Vol. 58, No. 11
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1992, p. 3488-3493
0099-2240/92/113488-06$02.00/0
Copyright X) 1992, American Society for Microbiology
Viability of Cryptosporidium parvum Oocysts: Correlation of In Vitro Excystation with Inclusion or Exclusion of Fluorogenic Vital Dyes A. T. CAMPBELL, L. J. ROBERTSON, AND H. V. SMITH*
Scottish Parasite Diagnostic Laboratory, Stobhill General Hospital, Springbum, Glasgow, Scotland G21 3UW Received 9 April 1992/Accepted 3 August 1992
A viability assay for oocysts of Cryptosporidium parnum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples. a group of experts have recommended that cryptosporidiosis in humans become a reportable disease (1). Following acceptance of the public health significance of both cryptosporidiosis and the waterborne route of infection, attention has focused on development of methods which indicate whether sporozoites contained within an individual oocyst are viable or nonviable. The development of a reproducible, sensitive, user-friendly viability assay which could be used to determine whether the sporozoites contained within oocysts are capable of excysting and which would parallel and be compatible with in vitro excystation would be of value. It should be applicable to individual or small numbers of oocysts, not only because oocysts may normally occur in low numbers in potable water (26), but also because currently recommended techniques for the isolation of oocysts from water are inefficient (9 to 59% recovery efficiency [26]). Such an assay would not only have an obvious applied use in laboratories concerned with the detection of protozoa in water but would also be a valuable tool in further research. A series of 27 fluorogenic dyes have been assessed (5) to determine whether inclusion or exclusion of them correlated with viability as assessed by in vitro excystation. Of these dyes, a simultaneous dual labeling with 4',6-diamidino-2phenylindole (DAPI) and propidium iodide (PI) showed significant promise. The aims of this communication are (i) to compare inclusion or exclusion of the fluorogenic vital dyes DAPI and PI with in vitro excystation, (ii) to assess the importance of known triggers of oocyst excystation such as incubation at 37°C with bile and acid induction on both efficiency of in vitro excystation and fluorogenic dye inclusion or exclusion, and (iii) to assess the usefulness of these dyes as surrogate indicators of viability for small numbers of C. parvum oocysts.
Transmission of protozoan parasites of the gastrointestinal tracts of humans, such as Giardia intestinalis and Cryptosporidium parvum, via water is well documented (12, 26), and surveys of the occurrence of transmissive stages of these parasites (cysts and oocysts, respectively) indicate that these stages occur commonly in the aquatic environment (9, 25, 29). The public health significance of dead transmissivestage organisms is minimal; however, when the organisms are infective, the risk to public health can be enormous. Hence, information about cyst and oocyst viability is of considerable importance. To this end, methods for determining the viability of cysts of Giardia spp. have received attention in recent years. Techniques used for assessing the viability and/or infectivity of Giardia cysts include in vitro excystation (3, 19), exclusion or inclusion of vital dyes (2, 11, 13, 14, 23, 24), and infectivity in animal models (17, 22, 23). In general, cysts and oocysts occur in low numbers in the aquatic environment, and the minimum infectious dose is likewise low (between 10 and 100 cysts or oocysts). It is therefore important to be able to assess whether individual organisms isolated from water are viable. Assessment of viability by in vitro excystation and infectivity of animals requires organisms in a relatively clean and concentrated suspension, and these methods are therefore unsuitable for assessing the viability of individuals or of small numbers of the transmissive stage. Thus, exclusion or inclusion of vital dyes and Nomarski differential interference contrast (DIC) microscopy are the techniques that have shown most promise for assessing viability of individual Giardia cysts isolated from water. Within the last 10 years, the importance of transmission of C. parvum in water has been acknowledged worldwide. Six outbreaks of waterborne cryptosporidiosis have been documented: four occurred in the United Kingdom, and two occurred in the United States (1). In the United Kingdom, *
MATERLALS AND METHODS Sources and purification of oocysts. C. parvum oocysts were obtained from the following sources. Purified cervine-
Corresponding author. 3488
VOL. 58, 1992
ovine oocysts (c-o oocysts) were purchased from the Moredun Research Institute (MRI), Edinburgh, Scotland. This strain, originally isolated from deer feces, has been passaged in sheep by MRI. Human oocysts were isolated from fecal samples submitted from individuals with cryptosporidiosis for routine examination to the Scottish Parasite Diagnostic Laboratory (SPDL), Stobhill Hospital, Glasgow, Scotland. Oocysts purified from each stool were kept as separate isolates. Bovine oocysts were isolated and purified from bovine fecal samples obtained either from a study farm by Glasgow Veterinary School or from MRI. Purified bovine oocysts were purchased from MRI. This isolate has been passaged in calves by MRI. Oocysts purchased from MRI had been purified by a semiautomated method which involved incubation of the oocysts in 1% sodium dodecyl sulfate and both acid sedimentation and sucrose flotation (30). Oocysts were obtained suspended in phosphate-buffered saline (PBS; pH 7.2) containing 100 U of penicillin and 100 pg of streptomycin per ml. Oocysts from bovine and human fecal samples were purified at SPDL as follows. Fecal samples (ca. 5 g) were made up to 50 ml with reverse osmosis (RO) water, emulsified by vortexing, and then centrifuged at 900 x g for 5 min, and the supernatant was removed by aspiration and discarded. This washing procedure was repeated three to five times until the supernatant was clear. The pellet was resuspended in 10 ml of RO water and overlaid with an equal volume of diethyl ether. After being thoroughly mixed by shaking, the samples were centrifuged at 900 x g for 5 min, and fat and supematant layers were discarded. The pellet was washed twice in RO water as described above to remove traces of diethyl ether. Further purification of oocysts was performed by resuspending the pellet in 10 ml of RO water which was underlayered with a sucrose solution (1.18 specific gravity when cold) and centrifuging it at 900 x g for 15 min. The interface was recovered, diluted with RO water, and washed. Oocyst purification by cold sucrose flotation was repeated until the oocyst suspension was free of excess contaminating matter. All oocyst suspensions were stored at 4°C in RO water, and aliquots were sampled for bacterial and fungal contaminants by routine culture on blood agar and Sabouraud agar plates. Incubation of oocysts with DAPI and PI. Working solutions of DAPI (2 mg/ml in absolute methanol) and PI (1 mg/ml in 0.1 M PBS, pH 7.2) were prepared and stored at 4°C in the dark. Purified oocysts were suspended in Hanks balanced salt solution (HBSS) (2 x 104 oocysts per ,ul of HBSS), and 100 ,ul of suspension was incubated simultaneously with 10 ,ul of DAPI working solution and 10 ,ul of PI working solution at 37°C. Initially, suspensions were sampled regularly to that oocysts in subsequent experiments were incubated with DAPI and PI for the length of time optimal for producing maximal dye uptake. Oocysts were washed twice in HBSS before being viewed by epifluorescence microscopy. Microscopy. Ten-microliter aliquots of oocyst suspension were viewed under both DIC (Nomarski) optics and epifluorescence with an Olympus BH2 microscope equipped with a UV filter block (350-nm excitation, 450-nm emission) for DAPI and a green filter block (500-nm excitation, 630-nm emission) for PI. Proportions of ruptured (ghost), PI-positive (PI+), DAPI-positive PI-negative (DAPI+ PI-), DAPIensure
negative PI-negative (DAPI- PI-) oocysts (Table 1) were quantified by enumerating more than 100 oocysts in each sample. Ghost oocysts were easily identified under Nomarski optics, being nonrefractile apart from the residual body.
