The gram-positive, spore forming bacteria Bacillus thuringiensis synthesize cytoplasmic crystalline inclusions, the Bt proteins, which are toxic to insect larvae. Although Bt proteins have been used as bioinsecticides for many years and a considerable number of studies have been carried out to investigate their mode of action, their precise mechanism of action still remains unclear. A 27 kDa protein from Bt subsp. israelensis (cytA27) is known for its broad cytolytic activity against a wide range of invertebrate and vertebrate cells in vitro as well as mosquitocidal activity in vivo. Given the interest in the mechanism of the membrane interaction of cytA27 and its proteolytic product, a 24 kDa protein (cytA24), we have undertaken a study of their interactions with model phospholipid membranes. The cytA27 and cytA24 proteins were purified by selective solubilization and ion exchange HPLC and characterized by SDS-PAGE, N-terminal sequencing and mass spectrometry. Fluorescence spectroscopy was used to characterize the cytA-induced release of fluorescence markers from vesicles and the cytA-vesicle affinity and stoichiometry. The results indicate that cytA proteins bind to lipids and affect the integrity of phospholipid vesicles. The binding is non-specific in that it does not require a receptor. CytA27 and cy8tA24 interact with PC-LUV with apparent binding constants of $\rm (0.34\pm0.02)\times10\sp5M\sp{-1}$ and $\rm (1.54\pm0.10)\times10\sp5M\sp{-1}$ respectively. Binding isotherm data indicate that a critical number of cytA molecules must associate with the membrane in order to induce vesicle leakage. Approximately 324 of cytA27 and 157 of cytA24 molecules are required to bind to one PC-LUV before the latter starts to release its contents. CytA-induced vesicle leakage follows an all-or-none mechanism, i.e. each LUV either releases all of its contents or remains intact. (Abstract shortened by UMI.)

Hepatitis A (HAV) continues to be a serious problem for human health. The present study was designed to (a) assess the vehicular role of human hands and environmental surfaces in the spread of HAV; (b) evaluate the efficacy of chemical disinfectants and handwashing agents to eliminate HAV from inanimate and animate surfaces; and (c) determine the efficacy of alkaline glutaraldehyde reuse against HAV and other microorganisms. Pressure and friction were found to significantly affect HAV transfer between hands and inanimate surfaces (F = 33.98; p 0.05) irrespective of the mode of transfer used. No statistically significant interaction was observed between mode of transfer and pressure or friction. The findings of this phase of the study suggest that human hands and inanimate surfaces may play an important role in the direct as well as indirect spread of HAV. HAV disinfection was assessed on experimentally-contaminated metal disks. No virus was transferred from disks treated with a 3% solution of Virkon. None of the eleven handwashing agents examined, however, was able to reduce the infectivity titer of HAV and PV to an undetectable level. The least reduction in HAV titer was shown by an unmedicated soap (77.96 $\pm$ 7.17%), while the highest level of reduction was given by Bacti-Stat Medicated Soap (92.04 $\pm$ 4.02%). Samples of 2% alkaline glutaraldehyde were collected over the 14-day reuse period from two manual and one automatic bath used for the disinfection of flexible bronchoscopes and gastrointestinal endoscopes at a neighboring hospital. The number of instruments put through each bath during the 14-day cycle was recorded. The broad-spectrum germicidal activity of the disinfectant lasted only up to six days. This suggests a review of alkaline glutaraldehyde reuse in the disinfection of semi-critical instruments such as flexible fiberoptic endoscopes. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their prevention and control. (Abstract shortened by UMI.)

