New Method Using Sedimentation and Immunomagnetic Separation ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2003, p. 6758–6761 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.11.6758–6761.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 11

New Method Using Sedimentation and Immunomagnetic Separation for Isolation and Enumeration of Cryptosporidium parvum Oocysts and Giardia lamblia cysts Jaime Massanet-Nicolau* Thames Water Utilities, Reading, Berkshire RG2 0JN, United Kingdom Received 16 January 2003/Accepted 17 August 2003

A new method for the isolation of Cryptosporidium parvum oocysts and Giardia lamblia cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (P < 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (P > 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.

merating C. parvum oocysts and G. lamblia cysts in bovine feces and in both raw and digested sewage sludge. The method combines a sedimentation step with immunomagnetic separation followed by detection with epifluorescence microscopy. The method is compared with sucrose flotation, which was used in a recent survey of pathogens in biosolids by UK Water Industry Research, Ltd. (3). The recovery efficiency of the method as a function of storage time is also investigated.

Cryptosporidium parvum and Giardia lamblia are protozoan parasites capable of infecting a variety of mammalian species, including humans (6). Consequently, these organisms can be found in both sewage and animal manures (2). As biosolids are recycled to land, there are potential transmission routes for these organisms via runoff and the contamination of water or foods cropped from land. Consequently, it is important to quantify the risks of contamination associated with this practice, which in turn requires accurate methods to quantify the levels of C. parvum and G. lamblia in biosolids. Several methods have been described to enumerate C. parvum and G. lamblia in various biosolids. These methods include NaCl flotation and differential density centrifugation with Percoll and sucrose (1, 4). Recovery efficiencies associated with these methods have been determined by a number of researchers, but the results exhibit a considerable degree of variation. In addition, most studies involve the addition of C. parvum oocysts and G. lamblia cysts to samples in quantities that are several orders of magnitude higher than background levels. This step may be undertaken to facilitate counting of the organisms (with a hemocytometer) or to prevent the levels of indigenous cysts and oocysts from influencing counts. Such high numbers of organisms influence estimates of recovery efficiency since the number of organisms recovered has been shown to vary with the quantity of organisms added to the sample (4). Adding prestained organisms (spike organisms) at a level equal to or less than that normally observed in a sample may provide a more realistic assessment of a recovery technique’s potential. In this paper a method is described for isolating and enu-

MATERIALS AND METHODS Preparation of stock spike solutions. Two stock spike solutions of approximately 103 organisms/ml were prepared, one containing C. parvum and the other containing G. lamblia. Cryptosporidium oocysts and Giardia cysts were obtained from the University of Arizona. C. parvum oocysts (Harley Moon strain, National Animal Disease Center, Ames, Iowa) were supplied at a concentration of 107 organisms ml⫺1. They were produced in neonatal Holstein calves and purified from feces by using discontinuous sucrose gradients. The Giardia cysts were supplied at a concentration of 106 organisms ml⫺1; they had been shed from gerbils (Meriones unguiculatur) and purified from feces by using discontinuous sucrose gradients. The C. parvum oocysts and G. lamblia cysts were nonviable and were preserved in 5% formalin. A total of 300 ␮l of the spike organisms was mixed in a microcentrifuge tube with 1 ml of 0.003 M sulforhodamine 101 acid chloride (Sigma) in methanol and incubated at 90°C for 60 min. The stained organisms were then centrifuged at 9,000 ⫻ g for 3 min, and the supernatant was aspirated, leaving a pellet of stained (oo)cysts and 100 ␮l of supernatant at the bottom of the tube. The organisms were then resuspended in 1 ml of deionized water and centrifuged again at 1,300 rpm for 3 min, and the supernatant was aspirated as described above. This wash step was repeated two more times. The stained organisms were enumerated with a hemocytometer, and their concentration was adjusted to 1,000 (oo)cysts ml⫺1 via serial dilutions with deionized water. For each organism, five replicates were analyzed to determine the exact concentration of organisms (see below for spike dose enumeration). Spike dose enumeration. Aliquots of 100 ␮l of the stock spike suspension were filtered through 2-␮m track-etched polyester membranes (Chemunex, MaisonsAlfort, France). Each membrane was wetted with Tween 20 (0.1%, wt/vol) (Sigma, Poole, United Kingdom), placed on an aliquot (100 ␮l) of staining solution (fluorescein isothiocyanate-labeled antibody with specificity for either Cryptosporidium or Giardia) (TCS Microbiology, Botolph Claydon, Buckingham, United Kingdom) and incubated in a humidity chamber (for 1 h at 37°C). After staining, the membrane was transferred to a sintered glass support (Millipore,

