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Maldi-Mis Investigations of Drinking Water Pathogens - Giardia and Cryptosporidium

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Abstract:The protozoan parasites, Cryptosporidium parvum and Giardia lamblia, have been responsible for numerous waterborne outbreaks of gastrointestinal illness in the United States. The 1993 cryptosporidiosis outbreak in Milwaukee affected approximately 400,000 people and resulted in over 100 death. Both Cryptosporidium parvum oocysts and Giardia lamblia cysts have been found in many surface waters and are generally resistant to treatment with the chemical disinfectants used by water utilities. At the time of the Milwaukee outbreak, the accepted method for measuring Cryptosporidum in water, beside being extremely labor-intensive, could not distinguish between viable and non-viable (or infective vs. non-infective) organisms. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) recently has shown promise for identifying bacteria and distinguishing between species. We conducted MALDI-MS investigations to determine if MALDI-MS can be used to identify pathogenic protozoans and also to determine if live organisms can be distinguished from dead ones.

The acquired spectrum of each of the organisms contained numerous protein marker ions, and the observed noise level was low. Careful inspection of MALDI-mass spectra of both pathogens revealed numerour marker proteins with molecular massdes ranging from 3-36 kDa. Mass spectra of Giardia lamblia and Giardia muris cysts revealed comon biomarkers at 662, 10365, 10571, 11321, and 12446 kDa, which may be indicators for genus Giardia. The marker proteins (>20) observed in MALDI spectrum of Giardia lamblia, ranging rom 3-36 kDa, may be representative of the lamblia species. Similary, proteins (>25; 3-32 kDa) detected only in the spectrum of Giardia muris may be representative of the muris species. Similar marker proteins for Cryptosporidium parvum and Cryptosporidium muris oocysts were also found, indicating that these species may be distinguished.

The solutions could be stored at 4 degrees C, at least over a period of two days, without noticeable decomposition. The spectral data could be generated reproducibly, and the observed detection limits for these microorganisms were also significantly low (~10 to 100 (oo)cysts). When the lyophilized organisms were suspended in PbS buffer and/or detergents, the observed signals were diminished either significantly or totally. However, clean up of such samples over a reverse phase cartridge (C8) nullified the ionization suppression caused by the salts and detergents. Investigations are presently underway to determine the reproducibility of the generated spectral data and the effect of variation in growth conditions and sample preparation on observed biomarkers. Similarly, sample concentration techniques are being developed to allow the detection of low levels of these organisms in larger volumes of water.
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Citation:Krishnamurthy, T., H. Nair, R. Jabbour, S. D. Richardson, D. D. Kryak, M. W. Ware, and F. W. Schaefer III. Maldi-Mis Investigations of Drinking Water Pathogens - Giardia and Cryptosporidium. Presented at 47th ASMS Conference on Mass Spectrometry & Allied Topics, Dallas, TX, June 13-17, 1999.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Immediate Office
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Product Type: Abstrct/Oral
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Presented: 06/14/1999
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