Grantee Research Project Results
1997 Progress Report: Protozoa in Risk Assessment of Legionellosis—Inadequacy of Guidelines and Monitoring
EPA Grant Number: R825352Title: Protozoa in Risk Assessment of Legionellosis—Inadequacy of Guidelines and Monitoring
Investigators: Berk, Sharon G. , Wells, Martha J.M. , Newsome, Anthony L.
Institution: Tennessee Technological University , Middle Tennessee State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1996 through September 30, 1999 (Extended to September 30, 2000)
Project Period Covered by this Report: October 1, 1996 through September 30, 1997
Project Amount: $359,300
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
There are four major objectives. One is to conduct new laboratory studies of protozoan infection with Legionella. Factors affecting production of free vesicles containing live legionellae will be examined. These include testing several species of protozoa and varying factors such as MOI, temperature, pH, and cooling tower biocides. Co-cultures of protozoa with mixed bacteria including legionellae will be made to determine whether protozoa become infected when feeding on different ratios of legionellae to other bacteria. A second objective is to determine whether amoebae or other protists contain Legionella in the natural habitat. A third objective is to test a new technique, a flow separation technique other than flow cytometry, to detect vesicles in cooling tower water. A lab study of refining the technique for vesicle-sized particles will be conducted, followed by field investigations. The fourth objective is to determine cooling tower biocide efficacy against protozoan host organisms and legionellae in vesicles.Progress Summary:
The following results have been acquired since the time of the last annual report, November, 1997.Objective I. Three dominant species of bacteria have been isolated from cooling towers and fed to Tetrahymena, a ciliate isolated from a cooling tower, and to Acanthamoeba polyphaga to determine whether they would be suitable food sources for the co-culture experiments with Legionella. Previously Escherichia coli was used in mixtures with L. pnuemophila, however, bacteria found in cooling towers should represent naturally occurring alternatives to legionellae. Two of the isolates supported population growth of the ciliate and amoeba, while the third did not. The two will be used in mixed culture experiments during the next phase of experiments. Recently another amoeba was isolated from a cooling tower, presumably a hartmannellid species. It has been rendered axenic by antibiotic treatment. It will be tested for its ability to produce vesicles containing live legionellae.
The effect of pH on vesicle production was tested with Tetrahymena, but results were not consistent as compared with those using A. polyphaga. When cultured at pH 8 (close to the pH of the cooling tower), Tetrahymena produced significantly more vesicles in water of pH 8 than in water of pH 7 or 9. When cultured at pH 7, however, they produced more vesicles at pH 7 than pH 8 or 9 on one occasion but did not differ from pH 8 or 9 on another occasion. Those cultured at pH 9 did not differ in vesicle production when tested in water of pH 7 or 8. This work will be repeated to determine whether such inconsistencies occur again.
Objective II. While attempting to isolate amoebae from a cooling tower, one graduate student observed amoebae infected with highly motile bacteria. The infected amoebae were isolated, and the bacteria were capable of infecting axenic cultures of A. polyphaga and A. castellanii. Although these bacteria are difficult to isolate, they have been passaged many times and are always infective. Infected A. polyphaga cultures have been examined with a gene probe to Legionella, and show a strong positive reaction. Additionally, the contents of infected cultures have been processed with the EnviroAmp PCR kit, and show a strong positive response for the genus-specific probe, but not the species probe, indicating that these are probably a Legionella species other than pneumophila. Attempts to isolate and sequence this bacterium are ongoing. Dr Newsome's team has stained samples attached to microscope slides submerged in groundwater and has found infected amoebae, based on Giemsa stains. Immunogold antibodies against Legionella also show what appear to be legionellae in food vacuoles within protozoa from natural samples. Previously his team has shown positive responses with the LEG 705 gene probe in amoebae from natural samples.
Objective III. Dr. Wells and a graduate student continue to develop the detection system for the flow, field-flow fractionation procedure. Fluorescent microspheres are used as a surrogate model for particles such as the protozoan vesicles. Such microspheres are easily detected by the process, and separation of small sized microspheres has been successful, however, the larger microspheres closer in size to the vesicles have not yet been separated adequately. Analytical conditions are being varied to optimize separation. The relationship between emergence time and cross flow rate, channel flow rate, ionic strength of the carrier solvent, pH of the carrier solvent, membrane type, and channel dimensions are currently under investigation
Objective IV. Legionella packaged within amoeba and/or vesicles survive exposure to isothiazolones and glutaraldehyde better than free bacteria outside of amoebae or vesicles. Compared with filter-sterilized cooling tower water, the TRIS buffered medium appeared to protect bacteria from biocide exposure, or the cooling tower water was more toxic. Future work with biocides should consider using biocide-free cooling tower water, although the chemical composition may vary over the operational period of the cooling tower.
Future Activities:
We will continue to co-culture legionellae with other natural bacteria to determine the threshold ratios for vesicle production, and the flow separation procedure will be refined. Environmental factors will be examined for their influence on vesicle production.Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 16 publications | 2 publications in selected types | All 2 journal articles |
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Berk SG, Ting RS, Turner GW, Ashburn RJ. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Applied and Environmental Microbiology 1998;64(1):279-286. |
R825352 (1997) R825352 (1999) R825352 (Final) |
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Supplemental Keywords:
LLAP, water quality, protozoology., Health, Scientific Discipline, Environmental Chemistry, Chemistry, Epidemiology, Disease & Cumulative Effects, Biology, health effects, microbiology, risk assessment, Legionellosis, bacteria, infective dose, waterborne disease, airway disease, cooling towers, protozoa, biocidesProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.