No description available.

The intestinal spirochaete Brachyspira hyodysenteriae is the causative agent of swine dysentery (SD), a diarrhoeal disease of pigs which has significant economic impact worldwide. Controlling SD remains problematic, particularly as there is no effective vaccine and there are few definitive diagnostic methods available. In this study, a partial genomic sequence of B. hyodysenteriae was screened in silico. A total of 19 putative open reading frames (ORFs) encoding outer-membrane proteins then were selected and these were subjected to a laboratory screening process. To select potential universal vaccines, a preliminary study was conducted using PCR to determine the distribution of the putative genes in 23 strains of B. hyodysenteriae. A total of 17 of the 19 ORFs were considered to be suitable for further testing as they were found to be present in the majority of strains investigated. After molecular cloning and protein expression and purification, of 19 cloned candidate molecules derived from 17 genes (one large gene was divided into two parts encoding N and C terminal proteins, respectively), 14 were expressed in E. coli and the recombinant proteins were successfully produced. A variety of sera from pigs naturally and experimentally infected with B. hyodysenteriae were tested for reactivity with the 14 recombinant proteins in an immunoblotting assay. Seven molecules from six genes reacted strongly with the tested sera, and therefore were selected and used to immunize mice. All these proteins generated a specific antibody response. Post-immunization sera raised against each recombinant protein had the capacity to agglutinate B. hyodysenteriae cells, and also recognized the cognate proteins of B. hyodysenteriae in cell extracts. Further sequencing analysis demonstrated that these molecules were highly conserved in the genomes of different B.hyodysenteriae strains. Therefore, from the genomic based study, the products of six genes were identified as promising candidates for vaccines or as diagnostic targets. Four genes were expressed on a large scale, the product (NAV-H7, NAV-H17 Cterminal, NAV-H34 and NAV-H42) were combined into one vaccine, and then this preparation was used to immunise pigs that subsequently were challenged with B. hyodysenteriae. These antigens generate systemic and colonic antibody responses, and vaccination tended both to delay the onset of clinical signs and attenuate lesion development. Hence these recombinant proteins showed promise as components for further SD vaccines. Recombinant proteins from the selected genes also were used as antigens in class-specific ELISAs used as serological assays for SD. Three antigens (NAV-H8, NAV-H42 and Bhlp29.7) were selected as good indicators of seroconversion in IgM ELISAs, and these were evaluated further using a large range of serum samples. The NAV-H8 IgM ELISA using a cut-off value 2.5 times the mean value of all negative pigs could be used as a herd test for SD, and both the NAV-H8 and NAV-H42 IgM ELISAs had potential for detecting exposure to B. hyodysenteriae at the pig level.

Swine dysentery (SD) is a severe mucohaemorrhagic colitis resulting from infection with the anaerobic spirochaetal bacterium, Serpulina hyodysenteriae. The disease affects weaner and grower pigs throughout the world, and causes significant financial losses due to mortality, decreased rate of growth, poor feed conversion, and expense of chemotherapy. Previous studies have shown that despite the presence of S. hyodysenteriae in pigs on many farms, clinical signs of SD do not always occur. This study was aimed at investigating the effect of diet on the clinical expression of SD. The ultimate aim was to identify diets that could be used to prevent or control the disease. One hundred and seventy-eight weaner pigs were purchased from specific-pathogen free farms and fed one of 16 diets based on: cooked riceanimal protein, cooked rice-dehulled lupin, wheat-lupin, wheat-animal protein, parboiled rice dehulled lupin, parboiled rice-animal protein, and processed (hammer-milled or steam-flaked) cereal grains (barley, groats, maize, sorghum and wheat) supplemented with animal protein. Eighty four pigs on these diets were slaughtered after one month to measure the influence of the diets on parameters in the large intestine, including organ sizes, and pH, VFA concentrations and dry matter content of the digesta in the caecum, and proximal and distal colon. The cooked rice-animal protein diet caused low levels of microbial fermentation in the large intestine of pigs as indicated by higher pH values, lower VFAs, smaller intestinal organ sizes, and drier contents in the colon and rectum, compared to pigs on the other diets. A limited amount of fermentative substrates from the cooked rice-animal protein diet entered the large intestine, and this led to a low microbial fermentation activity. Pigs fed diets containing cereal grains, parboiled rice and or dehulled lupins had greater fermentative activity in the large intestine. Parboiled rice unexpectedly was not easily digestible. Of the processed cereal grain diets, steam-flaked grains resulted in significantly higher (P<0.05) intestinal pH values than hammer-milled grains. This suggested that steam-flaking process made the nutrients (most likely starch) more available for digestion in the small intestine than did the hammer-milling process. Another 94 pigs fed on the various diets were orally challenged with broth cultures of S, hyodysenteriae and were monitored for faecal excretion of spirochaetes, and for the development of SD. Diseased pigs were slaughtered immediately, and healthy pigs were slaughtered after 4-6 weeks, and changes in the large intestine were recorded. None of 16 challenged pigs fed cooked rice-animal protein developed SD and it was assumed that the reduced fermentation with this diet inhibited colonisation by S. hyodysenteriae, and expression of SD. Disease occurred in varying numbers of pigs fed all the other diets, for example cooked rice-dehulled lupin (83.3%), wheat-dehulled lupin (62.5%) and wheat-animal protein (60%). The diseased pigs developed diarrhoea with blood and mucus, were depressed, lacked appetite and showed gross and microscopic evidence of severe mucohaemorrhagic colitis. When two pigs fed the protective cooked rice-animal protein diet were transferred to the wheat-dehulled lupin diet, one died of acute clostridial enterotoxaemia, whilst the other developed SD. This provided further evidence for the protective effect of the cooked rice-animal protein diet. Of the processed cereal grain types, steam-flaked maize and steam-flaked sorghum diets containing animal protein protected all pigs against SD, although small numbers of animals were used. All cereal-based diets resulted in greater fermentation than the cooked rice-animal protein diets, but fermentation was relatively reduced with steam flaked maize. The protective rice-animal protein diet was fed to pigs on a commercial piggery with SD. It resulted in good growth rate and carcass composition, but unfortunately no disease occurred amongst the control pigs during the experiment, so its efficacy against SD in the field could not be assessed. In conclusion, all protective diets were based on cooked cereal grains which had low levels of non-starch polysaccharides and resistant starch (cooked rice, steam-flaked maize and steam-flaked sorghum) and animal protein. It appears that reducing the availability of such fermentable substrate in the large intestine prevents colonisation by S. hyodysenteriae, and protects pigs from developing SD. This is a major new paradigm for the control of this important disease.

Call number: LD2668 .T4 1984 L33 / Master of Science

No description available.

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative –10 and –35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7. The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system. An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis. Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Health Sciences. Includes bibliographical references (leaves 196-248).

Thesis (Ph.D.)--Murdoch University, 2009. / Thesis submitted to the Faculty of Creative Technologies and Media. Includes bibliographical references (leaves 196-222)

Thesis (Ph. D.)--West Virginia University, 2005 / Title from document title page. Document formatted into pages; contains ix, 210 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet. / Härtill 5 uppsatser.