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Applied and Environmental Microbiology, October 2002, p. 5198-5201, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5198-5201.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Significance of Enhanced Morphological Detection of Cryptosporidium sp. Oocysts in Water Concentrates Determined by Using 4',6'-Diamidino-2-Phenylindole and Immunofluorescence Microscopy

H.V. Smith,* B. M. Campbell, C. A. Paton, and R. A. B. Nichols

Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, Scotland, United Kingdom

Received 15 May 2001/ Accepted 17 May 2002


   ABSTRACT
Top
 Abstract
Introduction
 Sampling, isolation, and...
 Confirmation of the presence...
 Microscopy.
 References
 
Of 2,361 water concentrates analyzed for the presence of Cryptosporidium spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-Cryptosporidium sp. monoclonal antibody and the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.


   INTRODUCTION
Top
 Abstract
 Introduction
Sampling, isolation, and...
 Confirmation of the presence...
 Microscopy.
 References
 
The importance of the waterborne route of transmission for Cryptosporidium was recognized some 12 years ago, and since then at least 50 waterborne outbreaks of cryptosporidiosis have been documented (4, 12, 15). Cryptosporidium sp. oocysts can occur commonly in the aquatic environment (15), and great interest is shown by the water industry, worldwide, in detecting waterborne oocysts and limiting the transmission of this waterborne protozoan parasite.

Determining reductions in the density of oocysts between raw and final waters can identify treatment processes that are effective in removing oocysts. Analysis of the water catchment and raw water samples for the presence of oocysts can identify not only the contributors of waterborne oocysts but also the likely risk of oocysts entering abstraction. The effectiveness of these procedures depends upon the ability of the analyst to identify oocysts accurately. Early methods recommended for the identification of Cryptosporidium oocysts by the United Kingdom Standing Committee of Analysts (UKSCA) of the Department of the Environment (1) and the American Society for Testing and Materials (5) relied on immunofluorescence, morphometry, and morphology to identify oocysts. Once objects of the correct size, shape, and fluorescence intensity are identified, differential interference-contrast (DIC) or phase-contrast microscopy was used to determine whether oocysts contain any identifiable contents such as sporozoites and nuclei.

While numerous surveys of occurrence have been published (15), few studies have addressed the internal morphology (im) of the oocysts detected. In a survey of raw and filtered drinking waters, LeChevallier et al. (10, 11) estimated, on the basis of phase-contrast and Nomarski DIC microscopy, that 32% of 242 Cryptosporidium oocysts detected in raw water concentrates and 9% of 23 oocysts detected in filtered drinking water concentrates contained sporozoites or densely packed cytoplasm. No indication was given either of the number of sporozoites present in individual oocysts or whether contaminating debris interfered with morphological assessment.

Grimason et al. (8) developed an enhanced fluorescent morphology method, using the fluorogen 4',6'-diamidino-2-phenylindole (DAPI), which intercalates with nuclei in sporulated oocysts, in conjunction with a fluorescein isothiocyanate-conjugated anti-Cryptosporidium monoclonal antibody (FITC-C-MAb) and fluorescence microscopy to visualize oocyst nuclei more readily than other methods in place at that time. Present United Kingdom regulatory (2), nonregulatory UKSCA (3), and U.S. Environmental Protection Agency (6) methods include DAPI staining in their protocols for identifying oocysts.

The usefulness of DAPI was assessed on only a small number of environmental water concentrates by Grimason et al. (8). Here we present data on the usefulness of DAPI as a fluorescent adjunct for the identification of Cryptosporidium oocysts in environmental water concentrates submitted to the Scottish Parasite Diagnostic Laboratory (SPDL) over a 6-year period.


   Sampling, isolation, and enumeration.
Top
 Abstract
 Introduction
 Sampling, isolation, and...
Confirmation of the presence...
 Microscopy.
 References
 
Raw and final water samples, sampled by various of the United Kingdom Water Companies, were submitted to the SPDL for Cryptosporidium sp. oocyst analysis. Final water samples consisted of potable water samples taken after treatment that, in some instances, was minimal (e.g., microstraining and/or chlorination). Up to 24,870 liters was filtered through a polypropylene microwynd depth filter (CUNO Europe SA; DPPPY) of 1-µm nominal pore size at a flow rate of 1.0 to <=4 liters min-1, and the UKSCA method (1), with minor modifications, was used to isolate and enumerate Cryptosporidium oocysts. At least 20% of the water concentrate was analyzed for every sample. Air-dried concentrates were fixed in absolute methanol for 5 min, which enhances oocyst attachment onto microscope slides (9), before being stained with FITC-C-MAb.

