Previous studies have shown that certain antigens can down-regulate immune responses of human peripheral blood mononuclear cells (PBMC) both in vivo and in vitro. This study demonstrates that tetanus toxoid (TT), in a dose-dependent fashion, suppresses the induction of a blastogenic response of PBMC by phytohemagglutinin (PHA), monoclonal anti-CD3, and anti-CD4 antibody. Pokeweed mitogen (PWM)-induction of IgG and IgM is suppressed as well. The suppression is partially reversed by indomethacin and IL-2, but not by IL-1 or tumor necrosis factor (TNF-$\alpha$). PBMC pre-incubated with TT could suppress the PHA blastogenic response of fresh autologous cells during co-incubation. The removal of CD4$\sp+$ cells prior to induction of suppression greatly diminished the suppression of PHA blastogenic response, whereas the elimination of CD8$\sp+$ cells had no effect. Therefore it is concluded that TT induces non-specific suppressor cells that can strongly dampen mitogenic responses, and that CD4$\sp+$ cells plays an important role in this suppression. CD4 $\sp+$ cell, together with monocytes, may suppress mitogenic responses involving a prostaglandin-dependent pathway.

Genomic analyses revealed Prospect Hill-1 (PH) virus has three RNA segments Large (L), Medium (M) and Small (S), like all Bunyaviridae, with relative molecular masses (M$\sb{\rm r}$) of 2.2 $\times$ 10$\sp6,$ 1.3 $\times$ 10$\sp6$ and 0.6 $\times$ 10$\sp6$ respectively. Complementary DNA representing the genomic M RNA segment of the PH virus was cloned and its nucleotide sequence determined. As with other Bunyaviridae the 3$\sp\prime$ and 5$\sp\prime$ termini are inversely complementary and could possibly allow the M RNA segment to form a circular structure. The PH virus M RNA segment has a single long open reading frame (ORF) in the viral complementary-sense RNA. This protein is the putative glycoprotein precursor that would be cleaved into the two viral envelope glycoproteins G1 and G2. The predicted gene product of the PH virus M segment was compared with the corresponding gene products of Hantaan virus, Sapporo rat virus strain SR-11 (SR) virus and Hallnas virus. There was 74% and 79% amino acid sequence similarity between the G1 and G2 proteins of PH virus and Hallnas virus respectively. In contrast, there was only 50% amino acid sequence similarity between the G1 proteins of PH virus and SR virus or Hantaan virus. However, the G2 proteins of SR virus and Hantaan virus were more closely related to the G2 protein of PH virus with amino acid sequence similarity of approximately 62%. Hydrophilicity plots of the four virus glycoproteins were very similar. The region of greatest hydrophilicity was conserved in the Hallnas virus, SR virus and Hantaan virus, and was located near the C-terminus of the G1 protein. In contrast, the region of greatest hydrophilicity in the PH virus glycoprotein precursor was located closer to the N-terminus of the G1 protein. Our data demonstrate that despite differences in the serotypic profiles and virulence of PH virus and Hallnas virus, their G1 and G2 proteins are closely related. Our data demonstrate that despite differences in the serotypic profiles and virulence of PH virus and Hallnas virus, their G1, G2 and N proteins are closely related. We conclude that PH virus and Hallnas virus may have evolved along a separate evolutionary pathway in the Hantavirus genus from SR virus and Hantaan virus. (Abstract shortened by UMI.)

The general objective of this thesis was to examine the effects of key environmental variables on fluorescence characteristics of picocyanobacteria using both culture and field experiments. Fluorescence ratios of phycoerythrin (PE) to phycocyanin (PC), and PE to Chl a, were found to be good surrogates of pigment ratios. The severity of nitrogen stress, as measured by the reduction in carbon to nitrogen ratios, PBP, and fluorescence ratios, appeared to be related to the pigment types of picocyanobacteria. Only slight changes in fluorescence ratios can be expected between high full spectrum and low monochromatic growth irradiance (PSII light) as a result of the small differences in PUR between these two environments. The fluorescence characteristics of low light acclimated picocyanobacteria can also be significantly influenced by growth rate. Depth patterns in fluorescence ratios in response to light and nitrogen availability were found to be consistent with differences in the seasonal and between-lake abundance of two populations of PE-picocyanobacteria. (Abstract shortened by UMI.)

No description available.

