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Unlike other common disinfectants, ammonia is a natural product. It occurs in natural environments as a product of urea hydrolysis and of the microbial degradation of proteins and other nitrogen-containing compounds (referred to as ammonification). Significant concentrations of ammonia may be present in decomposing manure. Several studies have documented NH₃ evolution from cow manure during storage (7, 11-13, 19, 21, 22), even when the pH of the manure was <6.8 (13). The pH of NH₃ evolving in manure generally ranged from 7.5 (10) to 8.8 (7). Although Cryptosporidium parvum oocysts can survive many months in naturally infected manure under static conditions at 4°C (9), inactivation may be enhanced under anaerobic conditions in which ammonia persists. The objective of this study was to determine the effects on inactivation of freshly purified oocysts of environmentally relevant ammonia concentrations, ranging from 0.007 to 0.148 M, associated with manure slurries (22).

Oocyst purification. Feces from 6- to 20-day-old Holstein calves with cryptosporidiosis were processed by a continuous-flow differential density flotation method, as described by Jenkins et al. (9). Purified oocysts were adjusted to a concentration of 10⁷ ml¹ and stored in distilled water containing 100 U of penicillin G sodium ml¹, 100 µg of streptomycin sulfate ml¹, and 0.25 µg of amphotericin B ml¹ of oocyst suspension. The viability of each lot was estimated with a fluorochrome dye permeability assay, and, for some lots, in vitro excystation was performed as described below. Batches of purified oocysts for all experiments ranged from 3 weeks to 4 months old and ranged in viability from 90 to 95%.

Dye permeability assay. The dye permeability assay, for determining viability and potential infectivity of oocysts from environmental water samples, has been described previously (1, 4, 9). Stock solutions of DAPI (4'-6-diamidino-2-phenylindole) (2 mg ml¹ in high-performance liquid chromatography-grade methanol) and propidium iodide (PI) (1 mg ml¹ in 0.1 M phosphate-buffered saline [PBS]) were added to aliquots of the sample in microcentrifuge tubes at a ratio of 1:10 (vol/vol); the sample was then mixed with a Vortex mixer and incubated in the dark at 37°C for 2 h. Each aliquot was stained with Hydrofluor antibody (Hydrofluor Combo; EnSys, Research Triangle Park, N.C.), washed twice with PBS, and resuspended to the original volume in 0.3 M DABCO (1,4-diazabicyclo[2.2.2]octane) in 0.1 M PBS (DABCO-PBS). The samples were stored in the dark at 4°C until being examined (within 72 h).

In vitro excystation assay. The acid pretreatment protocol was described previously (9, 16). Each 100-µl aliquot of oocyst suspension in microcentrifuge tubes was washed once with 1 ml of acidified Hanks' balanced salt solution (HBSS) (10 µl of 1 M HCl in 10 ml of HBSS [pH 2.5]), resuspended in 1 ml of acidified HBSS, and incubated at 37°C for 1 h in the dark. Suspensions were then sedimented by centrifugation at 11,300 × g for 30 s; the pellets were washed three times with warm (37°C) HBSS to remove any residual acid and then resuspended to 100 µl in warm HBSS. Ten microliters of 2.2% sodium bicarbonate in HBSS and 10 µl of 1% sodium deoxycholate in Hanks' minimal essential medium (Sigma Chemical Co., St. Louis, Mo.) were added to each sample. The suspensions were then mixed on a Vortex mixer and incubated for 3.5 h at 37°C in the dark. Ten microliters of primary Hydrofluor antibody was then added to each sample, and they were mixed on a Vortex mixer and returned to the incubator for 0.5 h, for a total excystation incubation time of 4 h. The samples were then washed in 1 ml of PBS, resuspended in 100 µl of PBS, and stained with 10 µl of Hydrofluor labeling reagent. After incubating at room temperature in the dark for 0.5 h, oocysts were washed with 1 ml of DABCO-PBS, suspended in 100 µl of DABCO-PBS, and stored at 4°C until being examined (within 72 h). The percentage of excysted oocysts was determined by subtracting the number of empty oocysts observed before excystation from the number of oocysts observed after excystation (9).

