The flagellated parasite, Trichomonas vaginalis, is one of the most commonly encountered genital pathogens. Despite its frequent incidence, pathogenic mechanisms of T. vaginalis are not well characterized. The first section of this project involved the ability of T. vaginalis to grow in serum-free medium. By employing a cell culture system, it was demonstrated that T. vaginalis can exhibit growth in the absence of serum. McCoy cells utilized in the system did not appear to secrete growth factors responsible for this proliferation. The second part of this project involved establishment of a mouse model for T. vaginalis infection and subsequent employment of the model in determining the effects of T. vaginalis on vaginal flora and pH. After the endemic flora and pH in mice were identified, other factors which may have influenced flora, were investigated. Both estrogenization and inoculation of media appeared to have no effect on individual vaginal species, and only appeared to increase the number of species found per mouse. T. vaginalis infection did not appear to alter vaginal pH or flora in vivo. In vitro studies of the interaction between T. vaginalis and Lactobacillus acidophilus indicated that indirect factors, such as secreted products, may mediate the alteration in flora and pH evident during infection.

A central composite design was employed to methodically investigate anaerobic treatment of aircraft deicing fluid (ADF) in bench-scale Upflow Anaerobic Sludge Blanket (UASB) reactors. A total of 23 runs at 17 different operating conditions were conducted in continuous mode. The development of four empirical models describing process responses (i.e., chemical oxygen demand (COD) removal efficiency, biomass specific acetoclastic activity, methane production rate, and methane production potential) as functions of ADF concentration, hydraulic retention time (HRT), and biomass concentration is presented. Model verification indicated that predicted responses (COD removal efficiencies, biomass specific acetoclastic activity, and methane production rates and potential) were in good agreement with experimental results. Biomass specific acetoclastic activity was improved by almost two-fold during ADF treatment in UASB reactors. For the design window, COD removal efficiencies were higher than 90%. Predicted methane production potentials were close to theoretical values, and methane production rates increased as the organic loading rate (OLR) was increased. ADF toxicity effects were evident for 1.6% ADF at medium specific organic loadings (SOLR above 0.5 g COD/g VSS/d). In contrast, good reactor stability and excellent removal efficiencies were achieved at 1.2% ADF for reactor loadings approaching that of highly loaded systems (0.73 g COD/g VSS/d). Acclimation to ADF resulted in an initial reduction in the biomass settling velocity. The fate of ADF additives was also investigated. There was minimal sorption of benzotriazole (BT), 5-methyl-1 H-benzotriazole (MeBT), and 5,6-dimethyl-1 H-benzotriazole (DiMeBT) to anaerobic granules. A higher sorption capacity was measured for NP. Active transport may be one of the mechanisms for NP sorption. Ethylene glycol degradation experiments indicated that BT, MeBT, DiMeBT, and the nonionic surfactant Tergitol NP-4 had no significant effects on acidogenesis and methanogenesis at the concentration levels studied. A significant inhibition of acetoclastic activity was observed for NP at 100 mg/L, with acetic acid consumption rate at 38% of that for controls. No evidence for anaerobic degradation of benzotriazole and its derivatives was observed; however, both batch and continuous experiments suggested that anaerobic degradation of NP occurred. Kinetic analysis of operational data obtained for the anaerobic treatment of ADF in UASB reactors indicated that the substrate utilization rate was independent of the reactor biomass concentration. The maximum rate of substrate utilization and the half-velocity constants for ADF treatment were 28.4 g COD/L/d and 648 mg COD/L, respectively. For 1.2% ADF, the biomass yield and endogenous decay coefficients were 0.027 g VSS/g COD and 0.012 d-1 , respectively.

Rabies viruses have very simple genomes made up of single-stranded, negative sense, non-segmented ribonucleic acid (RNA). Most, if not all RNA virus populations may exist as complex mixtures of genetic and phenotypic variants often referred to as quasispecies populations. A quantitative relative fitness assay has previously been used to demonstrate loss of fitness in RNA virus populations due to Muller's Ratchet and to show gains of fitness by natural selection during virus passages. Due to their mutation rates, rapid replication, large population sizes and controlled (variable or constant) host cells, RNA viruses are useful for examining evolutionary processes. This study uses rabies virus as a model to examine virus evolution and virus population biology. The fate of two closely related rabies virus variants (the Western Skunk and Eastern Artic Fox viruses), cloned using end-point dilution techniques, passaged by themselves and in competition to each other in mouse neuroblastoma (MNA) cells was investigated. (Abstract shortened by UMI.)

