adultes demeure inconnu. Cet objectif a constitué le premier but de cette étude en particulier en essayant de déterminer quelle part jouent les effets directs et indirects dans l’action des PRT sur les populations de Nématodes adultes dans le tractus digestif des petits ruminants. Un effet direct correspond à une action de type pharmacologique sur la biologie, la structure et l’ultrastructure des nématodes liés à des composés biochimiques présents dans les PRT. L'hypothèse d’un effet indirect correspondrait une modification de la réponse cellulaire dans les muqueuses gastro-intestinales de l'hôte, affectant la biologie des NGI. Cette thèse est divisée en deux parties: 1) Deux expériences in vivo réalisées respectivement, au Mexique et en France, visant à déterminer les effets directs ou indirects sur la biologie des NGI liés à la consommation de PRT tel le tzalam (Lysiloma latisiliquum), une Légumineuse arbustive du Yucatan, le sainfoin (Onobrychis viciifolia) une Légumineuse fourragère tempérée, et ainsi que le quebracho(Schinopsis spp.), un extrait riche en tannins condensés Les agneaux ont été infestés par Haemonchus contortus (au Mexique) ou H. contortus et Trichostrongylus colubriformis (en France). La consommation de tzalam a provoqué une perturbation de croissance des vers ou de fertilité des femelles d’H. contortus. Selon la durée de consommation, le quebracho a réduit la population de H. contortus et T. colubriformis, et affecte également la fertilité de T. colubriformis. Le sainfoin a affecté la fécondité de H. contortus sans affecter celle de T. colubriformis. Le comptage de cellules effectrices dans les muqueuses pour évaluer l’implication d’un effet indirect n'a pas montré de différences significatives entre les lots recevant ou non des PRT. La seconde partie s’est fondée sur un test in vitro et deux essais in vivo chez des chèvres infestées par H. contortus pour mieux comprendre l'effet de la consommation de PRT (tzalam, au Mexique ou sainfoin, en France) sur la structure et l’ultrastructure d’H. contortus. Les principales altérations ont été trouvées dans la cuticule et la région céphalique des vers. Des agrégats de matériel végétal autour de la vulve n’ont été trouvé qu’in vitro. L’examen de l’ultrastructure des H. contortus a montré des signes de vacuolisation, surtout visible dans les tissus intestinaux et musculaires. Ces lésions suggèrent que des composés secondaires présents dans le feuillage des PRT peuvent être impliqués dans les fonctions vitales d’ H. contortus, telles que la mobilité, la nutrition et éventuellement la reproduction. / Gastrointestinal nematodes (GIN) represent a major health problem worldwide in grazing sheep and goat production systems. The control of these parasites has been complicated by the emergence of nematodes which are resistant to the commercially available anthelmintics (AHs). The use of tannin rich plants (TRP), as non-conventional Ahs, represents an alternative for the control of these parasites. However, the mechanism of action of such plants against adult populations of GIN has not been determined. The objective of the study was to determine the direct and indirect effect of TRP against adult populations of GIN in small ruminants. A direct effect is considered to be any action against the biology, structure and/or ultrastructure of the nematodes which is similar to a chemical AH. The indirect effect is a modification of the immune response of the host at the level of the gastrointestinal mucosa which affects the biology of the GIN. This study is divided into two stages: First stage) Two in vivo experiments conducted in Mexico and France respectively, determined the direct and indirect effects on the biology of GIN due to the consumption of the TRP forage of tzalam (Lysiloma latisiliquum), sainfoin (Onobrychis viciifolia) as well as a tannin-rich quebracho extract (Schinopsis spp). Lambs were artificially infected with Haemonchus contortus (Mexico) or H. contortus and Trichostrongylus colubriformis (France). The consumption of tzalam affected the length and fertility of H. contortus females. Meanwhile, the quebracho extract reduced the population of H. contortus and T. colubriformis also affecting the fecundity of T. colubriformis. The sainfoin affected the fecundity of H. contortus without affecting T. colubriformis. The indirect effect was not evident. Second stage) An in vitro assay and two in vivo experiments with goats infected with H. contortus allowed to identify the effect of the consumption of TRP (tzalam or sainfoin) on the structure and ultrastructure of H. contortus. Alterations in cuticle and cephalic region were found. The aggregates in the vulva were only found in vitro. The ultrastructure of H. contortus showed a vacuolization process in the intestinal and muscular tissues of these nematodes. The lesions suggest that the compounds contained in the foliage of TRP may be involved in vital functions of H. contortus such as mobility, nutrition and possibly reproduction. This study is a contribution towards the understanding the mechanisms of AH action of the TRP against GIN.

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.

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.