* Mailing address: Thames Water Utilities, Spencer House, Manor Farm Rd., Reading, Berkshire RG2 0JN, United Kingdom. Phone: 0118 923 623 1. Fax: 0118 923 631 1. E-mail: jaime.massanet-nicolau @thameswater.co.uk. 6758

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TABLE 1. Numbers of spike organisms detected in 100-␮l replicates of each stock suspension Replicate

Cryptosporidium

Giardia

1 2 3 4 5

149 129 139 137 150

142 105 106 78 104

Mean Standard deviation

140.8 8.78

107 22.8

Billerica, Mass.), and the excess antibody was drawn through the membrane with a ⫺10-kPa vacuum source. The membranes were placed onto glass microscope slides and a drop of immunofluorescence assay mounting fluid (TCS) was placed on each. The membranes were then covered with glass coverslips, and the numbers of (oo)cysts on each membrane were determined by epifluorescence microscopy; cysts and oocysts fluoresced apple green when viewed under light with a wavelength of 340 to 380 nm. Biosolids. The biosolids used in this experiment were primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. The sludges were collected from a sewage treatment facility that served a population of 276,000 and that also received both agricultural and industrial inputs. One-gram samples of the biosolids were deposited in 50-ml plastic centrifuge tubes (Corning). Each of these biosolid samples was spiked with approximately 102 (oo)cysts of both G. lamblia and C. parvum by adding 100-␮l aliquots of each of the stock spike suspensions to the centrifuge tubes containing the biosolids and vortexing for 5 s. Isolation by sedimentation. A 25-ml volume of 0.1 M phosphate-buffered saline (Sigma) was added to the centrifuge tube containing the spiked biosolid sample and vortexed for 60 s. Another 25 ml of phosphate-buffered saline was then added to the sample, and the tube was inverted five times. The sample was left to stand for 60 min at room temperature, during which time the heavier particles of debris settled at the bottom of the tube. At the end of the 60-min period, a 10-ml disposable pipette was used to transfer the top 45 ml of liquid to a clean 50-ml centrifuge tube. During this step, care was taken not to disturb the solids that had accumulated at the bottom of the tube. The 45 ml of liquid was then brought to a final volume of 50 ml with filtered deionized water and centrifuged for 5 min at 1,050 ⫻ g (with no braking during the deceleration phase). The sample was then further purified by immunomagnetic separation as described below.

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Isolation by sucrose flotation. A 10-ml volume of 0.01% Tween 20 was added to the tube containing the spiked biosolid and vortexed for 60 s. A 30-ml solution of sucrose dissolved in deionized water to a density of 1.18 g/ml was then deposited under the sample. This process was carried out by injecting the sucrose into the bottom of the tube with a 50-ml syringe and a 15-cm 19-gauge steel syringe to avoid disturbing the biosolid layer. The sample was then centrifuged at 1,050 ⫻ g for 10 min (with no braking during the deceleration phase). After centrifugation, the top 10 ml of the sample, the interface between the layers, and the top 10 ml of sucrose were carefully transferred with a 10-ml pipette to a new 50-ml centrifuge tube. The contents of this tube were brought to a final volume of 50 ml with deionized water, and the sample was then centrifuged once more at 1,050 ⫻ g for 10 min. The sample was then purified by immunomagnetic separation as described below. Purification by immunomagnetic separation. Immediately after centrifugation, the top 45 ml of liquid was removed with a 10-ml pipette and discarded. Care was taken to ensure that the pellet at the bottom of the tube was not disturbed. The sample was resuspended by vortexing for 10 s and then transferred with a disposable 10-ml pipette to a Leighton tube (Dynal, Oslo, Norway) and brought to a final volume of 10 ml with filtered deionized water. The sample was further purified with a commercial kit for the immunomagnetic separation of Giardia and Cryptosporidium (oo)cysts (Dynal) in accordance with the manufacturer’s instructions. In brief, the samples were incubated with paramagnetic beads which were coated with antibodies raised against Cryptosporidium or Giardia. The resulting complex of (oo)cysts and beads was then isolated from the sample with magnets and washed; the washing step described in the instructions was repeated to further reduce the amount of particulate matter in the sample. The (oo)cysts were then dissociated from the beads, and the beads were removed from the purified sample with magnets. After purification, the sample was transferred to a 12-mm-well slide (Dynal) containing 10 ␮l of 1 M NaOH and air dried at 37°C. The sample was then fixed by applying a drop of methanol and allowed to air dry at room temperature. The slide was stained with 50 ␮l of fluorescein isothiocyanate-labeled Cryptosporidium and Giardia monoclonal antibodies (Waterbourne, Inc., New Orleans, La.). Cysts and oocysts in the purified sample were identified by epifluorescence microscopy; these fluoresced apple green under light with a wavelength of 340 to 380 nm. Spike (oo)cysts were differentiated by checking for the inclusion of the sulforhodamine stain, which glowed red under light with a wavelength of 546 nm. Comparison of sedimentation and sucrose flotation. In order to compare the recovery efficiencies associated with both sedimentation and sucrose flotation, five replicates of each of the spiked biosolid samples were processed by the two different isolation methods. Samples were then further purified by immunomagnetic separation and enumerated by using epifluorescence microscopy. The numbers of spike (oo)cysts recovered from each replicate were recorded, and the mean recovery efficiencies from each method were compared by an unpaired t test.