Two commercially available FITC-C-MAbs (Shield Laboratories, Dundee, United Kingdom; and Crypt-a-glo; Waterborne Inc., New Orleans, La.), diluted optimally, were used during this study. Both FITC-C-MAbs have been shown to be equally effective in detecting Cryptosporidium oocysts in positive controls and duplicate environmental samples. DAPI (Sigma Chemical Co., Poole, United Kingdom) was used at 0.4 µg ml-1 according to the method described by Grimason et al. (8) with the minor modifications described below.

The following modifications were undertaken to reduce oocyst loss further, minimize the potential for cross-contamination, simplify the FITC-C-MAb removal procedure, and minimize exposure to the DNA intercalator, DAPI: FITC-C-MAb was aspirated by tilting a four-well slide to an angle of about 45° from the horizontal towards the operator and by aspirating the fluid that collected at the bottom of the wells by placing the tip of an aspirator close to, but not touching, the fluid. One drop (ca. 50 µl) of 150 mM phosphate-buffered saline (PBS), pH 7.2, was then dispensed from an eye dropper onto each well and allowed to stand for 2 min. Excess fluid was aspirated as described above, and the washing procedure was repeated twice. One drop of DAPI was dispensed onto each horizontal well, allowed to stand for 2 min, and then aspirated, as described above. Finally, a drop of deionized water was placed on each well for 1 to 3 s, to ensure removal of residual PBS and DAPI, and the excess was aspirated as described. Samples were mounted in 60:40 glycerol-PBS containing 2% (wt/vol) of the antifadant 1,4-diazabicyclo(2,2,2)octane and viewed within 30 min of preparation.


   Confirmation of the presence or absence of oocyst contents.
Top
 Abstract
 Introduction
 Sampling, isolation, and...
 Confirmation of the presence...
Microscopy.
 References
 
Confirmation of the presence or absence of oocyst contents was conducted using both DIC microscopy and the visualization of DAPI-positive fluorescent nuclei (Fig. 1), according to the method of Grimason et al. (8). Where possible, structures such as sporozoites and nuclei were noted.



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  		FIG. 1. Benefit of using DAPI staining as an adjunct to identifying Cryptosporidium spp. oocysts. (A) Cryptosporidium parvum oocyst stained with a commercially available FITC-C-MAb and DAPI and viewed under the FITC filter block described. Magnification, x1,250. (B) C. parvum oocyst (same as in panel A) stained with a commercially available FITC-C-MAb and DAPI and viewed under the UV filter block described. Magnification, x1,250. Note the presence of four sporozoite nuclei within the fluorescing oocyst.

 

   Microscopy.
Top
 Abstract
 Introduction
 Sampling, isolation, and...
 Confirmation of the presence...
 Microscopy.
References
 
An Olympus BH-2 fluorescence microscope equipped with Nomarski DIC optics was used for the analysis of prepared slides. A blue filter block (excitation, 490 nm; emission, 510 nm) was used to visualize FITC-C-MAb localization, and a UV excitation (excitation, 355 nm; emission, 450 nm) was used to determine the presence of DAPI-stained sporozoite nuclei. Nomarski DIC optics were used to determine the presence of im. All assessments of the presence of fluorescent nuclei and im were undertaken at either x400 or x1,000 magnification.

Of 2,361 environmental water samples analyzed by the SPDL between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw water concentrates and 34 were final water concentrates. A total of 740 oocysts was enumerated in these 269 positive samples, with 656 oocysts (88.7%) detected in raw water samples and 84 (11.3%) oocysts detected in final water samples. Of the 235 raw water positives, 157 samples (66.8%) had oocysts that contained nuclei, while 20 samples (58.8%) of the final water samples had oocysts that contained nuclei.

The 740 oocysts observed in the 269 positive samples were divided into two primary criteria, namely, those with DAPI-stained nuclei and those without DAPI-stained nuclei. Confirmation of the presence or absence of oocyst contents was by DIC microscopy. Those oocysts with DAPI-stained nuclei were further subdivided into oocysts with 1, 2, 3, or 4 identifiable nuclei (Table 1). Using these criteria, the oocysts most frequently identified had either no nuclei detectable (0 x n) with DAPI and no im (nim) by Nomarski DIC microscopy or four DAPI-stained nuclei (4 x n) together with internal contents as determined by DIC (im) and accounted for 259 of 656 (39.5%) and 215 of 656 (32.8%) of raw water positive samples and 36 of 84 (42.9%) and 26 of 34 (30.9%) of final water positive samples, respectively (Table 1).