VIABILITY OF C. PARVUM OOCYSTS
3489
TABLE 1. Correlation of oocyst viability, visualization of oocyst contents by Nomarski (DIC) microscopy, and exclusion or inclusion of DAPI and PI Type of
Ghost PI+ DAPI+ PIDAPI- PI-
Contents
seen
by Nomarski
Inclusion
of:
microscopy
PI
DAPI
No Yes Yes Yes
No Yes No No
No Yes Yes No
Viability Dead Dead Viable at assay Viable after further trigger
a DAPI- PI- oocysts can be converted to DAPI+ Pl- oocysts and vice versa.
PI+ oocysts fluoresced bright red under the green filter block, and this red fluorescence varied from distinct points of intense fluorescence corresponding to the locations of sporozoite nuclei to a more-diffuse fluorescence within the oocyst. Oocysts were considered DAPI+ PI- only if they did not include PI and if the nuclei of the sporozoites fluoresced a distinctive sky blue under the UV filter block. Those oocysts which were neither PI+ nor ghosts and which showed either a rim fluorescence or an absence of fluorescence under the UV filter block were considered DAPIPI- (Table 1). Excystation. Two similar excystation protocols, S and M, were used on oocyst suspensions following either preincubation treatment or incubation with vital dyes. For excystation protocol S, 200 p.l of bile solution (1% bovine bile in Hanks minimal essential medium) and 50 .1l of sodium hydrogen carbonate solution (0.44% sodium hydrogen carbonate in RO water) were added to 100 p,l of purified oocyst suspension. The reactants were mixed thoroughly prior to incubation at 37°C. For excystation protocol M, 10 ,ul of sodium deoxycholate solution (1% sodium deoxycholate in Hanks minimal essential medium) and 10 ,ul of sodium hydrogen carbonate (2.2% sodium hydrogen carbonate in HBSS) were added to 100 p.l of purified oocyst suspension. The reactants were mixed thoroughly prior to incubation at 37°C. For both excystation protocols, reagents were made up freshly (il of oocyst suspension and 1 ml of HBSS acidified to pH 2.75 with HCI (20 ml HBSS:200 ,ul of 1 M HCI) at 37°C; (v) 100 ,ul of oocyst suspension and 1 ml of HBSS at 37°C (control for iv). Statistics. Calculation of correlation coefficients, linear regression analysis, analysis of covariance, and Mann-Whitney U tests were performed with a MINITAB statistical package. RESULTS Uptake of DAPI and PI. Uptake of PI by oocysts was maximal after a 5-min incubation at 37°C; however, uptake of DAPI did not maximize until oocysts had been incubated for 2 h or longer at 37°C. Therefore, in all subsequent experiments described here, both DAPI and PI were incubated simultaneously at 37°C for 2 h. The proportion of oocysts which took up the dyes varied between oocyst isolates; c-o isolates contained a considerably greater proportion of DAPI+ PI- oocysts than the other isolates tested. Excystation dynamics and correlation of excystation protocols. For human, bovine, and c-o isolates of C. parvum oocysts excysted by excystation protocol S or M, the excystation efficiency was maximal after 4 h (Fig. 1). Excystation efficiencies varied between oocyst isolates, with the c-o isolates having a considerably higher maximal excystation efficiency than the other isolates tested. Correlation of excystation protocol S with excystation protocol M gave a correlation coefficient of 0.996. This correlation also held for oocyst suspensions that had been preincubated with acidified HBSS before being subjected to the excystation protocols (Fig. 2). Prediction of maximal (4-h) excystation efficiency with DAPI and PI. It was hypothesized that inclusion or exclusion of DAPI and PI following a 2-h incubation at 37°C could be used to predict the viability of C. parvum oocysts, the proportion of DAPI+ PI- oocysts being the predicted viability and the observed or actual viability being calculated from maximal in vitro excystation. Oocysts were considered
10
0
FIG. 1. Excystation of different isolates of C. parvum oocysts over time.
100
20 30 40 50 60 70 80 90 % excystation using excystation protocol S
FIG. 2. Correlation of excystation protocols S and M.
