Working with transmissible spongiform encephalopathy agents.
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The family of illnesses called transmissible spongiform encephalopathies (TSEs), or "prion" diseases, is composed of a small number of human and animal neurodegenerative diseases caused by unique pathogenic agents that are still not fully defined. They are best considered as "protein-misfolding diseases" (together with Alzheimer's disease, Parkinson's disease, and a few other rare examples) resulting from the conversion of a normal body protein into a misfolded amyloid multimer. The pathogenic agents display a unique resistance to conventional disinfection methods and an extraordinary environmental durability, which has led the US Department of Agriculture to designate the causative agent of bovine spongiform encephalopathy as a bioterrorism security threat. In this review, precautions and regulations concerning the handling of TSE agents are discussed in relation to personnel and environmental biosafety. (+info)
Containment of arthropod disease vectors.
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Effective containment of arthropod vectors of infectious diseases is necessary to prevent transmission of pathogens by released, infected vectors and to prevent vectors that escape from establishing populations that subsequently contribute to increased disease. Although rare, past releases illustrate what can go wrong and justify the need for guidelines that minimize risks. An overview of recommendations for insectary facilities, practices, and equipment is provided, and features of four recently published and increasingly rigorous arthropod containment levels (ACLs 1-4) are summarized. ACL-1 is appropriate for research that constitutes the lowest risk level, including uninfected arthropods or vectors that are infected with micro-organisms that do not cause disease in humans, domestic animals, or wildlife. ACL-2 is appropriate for indigenous and exotic arthropods that represent a moderate risk, including vectors infected or suspected of being infected with biosafety level (BSL)-2 infectious agents and arthropods that have been genetically modified in ways that do not significantly affect their fecundity, survival, host preference, or vector competence. ACL-3 is recommended for arthropods that are or may be infected with BSL-3 infectious agents. ACL-3 places greater emphasis on pathogen containment and more restricted access to the insectary than ACL-2. ACL-4 is intended for arthropods that are infected with the most dangerous BSL-4 infectious agents, which can cause life-threatening illness by aerosol or arthropod bite. Adherence to these guidelines will result in laboratory-based arthropod vector research that minimizes risks and results in important new contributions to applied and basic science. (+info)
Public response to infectious disease research: the UC Davis experience.
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This summary of the experience of the University of California, Davis, in public communications describes the course of applying for funds to build a National Biocontainment Laboratory. Opponents of the project put forward a wide range of arguments falling into two main areas: (1) the safety of the facility and the perceived risk of release of biological agents by accident, theft, or terrorist acts; and (2) concerns that the laboratories would be used for military or secret research beyond the control of the university. The communications strategy in support of the proposal used a number of different tools, including public workshops, direct mail, web sites, and proactive media relations. Communicating in this type of environment is challenging and requires long-term commitments of time and effort, as well as efficient cooperation across departments within the university and externally with local, county, and regional governments, agencies, elected officials, and community members. (+info)
Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a containment level-III laboratory as part of a Laboratory Risk Assessment Program.
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BACKGROUND: In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating/chemical fixation) may not consistently kill MTB organisms. METHODS: An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. RESULTS: Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80 degrees C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. CONCLUSIONS: This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures. (+info)
Extraction of Mycobacterium tuberculosis DNA: a question of containment.
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DNA fingerprinting of Mycobacterium tuberculosis by IS6110 restriction fragment length polymorphism analysis requires substantial high-quality DNA. We demonstrated that, despite extraction treatments that might be expected to inactivate this organism, M. tuberculosis remained viable during this process. These data suggest that the extraction of M. tuberculosis DNA should be performed within containment until complete. (+info)
Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet.
