Browsing by Author "Flint, Stephen Harry"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Formation and control of biofilms of thermo-resistant streptococci on stainless steel : a thesis presented in partial fulfilment of the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 1998) Flint, Stephen Harry; Flint, Stephen Harry
    The aim of this study was to develop improved methods of controlling biofilms of thermo-resistant streptococci in dairy manufacturing plant. A method to rapidly and accurately detect viable cells of thermo-resistant streptococci on stainless steel surfaces involving the use of the Malthus microbiological growth analyser was developed. A modified Robbins device was designed and installed in a dairy manufacturing plant to monitor biofilm development and obtain isolates for study. These studies confirmed that routine cleaning programmes were not eliminating biofilms of thermo-resistant streptococci from the stainless steel surface. The isolates obtained were identified using biochemical and molecular techniques. As well as the expected Streptococcus thermophilus, a new species, S. waiu representing 24% of the isolates was also described. Molecular techniques (polymerase chain reaction and fluorescent in situ hybridisation) were developed to rapidly identify the bacteria. The cell surface hydrophobicity of all isolates was determined, with those obtained from dairy manufacture being highly hydrophobic compared with mixed hydrophobicity in the general population. There was no correlation between many factors often associated with adhesion (such as hydrophobicity, polysaccharide production, surface charge) and the rate of cell adhesion. However, treatment of the bacteria with proteolytic agents reduced the number of all isolates adhering to stainless steel by approximately 100-fold. A 55 kDa protein with an N-terminal sequence matching that of β-lactoglobulin was identified as being associated with adhesion, through comparisons between cell proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis before and after treatment with proteolytic agents. Further evidence of the involvement of this protein in adhesion was the reduction in adhesion following treatment of the cells with specific antiserum to the 55 kDa "adhesion protein". The presence of the protein on the surface of the cells was demonstrated by immunolabelling. A continuous flow laboratory reactor was developed to generate biofilms of thermo-resistant streptococci on stainless steel surfaces in the presence of skim milk. Trials using biofilms developed in laboratory reactors and on the surface of coupons in pilot plants, indicated that chemicals routinely used in dairy manufacturing plants were inadequate to remove or inactivate thermo-resisant streptococci. Proteolytic enzyme treatments removed more bacteria from the surface than any other treatment, reducing the total number of cells by at least 100-fold. This was confirmed in a pilot-scale trial using a commercial proteolytic-enzyme-based cleaner. In addition, no viable cells were detected following treatment with this cleaner. Proteolytic enzyme cleaners may be more effective than the caustic and acid cleaners for the routine cleaning of biofilms of thermo-resistant streptococci from dairy manufacturing plants.
  • Thumbnail Image
    Item
    A study of Leptospira interrogans infection in deer and goats in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1988) Flint, Stephen Harry
    In order to determine the prevalence of leptospirosis in deer and goats both serological tests and the culturing of bacteria from urine samples were used. The serological tests enabled an assesment to be made as to the nature and extent of antibody levels. To ensure confidence in the serological results, it was necessary to validate the standard microscopic agglutionation test (MAT). Repeat tests demonstrated reproducible results that were within one two-fold serial dilution. Enzyme linked immunosorbent assays (ELISA) were investigated as alternatives for both the detection of antibodies and Leptospira antigens. These assays demonstrated greater sensitivity than the MAT for the detection of antibodies, but were less sensitive than standard methods for the detection of antigen. Using the MAT, antibodies to australis, ballum, bratislava, copenhageni, hardjo, pomona, and tarassovi were detected in serological surveys of deer and goats. In deer, the most frequently recorded antibody titres were to ballum, bratislava and copenhageni. As 87% of the antibody titres <80, there appears to be a low level of active infection. In some areas there was a high prevalence of antibody titres to hardjo. In goats, 70% were found to have antibody titres ≥10 to one or more serovars with antibodies to ballum and bratislava the most frequently recorded. As 90% of the antibody titres were <80, there appears to be a low level of active infection in goats. Antibodies to bratislava, a serovar that has not been isolated in New Zealand, were widespread in both deer and goats. The possibility that these resulted from mixed infections was considered but not resolved. Unsucessful attempts were made to purify mixed cultures using specific antisera. The possibility of serological cross reactions of antibodies to other serovars with bratislava was supported by the increased serological response of deer and goats to vaccination with hardjo and pomona antigens. Western blot studies identified several common antigens between bratislava and pomona. A study of a deer farm showed a high prevalence of antibody titres to hardjo corresponding to a similar prevalence of antibody titres to balcanica believed to be due to the antigenic similarity of these two serovars. Balcanica was isolated from urine samples from these deer and is believed to be the first isolation of this serovar from deer. Studies of six goat, farms showed low antibody levels and no Leptospira were isolated from urine samples.