viable if at least one sporozoite was ejected (partially or totally excysted) during the in vitro excystation. For human, bovine, and c-o oocysts, excystation efficiency was maximal after 4 h. The predicted viabilities (after 2 h of incubation with DAPI and PI at 37°C) were comparable to the maximum observed excystation efficiencies (Fig. 1). Correlation of this excystation efficiency (observed viability) with predicted viability (inclusion or exclusion of DAPI and PI) gave a correlation coefficient of 0.997. Linear regression analysis provided the following mathematical relationship between predicted viability (x) and observed viability (y): y = 1.85 + 0.936x (d2 adjusted for degrees of freedom = 99.3%). Analysis of covariance demonstrated no significant difference between this relationship (y = 1.85 + 0.936x) and the simpler mathematical relationshipy = x (Fig. 3). Since a simple correlation between predicted viability (percentage of DAPI+ PI- oocysts after a 2-h incubation with vital dyes) and observed viability (maximal excystation efficiency) had been derived, it was suggested that those oocysts which excysted during a 4-h excystation protocol would be those which were DAPI+ PI-. Excystation time trials showed that for all oocyst isolates, there was a marked decrease in the proportion of DAPI+ PI- oocysts with time. The proportion of DAPI- PI- oocysts fluctuated but did not decrease significantly with time (Fig. 4). The sporozoites seen in the excystation medium all contained fluorescent 100O
cernate/ovine isolate
* bovine isolates
80
o3
/ human isolates
0.3). In the suspension of c-o oocysts, the 10); --@- DAPI+ human isolate; --0--, DAPI- human isolate; proportion of viable (DAPI+ PI-) oocysts was not signifi-0--, DAPI+ bovine isolate; -- O --, DAPI- bovine isolate. cantly different after preincubation treatment (P > 0.3), and the mean proportion of viable oocysts remained higher than 70% (Table 4). For bovine oocysts, the suspension which (DAT?I+ PI-) nuclei. Free sporozoites with nonfluorescent had not been acidified had relatively low proportions of nucle i were never observed. DAPI+ PI- oocysts, but this proportion was significantly Pteincubation treatments. Preincubation treatments perincreased (Table 4; P c 0.009) by incubation in acidified form(ed on oocysts (see below) had a significant impact on HBSS. viabi]lity, both observed (maximal excystation) and predicted In both the c-o isolate and the bovine isolate, the propor(incu bation with DAPI and PI). For all preincubation treattion of DAPI- PI- oocysts was reduced significantly by ment;s, a good correlation between observed and predicted preincubation with acidified HBSS (Table 4; c-o isolate, P < viabillities was observed (Fig. 5), with a correlation coefficient of 0.989. 0.006; bovine isolate, P < 0.009). Sporozoite ratios. Sporozoite ratios were enumerated dur(i):Preincubation with 0.1 M NaOH and 0.1 M HCI. In both c-o a:nd human isolates, preincubation with 0.1 M NaOH at ing excystation of c-o and bovine (purified at SPDL) oocyst isolates (Table 5). High variability was observed, particuroom temperature was associated with a significant increase in oo cyst death (Tables 2 and 3; c-o isolate, P < 0.01; human larly in the c-o isolate, as demonstrated by the relatively high standard deviations (Table 5). Sporozoite ratios were also isolatte, P c 0.05). In the c-o isolate after incubation with enumerated during excystations of oocyst suspensions that NaO]H, a significant reduction in the proportion of viable had been preincubated in acidified HBSS. For both isolates, (DATPI+ PI-) oocysts was observed (Table 2; P c 0.001). However, in the human isolate, a dynamic situation was sporozoite ratios tended to fall over the duration of the obsexrved, with a significant decrease in DAPI- PI- oocysts excystation, presumably because of lysis of the sporozoites. After 4 h, the sporozoite ratio never exceeded 1 (Table 5). and al significant increase in DAPI+ PI- oocysts (Table 3; P For both isolates, preincubation in acidified HBSS tended to be associated with an enhanced sporozoite ratio during the initial stages of excystation, and this association was signif100] icant (P < 0.009) in the bovine isolate (Table 5). /Z a.
2201t
80 :5
DISCUSSION The results of this study demonstrate that oocysts whose walls are permeable to DAPI but not to PI (DAPI+ PI-)
60 40
0
0
201 0
TABLE 3. Inclusion or exclusion of DAPI and PI in human
20
40 60 Prdccted viabty
80
100
FIG. 5. Correlation of observed and predicted viabilities after preincubation treatments. Symbols: 0, RO water at room temperature; A, 0.1 M NaOH at room temperature; 0l, 0.1 M HCO at room temperature; 0, HBSS at 37C; A, acidified HBSS at 37°C; M, 1% trypsin-HBSS at 37'C; x, 1% trypsin-acidified HBSS at 37°C; =y.
x
oocysts following preincubation treatment % Inclusion (mean + SD)' Preicubation treatment
DAPI+ PI-
DAPI- PI-
RO water 0.1 M HCI 0.1 M NaOH
18.6 ± 1.9 53.6 ± 3.1 58.0 ± 1.1
76.8 ± 1.8 23.9 ± 4.6 20.5 ± 1.7
an =5.
% Dead
4.6 ± 0.6 22.5 ± 2.0 21.5 ± 2.9
APPL. ENvIRON. MICROIBIOL.
CAMPBELL ET AL.
3492
TABLE 4. Inclusion or exclusion of DAPI and PI in c-o and bovinea isolates of oocysts following preincubation in HBSS or acidified HBSS
% Inclusion (mean + SD)b Isolate and treatment DAPI+ PlDAPI- PItreatment C-o HBSS Acidified HBSS Bovine HBSS Acidified HBSS
Dead ~~~~~~~%
73.2 ± 0.8 74.1 + 4.6
3.4 ± 2.3 0.3 ± 0.5
23.4 ± 2.4 25.6 ± 5.0
11.1 ± 1.2 64.0 ± 2.2
60.3 ± 4.2 5.3 ± 1.5
28.6 ± 3.1 30.7 ± 1.2
a Purified at SPDL. bn = 3.
after a 2-h incubation at 37°C will excyst in an excystation protocol during a 4-h incubation. Such oocysts are described as viable at assay (Table 1). DAPI is an AT-selective DNA stain, and when binding to DNA occurs, there is an approximately 20-fold enhancement in fluorescence (16).
The correlation of inclusion of DAPI and exclusion of PI with excystation during a 4-h excystation protocol was demonstrated to hold for oocysts obtained from various sources. This correlation held if excystation dynamics were altered by a variety of preincubation treatments. The high viability observed in suspensions of c-o oocysts (low numbers of DAPI- PI- oocysts) could be due to the oocyst purification method used at MRI, which involves contact between the oocysts and dilute acid; preincubation of SPDLpurified oocysts with acid was found to increase their excystation efficiencies to similar levels. PI+ oocysts have sporozoites with disrupted or broken membranes and are considered dead (Table 1). It has been reported (10) that only cells with disrupted or broken membranes can be stained with PI. In our experience, motile sporozoites never include PI. Oocysts are also considered dead if they have ruptured and lost their contents (ghost oocysts) before being subjected to an excystation protocol
(Table 1).
Oocysts which exclude both DAPI and PI but appear by Nomarski microscopy to be morphologically intact and undamaged (Table 1) require a further trigger before they will excyst in one of the protocols described above. Inclusion of a selected preincubation treatment (e.g., incubation with acidified HBSS for 1 h at 37°C) converts the majority of these DAPI- Pl- oocysts to DAPI+ Pl- oocysts, regardless of source of oocysts or purification technique. Following such a preincubation treatment, the oocyst wall becomes TABLE 5. Sporozoite ratios during excystation of c-o isolate and bovine isolatea of oocysts following preincubation in HBSS or acidified HBSS Isolate and treatment
C-o HBSS
Acidified HBSS Bovine HBSS Acidified HBSS a Purified bn = 3.
at SPDL.
