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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Aims: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water.

Methods and Results: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38·8%) compared with ferric sulphate (61·5%) and aluminium sulphate (58·1%) for the recovery of 2·5 × 105 oocysts l−1; 2·5 oocysts l−1 and 1 oocyst l−1 were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts.

Conclusions: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water.

Significance and Impact of the Study: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Numerous water-borne outbreaks of cryptosporidiosis have been reported in North America, Europe and Japan (Craun et al. 1998; Smith and Rose 1998; Barwick et al. 2000). This has led to increased research efforts for the establishment of an efficient method to detect Cryptosporidium oocysts in water samples using various types of filtration (USEPA 1999). With some methods, considerable numbers of oocysts are likely to become embedded in the filter pores and subsequent elutions of the material are demanded, resulting in additional cost, labour and time (Shepherd and Wyn-Jones 1996).

As an alternative to the suggested techniques, Vesey et al. (1993) developed the flocculation method for the recovery of Cryptosporidium oocysts from water. They used calcium carbonate as flocculant and demonstrated recovery rates of more than 70% for Cryptosporidium oocysts seeded into various water types. However, Campbell et al. (1994) pointed out that this method reduces oocyst viability because of their exposure to high pH values for several hours during processing.

In the present study, three flocculants (ferric sulphate, aluminium sulphate and calcium carbonate) were compared for their recovery efficiency of C. parvum oocysts from tap water replicates. In addition, C. parvum oocyst viability after ferric sulphate flocculation was tested.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Source of C. parvum oocysts

Fresh faeces from infected calves were supplied by PD Dr Bauer, Veterinary School of University of Giessen, Germany (isolates had no further characterization and oocysts were used 4 weeks after purification from calves). Faeces from SCID-mice infected with C. parvum HNJ-1 isolate were also used (provider: Professor Dr M. Iseki, Kanazawa University School of Medicine, Japan). The oocysts were purified from faeces by the sucrose flotation technique (Kimura et al. 2000), counted 10 times using a Neubauer Cell Counter (NCC; Blaubrand®, Brand GmbH & Co, Wertheim, Germany) and stored in 2·5% potassium dichromate solution at 4°C until use. For all the experiments in this study, oocysts of approximately one month old were used.

Comparative study of ferric sulphate, aluminium sulphate and calcium carbonate flocculation for the recovery of high numbers of oocysts

Tap-water replicates of 1 litre were contaminated with either 1 × 106 or 2·5 × 105 oocysts. Contamination occurred through seeding a certain volume from the stock solution (<100 μl) into the water replicates with a micropipette, under constant gentle mixing. Counting of the stock solution was performed using the NCC, and oocyst concentration was determined by averaging five independent counts just prior to the seeding procedure. The contaminated tap-water samples were further treated using one of the flocculation procedures described below.

Ferric sulphate flocculation.

For the experiments using 1 × 106 oocysts, three replicate trials were performed, whereas for the experiments using 2·5 × 105 oocysts, there were seven replicates (according to previous experience, more homogenous results were obtained by higher numbers of oocysts and more trials were required by lower numbers to reduce the S.D.). Oocysts were seeded into 1 litre tap-water replicates as described above. Ferric sulphate solution (9·37 g ferric sulphate l−1 distilled H2O) was added to each 1 litre glass flask (543 ml l−1) and the pH adjusted to 6·0 ± 0·05. Samples were left overnight (approximately 24 h) at room temperature, so that the floc precipitation could be completed. The supernatant fluids were carefully removed the next day using a vacuum pump without disturbing the sediment. The resultant pellets (200 ml) were transferred to 50 ml sterile polypropylene tubes and centrifuged at 2000 g for 10 min (4°C). After discarding the supernatant fluids, the 1 ml pellets from the same trial were combined and re-centrifuged. The supernatant fluids were discarded for a third time and the final pellets (1 ml) were resuspended in 1 ml citric acid buffer (8·4 g citric acid monohydrate, 17·64 g tri-sodium-citrate-dihydrate, distilled H20 up to 100 ml; pH 4·7). The pellets were left to settle with the buffer for 1 h (vortexed every 15 min). At the end of the settlement, the samples were washed twice more with distilled H20 by centrifugation at 2000 g for 10 min. Oocysts in the final pellets (1 ml) were counted by the NCC (five independent counts per trial).

Aluminium sulphate flocculation.

For the experiments using 1 × 106 oocysts, three replicate trials were performed, whereas for the experiments using 2·5 × 105 oocysts, there were 11 replicates. A 2 ml volume of aluminium sulphate solution (10·13 g aluminium sulphate 100 ml−1 distilled H20) was added to each glass flask and the pH adjusted to 5·4–5·8. As with the ferric sulphate flocculation procedure, the samples were left overnight and the supernatant fluids discarded the next day. The resultant pellets (200 ml) were treated exactly as in the ferric sulphate flocculation process, and the same lysis buffer was used for the resuspension of the sediments. After settlement for 1 h, the samples were washed twice as in the ferric sulphate flocculation, the final pellets (1 ml) were obtained and the oocysts recovered were counted using the NCC (five independent counts/trial).