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

A reduction in voluntary feed intake is a major factor in the lost productivity of grazing lambs infected by gastrointestinal parasites yet the mechanisms involved are poorly understood. Potential pathways involved in parasite-induced feed intake depression were investigated in lambs with minimal previous exposure to parasites and artificially infected by the small intestinal parasite Trichostrongylus colubriformis. Six in vivo experiments were conducted on lambs housed in individual pens or metabolism crates with similar feeding and experimental procedures. In Experiment 1 (Chapter 4) the effect of T. colubriformis infection on short term feed intake in lambs and of some pharmacological agents on feed intake depression were investigated. Prior to and for the duration of infection, lambs were fed once per day and feed intake recorded at regular intervals over the day (8 h). Following the onset of feed intake depression in the infected group (9 weeks after commencing dosing), all animals were treated with an analgesic (codeine phosphate per os), an anti-inflammatory agent (indomethacin per os), a CCK antagonist (L364-718 by subcutaneous injection) or saline (control) in a replicated Latin square design (n = 8). Although the pattern of feed consumption was similar in infected and non-infected lambs, average daily intake was reduced 32 % and short term intake (recorded at 10 minute intervals for the first hour of feeding, 15 minute intervals for the second hour and hourly for the next 6 hours of feeding) reduced 40 % by infection. This identified the key component by which intake was depressed and enabled the use of a short term intake model and short duration of action compounds to identify the pathways involved in intake depression in this sequence of experiments. None of the pharmacological treatments increased intake in the infected group. These results suggest a reduction in the rate of consumption due to reduced hunger signals, rather than change of meal eating patterns, is the major cause of feed intake depression. Specific conclusions about the pathways investigated using the pharmacological agents could not be obtained. Experiment 2 (Chapter 5) was designed to investigate the roles of pain and osmolality on feed intake depression. Digesta samples collected prior to and during parasite infection and before and after feeding had similar osmolalities (240-260 mosmol/l) which indicated that feeding or infection had no effect on osmolality of digesta. Following the onset of feed intake depression in infected animals, all animals were treated in a Latin square design (n = 4) with no treatment, saline, local anaesthetic (xylocaine) or analgesic (codeine phosphate) solution 15 minutes before feeding, by slow injection into the duodenum. There was no effect of these treatments on food intake. In the second part of the experiment, hyperosmotic solutions (mannitol and NaCI) markedly depressed short term intake in non-infected animals, suggesting a role for osmoreceptors in intake regulation. However these effects were not blocked by local anaesthetic so the depressed intake may have resulted from generalised malaise rather than from specific osmoreceptor effects. In Experiment 3 (Chapter 6) the role of peripheral CCK on intake depression was examined by a dose-response study utilising the CCK antagonist, loxiglumide. Intravenous injection of 5, 10 or 20 mg/kg LW of loxiglumide to infected lambs 10-15 minutes before feeding (n = 6) had no effect on feed intake at any of the dose levels. In experiment 4 (Chapter 7) loxiglumide was infused intravenously for 10 minutes (30 mg/kg/h) before feeding and for the first 2 h (10 mg/kg/h) after feed was offered to minimise any effect of the rate of clearance of loxiglumide on the lack of feed intake response. As well, the rate of marker disappearance from the abomasum was recorded in both infected and non-infected animals. Continuous infusion of loxiglumide did not attenuate parasite induced intake depression nor did it have any effect on abomasal emptying. Abomasal volume was reduced by infection (66.3 vs 162 ml) as was the fractional outflow rate (2.2 vs 2.8 ml/min) but these differences were accounted for by the lower level of feed intake in the infected animals. In Experiment 5 (Chapter 8) brotizolam, a benzodiazepine appetite stimulant, thought to act on the hypothalamus, was administered in a dose-response study to infected and non-infected animals (n = 4) immediately prior to feeding or following termination of the first meal (45 minutes after feeding) and the feed intake response recorded. Brotizolam elevated both the short term (0-0.75 h), daily (22 h) intake and all time intervals in the first 5 h after feeding in infected and non-infected animals when administered after the first meal but when administered prior to feeding elevated intake only over the first 6 h of feeding. In both cases the magnitude of the response was greater in infected animals than in non-infected animals. Brotizolam appeared to increase the rate of eating without having a major impact on meal eating patterns when administered before feeding. Where administration was after the first meal, the effect was due to an "extra" meal being consumed. These findings showed that infected animals can respond to central stimulators of intake although the mechanism of the response is not known. Opioids were implicated in intake depression as the rate of intake rather than meal patterns appeared to be the major parameter depressed under