Grantee Research Project Results
1999 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, 1998 through September 30, 1999
Project Amount: $359,300
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences
Objective:
There are four major objectives. One is to study factors affecting production of free vesicles containing live Legionellae. The factors include temperature, pH, and cooling tower biocides. Also, different ratios of Legionellae to other bacteria will be used to determine whether protozoa become infected when feeding on a mixture of 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 flow separation technique to detect vesicles in cooling tower water. The fourth objective is to determine cooling tower biocide efficacy against protozoan host organisms and Legionellae in vesicles.Progress Summary:
Objective I. Several factors affecting vesicle production were studied. Higher temperatures resulted in more vesicles released from the amoeba Acanthamoeba polyphaga; however, temperature made no difference with the ciliate Tetrahymena. Three biocides, thiocarbamate, QACs, and isothiazolones did not affect vesicle production with the amoeba; however, the isothiazolones inhibited vesicle production by the ciliate. No difference was observed in vesicle production for amoebae feeding on biofilms of bacteria compared with bacteria added in suspensions. Surface to volume ratio of the tissue culture flask made a difference in vesicle production for the amoeba. 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. Approximately 80 to 100 percent of vesicles from Tetrahymena contained viable Legionella pneumophila or L. micdadei. Only 6 to 22 percent of vesicles contained viable E. coli when the ciliate fed on these bacteria.Experiments to determine the lowest infectious dose of L. pneumophila that would result in infected amoebae and multiplication of the Legionellae showed that a ratio as low as 0.001:1 (bacteria to amoebae) resulted in a 7,000-fold increase in the bacteria. Addition of other non-Legionella species did not alter the number of recoverable L. pneumophila from amoeba co-cultures. Also, addition of Tetrahymena to an amoeba/Legionella co-culture resulted in concentration and packaging of the Legionella into Tetrahymena vesicles released to the medium. The presence of amoebae was necessary to initially amplify the numbers of Legionellae
for the ciliates to concentrate. This suggests that various groups of protozoa may function sequentially to affect the number and form of Legionella in water.
Objective II. 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 reveal Legionellae in food vacuoles within protozoa from natural samples. A paper on methods for assessing associations of amoeba and Legionella has been published, emphasizing the use of immunogold and in situ hybridization techniques. This work should elucidate the nature and extent of associations of the bacteria and their natural hosts.
Objective III. Dr. Wells and a graduate student continued to develop the flow-field-flow fractionation procedure. Some of the difficulties encountered earlier had been resolved, and the detection limits of the system were determined for microspheres in the size range of the protozoan vesicles. However, detection at the present time requires approximately 104 microspheres per 30 µl. It is unlikely at this time that natural or cooling tower waters could be concentrated to provide the high number of vesicles for detection. The technique still holds promise, as investigators continue to improve the detection limits.
Objective IV. Legionella packaged within amoeba and/or vesicles
survive exposure to isothiazolones and glutaraldehyde better than free bacteria
outside of amoebae or vesicles. The biocide BCDMH was equally toxic to free or
vesicle-bound bacteria. Also, glutaraldehyde and BCDMH were tested for
cross-resistance by protozoa. Exposure to each of these biocides did not confer
resistance to the other, indicating that these may be good biocides to use
sequentially.
Future Activities:
Experiments with biocides and vesicle-bound bacteria will continue, as well as work on various ratios of natural bacteria mixed with Legionella. Other potential host species will be examined to reveal the scope of natural hosts for the bacteria.Journal Articles on this Report : 2 Displayed | Download in RIS Format
| Other project views: | All 16 publications | 2 publications in selected types | All 2 journal articles |
|---|
| Type | Citation | ||
|---|---|---|---|
|
|
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) |
Exit Exit |
|
|
Desai R, Welsh C, Summy M, Farone M, Newsome AL. The potential of in situ hybridization and an immunogold assay to identify Legionella associations with other microorganisms. Journal of Microbiological Methods, Volume 37, Issue 2, August 1999, Pages 155-164. |
R825352 (1999) R825352 (Final) |
not available |
Supplemental Keywords:
Legionella, water quality, protozoology, cooling towers, biocides., Health, Scientific Discipline, Chemistry, Biology, Environmental Chemistry, Epidemiology, Disease & Cumulative Effects, health effects, Legionellosis, protozoa, risk assessment, biocides, waterborne disease, infective dose, microbiology, cooling towers, airway disease, bacteriaProgress 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.