FIG. 1. Mean recoveries associated with each isolation method. Error bars indicate standard deviations of 5 replicate samples.

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FIG. 2. Recovery efficiencies associated with sedimentation after storage of the spiked biosolid samples ar 2 to 8°C. Error bars indicate standard deviations of 3 replicate samples.

Effect of storage time on recovery efficiency. The effect of storage time on the recovery efficiency associated with sedimentation was investigated. Three replicates of the spiked biosolid samples of raw and digested sewage sludge were stored at 2 to 8°C for 0, 7, 14, and 21 days. These samples were then analyzed by sedimentation and immunomagnetic separation. The numbers of spike organisms recovered from each sample were recorded, and a simple rectilinear regression was employed to characterize changes in the mean recovery efficiency from each biosolid as a function of storage time.

RESULTS Spike dose enumeration. Table 1 shows the number of organisms in 5,100-␮l aliquots of each of the stock suspensions. The mean concentration of oocysts in the stock suspension of C. parvum was 1,408 oocysts ml⫺1, and the mean concentration of cysts in the stock suspension of G. lamblia was 1,070 cysts ml⫺1. Comparison of sedimentation and sucrose flotation. Figure 1 shows the recovery efficiencies associated with sedimentation and sucrose flotation for each combination of the biosolids and organisms investigated. In each case the recovery efficiencies for both isolation methods were compared by an unpaired t test. The results of this analysis are shown in Table 2. Effect of storage time on recovery efficiency. Figure 2 shows recovery efficiencies after each storage period for each combination of the biosolids and organisms investigated. In each case TABLE 2. Comparison of recovery efficiencies associated with sedimentation and sucrose flotation Biosolid

Raw sludge Digested sludge Manure a

Organism

Difference in mean recovery efficienciesa

C. parvum G. lamblia C. parvum G. lamblia C. parvum G. lamblia

43.7 50.8 33.5 55.1 54.3 57.2

Differences between the sedimentation and sucrose methods were significant at P values of ⬍0.001 based on the results of an unpaired t test.

the change in recovery efficiency as a function of storage time was characterized by a simple rectilinear regression. The results of this analysis are shown in Table 3. DISCUSSION Use of prestained organisms. Prestaining Cryptosporidium and Giardia organisms with sulforhodamine allowed the enumeration of (oo)cysts at low levels [100 to 140 (oo)cysts], and indigenous organisms did not influence estimates of recovery efficiencies. In practice, both prestained organisms could readily be distinguished from those naturally present in the biosolids. However, the prestained Giardia cysts fluoresced slightly less brightly, increasing the time needed to enumerate them. Comparison between sedimentation and sucrose flotation. The recovery efficiencies associated with sedimentation were higher than those associated with sucrose flotation for every combination of biosolids and organisms investigated. Recoveries associated with sedimentation ranged from 38.9 to 58.5%, whereas those associated with sucrose flotation were between 1.3 and 5.4%. Evaluation of these differences with an unpaired t test indicated that they were significant in every case (P ⬍ 0.001). The recovery efficiencies for sucrose flotation were somewhat lower than expected. In previous work conducted by this laboratory, recoveries of approximately 30 to 40% were ob-

