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Project objectives were to develop an improved plating medium to achieve reproducible colony counts and accurate quantification for growth or survival of Spirillum volutans; and to develop an improved method for selection of S. volutans from nature. Growth enhancement studies began with casein hydrolysate-succinate-salts medium (CHSS). Casein hydrolysate concentration, type of casein hydrolysate, pH, and phosphate concentration optima were determined. Growth of S. volutans doubled after 24 h with the addition of potassium phosphate buffer (1 mM final concentration) to CHSS medium (CHSS-P). Addition of supplements to CHSS-P medium, and modified Bordetella and Brucella media failed to increase the cell yields of S. volutans. Punctiform colonies did form in CHSS-P medium (0.7% agar) pour-plates, yet at a recovery rate much lower than reported by Padgett et at. (1982) for surface growth on spread plates. The role of amino compounds as possible growth factors was investigated. Thin-layer chromatography, gas chromatography, and amino acid analysis were employed to identify those amino compounds (if any) that decreased or disappeared in the medium after 48 h of growth. None could be found. Numbers of viable cells were not greatly increased, however, viability was prolonged by growing S. volutans in dialysis sacs suspended in CHSS-P medium. The tolerances of S. volutans to several antimicrobial compounds were determined. Attempts at using hay infusion, Pringsheim, and CHSS-P media supplemented with various compounds as selective agents did not result in predominance of S. volutans. / M.S.

When estimating the degree of oxygen tolerance of Campylobacter spp. on Brucella medium, the brand of tryptone (pancreatic digest of casein) used in the medium greatly influenced the results. In fact, with some brands and batches of tryptone, C. jejuni could grow at 21 % O₂ without any additional scavengers of reactive oxygen intermediates (ROIs), although CO₂ was still required for growth. When the medium was prepared with other brands of tryptone, it did not allow growth of C. jejuni beyond 10% O₂. The dependence of the growth response on different types and brands of tryptone-based media makes it impossible to achieve reproducibility in oxygen tolerance studies. To eliminate such variation in complex media, we developed a chemically defined medium for Campylobacter spp. This medium allows reproducible colony counts to be obtained. The medium was used to assess the effect of ROI scavengers on the oxygen tolerance of various Campylobacter species. Allopurinol, azelaic acid, caffeine, cimetidine, and pyruvate when used singly were the most effective in enhancing oxygen tolerance. When ROI scavengers were combined with dimethyl sulfoxide, the effects of allopurinol, azelaic acid, caffeine, cimetidine, and pyruvate were even more pronounced than when they were used alone. A combination of tetramethylpiperidine-1-oxyl (1EMPOL), a superoxide dis mutase mimic, with pyruvate also enhanced oxygen tolerance effectively. A survey of the literature dealing with the types of ROIs destroyed by scavengers used in our study suggests that hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH.) are the most toxic ROIs for Campylobacter species. / Master of Science

The relationships of the fourteen (14) species of Campylobacter were derived by comparison of the partial 16S ribosomal RNA sequences determined using reverse transcriptase and oligonucleotide primers specific for regions in the 16S rRNA molecule. These species formed three distinct RNA sequence homology groups. We propose that the following species remain in the genus Campylobacter (homology group I): C. coli, C. jejuni, C. laridis, C. fetus, C. hyointestinalis, C. concisus, C. mucosalis, C. sputorum and the "catalase-negative to weakly-positive" (CNW) strains. C. pylori, "C. cinaedi" and "C. fennelliae'' were not related to the true campylobacters at the genus level. However, they were related to each other and to Wolinella succinogenes at the genus level and constituted homology group II. These four species should be reclassified and placed within a single genus based on 16S rRNA sequence similarity. C. cryaerophila and C. nitrofigilis also exhibited a high level of RNA sequence homology with each other but not with any other species tested; they constituted homology group III and should be considered as being a single genus. A comparison of the 165 rRNA sequence data from the three homology groups with the same sequences from representatives of the three major phylogenetic branches of the purple bacteria indicated that these genera form a single phylogenetic branch which is only distantly related to the purple bacteria. / Master of Science