Sporozoite ratio (mean + SD) at': 30 min
1h
4h
1.8 + 2.2 4.0 ± 1.0
2.2 ± 1.6 2.3 ± 1.2
0.5 + 0.2 0.4 + 0.3
0.2 ± 0.3 2.8 + 0.3
1.2 ± 0.5 2.2 + 0.8
0.6 ± 0.2 0.6 + 0.2
permeable to DAPI. The DAPI can then be incorporated into sporozoite DNA through intact sporozoite membranes. The opposite conversion, of oocyst walls permeable to DAPI becoming impermeable to DAPI, has also been demonstrated to occur (21), and such a conversion has been shown to be reversible (21). The requirement of an acid trigger, which would normally occur in vivo, to convert a proportion of the oocyst population from being DAPI impermeable to being DAPI permeable is important for two reasons. First, for optimized in vitro excystation, the following series of stimuli should be included: (i) temperature elevation to 37°C, as little excystation is detected at lower temperatures (8, 28), and above 37°C, sporozoites do not survive (17a); (ii) incubation in dilute acid for 1 h; and (iii) incubation with bile salt or sodium deoxycholate in sodium hydrogen carbonate for 4 h. Incubation with chemicals other than acid might have the same trigger effect, although whether this is achieved by the same mechanism is unknown. For example, one recommended excystation protocol (7) involves a 10-min preincubation of oocysts in 1.05% sodium hypochlorite on ice. We suggest that dilute acid is more appropriate, as it more closely resembles the environment expected to be encountered by ingested oocysts in vivo (21). Second, permeability of oocyst walls varies within an isolate. The degree of permeability may affect the abilities of individual oocysts to withstand environmental pressures; the proportion killed by incubation in either 0.1 M NaOH or 0.1 M HCl was significantly lower in the isolate whose oocysts were originally impermeable to DAPI. We suggest that oocyst permeability should be considered when results from environmental and disinfection studies are interpreted (4-6, 15, 18, 20, 27). DAPI staining of sporozoite nuclei gives a characteristic sky blue fluorescence under a UV filter block. Besides indicating oocyst viability, visualization of the sporozoite nuclei provides a useful adjunct for more-definitive techniques of recognition and identification of Cryptosporidium oocysts. Visualization of sporozoite nuclei can be used in conjunction with the more-conventional techniques involving fluorescence-labeled monoclonal antibodies. It has been recommended that recognition of cysts of Giardia spp. in water rely on the identification of three morphological features (morphometry and the presence and distribution of at least two internal organelles) (12). The use of DAPI in conjunction with a fluorescence-labeled monoclonal antibody appears to fulfill such criteria for viable oocysts (5). To maximize the proportion of oocysts which include DAPI and minimize the proportion of DAPI- PI- oocysts, a 1-h preincubation in acidified HBSS at 37°C is recommended. The other preincubation treatments investigated here increased the proportion of dead oocysts in either all (0.1 M NaOH) or some (0.1 M HCI) of the isolates of C. parvum oocysts tested. It should be borne in mind that oocysts detected in environmental or water samples may have already been exposed to alkaline or acid pHs. Enumeration of sporozoite ratios at maximized excystation (4 h) provides information of little value, as most of the sporozoites appear to have lysed. The fragility of sporozoites necessitates that any interpretation of sporozoite ratio data be treated with caution. The correlation of viability and in vivo infectivity is an important issue that the research described here did not address. A correlation between in vitro excystation and infection of mice with large inocula (105 oocysts per mouse)
VIABILITY OF C. PARVUM OOCYSTS
VOL. 58, 1992
of Cryptosponidium oocysts has, however, been reported (4). In conclusion, simultaneous staining with DAPI and PI is an excellent, reproducible, user-friendly indicator of viability of C. parvum oocysts as defined by in vitro excystation and correlates well when excystation is increased by an acidic preincubation step or when excystation is reduced by harsher preincubation treatments that are lethal to some oocysts. A study of the permeability of oocyst walls to DAPI might provide interesting data on the degree of viability and the hardiness of oocysts. The use of DAPI in conjunction with fluorescence-labeled monoclonal antibodies could also be useful in the identification of oocysts in environmental and water samples (5). ACKNOWLEDGMENTS This work was supported by the U.K. Department of the Environment, with administration of funds through the Water Research
Centre.