parasitism. This was examined in experiment 6 (Chapter 9) where animals (n = 6) were fasted for 26 h or not fasted, then treated with saline (control), brotizolam (intake stimulant) or naloxone (opioid antagonist) immediately prior to feeding. Fasting stimulated feed intake in the short term (100 % increase in 75 min) and over the day (12 % increase) in both infected and non-infected animals. Following fasting, infected animals ate a similar amount of feed to the non-infected, fasted animals and more than the non-infected, non-fasted animals. The signals resulting from a one day fast were sufficient in the short term to override parasite induced mechanisms causing feed intake depression. Naloxone suppressed the intake stimulatory effects of a 26 h fast in both infected and non-infected animals, which supports a role for endogenous opioids as hunger signals. Where animals were not fasted, naloxone reduced intake only in the non-infected animals which suggested endogenous opioid levels may be lower in infected animals than in non-infected animals. In the final experiment (Experiment 7, Chapter 10) the role of central hunger and satiety mechanism were investigated. Infected and non-infected animals (n = 6) were treated with naloxone or saline by intravenous injection, or saline and met-enkephalinamide (an opioid analogue) by intracerebral infusion, or naloxone and the opioid analogue simultaneously to investigate the role of central opioids in feed intake depression. To determine the role of CCK induced satiety signals on feed intake at a central level, loxiglumide and CCK were infused separately and in combination for 30 minutes prior to feeding and for the first 60 minutes of feed on offer, into a lateral cerebral ventricle of the brain of infected and control animals (n = 6). The opioid analogue tended to increase intake in infected animals but the effect was not significant probably because the dose used was too low to elicit a response in sheep. Naloxone depressed intake only in the infected animals, which conflicted with the results of Experiment 4. As a consequence these results were inconclusive because of the single low dose of opioid analogue used and the conflicting naloxone responses. CCK alone depressed intake by 39-52 % only in infected animals and this effect of the 90 minute infusion was evident over the 8 h short term recording period. Loxiglumide attenuated the feed intake depressive effects of CCK in the infected animals to the extent that intake was elevated above control levels. Loxiglumide alone was an intake stimulant in both infected and non-infected animals. Intake was increased over the entire 8 h but mostly in the second hour when intake was increased by 188 % in infected animals and by 16 % in the non-infected animals and resulted in almost continuous eating. These results showed loxiglumide will temporarily block the effect of parasite infection on feed intake in sheep when administered centrally and the fact that it blocked the effects of exogenous CCK on intake indicated that the effect is mediated via CCK receptors. In conclusion GIT parasite infection reduced both short term and daily feed intake apparently by a change in rate of intake rather than any alteration in meal patterns. It was further suggested that anyone of a number of potential peripheral pathways, including changes to osmolality, gut emptying, pain and inflammation of the gut, alone is not involved in anorexia in sofar as the compounds used could block these factors and the results support the idea that intake depression is mediated via a central mechanism. Intake in infected animals responded to a much greater extent when fasting, i.c.v. loxiglumide or brotizolam were employed. Feed intake thus appears to be regulated through the same mechanisms in infected and non-infected animals. The results from compounds affecting the central mechanism suggest central CCK receptors are important in parasite induced anorexia, possibly by changing the onset of satiety or by interacting with endogenous opioids to reduce the rate of feed intake. Secondly reduced endogenous opioids may be causing the reduction in the rate of feed consumption alone or as a result of other interactions. It was concluded that intake in parasitised animals could be increased to that of control animals by employing procedures and compounds thought to act on the hypothalamus.

Les nématodes gastrointestinaux (NGIs) représentent une contrainte majeure en élevage des ruminants. Jusqu’à présent, la maîtrise de ces parasitoses a reposé essentiellement sur l’emploi répété de traitements anthelminthiques (AH) de synthèse. Cependant, le développement constant de résistances aux AH dans les populations de nématodes chez les petits ruminants conduit à rechercher des méthodes alternatives ou complémentaires de lutte contre ces parasitoses. Au cours des deux dernières décennies, les données se sont accumulées montrant que certaines plantes étaient dotées de propriétés anthelminthiques, ce qui a été associé à la présence de tannins condensés (TCs) et de flavonoïdes. Ces ressources contenant des TCs, exploitées comme nutricaments, représentent donc une alternative attractive à la chimiothérapie, en combinant des effets bénéfique sur la santé et la nutrition des ruminants. Toutefois, une des difficultés à résoudre, pour un usage pertinent de ces nutricaments, est de comprendre l’origine de la variabilité des résultats observés dans l’activité AH. A côté de facteurs quantitatifs, la qualité (la structure) des TC semble également jouer un rôle dans les propriétés