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BACKGROUND: The jet-in-air cell sorters currently available are not very suitable for sorting potentially biohazardous material under optimal conditions because they do not protect operators and samples as recommended in the guidelines for safe biotechnology. To solve this problem we have adapted a cell sorting system to a special biosafety cabinet that satisfies the requirements for class II cabinets. With aid of this unit, sorting can be performed in conformance with the recommendations for biosafety level 2. METHODS: After integrating a modified fluorescence-activated cell sorter (FACS) Vantage into a special biosafety cabinet, we investigated the influence of the laminar air flow (LAF) inside the cabinet on side stream stability and the analytical precision of the cell sorter. In addition to the routine electronic counting of microparticles, we carried out tests on the containment of aerosols, using T4 bacteriophage as indicators, to demonstrate the efficiency of the biosafety cabinet for sorting experiments performed under biosafety level 2 conditions. RESULTS: The experiments showed that LAF, which is necessary to build up sterile conditions in a biosafety cabinet, does not influence the conditions for side stream stability or the analytical precision of the FACS Vantage cell sorting system. In addition, tests performed to assess aerosol containment during operation of the special biosafety cabinet demonstrated that the cabinet-integrated FACS Vantage unit (CIF) satisfies the conditions for class II cabinets. In the context of gene transfer experiments, the CIF facility was used to sort hematopoietic progenitor cells under biosafety level 2 conditions. CONCLUSIONS: The newly designed biosafety cabinet offers a practical modality for improving biosafety for operators and samples during cell sorting procedures. It can thus also be used for sorting experiments with genetically modified organisms in conformance with current biosafety regulations and guidelines. (+info)
Efficacy of burning, tillage, and biocides in controlling bacteria released at field sites and effects on indigenous bacteria and fungi.
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Decontamination treatments of burning and biocide application, alone and in combination with tillage, were evaluated for their ability to reduce populations of bacteria applied to the leaves of plants in field plots. In addition, the effects of these control methods on indigenous leaf and soil bacteria and fungi were assessed. Field plots of bush beans (Phaseolus vulgaris), sprayed with the bacterium Pseudomonas syringae, Pseudomonas fluorescens, or Erwinia herbicola, received the following treatments: (i) control, (ii) tillage, (iii) burning, (iv) burning plus tillage (burn-tillage), (v) Kocide (cupric hydroxide), (vi) Kocide plus tillage, (vii) Agri-Strep (streptomycin sulfate), and (viii) Agri-Strep plus tillage. Leaves and soil from the plots were sampled at 1 day before and at 1, 3, 7, 10, 14, 21, and 30 days after application of the decontamination treatments. The burn and burn-tillage treatments produced the most significant reductions in bacterial populations. The Agri-Strep treatment was more effective than the Kocide treatment in eliminating applied bacteria, but neither biocide produced consistent or persistent control. In contrast, the tillage treatment, alone or in combination with the Agri-Strep or Kocide treatments, had a short-term stimulatory effect and increased populations of applied bacteria and also levels of indigenous fungi and bacteria. Agri-Strep and Kocide treatments caused significant reductions in indigenous bacterial populations up to 14 days after application and in indigenous fungal populations on day 7 after application. Our results suggest that conventional plant disease control methods may not provide satisfactory control of genetically engineered microorganisms and indicate a need for further development of effective and selective methods to control release microorganisms at field sites. (+info)
Evaluation of aquatic sediment microcosms and their use in assessing possible effects of introduced microorganisms on ecosystem parameters.
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In this paper we describe a sediment microcosm system consisting of 20 undisturbed, layered sediment cores with overlying site water which are incubated under identical conditions of temperature, light, stirring rate of overlying water, and water exchange rate. Ecosystem parameters (nutrient level, photosynthetic potential, community structure of heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients) of the laboratory microcosms and the source ecosystem were compared and shown to be indistinguishable for the first 2 weeks. In weeks 3 and 4, small differences were detectable in the nutrient level, community structure of heterotrophic bacteria, and thymidine incorporation rate. However, the photosynthetic potential, depth profiles of heterotrophic bacterial community structure, and oxygen microgradients were maintained throughout the incubation period and did not differ between laboratory microcosms and the source ecosystem. The microcosm system described here would thus appear to be a valid model of aquatic sediments for up to 4 weeks; the actual period would depend on the sediment source and incubation temperature. The validated systems were used with Rhine river sediment to assess possible effects on ecosystem parameters of Pseudomonas sp. strain B13 FR1(pFRC20P), a genetically engineered microorganism (GEM) that had been constructed to degrade mixtures of halo- and alkylbenzoates and -phenols. The GEM survived in the surface sediment at densities of 5 x 10(4) to 5 x 10(5)/g (dry weight) for 4 weeks and degraded added chloro- and methylaromatics. The GEM did not measurably influence ecosystem parameters such as photosynthesis, densities of selected heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients. Thus, the microcosm system described here would seem to be useful for the study of the ecology of biodegradation and the fate and effect of microorganisms introduced into the environment. (+info)