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Meyer (ed.), Giardia and giardiasis. Biology, pathogenesis, and epidemiology. Plenum Press, New York. 13. Jones, K. H., and J. A. Senft. 1985. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate and propidium iodide. J. Histochem. Cytochem. 331:77-79. 14. Kasprzak, W., and A. C. Majewska. 1983. Infectivity of Giardia sp. cysts in relation to eosin exclusiosion and excystation in vitro. Tropenmed. Parasitol. 34:70-72. 15. Korich, D. G., J. R. Mead, M. S. Madore, N. A. Sinclair, and C. R. Sterling. 1990. Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum ocyst viability. Appl. Environ. Microbiol. 56:1423-1428. 16. Kubista, M., B. Akerman, and B. Norden. 1987. Characterisation of interaction between DNA and 4',6-diamidino-2-phenylindole by optical spectroscopy. Biochemistry 26:4545-4553. 17. Labatiuk, C. W., F. W. Schaefer Ill, G. R. Finch, and M. Belosevic. 1991. Comparison of animal infectivity, excystation and fluorogenic dye as measures of Giardia muris inactivation by ozone. Appl. Environ. Microbiol. 57:3187-3192. 17a.McDonald, V. Personal communication. 18. Peeters, J. E., E. A. Mazas, W. J. Masschelein, I. V. M. de Maturna, and E. Debacker. 1989. Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 55:1519-1522. 19. Rice, E. W., and F. W. Schaefer III. 1981. Improved in vitro excystation procedure for Giardia lamblia cysts. J. Clin. Microbiol. 14:709-710. 20. Robertson, L. J., A. T. Campbell, and H. V. Smith. 1992. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl. Environ. Microbiol. 58:34943500. 21. Robertson, L. J., A. T. Campbell, and H. V. Smith. In vitro excystation of Cryptosporidium parvum. Parasitology, in press. 22. Roberts-Thomson, I. C., D. P. Stevens, A. A. I. Mahmoud, and K. S. Warren. 1976. Giardiasis in the mouse: an animal model. Gastroenterology 71:57-61. 23. Schupp, D. J., and S. L. Erlandsen. 1987. A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity. Appl. Environ. Microbiol. 53:704-709. 24. Smith, A. L., and H. V. Smith. 1989. A comparison of fluorescein diacetate and propidium iodide staining and in vitro excystation for determining Giardia intestinalis cyst viability. Parasitology 99:329-331. 25. Smith, H. V., A. M. Grimason, C. Benton, and J. F. W. Parker. 1991. The occurrence of Cryptosporidium spp. oocysts in Scottish waters and the development of a fluorogenic viability assay for individual Cryptosporidium spp. oocysts, p. 169-172. In W. 0. K. Grabow, R. Morris, and K. Botzenhart (ed.), Health related water microbiology 1990. Pergamon Press, Oxford. 26. Smith, H. V., and J. B. Rose. 1990. Waterborne cryptospiridiosis. Parasitol. Today 6:8-12. 27. Smith, H. V., A. L. Smith, R. W. A. Girdwood, and E. G. Carrington. 1989. The effect of free chlorine on the viability of Cryptosporidium spp. oocysts. WRC report PRU 2023-M. Marlow, Buckinghamshire, United Kingdom. 28. Speer, C. A., and D. W. Reduker. 1986. Oocyst age and excystation of Cryptosporidiumparvum. Can. J. Zool. 64:12541255. 29. Sterling, C. R. 1990. Waterborne cryptosporidiosis, p. 51-58. In J. P. Dubey, C. A. Speer, and R. Fayer (ed.), Cryptosporidiosis of man and animals. CRC Press, Boca Raton, Fla. 30. Wright, S. Personal communication.
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0099-2240/92/113488-06$02.00/0
Copyright X) 1992, American Society for Microbiology
Viability of Cryptosporidium parvum Oocysts: Correlation of In Vitro Excystation with Inclusion or Exclusion of Fluorogenic Vital Dyes A. T. CAMPBELL, L. J. ROBERTSON, AND H. V. SMITH*
Scottish Parasite Diagnostic Laboratory, Stobhill General Hospital, Springbum, Glasgow, Scotland G21 3UW Received 9 April 1992/Accepted 3 August 1992
A viability assay for oocysts of Cryptosporidium parnum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples. a group of experts have recommended that cryptosporidiosis in humans become a reportable disease (1). Following acceptance of the public health significance of both cryptosporidiosis and the waterborne route of infection, attention has focused on development of methods which indicate whether sporozoites contained within an individual oocyst are viable or nonviable. The development of a reproducible, sensitive, user-friendly viability assay which could be used to determine whether the sporozoites contained within oocysts are capable of excysting and which would parallel and be compatible with in vitro excystation would be of value. It should be applicable to individual or small numbers of oocysts, not only because oocysts may normally occur in low numbers in potable water (26), but also because currently recommended techniques for the isolation of oocysts from water are inefficient (9 to 59% recovery efficiency [26]). Such an assay would not only have an obvious applied use in laboratories concerned with the detection of protozoa in water but would also be a valuable tool in further research. A series of 27 fluorogenic dyes have been assessed (5) to determine whether inclusion or exclusion of them correlated with viability as assessed by in vitro excystation. Of these dyes, a simultaneous dual labeling with 4',6-diamidino-2phenylindole (DAPI) and propidium iodide (PI) showed significant promise. The aims of this communication are (i) to compare inclusion or exclusion of the fluorogenic vital dyes DAPI and PI with in vitro excystation, (ii) to assess the importance of known triggers of oocyst excystation such as incubation at 37°C with bile and acid induction on both efficiency of in vitro excystation and fluorogenic dye inclusion or exclusion, and (iii) to assess the usefulness of these dyes as surrogate indicators of viability for small numbers of C. parvum oocysts.
Transmission of protozoan parasites of the gastrointestinal tracts of humans, such as Giardia intestinalis and Cryptosporidium parvum, via water is well documented (12, 26), and surveys of the occurrence of transmissive stages of these parasites (cysts and oocysts, respectively) indicate that these stages occur commonly in the aquatic environment (9, 25, 29). The public health significance of dead transmissivestage organisms is minimal; however, when the organisms are infective, the risk to public health can be enormous. Hence, information about cyst and oocyst viability is of considerable importance. To this end, methods for determining the viability of cysts of Giardia spp. have received attention in recent years. Techniques used for assessing the viability and/or infectivity of Giardia cysts include in vitro excystation (3, 19), exclusion or inclusion of vital dyes (2, 11, 13, 14, 23, 24), and infectivity in animal models (17, 22, 23). In general, cysts and oocysts occur in low numbers in the aquatic environment, and the minimum infectious dose is likewise low (between 10 and 100 cysts or oocysts). It is therefore important to be able to assess whether individual organisms isolated from water are viable. Assessment of viability by in vitro excystation and infectivity of animals requires organisms in a relatively clean and concentrated suspension, and these methods are therefore unsuitable for assessing the viability of individuals or of small numbers of the transmissive stage. Thus, exclusion or inclusion of vital dyes and Nomarski differential interference contrast (DIC) microscopy are the techniques that have shown most promise for assessing viability of individual Giardia cysts isolated from water. Within the last 10 years, the importance of transmission of C. parvum in water has been acknowledged worldwide. Six outbreaks of waterborne cryptosporidiosis have been documented: four occurred in the United Kingdom, and two occurred in the United States (1). In the United Kingdom, *