Lyssavirus specific monoclonal antibodies (Mabs) were used in competitive ELISAs to topographically map two major structural proteins of representative viruses of each of the six lyssavirus genotypes. This is the first description of topographical mapping of lyssavirus proteins based upon competitive Mab binding. A panel of anti-glycoprotein Mabs, all generated against rabies viruses, was used to map antigenic sites on lyssavirus glycoproteins. These Mabs identified thirteen unique epitopes that define four major antigenic sites and two antigenic sub-sites. Glycoprotein antigenic sites were found to be highly conserved throughout all lyssavirus genotypes. Thirty-four unique epitopes were identified on lyssavirus nucleoproteins using a panel of Mabs comprised of representative Mabs generated against nucleoproteins of all lyssavirus genotypes. The panel of anti-nucleoprotein Mabs used in this study revealed five major antigenic sites that again were largely conserved in all lyssavirus genotypes. The epitope variability of the nucleoprotein was much greater than that of the glycoprotein, confirming previous Mab studies of lyssaviruses. Mabs to several epitopes of both structural proteins studied were lyssavirus specific, while at least one anti-nucleoprotein Mab for each lyssavirus was genotype specific with the exception of genotype 5 (European bat lyssavirus type 1). Most of the epitopes identified on both proteins were conformational, however, certain linear epitopes identified in this study that are lyssavirus specific may be useful for diagnosis and vaccine development. Genotype specific epitopes will be useful epidemiologically. The competitive antigenic analysis of the glycoprotein and nucleoprotein support the published phylogenetic relationships of the lyssavirus genotypes. Two of the African lyssaviruses (Mokola virus and Lagos Bat virus) are the furthest diverged of the lyssaviruses. The nearly identical topographical maps of the antigenic sites of both the glycoprotein and nucleoprotein strongly suggest that lyssaviruses have evolved from a common rhabdovirus ancestor. Further, this ancestral rhabdovirus was probably a bat virus.

Waste tires pose an ever-growing disposal and environmental problem. In the United States alone it is estimated that there are 2.5 billion waste tires sitting in landfills and this number is growing by 200 million every year. In order to recycle the rubber in these tires the rubber must be effectively devulcanized by breaking the carbon sulfur cross-links that are present. The need for a more cost-effective devulcanization process that produces rubber of higher quality has driven research in this field. It was the goal of my project to develop rubber recycling methods involving sulfur oxidizing Archaea and to determine the usefulness of these methods. In order to accomplish this goal two major methods were investigated: one using whole cells from the species Sulfolobus solfataricus P2, another using an enzyme, the sulfur oxygenase/reductase (Sor) from Acidianus ambivalens. My results have shown that both the whole cell method and the enzyme method oxidize the sulfur found in rubber crumb, thereby, causing some degree of devulcanization. An attempt was made to locate homologues to sor found in a number of species related to Acidianus ambivalens. Interestingly, after exhausting a number of methods, this search resulted in no homologue being found. The possibility of a novel form of sulfur metabolism is discussed.

Various outbreaks of hepatitis A have been associated with the consumption of foods contaminated with hepatitis A virus (HAV). In this study, experiments were designed to develop rapid molecular methods to detect HAV in produce, to investigate its heat inactivation in dairy products and gamma irradiation resistance in produce, to assess its survival under modified atmosphere packaging (MAP) and to determine the amount of virus transferred from human hands to foods. The results demonstrated the following: (1) Immunomagnetic beads-PCR (IM-PCR), positively-charged 1MDS filters (F), or a combination of both (F-IM-PCR) methods were used to capture, concentrate and rapidly detect HAV in experimentally-contaminated lettuce and strawberries. Although the detection limit (10 PFU) of the F-IM-PCR was slightly less sensitive than that by the IM-PCR (0.5 PFU), the F-IM-PCR method has the potential to be of a greater sensitivity when detecting the virus in larger analytical units of foods (>50 mL) than the IM-PCR which is restricted to ≤20 mL, volumes. (2) Studies on the thermal resistance of HAV in three types of dairy products containing increased amounts of fat content (skim milk, homogenized milk; 3.5% MI7G, and table cream; 18% MFG) indicated that increased fat content seems to protect HAV and render it more heat-resistant as compared to those of lesser fat content. A list of time and temperature combinations required to achieve a 1 to 5 log 10 reduction in HAV titre is presented. (3) Exposure of HAV to gamma irradiation (60Co) doses ranging between 1 and 10 kGy showed that DIO values of 2.73 and 2.97 kGy were needed to achieve a 1-log10 reduction in HAV inoculated onto lettuce and strawberries, respectively. (4) Various MAP environments did not influence HAV survival on lettuce stored at 4°C. At room temperature, however, an atmosphere consisting of 70% CO2: 30% N2 slightly (p = 0.06) enhanced virus survival as compared to other storage conditions. (5) Approximately 10% of HAV inoculated onto the fingerpads of human volunteers was transferred to lettuce by touching. Treatment of the fingerpads with water, topical agents, or alcohol significantly (p < 0.05) reduced virus transfer from 10% down to ≤0.64%.