Microscopy. All samples were examined with a Zeiss LSM-210 microscope (1) in conventional DIC and epifluorescence mode with a triple excitation-emission filter set (catalog no. 61001; Chroma Technology Corp., Brattleboro, Vt.) with excitations at 390 to 410, 485 to 510, and 555 to 585 nm and emissions at 450 to 475, 510 to 550, and 595 to 660 nm and a separate UV filter combination (excitation wavelength, 310 to 395 nm). Except for enumeration studies, a Zeiss 100×/1.3 plan-neofluor objective with 10× eyepieces was used for all other microscopy.

Experimental design. The source of ammonia for experimentation was a commercial ammonia solution containing distilled water and ammonia. The NH₃ concentration was proprietary and therefore was not given. All treatments were in triplicate. Freshly purified oocysts were suspended in distilled water at a concentration to 10⁷ ml¹. A 0.1-ml aliquot of the oocyst suspension was pipetted into 1.5-ml microcentrifuge tubes. Additional quantities of distilled water were added to the tubes and corresponded to the ammonia dilutions used as treatments: 90, 50, 25, 10, 5, 1, and 0% (vol/vol). The total volume of oocyst suspension per tube was 1 ml. The pH of each dilution was measured with a standard, calibrated calomel electrode, and the total NH₄ concentration was determined by the automated phenate method at the Soil Testing Laboratory of Cornell University. The concentrations of NH₃ were calculated for each dilution with the equation pOH = pK_b + log ([NH₄]/[NH₃]) (6), where K_b is the base dissociation constant. In descending order, final concentrations were 0.148 M (2,516 mg/liter), 0.103 M (1,751 mg/liter), 0.060 M (1,020 mg/liter), 0.039 M (663 mg/liter), 0.026 M (443 mg/liter), and 0.007 M (119 mg/liter). The tubes were incubated at room temperature (24 ± 1°C) in the dark. The tubes were labeled so that analysis would be performed in a blinded fashion. Tubes were sampled after 10 min, 1 h, and 24 h of incubation. The suspensions were washed four times in PBS and resuspended in 0.1 ml of PBS. For each sampling time, one set of tubes was assayed for viability with the dye permeability assay, and one set underwent in vitro excystation. Two 10-µl subsamples per replicate sample were observed microscopically, and 100 oocysts per subsample were characterized.

Statistical analysis. Except where otherwise stated, all statistical analyses of data were performed with Minitab Statistical Software (Minitab Inc., State College, Pa.).

