Two bacteriophages, phi6 and phi8, were investigated as potential surrogates for H5N1 highly pathogenic avian influenza virus in persistence and chlorine inactivation studies in water. In the persistence studies, phi6 and phi8 remained infectious at least as long as the H5N1 viruses at both 17 and 28 degrees C in fresh water, but results varied in salinated water. The bacteriophage phi6 also exhibited a slightly higher chlorine resistance than that of the H5N1 viruses. Based upon these findings, the bacteriophages may have potential for use as surrogates in persistence and inactivation studies in fresh water.

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.The use of spores of Bacillus anthracis as a bioterrorist weapon has prompted renewed interest in the study of the inactivation of Bacillus spores by chemical disinfectants. Information regarding the resistance of B. anthracis spores to chlorination is of particular interest in reference to drinking water treatment. There has been a growing awareness, due to restrictions of working with select agents, of the need to evaluate the resistance to disinfectants of spores of attenuated strains of B. anthracis as well as other closely related species of Bacillus which might serve as surrogates for the overt pathogenic agent. The three species evaluated as surrogate organisms in this study were chosen based upon their genetic homogeneity (9). Indeed, some investigators have suggested that the three organisms comprise a single species (5).The sporicidal effectiveness of free available chlorine was determined at two pH levels and two temperatures. The inactivation experiments were conducted under chlorine-demandfree (CDF) conditions. CT values (C is the concentration of chlorine in mg/liter, and T is the exposure time in minutes) were determined for each Bacillus species for the various experimental conditions. The sporulation and purification procedures were performed in the same manner for each species.Three species of Bacillus were used in this study: B. anthracis Sterne (34F2; Colorado Serum Co., Denver, CO), Bacillus cereus (ATCC 7039), and Bacillus thuringiensis subsp. israelensis (ATCC 35646). B. anthracis Sterne is an attenuated strain of B. anthracis. Endospores were produced in a broth sporulation medium (3). The cultures were grown at 35°C with agitation on a rotary shaker for 5 days. Spores were purified by gradient separation using RenoCal-76 (Bracco Diagnostics, Princeton, NJ) and washed three times by centrifugation with distilled water, as previously described (7). Purified spore preparations (approximately 1 ϫ 10 7 CFU/ml) were examined using phasecontrast microscopy and stored in 40% (vol/vol) ethanol at 5°C until the time of use. Microscopic examination of the purified spore preparations exhibited Ͻ0.1% vegetative cells.Sterile CDF buffer (0.05 M KH 2 PO 4 ) was used in the experiments. The buffer was made chlorine demand free by adding reagent-grade sodium hypochlorite (4 to 6%) to the buffer to achieve a free-chlorine residual of approximately 3.0 mg/ liter. The pH of the buffer was adjusted by the addition of 10 M sodium hydroxide. The buffer was boiled for 5 minutes, transferred to 4-liter beakers, and exposed to short-wave UV irradiation in a biological-safety cabinet for 48 hours to remove the chlorine. The buffer was then sterilized by autoclaving and...

To determine resistance of highly pathogenic avian influenza (H5N1) virus to chlorination, we exposed allantoic fluid containing 2 virus strains to chlorinated buffer at pH 7 and 8, at 5°C. Free chlorine concentrations typically used in drinking water treatment are sufficient to inactivate the virus by >3 orders of magnitude.

Recent events in which spores of Bacillus anthracis have been used as a bioterrorist weapon have prompted interest in determining the resistance of this organism to commonly used disinfectants, such as chlorine and ozone. This work was undertaken to study the effect of temperature over the range of SX to 30°C for pH levels of 7 or 8, on the inactivation kinetics of the spores of Bacillus globigii and to evaluate whether these spores could serve as a surrogate for the spores of B. anthracis in chlorine inactivation studies in water. The delayed Chick-Watson model, i.e. a lag phase followed by pseudo-first order rate of inactivation, was found to adequately describe the inactivation kinetics of s. giobigii. Markov Chain Monte Carlo (MCMC) simulation method was used to estimate the length of the lag phase and the post-lag phase rate constant-As expected, the length of the lag phase decreased with increasing temperature and the post-lag phase rate constant increased with increasing temperature. A hierarchical Bayesian modelling approach was used to model the kinetic parameters of the inactivation mode! as functions of temperature. The MCMC simulation method was used to estimate the minimum CT requirement (with safety factor) for 99% inactivation of s, giobigii.

Francisella tularensis has been associated with naturally occurring waterborne outbreaks and is also of interest as a potential biological weapon. Recovery of this pathogen from water using cultural methods is challenging due to the organism's fastidious growth requirements and interference by indigenous bacteria. A 15-min acid treatment procedure prior to culture on a selective agar was evaluated for recovery of F. tularensis from seeded water samples. Mean levels of reduction of virulent strains of F. tularensis subsp. holarctica and subsp. tularensis were less than 20% following acid treatment. The attenuated live vaccine strain (LVS) was less resistant to acid exposure. The acid treatment procedure coupled with plating on cystine heart agar with rabbit blood and antibiotics (CHARBab) allowed the isolation of F. tularensis seeded into five natural water samples.