Infections caused by members of the genus Mycobacterium , particularly Mycobacterium tuberculosis (TB), are responsible for significant morbidity and mortality worldwide. On the other hand, nontuberculous mycobacteria (NTM), which are found in the environment, are increasingly being implicated in human disease, especially in the immunocompromised. There is concern about the escalating risk of spread of M. tuberculosis and NTM through the increasing use of semi-critical medical devices such as flexible endoscopes and several cases of iatrogenic infection have already been documented in recent years. This concern is further reinforced by the realization that current methods for testing the mycobactericidal activity of high level disinfectants are slow, labor-intensive, non-quantitative and use unsuitable surrogates. Therefore, the first objective of this study was to adapt a quantitative carrier test for sporicidal activity developed in our laboratory to assess the mycobactericidal activity of chemical germicides. The second objective of this study was to develop a fluorescence assay utilizing mycobacteria expressing GFP that would reduce the turn-around time in the testing of mycobactericidal activity of germicides. (Abstract shortened by UMI.)

This laboratory study was undertaken to investigate the natural mechanisms of Fe-oxide formation onto bacteria and their ageing under laboratory conditions simulating the natural aquatic environment. Fe-oxides were formed the oxidation of Fe(II) at pH 5.75 at two different Fe concentrations (i.e. 10-5 M and 10-4M) in the presence of four different bacterial strains (Escherichia coli, Bacillus subtilis, Bacillus licheniformis and Pseudomonas aeruginosa). Various dissolved species (SO4 (10-4M), PO 4 (10-5M), Al (10-7 M) and Si (10-3M)) were also added to the systems in order to assess their role on the formation and ageing of biogenic Fe-oxides. All systems were left in the dark at 4°C for a period of six months to simulate lake diagenesis. Abiotic systems were formed under identical conditions for further comparisons. Each month, a filtered aliquot of each system was analyzed for its content in dissolved Fe, SO4, PO4 and Si, and for pH and Eh. Sub-samples were also taken for transmission electron microscopy (TEM) observations and X-ray diffraction. (Abstract shortened by UMI.)

This work represents a comprehensive analysis of the precise structural relationship and organization existing amongst the gonococcal beta-lactamase-producing plasmids. The second major aspect of this work deals with investigations into the genetic and molecular characterization of the multiple origins of replication of the gonococcal beta-lactamase-producing plasmids. The Asia-plasmid, pJD4, a prototype gonococcal beta-lactamase-producing plasmid, is 7,426 bp and contains two large, direct repeats (DR-30A, 507 bp and DR-30B, 509 bp) which are implicated in the formation of deletion variant plasmids, such as the naturally-occurring Africa-type plasmid. The deletion observed in Africa-type plasmids, represented by pJD5, is 1,827 bp. One of the DR-30 repeats is also missing in the formation of Africa plasmids. The deletion in the Rio-type and several Toronto-type is 2,273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. Thus, the Rio and Toronto-type plasmids are identical. The Nimes-type plasmid is proposed to be identical to the Africa-type but contains an IS 5 insertion sequence. Since IS5 has not been identified in gonococcal isolates and is not present in the gonococcal genome, it is suggested that this sequence was inserted after the original gonococcal plasmid was transformed into a strain of Escherichia coli. The New Zealand plasmid was shown to be an Asia-type plasmid which contains an endogenous tandem duplication of 1,883 bp. The direct repeat DR-2 is implicated in this duplication. Branch-point analysis by electron microscopy indicated that the Asia-type plasmid contains three origins of replication, named ori1, ori2, and ori3. Although pJD4 belongs to the incompatibility (Inc) group IncW, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid was shown to carry only oril, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I for replication. Plasmid constructs from pJD4 lacking oril but carrying ori2 and ori3 are incompatible with IncW plasmids, suggesting the ori2/ori3 region contains the IncW determinant. A novel replication initiation protein, RepB, was identified and shown to be necessary for ori2 and ori3 to function. The RepB is distinct from RepA, the replication initiation protein required for plasmids carrying ori1. (Abstract shortened by UMI.)