Epstein-Barr virus (EBV) and its oncogene, the latent membrane protein 1 (LMP1), are associated with many human malignancies. Likewise, EBV-like viruses have been associated with lymphomas in immunosuppressed cynomolgus monkeys. This study characterizes the biology of Cyno-EBV, a cynomolgus monkey EBV-like virus, including the structural proteins, antigenic relatedness to EBV and molecular and functional analysis of its LMP1 homologue. Cyno-EBV infection is ubiquitous in its host species. In vitro , Cyno-EBV immortalized cynomolgus monkey but not human B cells. The electrophoretic profile of Cyno-EBV structural proteins was almost identical to that of EBV proteins, and cynomolgus monkey and human sera showed reciprocal reactivities for several of these proteins, including the EBV receptor binding protein gp350. The coding region of the Cyno-EBV LMP1 gene was cloned and expressed. The sequence predicted a 588 amino acid (a.a.) protein with a 19 a.a. N-terminus, 6 transmembrane domains and a carboxy tail of 404 a.a. containing a 8-histidine cluster used as a natural protein tag in expression studies. Western blot analysis revealed a major polypeptide of 110 kDa. Cyno-EBV LMP1 contained series of repeats that shared no homology with other LMP1s. However, a proline-rich sequence GPXXPX6 found within the 11 a.a.-repeats of EBV LMP1 was conserved in a non-repeat region of Cyno-EBV LMP1 and contained two Janus kinase (JAK) binding motifs PXXPXP. Cyno-EBV LMP1 harbored 4 motifs PXQXT/S, predicted to interact with TRAFs, adapter proteins of the tumor necrosis factor receptor signaling pathway, which lead to activation of the nuclear factor kappaB (NFkappaB). Cyno-EBV LMP1 shared the same ability as EBV LMP1 to induce a NFkappaB driven reporter gene. An apparent increased toxicity of Cyno-EBV LMP1 for rodent Rat-1 cells impeded the establishment of LMP1 expressing cell lines whereas a reduced toxicity was observed for transiently expressing 293 cells when compared to EBV LMP1. Cyno-EBV is structurally and antigenically closely related to EBV. Cyno-EBV LMP1 harbored several features and motifs found in EBV LMP1. New conserved sequences were reported including a histidine cluster and a proline-rich sequence encompassing two Janus kinase binding motifs. The increased numbers of TRAF motifs found in Cyno-EBV LMP1 did not result in increased ability to activate NFkappaB in 293 cells and as for EBV LMP1, the protein appeared to have specific cell type toxicity.

An effective vaccine against chancroid could take the place of therapeutic control programs, offering long-lasting protection without the risk of widespread drug resistance. Orally administered recombinant attenuated Salmonella strains are used as vaccine vectors to deliver heterologous, pathogen-derived antigens to intestinal mucosal associated lymphoid tissue, and to provide vaccine adjuvancy. Chancroid vaccines are tested in a temperature-dependent rabbit model of experimental H. ducreyi infection. However, testing of recombinant attenuated Salmonella strains as vaccine vectors has never been done in rabbits; it is usually done in mice. Anatomic and physiologic differences may limit this approach to the demonstration of vaccine feasibility in rabbits. A three-part study was designed to assess the feasibility of testing attenuated Salmonella vector vaccines in rabbits. The questions asked were, (1) what is the maximum tolerated oral dose and minimum immunogenic oral dose of attenuated Salmonella in rabbits, (2) can a recombinant antigen expressed in the attenuated vector be recognized by the rabbit immune system, and (3) will experimental H. ducreyi infection in rabbits after oral Salmonella vaccination function as a comparative quantitative virulence assay to permit vaccine evaluation? (Abstract shortened by UMI.)