Calcium carbonate flocculation.

This method was adapted by Vesey et al. (1993), with some modifications. For the experiments using 1 × 106 oocysts, three replicate trials were performed, whereas for the experiments using 2·5 × 105 oocysts, there were 13 replicates. A 10 ml aliquot of a 10% CaCl2 solution and 10 ml of a 10% NaHCO3 solution were added, under constant stirring, to each glass flask and the pH was adjusted to 10 ± 0·05. After 24 h of settlement, the supernatant fluids were carefully removed and the resultant residues (200 ml) were dissolved in 10% w/v sulphamic acid solution. The residues were then centrifuged (2000 g,10 min) in sterile 50 ml polypropylene tubes. After removal of the supernatant fluids, the 1 ml pellets from the same trial were combined and washed twice in distilled H20 by centrifugation at 2000 g for 10 min. The final pellets (1 ml) were obtained and the oocysts recovered were counted (NCC; five independent counts per trial).

Evaluation of the oocyst recovery efficiency of the ferric sulphate flocculation method using low numbers of oocysts

Low numbers of oocysts (5 × 102) were seeded into 1 litre tap-water replicates (n=10) and the ferric sulphate flocculation method was performed as described above. The specific oocyst number was estimated using the ImmunoFluoresenceTest (IFT) as follows. Certain volumes from the oocyst stock solution were transferred to Eppendorf tubes and resuspended with 100 μl phosphate-buffered saline (PBS, pH 7·2). These tubes were centrifuged in an Eppendorf centrifuge (6000 g, 5 min), the supernatant fluids discarded and the pellets (30–50 μl) treated with 30 μl FITC-labelled monoclonal antibody (MoAb). MoAb was obtained either from Crypto-a-Glo, Waterborne Inc., New Orleans, LA, USA, or from Crypto-Cel, Cellabs Ltd, Brookvale, NSW, Australia. The samples were vortexed and left to incubate at 37°C for 45 min. After washing with 1 ml PBS, they were vortexed and centrifuged again in an Eppendorf centrifuge (4000 g, 3 min). The supernatant fluids were discarded and, for each sample, a final pellet of approximately 50 μl was dispensed onto glass multi-well slides, dried, and examined by epifluorescent microscopy. The slides were scanned at 400× with a Leitz microscope (LEITZ DIALUX 20 EB) equipped with PL Fluotar objectives, followed by counting of the fluorescing oocysts. After the flocculation procedure, the oocysts recovered from each trial were counted using exactly the same process, and the mean recovery efficiency for all 10 trials was estimated.

Lower numbers of oocysts were seeded into 3 l tap-water replicates (2·5 or 1·0 oocysts l−1) followed by the ferric sulphate flocculation method; 10 separate trials were performed for each concentration. Seeded and recovered oocyst numbers were both determined by IFT, as described above. Prior to each series of trials, tap water from the same source (3 l) was examined three times by the same method, to check for possible natural contamination.

In vitro excystation assay

Oocyst viability after the ferric sulphate flocculation procedure was tested by in vitro excystation assay. About 100–150 oocysts were seeded into 1 litre tap-water replicates (n=3) and concentrated by the ferric sulphate flocculation method, as described above. The viability of the recovered oocysts was estimated using the in vitro excystation assay, as described by Jenkins et al. (1997). As negative controls, about 100–170 oocysts of the same origin were tested for viability using the same method (n=3) but without any preceding flocculation treatment. At the end of the excystation assay, the samples were stained with FITC-labelled MoAb and DAPI (4·6-diamidino-2-phenylindole; Wako Pure Chemicals Industries, Ltd, Osaka, Japan), and at least 100 oocysts per trial were examined and counted under epifluorescent and differential interference contrast (DIC) microscopy. Empty oocysts from the original stock solution, which had not undergone any flocculation or excystation procedure, were also examined by epifluorescent and DIC microscopy for accurate estimation of the numbers of excysted oocysts after the assay. The oocyst excystation rate was determined for each experiment according to the following formula:

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RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

Results from the comparative study of the ferric sulphate, aluminium sulphate and calcium carbonate flocculation methods

Table 1 shows the results of the comparative study of the ferric sulphate, aluminium sulphate and calcium carbonate flocculation methods. For 1 × 106 oocysts l−1, no obvious difference among the mean recovery efficiencies achieved by the three methods was observed (ferric sulphate 68·1%, S.D. ± 2·0; aluminium sulphate 67·8%, S.D. ± 2·8; calcium carbonate 65·2%, S.D. ± 1·1). For 2·5 × 105 oocysts l−1, a lower recovery efficiency was achieved using calcium carbonate as flocculant (38·8%, S.D. ± 12·1), compared with those achieved with ferric sulphate (61·5%, S.D. ± 7·5) and aluminium sulphate (58·1%, S.D. ± 17·0) as flocculants.

