Pacific shrimp (Pandalus jordani) was washed repeatedly and the eluted bacteria were enumerated and identified. Selected isolates were tested for their adhesive properties. Washing reduced the microbial load by 3.84 to 42.04%. The bacteria which most resisted wash-off were Staphylococcus and Pseudomonas spp. The easiest to wash off was Flavobacterium spp. In higher-count samples, Moraxella and/or Lactobacillus spp. washed off readily, but they still constituted large proportions of the residual bacteria on shrimp. Adhesiveness, measured by hydrophobic interaction with octane, showed 43.3% change in absorbance by Staphylococcus spp., followed by 21.5% by Moraxella spp., and Arthrobacter spp. at 13.5%. Pseudomonas spp. showed only 5.7% change in absorption. Attachment, measured by hanging glass cover slips in broth, however, showed Pseudomonas and Staphylococcus spp. to have the greatest ability to adhere, with 0.47 and 0.46% attachment, respectively. Moraxella spp. showed the least ability to adhere to glass (.02%), followed by Lactobacil lus spp. at 0.11%. Arthrobacter and Flavobacterium spp. adhered at 0.30 and 0.37% levels, respectively. Attachment of Pseudomonas spp. to glass was the least affected by media composition, temperature, or presence of a surface-active agent (sodium hexametaphosphate). Staphylococcus spp., on the other hand, attached most strongly under optimum growth conditions but were most affected by varying growth conditions, temperature, and presence of a metabolic inhibitor (sodium hexametaphosphate). This indicates that the adhesive ability of Staphylococcus spp. is directly related to its metabolic activity, while Pseudomonas spp. is less sensitive to changes in metabolism and may depend on motility for adhesion. Bacteria that could adhere strongly on solid surfaces (Pseudomonas and Staphylococcus spp.) tend to be found in greater proportions and, hence, contribute more to the spoilage of shrimp. / Graduation date: 1983

by Lap-Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 90-101). / Abstract also in Chinese. / Abstract --- p.i / Contents --- p.iv / List of tables --- p.ix / List of figures --- p.x / Abbreviations --- p.xi / Acknowledgements --- p.xii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- The biology of hepatitis A virus --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- General characteristics of HAV --- p.2 / Chapter 1.1.3 --- Stability and disinfection of HAV --- p.3 / Chapter 1.1.4 --- Molecular biology of HAV --- p.5 / Chapter 1.1.4.1 --- Genomic organization of HAV --- p.5 / Chapter 1.1.4.2 --- Antigenic sites on the capsid of HAV --- p.8 / Chapter 1.1.5 --- Laboratory diagnosis and methods of study for HAV --- p.8 / Chapter 1.1.5.1 --- Cell-culture propagation and antigen detection --- p.8 / Chapter 1.1.5.2 --- Nucleic acid detection --- p.10 / Chapter 1.1.6 --- Epidemiology of HAV --- p.12 / Chapter 1.1.6.1 --- Distribution of HAV infection --- p.12 / Chapter 1.1.6.2 --- Seasonal pattern of HAV infection --- p.13 / Chapter 1.1.6.3 --- Mode of transmission --- p.13 / Chapter 1.1.6.4 --- Molecular epidemiology --- p.15 / Chapter 1.1.7 --- Epidemiology of HAV infection in Hong Kong --- p.15 / Chapter 1.2 --- Transmission of viruses through contaminated food --- p.18 / Chapter 1.2.1 --- Active accumulation of water contaminants by shellfish --- p.20 / Chapter 1.2.2 --- Retention of viruses by shellfish in contaminate water --- p.21 / Chapter 1.2.3 --- Elimination of viruses in contaminated shellfish --- p.21 / Chapter 1.2.4 --- Indicators for contamination by enteric viruses --- p.21 / Chapter 1.3 --- Detection of viruses from foods --- p.23 / Chapter 1.3.1 --- Recovery of viruses from foods --- p.23 / Chapter 1.3.2 --- Detection of viral nucleic acid --- p.23 / Chapter 1.4 --- Objectives of the study --- p.26 / Chapter Chapter 2- --- Materials and methods / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Sera from patients with acute viral hepatitis --- p.27 / Chapter 2.1.2 --- Collection of shellfish samples --- p.28 / Chapter 2.1.3 --- Purified HAV preparations as positive control --- p.30 / Chapter 2.1.4 --- Control samples for the virion capture method --- p.30 / Chapter 2.1.5 --- Preparation of dissecting instruments and processing of shellfish samples --- p.30 / Chapter 2.1.6 --- Plasticwares and glasswares --- p.30 / Chapter 2.1.7 --- "Chemicals, reagents and commercial kits" --- p.31 / Chapter 2.1.7.1 --- Samples processing --- p.31 / Chapter 2.1.7.2 --- Reagents for RNA extractions --- p.31 / Chapter 2.1.7.3 --- Oligonucleotide primers synthesis --- p.33 / Chapter 2.1.7.4 --- Primers purification after synthesis --- p.34 / Chapter 2.1.7.5 --- Gel electrophoresis --- p.34 / Chapter 2.1.7.6 --- Reagents for hybridization --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Samples processing --- p.37 / Chapter 2.2.2 --- Artificially seeded HAV in shellfish --- p.37 / Chapter 2.2.3 --- RNA extraction methods --- p.37 / Chapter 2.2.3.1 --- Acid phenol method --- p.37 / Chapter 2.2.3.2 --- Spin cartridge method --- p.38 / Chapter 2.2.3.3 --- Virion capture method --- p.39 / Chapter 2.2.4 --- "Oligonucleotides used for RT, PCR and hybridization" --- p.40 / Chapter 2.2.4.1 --- Oligonucleotides used in HAV RT-PCR --- p.40 / Chapter 2.2.4.2 --- Primer set used for the evaluation of inhibitors of PCR in shellfish homogenates --- p.41 / Chapter 2.2.4.3 --- Preparation of oligonucleotide primers --- p.41 / Chapter 2.2.4.4 --- Detachment of the primer from the column --- p.42 / Chapter 2.2.4.5 --- Purification of the oligonucleotides --- p.42 / Chapter 2.2.4.6 --- Confirmation of synthesed oligonucleotide --- p.43 / Chapter 2.2.5 --- Reverse transcription of HAV genomic RNA template and PCR --- p.43 / Chapter 2.2.6 --- Human β-actin gene PCR for the evaluation of shellfish homogenates --- p.45 / Chapter 2.2.7 --- Analysis of PCR products --- p.46 / Chapter 2.2.7.1 --- Agarose gel electrophoresis for the analysis of PCR products --- p.46 / Chapter 2.2.7.2 --- Dot blot hybridization for the confirmation of PCR products --- p.46 / Chapter 2.2.7.3 --- Southern blot hybridization for the confirmation of PCR products --- p.47 / Chapter 2.2.7.4 --- 5'-end DNA labelling of oligonucleotide probe --- p.47 / Chapter 2.2.7.5 --- Hybridization in sodium chloride / sodium citrate --- p.48 / Chapter Chapter 3- --- Results / Chapter 3.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.52 / Chapter 3.2 --- Synthesis and yields of oligonucleotide primers --- p.54 / Chapter 3.3 --- Development of reverse-transcription polymerase chain reaction (RT-PCR) for HAV --- p.55 / Chapter 3.4 --- Sampling of shellfish from different markets in Hong Kong --- p.59 / Chapter 3.5 --- Quantitation of HAV RNA in stock virus preparations --- p.62 / Chapter 3.6 --- Comparison of RNA extraction methods and the detection limit of the established RT-PCR method for HAV --- p.62 / Chapter 3.7 --- Specificity of the RT-PCR in combination with virion capture method for the detection of HAV --- p.65 / Chapter 3.8 --- Detection of HAV RNA by RT-PCR in shellfish in Hong Kong / Chapter Chapter 4- --- Discussion / Chapter 4.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.76 / Chapter 4.2 --- Development of RT-PCR method for the detection of HAV --- p.78 / Chapter 4.3 --- Evaluation of RNA extraction methods for the detection of HAV in shellfish sample by RT-PCR --- p.78 / Chapter 4.4 --- Application of the established RT-PCR method for the detection of HAV contamination in locally available shellfish --- p.79 / Chapter Chapter 5- --- References --- p.90 / Appendix I Figures of agarose gel electrophoresis of RT-PCR products for all samples (I1 -I23 ) --- p.101 / Appendix II Figures of dot-blot hybridization assay (II2 -II4 ) --- p.114

Comparatively little work has been done to determine the ecology of Clostridium botulinum type E since its initial isolation in the nineteen-thirties. This spore forming, anaerobic microorganism is relatively heat labile and has been missed in ecological surveys in which heat was used to selectively screen for spore formers. Use of gentler methods has, however, facilitated its demonstration in marine sediments throughout the Northern Hemisphere. The type E organism elaborates a highly potent neurotoxin and has been isolated as the causative agent in recent fatalities involving the consumption of fish products. Until recently the organism was not believed to be present in the United States south of the Canadian border. The purpose of this investigation was to determine if the organism could be demonstrated in shellfish, inland lake fish, and sediment samples throughout the Northwest. Samples of coastal shellfish-- including various species of clams, crabs and oysters, varieties of smoked fish products, species of inland lake fish and inland lake, river and coastal sediment samples were examined for the presence of the type E organism. The organism was found, to some extent, in almost every type of sample tested. Type E toxin was demonstrated in incubated samples of shellfish and smoked fish products collected from eleven sites along the Oregon and Washington coast. The organism was found in shore sediments from the tidewater and