MATERLALS AND METHODS Sources and purification of oocysts. C. parvum oocysts were obtained from the following sources. Purified cervine-
Corresponding author. 3488
VOL. 58, 1992
ovine oocysts (c-o oocysts) were purchased from the Moredun Research Institute (MRI), Edinburgh, Scotland. This strain, originally isolated from deer feces, has been passaged in sheep by MRI. Human oocysts were isolated from fecal samples submitted from individuals with cryptosporidiosis for routine examination to the Scottish Parasite Diagnostic Laboratory (SPDL), Stobhill Hospital, Glasgow, Scotland. Oocysts purified from each stool were kept as separate isolates. Bovine oocysts were isolated and purified from bovine fecal samples obtained either from a study farm by Glasgow Veterinary School or from MRI. Purified bovine oocysts were purchased from MRI. This isolate has been passaged in calves by MRI. Oocysts purchased from MRI had been purified by a semiautomated method which involved incubation of the oocysts in 1% sodium dodecyl sulfate and both acid sedimentation and sucrose flotation (30). Oocysts were obtained suspended in phosphate-buffered saline (PBS; pH 7.2) containing 100 U of penicillin and 100 pg of streptomycin per ml. Oocysts from bovine and human fecal samples were purified at SPDL as follows. Fecal samples (ca. 5 g) were made up to 50 ml with reverse osmosis (RO) water, emulsified by vortexing, and then centrifuged at 900 x g for 5 min, and the supernatant was removed by aspiration and discarded. This washing procedure was repeated three to five times until the supernatant was clear. The pellet was resuspended in 10 ml of RO water and overlaid with an equal volume of diethyl ether. After being thoroughly mixed by shaking, the samples were centrifuged at 900 x g for 5 min, and fat and supematant layers were discarded. The pellet was washed twice in RO water as described above to remove traces of diethyl ether. Further purification of oocysts was performed by resuspending the pellet in 10 ml of RO water which was underlayered with a sucrose solution (1.18 specific gravity when cold) and centrifuging it at 900 x g for 15 min. The interface was recovered, diluted with RO water, and washed. Oocyst purification by cold sucrose flotation was repeated until the oocyst suspension was free of excess contaminating matter. All oocyst suspensions were stored at 4°C in RO water, and aliquots were sampled for bacterial and fungal contaminants by routine culture on blood agar and Sabouraud agar plates. Incubation of oocysts with DAPI and PI. Working solutions of DAPI (2 mg/ml in absolute methanol) and PI (1 mg/ml in 0.1 M PBS, pH 7.2) were prepared and stored at 4°C in the dark. Purified oocysts were suspended in Hanks balanced salt solution (HBSS) (2 x 104 oocysts per ,ul of HBSS), and 100 ,ul of suspension was incubated simultaneously with 10 ,ul of DAPI working solution and 10 ,ul of PI working solution at 37°C. Initially, suspensions were sampled regularly to that oocysts in subsequent experiments were incubated with DAPI and PI for the length of time optimal for producing maximal dye uptake. Oocysts were washed twice in HBSS before being viewed by epifluorescence microscopy. Microscopy. Ten-microliter aliquots of oocyst suspension were viewed under both DIC (Nomarski) optics and epifluorescence with an Olympus BH2 microscope equipped with a UV filter block (350-nm excitation, 450-nm emission) for DAPI and a green filter block (500-nm excitation, 630-nm emission) for PI. Proportions of ruptured (ghost), PI-positive (PI+), DAPI-positive PI-negative (DAPI+ PI-), DAPIensure
negative PI-negative (DAPI- PI-) oocysts (Table 1) were quantified by enumerating more than 100 oocysts in each sample. Ghost oocysts were easily identified under Nomarski optics, being nonrefractile apart from the residual body.
VIABILITY OF C. PARVUM OOCYSTS
3489
TABLE 1. Correlation of oocyst viability, visualization of oocyst contents by Nomarski (DIC) microscopy, and exclusion or inclusion of DAPI and PI Type of
Ghost PI+ DAPI+ PIDAPI- PI-
Contents
seen
by Nomarski
Inclusion
of:
microscopy
PI
DAPI
No Yes Yes Yes
No Yes No No
No Yes Yes No
Viability Dead Dead Viable at assay Viable after further trigger
a DAPI- PI- oocysts can be converted to DAPI+ Pl- oocysts and vice versa.
PI+ oocysts fluoresced bright red under the green filter block, and this red fluorescence varied from distinct points of intense fluorescence corresponding to the locations of sporozoite nuclei to a more-diffuse fluorescence within the oocyst. Oocysts were considered DAPI+ PI- only if they did not include PI and if the nuclei of the sporozoites fluoresced a distinctive sky blue under the UV filter block. Those oocysts which were neither PI+ nor ghosts and which showed either a rim fluorescence or an absence of fluorescence under the UV filter block were considered DAPIPI- (Table 1). Excystation. Two similar excystation protocols, S and M, were used on oocyst suspensions following either preincubation treatment or incubation with vital dyes. For excystation protocol S, 200 p.l of bile solution (1% bovine bile in Hanks minimal essential medium) and 50 .1l of sodium hydrogen carbonate solution (0.44% sodium hydrogen carbonate in RO water) were added to 100 p,l of purified oocyst suspension. The reactants were mixed thoroughly prior to incubation at 37°C. For excystation protocol M, 10 ,ul of sodium deoxycholate solution (1% sodium deoxycholate in Hanks minimal essential medium) and 10 ,ul of sodium hydrogen carbonate (2.2% sodium hydrogen carbonate in HBSS) were added to 100 p.l of purified oocyst suspension. The reactants were mixed thoroughly prior to incubation at 37°C. For both excystation protocols, reagents were made up freshly (il of oocyst suspension and 1 ml of HBSS acidified to pH 2.75 with HCI (20 ml HBSS:200 ,ul of 1 M HCI) at 37°C; (v) 100 ,ul of oocyst suspension and 1 ml of HBSS at 37°C (control for iv). Statistics. Calculation of correlation coefficients, linear regression analysis, analysis of covariance, and Mann-Whitney U tests were performed with a MINITAB statistical package. RESULTS Uptake of DAPI and PI. Uptake of PI by oocysts was maximal after a 5-min incubation at 37°C; however, uptake of DAPI did not maximize until oocysts had been incubated for 2 h or longer at 37°C. Therefore, in all subsequent experiments described here, both DAPI and PI were incubated simultaneously at 37°C for 2 h. The proportion of oocysts which took up the dyes varied between oocyst isolates; c-o isolates contained a considerably greater proportion of DAPI+ PI- oocysts than the other isolates tested. Excystation dynamics and correlation of excystation protocols. For human, bovine, and c-o isolates of C. parvum oocysts excysted by excystation protocol S or M, the excystation efficiency was maximal after 4 h (Fig. 1). Excystation efficiencies varied between oocyst isolates, with the c-o isolates having a considerably higher maximal excystation efficiency than the other isolates tested. Correlation of excystation protocol S with excystation protocol M gave a correlation coefficient of 0.996. This correlation also held for oocyst suspensions that had been preincubated with acidified HBSS before being subjected to the excystation protocols (Fig. 2). Prediction of maximal (4-h) excystation efficiency with DAPI and PI. It was hypothesized that inclusion or exclusion of DAPI and PI following a 2-h incubation at 37°C could be used to predict the viability of C. parvum oocysts, the proportion of DAPI+ PI- oocysts being the predicted viability and the observed or actual viability being calculated from maximal in vitro excystation. Oocysts were considered
10
0
FIG. 1. Excystation of different isolates of C. parvum oocysts over time.