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  		TABLE 1. The number and percentage of Cryptosporidium sp. oocysts with or without im and/or DAPI-stained nuclei detected in raw and final water concentratesa

 
Of the 284 DAPI-negative oocysts detected in raw water samples and the 41 DAPI-negative oocysts detected in final water samples, between 25 (8.8% of the former) and 5 (12.2% of the latter) had contents, as determined by DIC; however, no specific structures or organelles could be identified, the inclusions appearing amorphous. Oocysts containing an intermediate number of nuclei (i.e., 1 to 3 x n) accounted for 23.9 and 20.2% of the total number of oocysts detected in raw and final waters, respectively. The percentages of 1 x n, 2 x n, and 3 x n oocysts were similar in both raw and final water concentrates.

The percentage of empty oocysts (no DAPI/DIC-positive nuclei and no identifiable internal contents by DIC) detected in final water (42.9%) was marginally higher than that for raw water (39.5%).

Of a total of 2,361 environmental water samples analyzed by the SPDL between January 1992 and May 1998, 269 (11.4%) contained Cryptosporidium oocysts (range, 0.002 to 0.67 oocyst liter-1 [data not shown]) with 13.3% of raw water samples and 5.8% of final water samples tested being positive (data not shown). While these data fall within published ranges (15), they are probably underestimates, given the inherent inefficiencies and variability of the methods used (UKSCA method recovery efficiency <= 3 to 29% [7, 14]).

On the basis of the presence of sporozoites and/or granular contents, LeChevallier et al. (10, 11) concluded that 32 and 3% of oocysts detected in raw and final water concentrates, respectively, were "potentially viable." Although not stated in these publications, it must be assumed that such oocysts contain four sporozoites. Our data indicate that detecting four sporozoite nuclei within an oocyst occurred on more than 30% of occasions, irrespective of water type. While we would not ascribe the concept of "potentially viable" to such oocysts, we would observe that such oocysts were intact when air dried onto the microscope slide.

In this study, the two major categories of oocysts detected were those that contained four sporozoite nuclei (4 x n [im]) and those that were empty (0 x n [nim]). In the former category, the presence of occluding debris frequently made it impossible to determine organelle shape and number, reducing the usefulness of DIC microscopy. Similarly, for the 415 of 740 (56%) oocysts that contained DAPI-stained sporozoite nuclei, the interpretation of specific contents by DIC was also hindered by debris. In these instances where DIC was unhelpful, DAPI-stained nuclei were readily detected at x400 magnification.

Both United Kingdom (1) and U.S. (6) immunofluorescence methods advocate the use of DIC and/or phase-contrast microscopy to determine whether a fluorescent sphere or subsphere of 3 to 4 by 6 to 7 µm contains sporozoites. The inclusion of DAPI, to highlight sporozoite nuclei, provides further adjuncts for the identification of oocysts and, in our opinion, parallels the criteria laid down in the UKSCA method (1) and reduces the requirement of attempting to determine structures within an oocyst by DIC microscopy. Furthermore, in the event of an oocyst being distorted, the demonstration of up to four fluorescent nuclei in an object of a comparable size to an oocyst will assist in its identification. Air-drying oocysts onto microscope slides is used in the United Kingdom methods, which can result in distortion of oocysts. In our study, DAPI was a more useful adjunct than DIC for determining the presence of oocyst contents on microscope slides. Even when an oocyst becomes distorted, the demonstration of up to four DAPI-positive nuclei in an object of a comparable size to an oocyst will assist in its identification, which reassures analysts.

Smith (13) suggested that the criteria for identifying Cryptosporidium oocysts should include oocyst wall fluorescence and the presence of at least two other identifiable organelles. We feel that DAPI-stained nuclei meet the criteria of "identifiable organelles" and believe that the criteria of Smith (13) can now be met. Presently, no guidance exists governing the minimum number of sporozoite nuclei required to identify oocysts in water concentrates. If we use the criteria of oocyst wall fluorescence and the presence of at least one nucleus (defined as a DAPI-positive [sky blue] inclusion 1 µm in diameter, approximately) for identifying oocysts for our raw water data, then 56.5% of oocysts can be identified, while increasing the number of nuclei necessary for identification to four reduces the percentage of oocysts identifiable to 32.8% (Table 2). Adopting similar criteria for identifying oocysts in final water concentrates provides the following data. Fifty-one percent of oocysts can be identified using the criteria of oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduces the percentage of oocysts identifiable to 30.9% (Table 2). By firming up our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we exclude ~20% of oocysts in either water type.