freshwater areas of the Columbia, Alsea, and Umpqua rivers but could not be demonstrated in sediments taken from saltwater beaches at the mouths of these rivers. The type E organism was also demonstrated in fish from inland lakes in the Oregon Cascade Mountains, in sediments from the shores of these lakes, and along the shores of a river and three reservoirs in this area. These samples were collected between 95 and 120 linear miles from the coast. The isolation of the type E botulinum organism in fish and shellfish products demonstrates that the organism does present a potential hazard which should be recognized by Northwest processors and distributors of these products. High concentrations of the spores of this organism, such as those found in bottom sediments of some of the inland lakes and reservoirs may serve to contaminate fish and other wildlife. Much of the data collected supports a terrestial distribution of the spores. / Graduation date: 1966

Seafood permits the transmission of many bacterial pathogens. In order to reconcile consumer demands with important safety standards, traditional means of regulating microbial spoilage and safety hazards in foods are combined with novel technologies. These include biological antimicrobial systems, such as the use of lactic acid bacteria (LAB) and/or their bacteriocins, such as Carnobacterium maltaromaticum CS526 and its bacteriocin piscicocin CS526. The aims of this study were to investigate the presence of Listeria monocytogenes in temperate seafood, namely fresh and smoked salmon, fresh and smoked haddock, and fresh mussels and oysters. Additionally, there was an aim to recover, characterise and use bacteriocin-like-substance to control Listeria monocytogenes in cold smoked haddock. Vibrio spp., Enterobacteriaceae representatives, total aerobic heterotrophic counts and Listeria monocytogenes were isolated from commercially prepared smoked and fresh Atlantic salmon, smoked and fresh haddock, live mussels and oysters using selective media and tryptone soya agar (TSA). Vibrio spp. occurred in high densities (>106 CFU gˉ1) in mussels and Enterobacteriaceae representatives were recorded at >106 CFU gˉ1 in fresh salmon. Total aerobic heterotrophic counts in fresh salmon, live mussels and oysters reached 107, > 107, and > 106 CFU gˉ1, respectively. Listeria monocytogenes was recorded at 5.0 x 104 CFU gˉ1 in mussels. In total sixty one bacterial isolates were recovered from the seafood examined. The results revealed 19 genera of bacteria, i.e. Acinetobacter, Aerococcus, Aeromonas, Bacillus, Brochothrix, Carnobacterium, Citrobacter, Corynebacterium, Enterobacter, Escherichia coli, Moraxella, Micrococcus, Pseudomonas, Psychrobacter, Serratia, Shewanella, Staphylococcus, Vibrio and Listeria. The prominent characteristics of fish spoilage isolates were demonstrated by the ability of the isolates to reduce trimethylamine oxide (TMAO) to trimethylamine, and to produce H₂S. Sh. baltica OS185, Aeromonas spp. HB-6, Sh. baltica, Sh. putrefaciens, A. hydrophila HX201006-3, A. salmonicida subsp. achromogenes, A. hydrophila, C. freundii, Enterobacter cloacae were strong producers of TMA and H₂S. The spoilage microorganisms were tested for potential pathogenicity. The result revealed that 6/15 of the spoilage microorganisms produced proteolytic, lecithinase, blood (β and α haemolysin) and elastinase activity, respectively, whereas 7/15 of the spoilage microorganisms showed lipolytic activity. Cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances of Carnobacterium maltaromaticum MMF-32 and KOPRI 25789 producing strains isolated from commercially prepared smoked salmon were investigated for their potential antimicrobial activity against potentially pathogenic and food spoilage microorganisms. Generally, a broad spectrum of activity was revealed against potentially pathogenic and food spoilage microorganisms in vitro. Cold-smoked haddock treated with bacteriocin producing C. maltaromaticum MMF-32, C. piscicola A9b bacˉ phenotype nonbacteriocin producing strain a mutant of C. piscicola A9b bac+, cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances was challenged with L. monocytogenes ATCC 19114 up to 103 CFU gˉ1, respectively. Samples were stored at 4 °C for 10 days. L. monocytogenes and total bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amines production as well as texture, colour and odour. Although the study on anti-listerial effects of C. maltaromaticum MMF-32 was not successful, this organism did have a positive effect on retention of firmness and sensory perception in cold smoked haddock.