Table 1.   Recoveries of high numbers of Cryptosporidium parvum oocysts by ferric sulphate, aluminium sulphate and calcium carbonate flocculation-comparative study Thumbnail image of

Results of the recovery experiments for high and moderate oocyst numbers using the ferric sulphate flocculation method

Table 2 shows the mean recovery rates for high and low numbers of oocysts seeded into tap water and recovered by the ferric sulphate flocculation method. Mean recovery rates in experiments using high numbers of oocysts ranged between 47·0% (S.D. ± 4·5; 1 × 105 l−1 seeded number; NCC) and 68·1% (S.D. ± 2·0; 1 × 106 l−1 seeded number; NCC). For low numbers of oocysts (5 × 102 l−1 seeded number; IFT), a mean recovery efficiency of 60·7% was observed (S.D. ± 10·6).

Table 2.   Recoveries of high and moderate numbers of Cryptosporidium parvum oocysts by ferric sulphate flocculation Thumbnail image of

Results of the recovery experiments for low oocyst numbers using the ferric sulphate flocculation method

Between one and 10 oocysts were seeded into 3 l tap-water replicates (mean: 2·5 oocysts l−1; n=10) and 3−10 oocysts were detected after flocculation by IFT (mean: 2·0 oocysts l−1; n=10). When 1–7 oocysts were seeded into 3 l tap-water replicates (mean=1·0 oocysts l−1; n=10), 0–4 oocysts were detected by IFT (mean: 0·8 oocysts l−1; n=10) (Table 3).

Table 3.   Recoveries of low numbers of Cryptosporidium parvum oocysts by ferric sulphate flocculation Thumbnail image of

No oocysts were detected in the tap-water replicates used for these experiments prior to seeding (data not shown).

Results from the in vitro excystation assay

The mean excystation rate for C. parvum oocysts after concentration by ferric sulphate flocculation was 65·1%, with a wide range of 57·8–78·4% (n=3). For the negative control experiments (excystation without the flocculation procedure), the mean excystation rate was 75·9%, with a smaller range of 73·8–79·4% (n=3) (Table 4).

Table 4.   Viability of Cryptosporidium parvum oocysts by in vitro excystation assay without and after performance of ferric sulphate flocculation Thumbnail image of

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

The present study aimed to develop a simple and effective method for the concentration of Cryptosporidium oocysts in water. Experiments were therefore carried out on the recovery efficiency of three flocculants commonly used by water treatment industries as a first step in drinking water preparation. However, flocculants with trivalent ions, i.e. aluminium and iron, are considered to be 50–60 times more effective than those with bivalent ions, i.e. calcium (AWWA 1995). Similarly, in the current experiments, higher recoveries were achieved using ferric sulphate and aluminium sulphate than using calcium carbonate.

Ferric sulphate flocculation proved very successful, even for the purification of 2·5 oocysts l−1 and 1 oocyst l−1 from tap water. Such low oocyst numbers can be present in drinking water and might constitute a public health risk. DuPont et al. (1995) reported that less than 30 oocysts caused infection in healthy adults. Therefore, sensitive concentration methods such as ferric sulphate flocculation could be a useful tool for routine drinking water surveillance. Following investigations with river water samples in Japan, Tsushima et al. (2001) concluded that 1·7–330 times more oocysts were detected by flocculation than by filtration. Although further studies are required to estimate the utility of the method, their data suggest that flocculation could also be useful for the concentration of Cryptosporidium oocysts from environmental raw water. Furthermore, the flocculation method requires only basic laboratory equipment and the procedures are much simpler and less labour-intensive than those for the filtration method.

Vesey et al. (1993) estimated that less than 10% of seeded C. parvum oocysts precipitate using ferric sulphate as flocculant. In those experiments, 103 oocysts were seeded into 1 litre of tap water and the pH was adjusted to 9·0. In contrast, higher recovery rates (47·0 and 68·1%) were achieved in the present studies by adjusting the pH to 6·0. The pH is one of the most important chemical factors influencing the success of a flocculant agent and, according to its value, might lead either to effective or to incomplete formation of the floc.

