INTRODUCTION

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Cryptosporidium parvum is an obligate protozoan parasite found in surface waters. It is the etiological agent for cryptosporidiosis, a parasitic infection that causes severe gastrointestinal illness which is potentially fatal among immunocompromised individuals (4, 6, 7, 15; http://www.awwa.org/pressroom /crypto.htm). The Milwaukee, Wis., outbreak of April 1993 infected as many as 400,000 individuals, making it the largest waterborne disease outbreak in U.S. history (16). Lawsuits arising from this incident are still in litigation. The quantification of Cryptosporidium in drinking water supplies is often problematic, and much research has focused on the detection and analysis of Cryptosporidium. Furthermore, Cryptosporidium is more resistant to conventional chlorine disinfection than most other microorganisms, so research in the development of effective treatment technologies for the removal and/or disinfection of Cryptosporidium has also been a matter of high priority for the drinking water industry.

An issue often neglected in research with C. parvum is that different researchers may use oocysts from different sources. This could make comparison among studies problematic. There can be observable differences in the resistance to disinfection of C. parvum organisms from different sources and of different lots from the same source (20; J. H. Owens, R. J. Miltner, T. R. Slifko, and J. B. Rose, Proc. Water Qual. Tech. Conf. 1999). An in-house production facility has been established to support Cryptosporidium research conducted by the U.S. Environmental Protection Agency. Production conditions have been quality controlled to minimize variation among oocyst lots. The present work investigates how matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) may be used to supplement quality controls by providing a spectral marker "fingerprint" of the C. parvum oocysts.

MALDI-TOF MS has been used to produce mass spectral responses to various biomolecules derived from microbiological samples to fingerprint various microbial species (5, 8, 10, 11, 14, 22; D. D. Kryack, F. W. Schaefer, M. Ware, T. Krishmamurthy, and S. Richardson, Proc. Int. Symp. Waterborn Pathog., 1999). It has been commonly applied to bacteria and has recently been extensively reviewed (2, 22). Spectral markers, sometimes referred to as biomarkers, may be dependent on the culture conditions for the bacteria, but species and genus level distinctions are possible when the conditions have been properly controlled. Software and algorithms may be used to aid in the analysis of such markers (10, 11).

MALDI-TOF MS has also been applied to Bacillus spores (8) and fungal spores (24), but to our knowledge the successful application of MALDI-TOF MS to protozoans, such as Cryptosporidium, has not been reported in the peer-reviewed literature. One preliminary investigation reported MALDI-TOF mass spectra for Giardia cysts and referred to its application to Cryptosporidium D. D. Kryack et al., Proc. Int. Symp. Waterborn Pathog.), and another reported experimental conditions for analysis of whole C. parvum oocysts (M. A. Claydon, D. J. Evason, K. Hall, and J. Watkins, Proc. 48th Am. Soc. Mass Spectrum Conf., 2000). The key to the success of MALDI-TOF MS analysis is sample preparation. In this paper, we review the way in which a freeze-thaw procedure may be used to prepare Cryptosporidium oocyst samples for fingerprint analysis. Freeze-thawed C. parvum oocysts were compared to Cryptosporidium muris oocysts. The effects of disinfection by chlorine and ozone of C. parvum were also investigated.

	MATERIALS AND METHODS

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Oocyst production. C. parvum and C. muris oocysts were propagated in house. A modified version of a protocol developed by Yang et al. (25) was used to propagate C. parvum oocysts in 6-week-old immunocompromised female C57BL/6 mice (J. Cicmanec and D. J. Reasoner, Proc. 1997 Int. Symp. Waterborne Cryptosporidium). One hundred twenty mice were administered dexamethasone phosphate (0.288 mg/liter) and tetracycline HCl (0.500 mg/liter) on alternating days via drinking water. On the eighth day of this dexamethasone-tetracycline regimen, each mouse was orally inoculated with approximately 1 million C. parvum oocysts obtained from the University of Arizona, where the Iowa strain, originally procured by Harley Moon (National Animal Disease Center, Ames, Iowa), has been propagated in Holstein bull calves. At 2 days postinfection (p.i.), the mice were placed in suspension cages over fecal collection pans. Feces were collected and washed through a series of sieves (mesh sizes, 10, 20, 60, and 100) with 0.01% (vol/vol) Tween 20. Oocysts were isolated from the fecal slurry on Sheather's sucrose and cesium chloride gradients (1) every 36 h until the number of oocysts shed/day/mouse declined to a point at which it was no longer reasonable to continue (~3 weeks p.i.). The RN66 strain of C. muris oocysts, originally obtained from M. Iseki (Osaka University Medical School, Osaka, Japan), was propagated in female CF-1 mice (9). Each mouse was orally inoculated with approximately 2 × 10⁵ oocysts in 200 µl of phosphate-buffered saline (PBS) and housed in a suspension cage (10 mice/cage). Two weeks (p.i.), feces were collected and washed through a series of sieves (mesh sizes, 10, 20, 60, and 100) with 0.01% (vol/vol) Tween 20. Approximately 150 ml of the sieved fecal slurry was underlaid with 75 ml of 1.0 M sucrose and centrifuged at 1,200 × g for 10 min at 4°C. Oocysts at the sucrose-Tween 20 interface were isolated and washed two times. The oocyst preparation was purified further by repeating this process with 0.85 M sucrose. The oocysts were stored in PBS with penicillin (100 U/ml) and streptomycin (100 µg/ml) at 4°C for up to 30 days.

