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Species and Genus Differentiation of Parasites (Giardia and Cryptosporidium) By Maldi Mass Spectrometry

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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 deaths. 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 Cryptosporidium 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 numerous marker proteins with molecular masses ranging from 3-36 kDa. Mass spectra of Giardia lamblia and Giardia muris cysts reveqled common biomarkers at 6662, 10365, 10571, 11321, and 12446 kDa, which may be indicators for the genus Giardia. The marker proteins (>20) observed in the MALDI spectrum of Giardia lamblia, ranging from 3-36 kDa, may be representative of the lamblia species. Similarly, 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 Cryptsporidium muris oocysts were also found, indicating that these species may be distinguished.

The solutoins could be stored at 40 degrees C, at least over a period of two days, without noticeable decomposition. The spectral data could be generated reproducibly, and observed detection limits for these mircoorganisms were also significantly low (~10 to 100 (oo)cysts). When 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. Species and Genus Differentiation of Parasites (Giardia and Cryptosporidium) By Maldi Mass Spectrometry. Presented at International Symposium on Waterborne Pathogens, Milwaukee, WI, August 29-September 1, 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: 08/30/1999
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