High resolution genotyping of Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat markers.

AIMS: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. METHODS AND RESULTS: Forty-seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi-locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. CONCLUSIONS: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.

A high resolution four-locus multiplex single nucleotide repeat (SNR) genotyping system in Bacillus anthracis.

The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis.

Use of staphylococcal enterotoxin A-induced lymphoproliferation and interleukin 2 production as indicators of immunotoxicity.

Suppression of mitogen-induced splenocyte lymphoproliferation and interleukin 2 (IL-2) production can be used as indicators of immunotoxicity. Staphylococcal enterotoxin A (SEA) is both a potent mitogen and the most potent in vitro inducer of IL-2 production that has been described. An in vitro system was used to measure impairment of SEA-induced lymphoproliferation and IL-2 production using splenocytes from female C57BL/6 mice dosed with either cyclosporin A (30 mg/kg/day, 14 days), benzene (220, 440, or 880 mg/kg/day, 14 days), or vehicle. Splenocytes were stimulated with either concanavalin A (con A) or SEA. Benzene- and cyclosporin A-treated mice demonstrated significant decreases in splenocyte proliferation. IL-2 production was determined by incubating splenocyte culture supernatants with IL-2 dependent cytotoxic T-cells (CTLL-2), pulsing with 3H-thymidine, and determining amount of incorporated label. Cell proliferation and IL-2 production were inhibited by both benzene and cyclosporin A, effects more clearly demonstrated using SEA than con A. SEA was a superior mitogen compared to con A in the assays evaluated here.

Differential ovotoxicity of 4-vinylcyclohexene and its analog, 4-phenylcyclohexene.

Vinylcyclohexene (VCH) is an industrial byproduct that is known to cause the destruction of ovarian follicles in mice. Its analog, 4-phenylcyclohexene (4PC), is a volatile product from latex-backed carpeting. These studies were undertaken to assess the structure-activity relationships of these compounds and the potential for 4PC to cause ovotoxicity. Female B6C3F1 mice were dosed with VCH (6 mmol/kg/day, ip) or 4PC (3 or 6 mmol/kg/day, ip) daily for 30 days. Treatment with VCH caused dramatic reductions in small and growing follicles as compared to those of vehicle controls. No treatment-related ovarian lesions were associated with 4PC administration. Plasma FSH concentrations were unaltered by treatment with either compound. These results indicate that in mice, the substitution of the phenyl for the vinyl group in the 4 position eliminates the ovotoxicity caused by this class of compounds. Presumably, the ability of the vinyl group to form an epoxide (or dihydrodiol) and/or its smaller size accounts for this difference in ovarian toxicity.

A comparison of controlled negative pressure and aerosol quantitative respirator fit test systems by using human subjects.

A quantitative respirator fit test system based on controlled negative pressure was evaluated by comparison testing with a computerized aerosol fit test system. Experiments ranged from multiple sequential tests of a single subject wearing a respirator equipped with a series of fixed leaks to sequential fit tests of 125 U.S. Air Force personnel using both systems. Throughout each test phase, measured negative pressure fit factors were consistently more conservative and less variable than aerosol fit factors. Comparison of subject and fixed leak fit factors indicated significant loss of aerosol during subject fit tests. Negative pressure system results did not show any effect from subject-related losses.

A comparison of controlled negative pressure and aerosol quantitative respirator fit test systems by using fixed leaks.

An automated version of a new method for quantitative respirator fit testing by controlled negative pressure was compared with a computerized aerosol fit test system. The controlled negative pressure technique eliminates many of the problems associated with aerosol and pressure decay fit test methods. A series of fixed leaks was used to compare the leak measurement capabilities of the controlled negative pressure system against a standard computerized aerosol fit test system. Negative pressure and aerosol fit factors determined for a series of fixed leaks through hypodermic needles were highly correlated with each other (r = 0.998) and with the cross-sectional areas of the leak needles (r greater than 0.995).

A feasibility study of quantitative respirator fit testing by controlled negative pressure.