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  		TABLE 2. Percentage of oocysts identifiable in raw and final water concentrates using the presence of one to four DAPI-stained nuclei as an adjunct

 
The above criteria rely on sporozoite nuclei being present for identification purposes and do not take into account the difficulty that arises if a fluorescent object is devoid of DAPI-stained nuclei. In this study, a significant number of oocysts (39.5% detected in raw water concentrates and 42.9% in final water concentrates) were empty. In addition, a subpopulation of DAPI-negative oocysts (0 x n [im]), whose contents were grossly contracted and contained no identifiable structure by DIC, was also present in both raw and final concentrates. This subpopulation probably represents a transitional state between oocysts containing one sporozoite nucleus and empty oocysts. Presently, recognition of these fluorescent rings as empty oocysts revolves around oocyst wall fluorescence, morphometry, and previous experience. We have suggested previously that MAbs specific to the internal oocyst wall may prove of value in discriminating empty oocysts from other similar-sized and -shaped contaminants.

It is pertinent to identify that, while sensitive molecular diagnostic techniques, such as the PCR and fluorescence in situ hybridization, have been developed for waterborne oocysts, our data, based on the presence of one or more fluorescent nuclei in a positive sample, indicate that 33.2% of our oocyst-positive raw and 41% of our oocyst-positive final water samples contained no DAPI-positive nuclei. Thus, ~40% of oocysts in these United Kingdom samples could not be detected by PCR.

The enhanced fluorogenic detection method can offer much-required assurance to the microscopist when determining whether an object of the correct size, which fluoresces with FITC-C-MAb, is, in fact, a sporulated oocyst and can decrease both false-positive and false-negative results. Reports from colleagues in the water industry indicate that this reproducible, user-friendly indicator of the presence of oocyst nuclei is readily assimilated into routine procedures in microbiology laboratories and has been used to good effect in waterborne-outbreak settings.


   ACKNOWLEDGMENTS
 
We acknowledge the assistance of staff of the United Kingdom Water Companies who provided samples of raw and final water for analysis and Z. Bukhari and S. Bukhari for technical assistance.

We are grateful to the SPDL and the United Kingdom Drinking Water Inspectorate (SPDL and Crown Copyright) for permission to reproduce Fig. 1A and B.


   FOOTNOTES
 
* Corresponding author. Mailing address: Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, Scotland, United Kingdom. Phone: 44 (0141) 201 3028. Fax: 44 (0141) 558 5508. E-mail: h.v.smith{at}spdl.org.uk. Back


   REFERENCES
Top
 Abstract
 Introduction
 Sampling, isolation, and...
 Confirmation of the presence...
 Microscopy.
 References
 

  1. Anonymous. 1990. Isolation and identification of Giardia cysts, Cryptosporidium oocysts and free living amoebae in water etc. 1989. Department of the Environment, Standing Committee of Analysis. Her Majesty's Stationery Office, London, United Kingdom.
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  3. Anonymous. 1999. United Kingdom statutory instruments no. 1524: the water supply (water quality) (amendment) regulations 1999. The Stationery Office, Ltd., London, United Kingdom.
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  8. Grimason, A. M., H. V Smith, J. F. W. Parker, Z. Bukhari, A. T. Campbell, and L. J. Robertson. 1994. Application of DAPI and immunofluorescence for enhanced identification of Cryptosporidium spp. oocysts in water samples. Water Res. 28:733-736.[CrossRef]
  9. Grimason, A. M. 1992. The occurrence and removal of Cryptosporidium sp. oocysts and Giardia sp. cysts in surface, potable and wastewater. Ph.D. thesis. University of Strathclyde, Glasgow, United Kingdom.
  10. LeChevallier, M. W., W. D. Norton, and R. G. Lee. 1991. Occurrence of Giardia and Cryptosporidium in surface water supplies. Appl. Environ. Microbiol. 57:2610-2616.[Abstract/Free Full Text]
  11. LeChevallier, M. W., W. D. Norton, and R. G. Lee. 1991. Giardia and Cryptosporidium spp. in filtered drinking water supplies. Appl. Environ. Microbiol. 57:2617-2621.[Abstract/Free Full Text]
  12. Slifko, T. R., H. V. Smith, and J. B. Rose. 2000. Emerging parasite zoonoses associated with water and food. Int. J. Parasitol. 30:1379-1393.[CrossRef][Medline]
  13. Smith, H. V. 1990. Environmental aspects of Cryptosporidium species: a review. J. R. Soc. Med. 83:629-631.[Medline]
  14. Smith, H. V., and C. R. Hayes. 1997. The status of UK methods for the detection of Cryptosporidium sp. oocysts and Giardia sp. cysts in water concentrates and their relevance to water management. Water Sci. Technol. 35:369-376.[CrossRef]
  15. Smith, H. V., and J. B Rose. 1998. Waterborne cryptosporidiosis: current status. Parasitol. Today 14:14-22.

Applied and Environmental Microbiology, October 2002, p. 5198-5201, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.5198-5201.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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