Aluminium sulphate is the most common flocculant used for water treatment in the USA. It has been suggested that alum works best in a pH range of about 5·8 to 8·5 (AWWA 1995). Vesey et al. (1993) evaluated the effectiveness of the specific agent at pH 9·0 and achieved a 59% flocculation efficiency for 103C. parvum oocysts seeded into 1 litre tap-water replicates. In the present studies, 67·8 and 58·1% recoveries were achieved, for 1 × 106 and 2·5 × 105 oocysts, respectively, by pH adjustment to 5·4–5·8.

Calcium carbonate has been considered as quite an efficient means of concentrating Cryptosporidium in water. Vesey et al. (1993) achieved a 73·7% mean recovery for 61 oocysts l−1 seeded into 10 l of tap-water replicates. Shepherd and Wyn-Jones (1996) obtained a mean recovery rate of 73·6% for 75 oocysts l−1 seeded into 10 l of tap water. Campbell et al. (1994) observed a 74·8% mean recovery rate for 5 × 105 oocysts l−1. Using the same method, lower mean recovery rates were achieved in the present studies; a 65·2% mean recovery was achieved for 1 × 106 oocysts l−1. For lower oocyst numbers (2·5 × 105 l−1), the mean recovery rate was significantly reduced (38·8%).

The in vitro excystation experiments did not show a significant reduction in oocyst viability following the ferric sulphate flocculation procedure, in contrast to data obtained using calcium carbonate flocculation. In the latter case, mean oocyst viability was reduced from 75·5% to 46·5% after flocculation (Campbell et al. 1994). In contrast, only a 5·5% reduction (from 75·9 to 65·1%) of viability was observed in the present study. It is hypothesized that this is due to the mild conditions of ferric sulphate flocculation compared with calcium carbonate flocculation, including the step of exposure of oocysts to pH 9·0 for 4 h. Ferric sulphate flocculation should be preferred to calcium carbonate flocculation when the viability of the detected oocysts is of crucial importance.

However, results obtained in different research laboratories cannot be directly compared for a variety of reasons, such as different researchers, source and age of Cryptosporidium oocysts, variable storage media and different counting techniques (Klonicki et al. 1997). The surface charge which probably differentiates different C. parvum isolates may affect the success of the flocculation method. In addition, the IFT method is likely to underestimate the number in the original seed, leading to significant errors in the recovery data. Despite all these difficulties, ferric sulphate flocculation could be a promising alternative for the detection of both high and low numbers of oocysts in contaminated water samples.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References

The authors are profoundly grateful to Professor Dr Iseki and PD Dr Bauer for kindly providing C. parvum isolates. They also thank Miss I. Taki for her technical assistance and Mr H. Doi for his valuable advice on the in vitro excystation experiments.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. References
  • 1
    AWWA (1995) Coagulation and flocculation. In Water Treatment. Principles and Practices of Water Supply Operations 2nd edn, ed. Von Huben, H. pp. 51–83. Denver: American Water Works Association.
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    Barwick, R.S., Levy, D.A., Craun, G.F., Beach, M.J. and Calderon, R.L. (2000) Surveillance for waterborne-disease outbreaks – United States. 1997–98. Morbidity and Mortality Weekly Report 49, 135.
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    Campbell, A.T., Robertson, L.J., Smith, H.V. and Girdwood, R.W.A. (1994) Viability of Cryptosporidium parvum oocysts concentrated by calcium carbonate flocculation. Journal of Applied Bacteriology 76, 638639.
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    Craun, G.F., Hubbs, S.A., Frost, F., Calderon, R.L. and Via, S.H. (1998) Waterborne outbreaks of cryptosporidiosis. Journal of American Water Works Association 90, 8191.
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    DuPont, H.L., Chappell, C.L., Sterling, C.R., Okhuysen, P.C., Rose, J.B. and Jakubowski, W. (1995) The infectivity of Cryptosporidium parvum in healthy volunteers. New England Journal of Medicine 30, 855859.
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    Kimura, A., Karanis, P., Maltezos, E. and Seitz, H.M. (2000) Bench scale experiments to evaluate the usefulness of sucrose flotation techniques for separation of Cryptosporidium oocysts from water. Journal of Protozoology Research 10, 155165.
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    Klonicki, P.T., Hancock, C.M., Straub, T.M. et al. (1997) Crypto research: are fundamental data missing? Journal of American Water Works Association 89, 97103.
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    Shepherd, K.M. and Wyn-Jones, A.P. (1996) An evaluation of methods for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water. Applied and Environmental Microbiology 62, 13171322.
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    Tsushima, Y., Karanis, P., Kamada, T. et al. (2001) Detection of Cryptosporidium parvum oocysts in environmental water in Hokkaido, Japan. Journal of Veterinary Medical Science 63, 233236.
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    Vesey, G., Slade, J.S., Byrne, M., Shepherd, K. and Fricker, C.R. (1993) A new method for the concentration of Cryptosporidium oocysts from water. Journal of Applied Bacteriology 75, 8286.