Reovirus has a segmented dsRNA genome enclosed in a nonenveloped double capsid shell. Reovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site for viral assembly. These inclusions contain the intermediate filament vimentin, in addition to viral proteins, RNA, mature and immature viral particles. However, the viral or host proteins involved in the formation of reovirus inclusions have not been identified, neither has the mechanism by which viral RNA and proteins are directed to these sites. In this study the reovirus M1 gene, which encodes the minor core protein mu2, was identified as the primary determinant of the rate of inclusion formation. Cellular localization studies of mu2 protein in cells infected with wild-type or ts mutants of reovirus as well as in M1 gene-transfected cells showed that mu2 protein may directly be involved in inclusion formation. Expression of mu2 protein in infected cells was shown to be strain-dependent with type 1 strain "Lang" (T1L) producing significantly more mu2 protein than type 3 strain "Dearing" (T3D). Protein stability was demonstrated to be partly responsible for the difference in mu2 protein expression between T1L and T3D, with T1Lmu2 being more stable than T3D mu2. Degradation of mu2 protein was shown to occur via the ubiquitin-proteasome pathway (UPP). Ubiquitinated mu2 protein was present in reovirus inclusions together with components of the UPP making reovirus inclusions structurally similar to aggresomes and suggesting that reovirus may be using the UPP and aggresome formation pathway for inclusion formation. In keeping with this theory, it is shown that both reovirus inclusion formation and replication are inhibited when the cellular pool of free ubiquitin (Ub) is depleted by using proteasome inhibitor and that replication maybe enhanced by mono-ubiquitination. Interestingly, the most abundant ubiquitinated mu2 protein species was mono-ubiquitinated mu2 suggesting a role for mono-ubiquitinated mu2 in reovirus inclusion formation and viral assembly. In addition, the amino-terminal end of recombinant mu2 protein, which contains a potential E3 Ub-ligase motif, was shown to be important for protein complex formation. Most importantly reovirus inclusions, like aggresomes, were shown to impair the normal functioning of the UPP resulting in inhibition of Ub-dependent proteolysis which presumably contributes to cytopathology and disease in infected animals. This study is the first to show the use of the UPP in the replication of a nonenveloped or dsRNA virus.

Campylobacteriosis is the most frequently reported type of acute bacterial gastroenteritis in Canada. In 2000 alone, 11,846 campylobacteriosis cases were reported in Canada. The majority of these cases are sporadic, and their causes remain unknown. An attempt was made to gain a better understanding of the epidemiology of campylobacteriosis, both by identifying foods and environments that harbor Campylobacter spp., and by characterizing clinical, food and environmental isolates using pulsed-field gel electrophoresis (PFGE). Spot maps were also used to determine the geographical relationship of these campylobacteriosis cases. A variety of raw and ready-to-eat foods were tested for the presence of Campylobacter spp. From the 300 samples analyzed, Campylobacter spp. were detected in four samples, one raw beef liver sample and three raw chicken samples. An isolation rate of 9.7% was observed among the raw chicken samples tested, a significantly-reduced percentage, as compared to a 1981 Canadian survey. The prevalence of Campylobacter spp. in a poultry foodservice operation in Ottawa, was also determined from March to August 2001. No Campylobacter spp. were detected in the 125 samples tested. Campylobacter clinical and food isolates were characterized using PFGE with two restriction enzymes, SmaI and KpnI. KpnI produced more complex banding patterns than SmaI, and proved to be more discriminatory. Among the 154 isolates assigned to clusters by SmaI, only 42% gave concordant results with KpnI. In contrast, among the 53 isolates assigned to 23 clusters by KpnI, 87% gave concordant results with SmaI. Five of the 20 concordant clusters represented isolates obtained from the same person, suggesting that some of these individuals may have become re-infected. Spot map analysis revealed a significant clustering of campylobacteriosis cases in the former city of Ottawa, most of which, did not belong to the same postal code. In contrast, very few cases were observed in outlying regions; however, most of these cases belonged to the same postal code, suggesting the possible presence of local outbreaks.