There is mounting concern on the efficacy of many germicides on the market because officially recognized germicidal tests for various classes of microorganisms vary widely and lack reproducibility and proper quantitation. This project outlines a novel carrier method for simultaneously and quantitatively assessing the efficacy of liquid chemical germicides against a mixture of microorganisms of varying degrees of resistance. In the test, glass cups were contaminated with 10 $\mu$l of a standardized mixture of Staphylococcus aureus, Mycobacterium bovis BCG, Trichophyton mentagrophytes spores, Sabin poliovirus type 1 and Bacillus stearothermophilus spores in 5% fetal bovine serum. The inoculum was dried for 60 min at room temperature (22 $\pm$ 2$\sp\circ$C), covered with 60 $\mu$l of the disinfectant under test or a balanced salt solution for the desired contact time. The carrier was then placed in 2,940 $\mu$l of an eluent and eluates assayed separately for the five microorganisms. The findings demonstrate the feasibility of a novel approach to the testing of chemical germicides. The combined carrier test allows for concurrent evaluation of efficacy claims and demonstrates the resistance patterns of various classes of microorganisms. This method also permits a more reliable means of classifying germicides based on their spectrum of activity. (Abstract shortened by UMI.)

The interaction of gentamicin with daptomycin and PI dispersions was investigated by FTIR spectroscopy. We found no evidence of a direct interaction involving the neutralization of the aspartate residues of daptomycin by gentamicin and the amide I band of daptomycin did not reveal significant conformational changes of its peptidic moiety. On the other hand, daptomycin readily inserts within bilayers of PI, dimyristoylphosphatidylglycerol or dipalmitoylphosphatidylcholine, as judged from its influence on the fluidity of these bilayers. The incorporation of daptomycin into PI bilayers has only a slight effect on the lipopeptide amide I band. The affinity of the aminoglycoside for PI is slightly increased in the presence of daptomycin, in agreement with the results of the dialysis study mentioned above. Gentamicin induces a slight narrowing of the amide I band of daptomycin bound to PI bilayers. The fluidity of the lipid bilayers corresponds to that seen in the absence of both drugs. It is proposed that in the presence of the lipopeptide antibiotic, the critical charge density and membrane fluidity required for optimal enzyme activity is restored, explaining its nephroprotective capabilities. The mechanism of nephroprotection by poly-L-aspartic acid is different from that of daptomycin. Dialysis studies have indicated an optimal binding between gentamicin and polyaspartic acid at acidic pH. We found no evidence of a direct interaction involving the neutralization of the carboxylates of polyaspartic acid by gentamicin and the amide I band of polyaspartic acid did not reveal significant conformational changes. On the other hand, polyaspartic acid had no effect on the spectral features of PI bilayers. A reduction in the changes induced by gentamicin in the lipid head group and interfacial region suggests that the affinity of the aminoglycoside antibiotic decreases in the presence of polyaspartic acid. (Abstract shortened by UMI.)

Enterovirus 70 (EV70), the causative agent of acute haemorrhagic conjunctivitis (AHC) is a picornavirus with a number of unique biological and pathogenic properties. In vivo, EV70 shows tropism for the conjunctiva and in rare cases, can infect the central nervous system, while in vitro, it has the ability to infect a wide range of mammalian cells in culture. The main objective of this study was to identify sites associated with neutralization of EV70. Identification of neutralization-associated sites is an initial stage for producing subunit vaccines, and neutralizing monoclonal antibodies produced against these sites may have direct application as therapeutic agents. Several different monoclonal antibodies which have neutralizing activity against the prototype EV70 J670 were tested. Two antibodies yielded resistant virus plaques. The nucleotide sequence of the capsid protein-coding region of each mutant was determined following cDNA synthesis, PCR amplification and cloning. The deduced amino acid sequences of the capsid protein-coding regions were compared with the corresponding sequences from the EV70 prototype and several wild isolates of EV70. Amino acid residues that correlated with escape from neutralization were identified. Two neutralization associated determinants were identified on VP1 and VP2. The locations of these sites were compared with neutralization associated sites of other picornaviruses.