Preparation of oocyst samples for MALDI-TOF mass spectrometric analysis. (i) Oocyst washing. For whole-oocyst analysis, oocysts suspended in PBS were placed in a 2-ml polypropylene centrifuge tube. The sample was desalted to prevent the formation of cation adducts, which tend to degrade the quality of the MALDI-TOF MS spectrum. The production of the oocysts, as detailed above, leaves the oocysts in a solution which contains a number of potential adduct-forming species. Washing also greatly reduces the quantity of residual chlorine following the disinfection studies. The following desalting procedure was used to prepare Cryptosporidium oocysts for analysis. Oocysts were centrifuged at 1,600 × g for 10 min, and the supernatant was aspirated with an automatic pipettor. A 200-µl volume of deionized water was added to the centrifuge tube, which was then capped and vortexed. The washing procedure was repeated three times. Samples were washed as many as five times; however, more than three washes did not improve the spectral quality. The washed oocysts were resuspended in 5 to 15 µl of deionized water and vortexed. The sample was then spotted on the MALDI-TOF target with a Gilson P-2 pipettor and analyzed. Samples were spotted immediately to prevent isotonic imbalances from disrupting the oocysts.

(ii) Freeze-thawing of washed oocysts. For the samples subjected to freeze-thawing, whole oocysts were first washed as described above. The samples were then alternately frozen with liquid nitrogen and thawed in a 60°C water bath for 1-min intervals. The freeze-thaw cycle was repeated five times. The sample was then centrifuged at 16,000 × g for 15 min. The sample (0.5 µl) for MALDI analysis was carefully removed with a Gilson P-2 pipettor, with care not to disturb the pellet.

MS study. A KOMPACT SEQ (Kratos Analytical, Ramsey, N.J.) MALDI-TOF mass spectrometer was used. Mass spectra were acquired in positive linear high-power mode, and pulsed extraction was used to improve resolution. The MALDI target was spotted with 0.5 µl of the sample solution. Before the sample dried, 0.25 µl of matrix solution (10 mg of 3,5-dimethoxy-4-hydroxy-cinnamic acid/ml in a water-acetonitrile mixture [various ratios of water-acetonitrile were investigated; mass spectral responses were similar for the various ratios, and 70:30 water-acetonitrile was chosen for convenience]) was applied on top of the sample. The resultant spot was allowed to air dry. Then, another 0.25 µl of matrix solution was applied. Mass spectra were acquired by taking 500 to 4,000 shots across the MALDI target. Results were similar for the range 500 to 4,000 shots but tended to degrade when there were less than 500 shots.

Disinfection procedure. Chlorination of oocysts was performed by mixing 5% commercial sodium hypochlorite solution with the oocysts in a 2-ml polypropylene centrifuge tube. After the reaction period, the chlorinating solution was removed in the first step of the freeze-thaw procedure. Thus, no neutralizing agent was added. Ozonation of the oocysts was performed by mixing ozonated, deionized water with the oocyst water in a 2-ml polypropylene centrifuge tube. The ozonated water was produced by sparging deionized water with ozone gas; the ozone concentration was monitored spectrophotometrically. After the reaction period, the ozonating solution was removed in the first step of the freeze-thaw procedure.