Levels and turnover of vitamin E ($\alpha$-T) were studied in guinea pigs placed for three weeks on a diet containing a scorbutic level of vitamin C and either a low level (LE group) or a high level (HE group) of hexadeuterium-labelled vitamin E (d$\sb6$-RRR-$\alpha$-T acetate). The levels of vitamin C in the ten tissues analyzed declined very rapidly at rates that were the same in both the LE and HE groups, indicating that the level of dietary vitamin E had no effect upon tissue vitamin C levels. On the vitamin C deficient diet, the total $\alpha$-T (d$\sb0$- + d$\sb6$-$\alpha$-T) declined significantly over 21 days in the HE group in two tissues with high P-450 enzyme activity and in one tissue with a high partial pressure of oxygen, whereas on a vitamin C-sufficient diet with the same concentration of vitamin E the levels of total $\alpha$-T remained steady in the same tissues. In the LE "scorbutic" group, the total $\alpha$-T declined only in heart and kidney, whereas in the vitamin C-sufficient LE group there was a decline of total $\alpha$-T in all tissues analyzed except brain. The results show that in guinea pigs, at least, vitamin C is indispensable for proper uptake of vitamin E from the gut and absorption into tissues. Changes of vitamin E levels also were studied in six anatomical regions of the brain of rats subjected to controlled ischemia/reperfusion. Analysis showed that ischemia/reperfusion caused statistically significant losses of vitamin E in all regions, except the pons-medulla, and the extent of loss correlated well with the previously determined deterioration of the blood-brain barrier in the corresponding regions. (Abstract shortened by UMI.)

Pichinde virus (Pic), like the other arenaviruses, possesses two glycoproteins, GP1 and GP2, that are derived by proteolytic cleavage from a precursor molecule, GPC. Within the arenaviruses, GP1 is the most heterogeneous protein, and GP1 of Pic differs from that of the other arenaviruses in having twice as many potential N-linked glycosylation sites, most of which appear to be utilized. In order to examine the effects of this heavy glycosylation on Pic GP1 structure and inmunogenicity, GPC of Pic was cloned and expressed in vaccinia virus. The recombinant vaccinia (vvGPC) expresses authentic Pic GPC as demonstrated by immunoprecipitation with MAb and several polyclonal anti-Pic sera. GPC expressed in vaccinia is fully glycosylated as it comigrates with Pic GPC. At the same time, sequence analysis of cDNA shows both nucleotide and amino acid changes compared to published sequences for Pic GPC, indicating that variation in the same strain of this virus occurs as virus is passaged in separate laboratories. Experiments to assess the ability of Pic GPC expressed in vaccinia to elicit anti-Pic antibody show that rabbit anti-vvGPC detects authentic Pic GPC and GP1. Site-directed mutagenesis was employed to remove a potential N-linked glycosylation site (aa 181-183) in Pic GPC. Attempts were made to produce recombinant vaccinia, vvGPC-183, harbouring the mutant Pic GPC.

One of the aims of this study was to determine whether the glycoproteins of Pichinde virus harbour any cytotoxic T-lymphocyte (CTL) epitopes on the murine haplotypes, H-2$\sp{\rm b}$ and H-2$\sp{\rm d}$ since, on neither of these MHC backgrounds are there any CTL epitopes on the nucleoprotein of this virus. Using a vaccinia virus recombinant, vvGPC, which expresses the full-length glycoprotein precursor (GPC) of Pichinde virus, standard chromium release CTL assays were performed. Three independent assays are shown for each of the haplotypes. In each of these assays for both of the haplotypes, it was observed that CTL derived against Pichinde virus did not recognize vvGPC-infected target cells nor did CTL derived against vvGPc recognize Pichinde-infected target cells. This indicates that no CTL were generated with either virus that might recognize the glycoproteins of Pichinde virus and, therefore, that the glycoproteins do not contain CTL epitopes on these murine MHC backgrounds. A second aim of this work was to compare the CTL epitopes of Pichinde wild-type virus with two temperature sensitive mutants derived from it. Both these mutants have been shown to be defective in their glycoprotein processing. The H-2$\sp{\rm d}$-restricted epitopes appear to be disrupted in TS13 as it is not recognized by Pichinde-specific CTL derived on this background. TS908 is not recognized by Pichinde-specific CTL on either haplotype suggesting that the wild-type virus' epitopes were probably disrupted during the derivation of this mutant. (Abstract shortened by UMI.)