The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics.

Thesis (M. App. Sc.)--University of Adelaide, Dept. of Animal Science, 1995? / Copies of the author's previously published articles inserted. Includes bibliographical references (leaves 142-159).

Thesis (MScAgric) -- Stellenbosch University, 2004. / ENGLISH ABSTRACT: The ruminant has the ability to utilize forages more efficiently than any other production animal. The utilization of forage fibre is an important aspect of ruminant production systems, as this is the main source of energy available to the animal. The availability of high-fibre forage nutrients is, however, restricted by cell wall degradability, and since low quality forages contribute a great deal to ruminant production systems worldwide, the improvement of this degradation process is of major economic importance. The use of exogenous fibre degrading enzymes has been proposed as a means of enhancing this process, with positive results being obtained from in vitro studies incorporating exogenous enzyme preparations. Positive in vivo results with regard to forage digestibility and other animal production parameters have consequently also been obtained following the addition of exogenous fibre-degrading enzyme preparations to the ruminant diet. Two initial screening experiments were undertaken in order to identify fungal enzyme preparations that may have a positive effect on in vitro fibre degradability. The initial screening employed an in vitro organic matter digestibility technique, and was successful in identifying at least six enzyme preparations displaying enhanced digestibility results that were statistically significant. A second in vitro gas production procedure was used to confirm results obtained from organic matter digestibility assays, as well as to increase screening capacity in order to evaluate new enzyme preparations more time-efficiently. Statistical analysis of results obtained from the secondary screening identified various enzyme candidates producing promising results. Only one of these, Abo 374, proved to be statistically superior to the control and other enzyme preparations. A growth trial was subsequently conducted to assess the performance of this enzyme in vivo. The trial involved individual feeding of 32 Dohne Merino ram lambs grouped according to weights into four groups consisting of 8 lambs each. Each group represented a specific application level of enzyme to the wheat straw component of a high fibre diet, amounting to 10, 5, or 1 ml enzyme supematant/kg straw. The enzyme was diluted with water at appropriate rates to obtain an application rate of 300ml/kg straw. The fourth (control) group was treated with water at the same application rate. The trial was conducted over a period of six weeks, during which feed intakes, weekly weight gains, as well as feed conversion efficiencies were recorded. Results suggested significant weight gains in the high (10ml/kg) and medium (5ml/kg) treatment groups, indicated by a P-value of 0.04. Similarly, feed conversion efficiencies were improved for above-mentioned groups (P=0.05), while feed intakes did not differ significantly between the four experimental groups. / AFRIKAANSE OPSOMMING: Die herkouer besit die vermoee om ruvoere beter as enige ander produksiedier te kan benut. Die gebruik van ruvoervesel is 'n belangrike aspek van herkouer produksiesisteme, aangesien ruvoere die hoof bron van energie aan die herkouer verskaf. Die beskikbaarheid van hoe-vesel ruvoer nutriente word egter beperk deur die degradeerbaarheid van die selwand, en aangesien lae kwaliteit ruvoere 'n groot bydrae tot wereldwye herkouer-produksiesisteme maak, is die moontlike verbetering van hierdie degraderingsproses van groot ekonomiese belang. In 'n poging om hierdie verteringsproses te help bevoordeel, is die gebruik van eksogene veselverterende ensieme ondersoek, en positiewe resultate is verkry wanneer hierdie ensieme in in vitro studies gebruik is. Goeie verbeterings ten opsigte van ruvoer verteerbaarheid en ander diereproduksie parameters is ook verkry deur middel van in vivo studies waar eksogene ensieme by die ruvoer van herkouers gevoeg IS. Twee eksperimente is ondemeem in 'n poging om ensiempreparate wat 'n moontlike positiewe effek op in vitro veselvertering mag he, te identifiseer. Die eerste, 'n in vitro organiese materiaal verteerbaarheid tegniek, was suksesvol in die identifisering van minstens ses ensiem preparate wat statisties betekenisvolle verbeterings ten opsigte van verteringsresultate geproduseer het. 'n Tweede in vitro gasproduksie prosedure is vervolgens gebruik om resultate verkry vanaf die eerste tegniek, te bevestig, asook om evalueringskapasiteit te vergroot en sodoende, nuwe ensiempreparate meer tydseffektief te evalueer. Statistiese evaluering van resultate verkry uit die tweede in vitro tegniek het 'n reeks ensieme met positiewe resultate opgelewer. Een van hierdie, Ab0374, het statisties betekenisvolle resultate ten opsigte van die kontrole, sowel as ander ensieme getoon. In 'n volgende eksperiment is 'n groeiproef gedoen om die effektiwiteit van hierdie ensiem in vivo te toets. In die proef is 32 Dohne Merino ramlammers op grond van hul gewig in vier groepe van agt skape elk verdeel, en individueel gevoer. Die groepe het verskillende toedieningsvlakke van die toetsensiem, toegedien tot die koringstrooi komponent van 'n hoe-vesel dieet, ontvang. Toedieningsvlakke was 10, 5, of lml ensiemkonsentraat/kg strooi. Elke groep se ensiemkonsentraat is verdun met die toepaslike hoeveelheid water om 'n toedieningsvlak van 300ml ensiemoplossing/kg koringstrooi te verkry. 'n Vierde groep is behandel slegs met water teen dieselfde toedieningsvlak, en het gedien as 'n kontrole. Die eksperiment is oor 'n periode van 6 weke uitgevoer. Tydens die proeftydperk is voerinnames, weeklikse gewigstoenames, sowel as voeromsetverhoudings, gedokumenteer. Resultate het betekenisvolle gewigstoenames in die hoe (lOml/kg) en medium (5ml/kg) groepe opgelewer, aangedui deur 'n P-waarde van 0.04. Voeromsetverhoudinge het ook verbeteringe getoon vir bogenoemde twee groepe (P=0.05), terwyl voerinnames nie merkbaar tussen die vier groepe verskil het nie.