100
20 30 40 50 60 70 80 90 % excystation using excystation protocol S
FIG. 2. Correlation of excystation protocols S and M.
viable if at least one sporozoite was ejected (partially or totally excysted) during the in vitro excystation. For human, bovine, and c-o oocysts, excystation efficiency was maximal after 4 h. The predicted viabilities (after 2 h of incubation with DAPI and PI at 37°C) were comparable to the maximum observed excystation efficiencies (Fig. 1). Correlation of this excystation efficiency (observed viability) with predicted viability (inclusion or exclusion of DAPI and PI) gave a correlation coefficient of 0.997. Linear regression analysis provided the following mathematical relationship between predicted viability (x) and observed viability (y): y = 1.85 + 0.936x (d2 adjusted for degrees of freedom = 99.3%). Analysis of covariance demonstrated no significant difference between this relationship (y = 1.85 + 0.936x) and the simpler mathematical relationshipy = x (Fig. 3). Since a simple correlation between predicted viability (percentage of DAPI+ PI- oocysts after a 2-h incubation with vital dyes) and observed viability (maximal excystation efficiency) had been derived, it was suggested that those oocysts which excysted during a 4-h excystation protocol would be those which were DAPI+ PI-. Excystation time trials showed that for all oocyst isolates, there was a marked decrease in the proportion of DAPI+ PI- oocysts with time. The proportion of DAPI- PI- oocysts fluctuated but did not decrease significantly with time (Fig. 4). The sporozoites seen in the excystation medium all contained fluorescent 100O
cernate/ovine isolate
* bovine isolates
80
o3
/ human isolates
0.3). In the suspension of c-o oocysts, the 10); --@- DAPI+ human isolate; --0--, DAPI- human isolate; proportion of viable (DAPI+ PI-) oocysts was not signifi-0--, DAPI+ bovine isolate; -- O --, DAPI- bovine isolate. cantly different after preincubation treatment (P > 0.3), and the mean proportion of viable oocysts remained higher than 70% (Table 4). For bovine oocysts, the suspension which (DAT?I+ PI-) nuclei. Free sporozoites with nonfluorescent had not been acidified had relatively low proportions of nucle i were never observed. DAPI+ PI- oocysts, but this proportion was significantly Pteincubation treatments. Preincubation treatments perincreased (Table 4; P c 0.009) by incubation in acidified form(ed on oocysts (see below) had a significant impact on HBSS. viabi]lity, both observed (maximal excystation) and predicted In both the c-o isolate and the bovine isolate, the propor(incu bation with DAPI and PI). For all preincubation treattion of DAPI- PI- oocysts was reduced significantly by ment;s, a good correlation between observed and predicted preincubation with acidified HBSS (Table 4; c-o isolate, P < viabillities was observed (Fig. 5), with a correlation coefficient of 0.989. 0.006; bovine isolate, P < 0.009). Sporozoite ratios. Sporozoite ratios were enumerated dur(i):Preincubation with 0.1 M NaOH and 0.1 M HCI. In both c-o a:nd human isolates, preincubation with 0.1 M NaOH at ing excystation of c-o and bovine (purified at SPDL) oocyst isolates (Table 5). High variability was observed, particuroom temperature was associated with a significant increase in oo cyst death (Tables 2 and 3; c-o isolate, P < 0.01; human larly in the c-o isolate, as demonstrated by the relatively high standard deviations (Table 5). Sporozoite ratios were also isolatte, P c 0.05). In the c-o isolate after incubation with enumerated during excystations of oocyst suspensions that NaO]H, a significant reduction in the proportion of viable had been preincubated in acidified HBSS. For both isolates, (DATPI+ PI-) oocysts was observed (Table 2; P c 0.001). However, in the human isolate, a dynamic situation was sporozoite ratios tended to fall over the duration of the obsexrved, with a significant decrease in DAPI- PI- oocysts excystation, presumably because of lysis of the sporozoites. After 4 h, the sporozoite ratio never exceeded 1 (Table 5). and al significant increase in DAPI+ PI- oocysts (Table 3; P For both isolates, preincubation in acidified HBSS tended to be associated with an enhanced sporozoite ratio during the initial stages of excystation, and this association was signif100] icant (P < 0.009) in the bovine isolate (Table 5). /Z a.
2201t
80 :5
DISCUSSION The results of this study demonstrate that oocysts whose walls are permeable to DAPI but not to PI (DAPI+ PI-)
60 40
0
0
201 0
TABLE 3. Inclusion or exclusion of DAPI and PI in human
20
40 60 Prdccted viabty
80
100
FIG. 5. Correlation of observed and predicted viabilities after preincubation treatments. Symbols: 0, RO water at room temperature; A, 0.1 M NaOH at room temperature; 0l, 0.1 M HCO at room temperature; 0, HBSS at 37C; A, acidified HBSS at 37°C; M, 1% trypsin-HBSS at 37'C; x, 1% trypsin-acidified HBSS at 37°C; =y.
x
oocysts following preincubation treatment % Inclusion (mean + SD)' Preicubation treatment
DAPI+ PI-
DAPI- PI-
RO water 0.1 M HCI 0.1 M NaOH
18.6 ± 1.9 53.6 ± 3.1 58.0 ± 1.1
76.8 ± 1.8 23.9 ± 4.6 20.5 ± 1.7
an =5.
% Dead
4.6 ± 0.6 22.5 ± 2.0 21.5 ± 2.9
APPL. ENvIRON. MICROIBIOL.
CAMPBELL ET AL.