Effect of ammonia. Based on the dye permeability assay, both increased time of exposure and the concentration of ammonia increased the inactivation rate of wild-type oocysts. Compared to the 1-h and 24-h exposure times, the 10-min exposure to the various concentrations showed the greatest discrepancies between the dye permeability assay and in vitro excystation, the results of which appeared not to be as time dependent as those of the dye permeability assay (Fig. 1). One-hour exposure showed better agreement, and 24-h exposure showed the best agreement, between the two assays. As previously observed (9), the dye permeability assay generally indicated a slightly higher survival rate than in vitro excystation. Results of the dye permeability assay indicated that exposure to all ammonia concentrations increased oocyst wall permeability (Fig. 2). The 10-min exposure showed generally increased frequencies of DAPI-positive, PI-negative (DAPI+ PI) oocysts with increased ammonia concentrations. Increased time of exposure resulted in increased frequencies of both DAPI+ PI and DAPI+ PI+ oocysts. After 1-h exposure, the frequency of DAPI+ PI oocysts appeared to reach a maximum between 24 and 36%, after which the frequency of DAPI+ PI+ oocysts increased. A 24-h exposure to 0.007 M ammonia at room temperature affected the permeability of the oocyst wall in more than 50% of the purified population; the proportion of DAPI+ PI+ (nonviable) oocysts was slightly less than the proportion of DAPI+ PI (potentially viable and/or infective) oocysts (Fig. 2). The progression of increased permeability of the oocyst wall from impermeable (DAPI PI) to partially permeable (DAPI+ PI) to lethally permeable (DAPI+ PI+) has been reported previously in relationship to increasing temperatures (9). The data indicated that increased times of exposure to the lowest concentrations of ammonia led to increases in the frequency of inactive (DAPI+ PI+) oocysts. Exposure to greater concentrations of ammonia also increased the percentage of inactivated oocysts. With the increased frequency of these inactivated oocysts, the correspondence between viability, as determined by the dye permeability assay (9), and the frequency of excysted oocysts, as determined by the in vitro excystation assay, increased. The principle difference between the two viability assays at the 1-h exposure was attributable to the number of partially permeable (DAPI+ PI) oocysts, which are considered viable and potentially infective (1, 4, 9). Untreated oocyst controls that paralleled these experiments showed no significant increase in oocyst wall permeability under the same conditions of time and temperature.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Viability of C. parvum oocysts exposed for 10 min, 1 h, and 24 h at 24 ± 1°C to various concentrations of ammonia (0.007, 0.026, 0.039, 0.060, 0.104, and 0.148 M), based on the dye permeability assay and in vitro excystation. The sum of the impermeable (DAPI PI) and semipermeable (DAPI+ PI) oocysts was the number of viable oocysts (9). Each datum point represents the mean ± standard error (SE) of at least six replicates of 100 oocysts observed per replicate. SE values not indicated are smaller than the symbols. , viable; , excysted.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Dynamics of dye permeability of oocysts as determined by the dye permeability assay. Each point corresponds to, and is from the same experiments as, the viability determinations of oocysts exposed to various concentrations of ammonia for 10 min, 1 h, and 24 h at 24 ± 1°C. Each datum point represents the mean ± standard error (SE) of at least six replicates of 100 oocysts observed per replicate. SE values not indicated are smaller than the symbols. , DAPI PI; , DAPI+ PI; , DAPI+ PI+; , empty.

Effect of high pH on oocyst viability. As indicated by the increased frequency of DAPI+ PI oocysts, there was a change in the permeability status of the oocyst wall with increasing pH (Fig. 3). However, there was only minor change in viability status, as indicated by the minimal change in frequency of DAPI+ PI+ oocysts. The in vitro excystation assay also showed little significant change in percent excystation with increasing pH (Fig. 4). These data strongly suggest that the high pH levels associated with ammonia solutions may have primarily time-dependent effects on oocyst wall permeability, whereas free ammonia, which has previously been reported to be lipid soluble and able to penetrate biological membranes (17), is able to permeate immediately the oocyst wall and sporozoite membranes and inactivate the sporozoites and/or enzymes involved in excystation. Based on the model proposed by Robin (17), the equilibrium equation NH₃  NH₄⁺ (6), and the nonpolar nature of NH₃, we hypothesize that an increased interoocyst NH₃ concentration raises the internal pH of the oocyst to deleterious levels. Because the components of pH, viz. H⁺ and OH, are charged, they remain external to the oocyst wall, thus explaining the inability of pH alone to inactive oocysts. The differential action of the elevated pH and free ammonia concentration on the oocyst may account for the initial differences (i.e., at 10-min exposure) between the dye permeability assay and in vitro excystation (Fig. 1). Increased oocyst wall permeability at elevated pHs associated with ammonia production would also lead to increased susceptibility to other forms of inactivation or disinfecting agents (15).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Results of the dye permeability assay on oocysts exposed to various pH levels (7, 9, 10, and 11) corresponding to the various concentrations of ammonia and incubated at 24 ± 1°C for 24 h. The sampling scheme and statistical analysis were the same as described in the legend to Fig. 1. , DAPI PI; , DAPI+ PI; , DAPI+ PI+.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Results of the dye permeability (A) and in vitro excystation (B) assays on oocysts exposed to various pH levels (7, 9, 10, and 11, corresponding to the various concentrations of ammonia) and incubated at 24 ± 1°C for 10 min, 1 h, and 24 h. The sampling scheme and statistical analysis were the same as described in the legend to Fig. 1. Squares, pH 7; diamonds, pH 9; circles, pH 10; triangles, pH 11.

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