Using the chemically defined medium designed by Smibert (1963), the stimulatory or inhibitory effect of test compounds on the growth and the production of acid precipitable material by seven strains of vibrio fetus was determined. The test compounds were Krebs cycle intermediates, lactate, pyruvate, fatty acid, amines, surface active agents, aminobutyrate, hydroxybutyrate, glutathione, asparagine, phenylacetyl chloride, and sodium bicarbonate. Experimental media consisted of the chemically defined medium containing various concentrations of the test compounds. An exact number of cells of each strain of V. fetus was inoculated into tubes of experimental broth media. The cultures were incubated at 37 °C in desiccator jars in an atmosphere containing 85% nitrogen, 10% carbon dioxide, and 5% oxygen. Cultures were observed daily for visible growth. After five days incubation, the optical density of each culture was read at 5/+0 mμ; in a spectrophotometer. The concentration of APM produced by each strain of V. fetus grown in experimental media was also determined. Five day old cultures of each strain were centrifuged, and the APM present in the supernatant fluid was precipitated with trichloroacetic acid. The optical density of the mixture was read at 380 mμ, in the spectrophotometer. Lactate increased the growth of four of five strains of V. fetus tested, and did not inhibit the growth of any strains. Lactate increased APM production of V. fetus due to the increased number of cells produced in its presence. Therefore, the addition of lactate to the 8 chemically defined mediums used for growth or APM production would be advantageous. Citrate and succinate, which increased the growth of V. fetus without affecting APM production, could be added to the chemically defined medium used for obtaining maximal growth of vibrios. Studies requiring a large cell crop of vibrios would be facilitated by the use of the chemically defined medium to which lactate, citrate, and/or succinate had been added. A large cell crop of V. fetus would be useful in preparation of V. fetus cellular antigen. Any study requiring large cell crops cf vibrios would be facilitated by the use of a medium which increased the growth of V. fetus. Alpha-ketoglutarate and tween 80 increased APM production of V. fetus, although these compounds neither increased nor decreased the growth of the vibrios. Therefore, the addition of α-ketoglutarate and tween 80 to the medium used for maximal production of APM would be advantageous. An increased production of APM by V. fetus would aid the study of the biosynthesis of APM. Also, the antigenic nature of APM could be studied more successfully by using a medium which increased the production of APM by vibrios. Compounds, such as glutathione and tergitol 7, which inhibited the growth and the production of APM of vibrios should not be added to the chemically defined medium. Glutamine and asparagine inhibited the growth of V. fetus and should also be avoided as additions to the medium. / Master of Science

Salmonella and Campylobacter are the leading causes of bacterial foodborne illness in the United States. Commonly found in the gastrointestinal tract of poultry, Salmonella and Campylobacter may show little to no signs of infection in birds. The objective of this dissertation was to evaluate the influence on mRNA abundance of nutrient transporters and host defense peptides (HDPs) during a Salmonella or a Campylobacter challenge in young commercial broilers. Comparisons were made between non-challenged and challenged (106, 107, or 108 colony forming units of Salmonella or Campylobacter) broilers on expression of nutrient transporters and host defense peptides in the duodenum, jejunum, ileum and cecum at various days after inoculation. During a Salmonella challenge, changes in mRNA abundance of nutrient transporters and avian beta-defensins (AvBD) vary by day, tissue and challenge dose. ZnT1 may play an important role during a Salmonella challenge as mRNA abundance of ZnT1 significantly increased (P<0.05) by day 7 in the 108 group compared to the control. Early changes in LEAP2 mRNA abundance were observed in the 106 group than the 107 and 108 groups. However, at a later time point post challenge, a lower abundance of almost all AvBD mRNA (P<0.05) was observed in the lower gastrointestinal tract especially in the 107 and 108 groups compared to the control group, indicating that the pathogen may be influencing intestinal expression of AvBD mRNA. In Campylobacter, analyses revealed that expression of zinc transporter 1 (ZnT1) increased (P<0.05) in the duodenum, ileum and ceca in the 106 group on day 7. An increase (P<0.05) in the expression of avian beta-defensins were observed on day 14 in the ileum and ceca in the 106 group compared to the control group. Pathogens like Salmonella and Campylobacter may have an influence on the mRNA abundance of nutrient transporters and HDPs. Manipulation of these genes may ensure the survivability of these pathogens. Through sequestration of nutrients, the pathogen would have the ability to colonize the host and replicate. However, it must evade the host immune system as well. The processing of infected poultry with these pathogens may lead to foodborne illness in humans. Further research is needed to investigate possible methods to counter the influence these pathogens have on host immunity genes. / Ph. D.

Campylobacter spp. have been isolated from live poultry, production environment, processing facility, and raw poultry products. The detection of Campylobacter using both quantitative and qualitative techniques would provide a more accurate assessment of pre- or post harvest contamination. Environmental sampling in a poultry grow-out house, combined with carcass rinse sampling from the same flock may provide a relative assessment of Campylobacter contamination and transmission. Air samples, fecal/litter samples, and feed pan/drink line samples were collected from four commercial chicken grow-out houses. Birds from the sampled house were the first flock slaughtered the following day, and were sampled by post-chill carcass rinses. Quantitative (direct plating) and qualitative (direct plating after enrichment step) detection methods were used to determine Campylobacter contamination in each environmental sample and carcass rinse. Campylobacter, from post-enrichment samples, was detected from 27% (32/120) of house environmental samples and 37.5% (45/120) of carcass rinse samples. All sample types from each house included at least one positive sample except the house 2 air samples. Samples from house 1 and associated carcass rinses accounted for the highest total of Campylobacter positives (29/60). The fewest number of Campylobacter positives, based on both house environmental (4/30) and carcass rinse samples (8/30) were detected from flock B. Environmental sampling techniques provide a non-invasive and efficient way to test for foodborne pathogens. Correlating qualitative or quantitative Campylobacter levels from house and plant samples may enable the scheduled processing of flocks with lower pathogen incidence or concentrations, as a way to reduce post-slaughter pathogen transmission. / Master of Science in Life Sciences