The Hollow Fiber Diffusion (HFD) system is a novel approach for the in situ study of the survival of bacteria and viruses in the aquatic environment. The HFD system employs a tangential flow, hollow fiber cartridge with a large area $(7 \times 10\sp3$ cm$\sp2$) of exchange surfaces for diffusion. When compared with diffusion chambers, the HFD system responded significantly faster and more accurately to changes in pH, Eh, nutrient concentrations and to the presence of disinfectants in the external aqueous environment. The T$\sb$ diffusion of low molecular weight substrates was 0.6 h for the HFD system but was 4.2 h for the diffusion chamber. The rate of diffusion or equilibration could be further improved by increasing the flow rate through the HFD system or reducing the volume of the sample reservoir. The HFD system was compatible with all test bacteria and viruses with the possible exception of tailed coliphages. The inactivation of tailed phages by the HFD system can be reduced or eliminated using a slower flow rate or larger diameter hollow fibers. Tailed coliphage inactivation in the HFD system was not apparent in natural waters. Neither adsorption of microorganisms to the hollow fiber membrane surfaces nor colonization of those surfaces was found to be a significant problem during its use in natural waters. A protocol for the decontamination and reuse of the hollow fiber cartridges using hydrogen peroxide was developed and applied successfully. The results of trials of the HFD system at five field sites suggests that the HFD system permits "real-time" accommodation to changes in the physicochemical parameters of the external aqueous environment which can influence the survival of microorganisms. Differences in the survival of microorganisms in the HFD system and in batch samples were shown. The HFD system demonstrated regrowth of Escherichia coli in the Rideau River which is an eutrophic, temperate river. It also demonstrated a diurnal inactivation pattern for Enterococcus durans with the slower decay of E. durans numbers in the hours of darkness. For the other water sources tested (the Ottawa River, the Kennedy Burnett Stormwater Ponds, the Gombak River and the Kroh River), the general order of survival of test microorganisms was MS-2 coliphage $>$ poliovirus $>$ phage B $>$ E. coli and E. durans. In the Rideau River, the order of survival was E. coli $>$ poliovirus $>$ MS-2 coliphage $>$ E. durans. Surprisingly, there was no significant difference in the survival of microorganisms in the two equatorial rivers compared with the survival of the organisms in the oligotrophic, temperate Ottawa River. The HFD system will be very useful in studying the survival and natural ecology of microorganisms in the aquatic environment. It can be applied to model the behavior of water quality indicators, pathogenic organisms and genetically engineered microorganisms. It also has potential for ecotoxicological studies, monitoring for toxins or pollutants in the environment and for the in-line monitoring of the efficiency of water treatment processes such as chlorination.

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No description available.

Recent work with a mouse model of T. vaginalis indicated that only 25% of mice harbour Lactobacillus spp. and T. vaginalis introduced in the vagina persisted for about 7 days after which it was rapidly eliminated. In an attempt to establish a better model of intravaginal infection that resembles human disease we established Lactobacillus acidophilus in estrogenized Balb/c mice. Rates of T. vaginalis infection in this group were then compared to a control group of mice who were not treated with L. acidophilus. The addition of L. acidophilus did not significantly alter the resident mouse vaginal flora. The interaction observed in the model between T. vaginalis and L. acidophilus was then examined in in-vitro competitive assays between the two organisms. Interesting was the deleterious effect of T. vaginalis on L. acidophilus seen in the combined cultures with the reduction in bacteria seen at a much greater rate than in matched controls. Finally, this modified model was employed in preliminary immunological studies of T. vaginalis infection. Mice immunized with T. vaginalis prior to intravaginal challenge were less likely to develop infection than controls. (Abstract shortened by UMI.)

The recombinant human adenovirus type 5 (rHAd5-RG1) tested in this study contains the rabies virus glycoprotein gene. This virus is being considered as a possible substitute for the attenuated rabies virus in the oral immunization of wildlife in Ontario. This study examined the stability of the virus indoors and outdoors, and tested technically simple ways of concentrating the virus and its microencapsulation. The findings of this study show that the recombinant virus has the potential for release into the environment and there is no evidence that the virus will rapidly decay under the outdoor conditions experienced during the fall season. The virus can probably be microencapsulated and packaged for oral delivery. However, more studies are needed to assess the actual immunizing potential of these microencapsules. The use of detergents for increasing the virus yield has potential. (Abstract shortened by UMI.)