TABLE 3. Results of a regression analysis characterizing the recovery efficiency associated with sedimentation as a function of storage time (days) Biosolid

Raw sludge Digested sludge

Organism

Coefficient of variation

Significance of coefficient’s deviation from zero (P)

C. parvum G. lamblia C. parvum G. lamblia

0.64 0.34 ⫺0.01 ⫺0.04

0.06 0.42 0.98 0.91

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tained with sucrose flotation. However, previous recovery efficiencies were estimated by determining the mean numbers of background cysts and oocysts and then deducting these values from the total counts. Because of the heterogeneity of biosolid samples and the small sample sizes (5 replicates), this method may have led to an overestimation of recovery efficiencies. The recovery efficiencies for sedimentation compares favorably with the recovery efficiencies associated with other isolation methods. Kuczynska and Shelton (4) evaluated eight methods for the detection of Cryptosporidium and found that the greatest recovery efficiency was 18.7% ⫾ 5.9%. The higher recovery efficiencies associated with the method presented are possibly due to the smaller number of manipulation steps involved. It is important to note, however, that the recovery efficiencies associated with live cysts and oocysts or with organisms that have been environmentally stressed may be different than recovery efficiencies associated with fixed cysts and oocysts. Recovery efficiency of sedimentation as a function of storage time. The sedimentation method is based in part on the difference in sedimentation rates of cysts and oocysts from rates of other particulates in the biosolids. Therefore, if prolonged contact between cysts and oocysts and the biosolids resulted in adherence to particulates, then the recovery efficiency of the method could be reduced. To test this hypothesis, a simple rectilinear regression was used to investigate the effect of storage time on the recovery efficiency of the sedimentation method. No significant association between these two variables was detected for any of the combinations of organisms and biosolids investigated (P ⬎ 0.1). The absence of any significant effect of storage time on recovery efficiency may be because Cryptosporidium and Giardia did not readily adhere to particles of biosolids. Alternatively, adherence may have occurred rapidly, and so the effect

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of adhesion was manifested equally at all time points. This hypothesis is consistent with the observations of Medema et al. (5) that the majority of cysts and oocysts become attached to particulates within the first 5 h. It is also possible that storage time has an effect on recovery efficiency but that the effect is small in comparison with other sources of variation affecting the method and cannot, therefore, be detected statistically. In conclusion, sedimentation in conjunction with immunomagnetic separation resulted in good (38.9 to 58.5%) recovery of Cryptosporidium and Giardia. The use of prestained cysts and oocysts allowed for accurate estimates of recovery efficiencies at spike concentrations equal to or less than background levels. Recovery efficiencies remained stable across a range of biosolids and were not affected by storage times of up to 21 days. ACKNOWLEDGMENTS I thank Thames Water Utilities for permission to present this paper. The views expressed are those of the author and not necessarily those of Thames Water Utilities. REFERENCES 1. Bukhari, Z., and H. V. Smith. 1995. Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces. J. Clin. Microbiol. 33:2592–2595. 2. Fricker, C. R., and J. H. Crabb. 1998. Water-borne cryptosporidiosis: detection methods and treatment options. Adv. Parasitol. 40:241–276. 3. Horan, N., and P. Lowe. 2001. Pathogens in biosolids—the fate of pathogens in sewage treatment. UK Water Industry Research (UKWIR) report no. 02/SL/06/6. UK Water Industry Research, Ltd., London, United Kingdom. 4. Kuczynska, E., and D. R. Shelton. 1999. Method for detection and enumeration of Cryptosporidium parvum oocysts in feces, manures, and soils. Appl. Environ. Microbiol. 65:2820–2826. 5. Medema, G. J., F. M. Schets, P. F. M. Teunis, and A. H. Havelaar. 1998. Sedimentation of free and attached Cryptosporidium oocysts and Giardia cysts in water. Appl. Environ. Microbiol. 64:4460–4466. 6. O’Donoghue, P. J. 1995. Cryptosporidium and cryptosporidiosis in man and animals. Int. J. Parasitol. 25:139–195.