	RESULTS AND DISCUSSION

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Effect of sample preparation. Figure 1a shows the positive-ion MALDI mass spectrum of whole C. parvum oocysts washed three times with deionized water. The mass spectrum has several discernible features, which are summarized in Table 1. Figure 1a represents ~4 million oocysts contributing to the spot deposited on the MALDI target. Figure 1a shows the range of the mass spectrum from m/z 6,000 to 13,000. Portions of the mass spectrum in m/z 300 to 1,000 contained numerous peaks (>60), similar to another report (Claydon et al., Proc. 48th Am. Soc. Mass Spectrom. Conf.; M. A. Claydon, personal communication) of the MALDI-TOF mass spectrum of whole oocysts. A high signal/noise ratio in the region made mass assignments problematic. Therefore, the region m/z 6,000 to 13,000 was used in further studies, since these peaks were better resolved (more separated in the m/z range) and in a region of the mass spectrum with a lower signal/noise ratio.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   MALDI-TOF mass spectra of C. parvum and C. muris oocysts. The portion of the mass spectra containing the spectral markers is shown. Other regions were not interesting. (a) Whole C. parvum oocysts washed in deionized water. (b) C. parvum oocysts washed and freeze-thawed. (c) C. muris oocysts washed and freeze-thawed. Note that the region between m/z 6,000 and 12,500 has been vertically expanded by a factor of 10.

Spectral markers from several batches of freeze-thawed oocysts. In order to compare the variabilities of oocysts produced in our laboratory, several oocyst lots were collected over a 5-month period. Oocysts from each batch were subjected to MALDI-TOF MS using the freeze-thaw procedure, and their mass spectral peaks were tabulated. These peaks appear in multiple preparations, i.e., desalting, freeze-thaw, and sample spotting, of the MALDI targets from each lot. The peaks may be organized into a MALDI fingerprint, defined by (11)

(1)

where for each peak i, l_i is the average peak location, s_li is the standard deviation in peak location, h_i is the average peak height, s_hi is the standard deviation of peak height, and p_i is the fraction of replicates in which peak i appears. In an algorithm for the fingerprint analysis of bacteria (11), the peak height, h_i, was not used because the variability in the peak intensities could not be dealt with objectively. Variability was experienced in the analysis of C. parvum. For instance, there was ~75% variability in the relative intensity of the spectral marker at m/z 9,243; the cause of this variability is unknown. Since marker intensities for bacteria did not generally affect the success of the algorithm (11), they were not considered in the MALDI fingerprint (equation 1) of C. parvum.

Effect of disinfection on spectral-marker patterns. It would be interesting to relate changes in the fingerprint (Table 2) to practical C. parvum issues. While many endpoints may be studied, one relevant issue is how different lots and sources of C. parvum resist disinfection to different degrees (20; Owens et al., Proc. Water Qual. Tech. Conf.). As a first step in this direction, oocysts were subjected to both chlorine and ozone to investigate the effect of chemical disinfection on the spectral-marker patterns of C. parvum. Figure 2a shows the MALDI-TOF MS spectrum obtained from oocysts exposed to ozone (12 mg/liter for 20 min). Compared to the nondisinfected sample (Fig. 1b [1/10 the vertical scale of Fig. 2a]), there is a marked reduction in the intensity of the spectral markers, indicating that the spectral markers were destroyed along with the organism. Most were no longer detectable, with the region m/z 6,000 to 6,400 containing most of the remaining signal. For comparison, the same number of oocysts were subjected to 250 mg of chlorine/liter (a much higher chlorine concentration is required to achieve inactivation rates comparable to those achieved with ozone [12]). The mass spectrum for these oocysts (Fig. 2b) was similar to that of ozonated oocysts (Fig. 2a). When the chlorine concentration was increased to 25,000 mg/liter (far in excess of the amount needed), no spectral-marker-type features were apparent (Fig. 2c).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   MALDI-TOF mass spectra of freeze-thawed C. parvum oocysts subjected to disinfection. The portion of the mass spectra containing the spectral markers is shown. Other regions were not interesting. (a) Ozone (12 mg/liter) added and reacted for 20 min. (b) Chlorine (250 mg/liter) added and reacted for 20 min. (c) Chlorine (25,000 mg/liter) added and reacted for 20 min. The vertical scale has been adjusted to 10 times the intensity of that in Fig. 1b, which does not contain disinfectant.

Conclusion. This study may have led to the first MALDI-TOF MS fingerprint of Cryptosporidium oocysts. The mass spectrum of C. parvum differs from that of C. muris, increasing confidence in the specificity of the fingerprint analysis. The analysis was relatively rapid and simple to reproduce. Oocysts from several different lots obtained at the Environmental Protection Agency produced similar mass spectra, suggesting that the technique may be used to supplement quality controls for C. parvum oocyst production. Maintaining the quality of the mass spectral fingerprint generated by the MALDI-TOF MS technique may also lead to more tightly controlled Cryptosporidium disinfection and removal experiments. The disinfection results obtained in this study suggest that further exploration of MALDI-TOF MS for C. parvum analysis is warranted, particularly an investigation of the relationship between spectral markers and the biocidal potentials of various chemical disinfectants, i.e., chlorine, ozone, and chlorine dioxide.