antiparasitaires mais ce facteur demeure peu exploré. En utilisant Haemonchus contortus comme modèle expérimental de nématode et le sainfoin (Onobrychis viciifolia) comme modèle de plantes contenant des tannins, l’objectif général de cette thèse a été de mieux comprendre les relations structure/activité des TCs. Les objectifs spécifiques des diverses études menées ont été 1) d’ examiner in vitro (par la méthode LEIA) l’activité AH de 36 fractions purifiées de TCs présentant une large diversité de caractéristiques structurelles [ex: la taille (mDP), les proportions prodelphinidols /procyanidols (PD/PC) et trans/cis ], et 2) le rôle possible d’interactions entre tannins et flavonoïdes, 3) d’ évaluer et comparer in vivo chez des agneaux, les conséquences sur les populations de vers et sur la résilience de l’hôte de la distribution de 2 ressources contenant des TCs de qualité contrastée pour les valeurs PD/PC [fort PD/PC, sainfoin (O. viciifolia); bas PD/PC, pellicules de noisettes (Corylus avellana)], 4) d’ examiner si les résultats varient en fonction de l’espèce et de la localisation des parasites (abomasum, H. contortus vs intestin grêle, Trichostrongylus colubriformis), 5) d’évaluer les concentrations, la qualité et la bio-disponibilité des TCs le long du tube digestif chez les ovins. Les résultats acquis ont montré: 1) des relations in vitro entre certaines caractéristiques des TCs et l’activité AH : un rapport élevé PD/PC pour les 2 espèces de nématodes auquel s’ajoute un rôle de la taille des tanins (mDP) pour H. contortus sont les facteurs identifiés. De manière générale, H. contortus s’est avéré plus sensible (valeurs EC50 plus faibles) aux effets des diverses ressources que T. colubriformis. Des synergies d’effets AH ont été observés entre tannins et flavonoides (quercétine et lutéoline), tout particulièrement dans le cas de TCs avec un faible rapport PD/PC. Dans l’étude in vivo, les agneaux consommant du sainfoin ont montré des effets AHs et une amélioration de la résilience de l’hôte alors que les effets de la noisette ont été beaucoup plus discrets. Les analyses menées sur les contenus des divers organes digestifs et les fèces, pour les deux types de ressources ont montré de fortes réductions de TCs détectés par rapport aux teneurs initiales dans la ration alors que les caractéristiques de structure étaient globalement préservées au long du tube digestif. En conclusion, le rôle de la qualité (structure) des TCs dans l’activité AH a été confirmé à la fois par les études in vitro et in vivo. Ces résultats suggèrent l’importance de prendre en compte ces facteurs dans la recherche de ressources pouvant être exploitées comme futurs nutricaments chez les ruminants. / Gastrointestinal nematodes (GINs) represent a major threat for the breeding and production of grazing ruminants. So far, their control has been based mainly on the repeated use of synthetic AH drugs. However, worm populations in small ruminants have consistently developed resistance against all AH drugs. In the search for alternative solutions to such drug treatments, the last two decades have provided evidence that some plants possess natural AH bioactivity. This has been related to the presence of condensed tannins (CTs) and flavonoids. Therefore, CT-containing resources represent a promising alternative to chemotherapy especially when used as nutraceuticals that combine beneficial effects on health and nutrition in small and large ruminants. However, one of the main constraint to use these nutraceuticals relates to the variations in AH results. The structural features (quality) that contribute to the AH activities of CTs remain elusive. By using the GIN Haemonchus contortus as a experimental model of nematode and Onobrychis viciifolia as a model of CT-containing plant, the general aim of this PhD was to better understand the structure/activity relationship of CT. The specific objectives were i) to examine in vitro (LEIA) the AH activity of 36 purified CT fractions with a wide range of structural features [i.e. size (mDP), prodelphinidin/procyanidin (PD/PC) and trans/cis ratios], and ii) the possible role of interactions between tannins and flavonoids, iii) to evaluate in vivo the AH activity and host resilience of two CT-resources with contrasting PD/PC ratios (i.e. high PD/PC, O. viciifolia; low PD/PC, Corylus avellana), iv) to examine whether these results vary depending on the parasites species (abomasal, H. contortus vs intestinal, Trichostrongylus colubriformis), v) to examine the CT availability (quantity and quality) along the digestive tract in sheep. In regard of CT features and AH activity, in vitro correlations were observed between AH activity and high PD/PC for both species. mDP was an additional criteria for H. contortus. Lower EC50 values were assessed for H. contortus. Synergistic AH effects were observed between CT and flavonoids (quercetin and lutein), and they were higher with fractions of low PD/PC ratios. In vivo, the lambs consuming sainfoin pellets showed consistently higher AH effects and improvement in the host’s resilience. Analyses of digesta samples and faeces, for both diets showed large reduction of detected CT related to the feed, whereas the CT structures seem preserved along the gut. In our studies, the role of CT structural features in the AH activity was observed both in vitro and in vivo. These results confirmed the influence of CT quality in the AH effects, and underline the relevance of taking these factors into account when screening CT resources as potential nutraceuticals in small ruminants.