Copies of the author's previously published articles inserted. Bibliography: leaves 142-159. Provides information on the significance of pea straw as a ruminant feed and the potential of two biological techniques for improving its feeding value.

Call number: LD2668 .T4 1978 C38 / Master of Science

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.

A replicated 3 x 3 latin square design (3 periods and 6 rumen fistulated steers) was utilized to investigate the effect of dietary concentrate levels (30, 60 and 90%) on the in situ disappearance of dry matter (ISDMD) and neutral detergent fiber (NDF) and kinetics of fiber digestion of alfalfa hay, wheat straw and steam processed and flaked milo (SPFM). Concentrate levels of 30 and 60% did not affect (P > .05) ISDMD or NDF disappearance from any of the substrates. However, ISDMD and NDF disappearance of all substrates were depressed (P < .05) by the 90% concentrate diet. The degradation of the potentially digestible fiber in these substrates followed first order kinetics at all concentrate levels. Fiber digestion kinetics were not affected (P > .05) by dietary concentrate levels of 30 or 60%. Incubation of substrates in animals receiving the 90% concentrate diet resulted in lower potential extent of digestion (P > .05) and decreased rate of degradation (P > .05) for all substrates. However, digestion was not complete even after incubations of 168-h, and thus, it is possible that the potential extent of digestion was underestimated. For all substrates lag time of digestion appeared to increase in the 90% concentrate diet. However, influx of NDF-like material into the bag during incubation makes the validity of the lag times obtained in situ questionable. Ruminal pH was depressed in animals consuming the 90% concentrate diet and could be at least one of the factors responsible for the decreased (P > .05) fiber digestibility in this diet. Mean ruminal pH of 6.5 (30 and 60% concentrate diets) did not depress (P > .05) fiber digestibility while a mean pH of 6.2 resulted in a lower (P > .05) fiber digestibility in the rumen.

A series of studies was conducted using SEM in conjunction with chemical analysis, in situ and in vitro digestion techniques, to characterize the anatomical components from barley, corn, sorghum and wheat grains which constitute "fiber" and investigate their susceptibility to rumen microbial digestion. Fractured grains were used to identify anatomical features and cell types prior to and after extraction or digestion. Certain anatomical features, including pericarp tissue, aleurone cells, endosperm cell walls, corneous and floury endosperm tissue and lemma and palea from barley, were easily identifiable in fractured and ground grains, and in neutral detergent extracted or digested residues. In situ and in vitro incubation conditions were varied to assess the effect of concentrate and/or reduction of pH on the disappearance of identifiable grain fractions. In situ incubations were conducted using steers adapted to 0-, 30- and 90% concentrate diets. In vitro inoculum buffered at pH 7 or 6 was provided by a steer fed 0- or 90% concentrate. Tissues resistant to rumen microbial digestion during extended (144-h) in situ incubations and shorter term (12- to 48-h) in vitro incubations were primarily those identified in NDF, and included pericarp, lemma and palea, and small amounts of corneous endosperm. Remaining tissues identified included barley lemma, palea and pericarp; corn pericarp, tip cap and small amounts of corneous endosperm; sorghum pericarp and corneous endosperm with matrix and protein bodies; and wheat pericarp. In vitro disappearance of isolated NDF after 48-h ranged from 43% for barley to 89% for corn. Labile structures included embryonic tissue and portions of endosperm cell walls, protein matrix and residual starch. Resistant tissues included pericarp, aleurone cell walls, tip cap and portions of the corneous endosperm. Relative rankings of NDF digestibility under all conditions studied were similar (corn > sorghum > wheat > barley) whether determined using isolated NDF or calculated from TIVDMD residues. Neither concentrate level fed to the host animal nor pH of the in vitro incubation flask affected rankings among grains, although increasing concentrate level and/or reducing pH appeared to reduce in vitro NDF disappearance. Evaluation of electron micrographs of fractured grains suggested that similar anatomical structures in the various grains differed in their resistance to microbial digestion. For example, pericarp from barley and wheat appeared to be more resistant than that from corn or sorghum. Endosperm of barley was less resistant than that of sorghum.

No description available.

No description available.