The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5 hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

Abstract:The efficacy of "gaseous" ozone in reducing numbers and re-growth of food-borne pathogens, (Escherichia coli and Listeria spp.), on leafy salads was investigated using spinach. A preliminary in vivo study showed 1-log reduction in six strains of E. coli and two species of Listeria spp. on spinach exposed to 1 ppm ozone for 10 min. A range of ozone treatments were explored to deliver optimal bacterial inactivation while maintaining the visual appearance (color) of produce. Exposure to a higher ozone concentration for a shorter duration (10 ppm for 2 min) significantly reduced E. coli and Listeria spp. viable counts by 1-log and the pathogens did not re-grow following treatment (over a nine-day storage period). Impacts of 1 and 10 ppm ozone treatments were not significantly different. Approximately 10% of the pathogen population was resistant to ozone treatment. We hypothesized that cell age may be one of several factors responsible for variation in ozone resistance. E. coli cells from older colonies demonstrated higher ozone resistance in subsequent experiments. Overall, we speculate that gaseous ozone treatment constitutes the basis for an alternative customer-friendly method to reduce food pathogen contamination of leafy produce and is worth exploring on a pilot-scale in an industrial setting.

We previously showed that a purE mutant (⌬purE201) of Brucella melitensis 16M is attenuated for growth in cultured human monocytes (E.

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