3492
TABLE 4. Inclusion or exclusion of DAPI and PI in c-o and bovinea isolates of oocysts following preincubation in HBSS or acidified HBSS
% Inclusion (mean + SD)b Isolate and treatment DAPI+ PlDAPI- PItreatment C-o HBSS Acidified HBSS Bovine HBSS Acidified HBSS
Dead ~~~~~~~%
73.2 ± 0.8 74.1 + 4.6
3.4 ± 2.3 0.3 ± 0.5
23.4 ± 2.4 25.6 ± 5.0
11.1 ± 1.2 64.0 ± 2.2
60.3 ± 4.2 5.3 ± 1.5
28.6 ± 3.1 30.7 ± 1.2
a Purified at SPDL. bn = 3.
after a 2-h incubation at 37°C will excyst in an excystation protocol during a 4-h incubation. Such oocysts are described as viable at assay (Table 1). DAPI is an AT-selective DNA stain, and when binding to DNA occurs, there is an approximately 20-fold enhancement in fluorescence (16).
The correlation of inclusion of DAPI and exclusion of PI with excystation during a 4-h excystation protocol was demonstrated to hold for oocysts obtained from various sources. This correlation held if excystation dynamics were altered by a variety of preincubation treatments. The high viability observed in suspensions of c-o oocysts (low numbers of DAPI- PI- oocysts) could be due to the oocyst purification method used at MRI, which involves contact between the oocysts and dilute acid; preincubation of SPDLpurified oocysts with acid was found to increase their excystation efficiencies to similar levels. PI+ oocysts have sporozoites with disrupted or broken membranes and are considered dead (Table 1). It has been reported (10) that only cells with disrupted or broken membranes can be stained with PI. In our experience, motile sporozoites never include PI. Oocysts are also considered dead if they have ruptured and lost their contents (ghost oocysts) before being subjected to an excystation protocol
(Table 1).
Oocysts which exclude both DAPI and PI but appear by Nomarski microscopy to be morphologically intact and undamaged (Table 1) require a further trigger before they will excyst in one of the protocols described above. Inclusion of a selected preincubation treatment (e.g., incubation with acidified HBSS for 1 h at 37°C) converts the majority of these DAPI- Pl- oocysts to DAPI+ Pl- oocysts, regardless of source of oocysts or purification technique. Following such a preincubation treatment, the oocyst wall becomes TABLE 5. Sporozoite ratios during excystation of c-o isolate and bovine isolatea of oocysts following preincubation in HBSS or acidified HBSS Isolate and treatment
C-o HBSS
Acidified HBSS Bovine HBSS Acidified HBSS a Purified bn = 3.
at SPDL.
Sporozoite ratio (mean + SD) at': 30 min
1h
4h
1.8 + 2.2 4.0 ± 1.0
2.2 ± 1.6 2.3 ± 1.2
0.5 + 0.2 0.4 + 0.3
0.2 ± 0.3 2.8 + 0.3
1.2 ± 0.5 2.2 + 0.8
0.6 ± 0.2 0.6 + 0.2
permeable to DAPI. The DAPI can then be incorporated into sporozoite DNA through intact sporozoite membranes. The opposite conversion, of oocyst walls permeable to DAPI becoming impermeable to DAPI, has also been demonstrated to occur (21), and such a conversion has been shown to be reversible (21). The requirement of an acid trigger, which would normally occur in vivo, to convert a proportion of the oocyst population from being DAPI impermeable to being DAPI permeable is important for two reasons. First, for optimized in vitro excystation, the following series of stimuli should be included: (i) temperature elevation to 37°C, as little excystation is detected at lower temperatures (8, 28), and above 37°C, sporozoites do not survive (17a); (ii) incubation in dilute acid for 1 h; and (iii) incubation with bile salt or sodium deoxycholate in sodium hydrogen carbonate for 4 h. Incubation with chemicals other than acid might have the same trigger effect, although whether this is achieved by the same mechanism is unknown. For example, one recommended excystation protocol (7) involves a 10-min preincubation of oocysts in 1.05% sodium hypochlorite on ice. We suggest that dilute acid is more appropriate, as it more closely resembles the environment expected to be encountered by ingested oocysts in vivo (21). Second, permeability of oocyst walls varies within an isolate. The degree of permeability may affect the abilities of individual oocysts to withstand environmental pressures; the proportion killed by incubation in either 0.1 M NaOH or 0.1 M HCl was significantly lower in the isolate whose oocysts were originally impermeable to DAPI. We suggest that oocyst permeability should be considered when results from environmental and disinfection studies are interpreted (4-6, 15, 18, 20, 27). DAPI staining of sporozoite nuclei gives a characteristic sky blue fluorescence under a UV filter block. Besides indicating oocyst viability, visualization of the sporozoite nuclei provides a useful adjunct for more-definitive techniques of recognition and identification of Cryptosporidium oocysts. Visualization of sporozoite nuclei can be used in conjunction with the more-conventional techniques involving fluorescence-labeled monoclonal antibodies. It has been recommended that recognition of cysts of Giardia spp. in water rely on the identification of three morphological features (morphometry and the presence and distribution of at least two internal organelles) (12). The use of DAPI in conjunction with a fluorescence-labeled monoclonal antibody appears to fulfill such criteria for viable oocysts (5). To maximize the proportion of oocysts which include DAPI and minimize the proportion of DAPI- PI- oocysts, a 1-h preincubation in acidified HBSS at 37°C is recommended. The other preincubation treatments investigated here increased the proportion of dead oocysts in either all (0.1 M NaOH) or some (0.1 M HCI) of the isolates of C. parvum oocysts tested. It should be borne in mind that oocysts detected in environmental or water samples may have already been exposed to alkaline or acid pHs. Enumeration of sporozoite ratios at maximized excystation (4 h) provides information of little value, as most of the sporozoites appear to have lysed. The fragility of sporozoites necessitates that any interpretation of sporozoite ratio data be treated with caution. The correlation of viability and in vivo infectivity is an important issue that the research described here did not address. A correlation between in vitro excystation and infection of mice with large inocula (105 oocysts per mouse)
VIABILITY OF C. PARVUM OOCYSTS
VOL. 58, 1992
of Cryptosponidium oocysts has, however, been reported (4). In conclusion, simultaneous staining with DAPI and PI is an excellent, reproducible, user-friendly indicator of viability of C. parvum oocysts as defined by in vitro excystation and correlates well when excystation is increased by an acidic preincubation step or when excystation is reduced by harsher preincubation treatments that are lethal to some oocysts. A study of the permeability of oocyst walls to DAPI might provide interesting data on the degree of viability and the hardiness of oocysts. The use of DAPI in conjunction with fluorescence-labeled monoclonal antibodies could also be useful in the identification of oocysts in environmental and water samples (5). ACKNOWLEDGMENTS This work was supported by the U.K. Department of the Environment, with administration of funds through the Water Research
Centre.
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