The feasibility of using a direct measure of respirator leakage flow rate as a quantitative index of respirator face seal fit has been explored through the use of a new controlled negative pressure method. The method is based on exhausting air from a temporarily sealed respirator facepiece at a rate sufficient to generate and then sustain a constant negative pressure inside the facepiece while the wearer holds his breath. The magnitude of the negative pressure is preselected to replicate the mean inspiratory pressure inside the mask during normal wear. With the air-purifying paths into the respirator temporarily blocked, measurement of the exhaust flow rate yields a synonymous measure of the leakage flow rate into the mask during inspiration under normal use conditions. The feasibility of using the new method to quantify respirator fit was assessed in a preliminary study that compared its performance with a quantitative fit test method based on the use of dichlorodifluoromethane as a challenge agent. Study data exhibit a high degree of correlation (r greater than 0.99) and no significant difference between the two methods over a range of controlled mask leakage rates. A major advantage of the new method is that a worker can be fit tested with his assigned respirator because the method does not require a destructive sampling probe. Other significant benefits compared to current methods used to quantify respirator fit appear to include (1) ease of test administration, (2) simplicity of test components, (3) lack of a potentially toxic challenge agent, (4) a straightforward calibration procedure, (5) multiple test capability, (6) immediacy of test results, and (7) field portability of the test system.

Physicochemical characterization of industrial effluents and their effect on fish survival.

Water samples taken from selected industrial drainages and receiving streams in and around Islamabad, Pakistan, during the summers of 1987 to 1990 showed disturbances in pH and low oxygen levels. Selected metals (Fe, Pb, Zn, Cd, Cu, Ni, Hg) were higher in all effluents than in receiving waters. Static bioassays of the undiluted industrial effluents from three sites caused 100% mortality in carp during the first 24 hrs. Fishes also suffered 30 to 60% mortality when exposed to other undiluted industrial effluents and some mortality when industrial effluents were diluted by 50%.

Worker exposure to chemical agents in the manufacture of rubber tires and tubes: particulates.

The Occupational Health Studies Group industrial hygiene studies at a group of 14 tire and tube manufacturing plants chosen to represent a cross-section of the industry include numerous evaluations of potential exposure to airborne particulate matter. Results of these environmental particulate sampling studies are reported by plant and by occupational groups within plants. High volume, open face and cyclone samplers were employed to evaluate both personnel and area particulate concentrations. The concentrations of particulates yielded by high volume and open face total particulate samplers are compared with those of comparison samples of respirable material. Personnel samples of particulates are compared with general air samples taken in the same work areas. An overall review and comparison is given of particulate exposures to workers in various occupational title groups where particulate materials are released to the air from processes or operations.

Worker exposures to chemical agents in the manufacture of rubber tires: solvent vapor studies.

Environmental sampling surveys have been conducted in ten large tire manufacturing plants across the U.S. to characterize the nature and intensity of current exposure to solvent vapors. These plants were chosen to represent a cross-section of the industry and include both old and new plants, plants of four different companies and plants with wide geographic distributions. A variety of organic solvents is used in the manufacture of tires and tubes; accordingly solvent vapors comprise one category of exposure for workers in specific Occupational Title Groups (OTGs). Approximately 1000 determinations of various solvent vapor components in air samples have been made with special emphasis on pentane, hexane, heptane, benzene and toluene vapor levels. Exposures stem from the widespread use of bulk materials including petroleum naphthas, gasoline and aliphatic and rubber solvents in various tire manufacturing operations.

The association of vinyl chloride exposures with morbidity symptoms.

A mail-out questionnaire examined symptoms previously associated with vinyl chloride exposure. Assessment of exposure levels was made by analytical measurement and observation. The resulting statistical analysis revealed a dose-response type of relationship between exposure and certain morbidity symptoms.

Gas-vacuolated strains of Microcyclus aquaticus.

Two strains of gas-vacuolated heterotrophic bacteria have been isolated from freshwaters by using 0.01% peptone enrichments. Both are nonmotile, vibrioid organisms that form rings before division. In terms of these morphological traits and other characteristics, including nutritional characters and deoxyribonucleic acid base composition, these new isolates are very similar to Microcyclus aquaticus. It is proposed that they be considered strains of this species.