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Applied and Environmental Microbiology, July 2004, p . 4035-4039, Vol . 70, No . 7

Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water

Isabelle Villena,1,2* Dominique Aubert,1,2 Philippe Gomis,2 Hubert Ferté,1,2 Jean-Christophe Inglard,3 Hélène Denis-Bisiaux,3 Julie-Muriel Dondon,4 Eric Pisano,1,2 Naïma Ortis,1,2 and Jean-Michel Pinon1,2

Laboratoire de Parasitologie-Mycologie, CHU Maison Blanche, UFR Médecine,1 EA 2070, IFR 53, Université de Reims Champagne-Ardenne, 51092 Reims,2 Communauté de Communes de I'Agglomération de Reims (CCAR), 51096 Reims,3 Direction Régionale des Affaires Sanitaires et Sociales, Service Santé Environnement, 51037 ChÂlons en Champagne, France4

Received 8 October 2003/ Accepted 6 April 2004


   ABSTRACT

 
Several recent outbreaks of toxoplasmosis were related to drinking water . We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S . Environmental Protection Agency method with the same sample . Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient . Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts . In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases . The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source . PCR was always more sensitive than mouse inoculation . This detection strategy was then applied to 139 environmental water samples collected over a 20-month period . Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition . Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%) . None of the samples were positive by mouse inoculation . This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method .


   INTRODUCTION

 
Toxoplasma gondii is a protozoan parasite capable of infecting a variety of birds and mammals, including humans (40) . Toxoplasmosis is a significant problem in congenitally infected infants and immunosuppressed patients . T . gondii is transmitted in three main ways: (i) from mother to fetus, (ii) by consumption of undercooked meat containing tissue cysts, and (iii) by ingestion of food or water contaminated by sporulated oocysts . Members of the Felidae are the only known hosts that can release oocysts into the environment in their feces (11) . Excreted oocysts are unsporulated and are not directly infectious . They become infective after 1 to 21 days in the external environment, depending on aeration and temperature (9) . Although cats excrete oocysts over only short periods (1 to 3 weeks), they release massive numbers of oocysts (107/108), which are highly resistant and can survive for months in the environment (13) .

Three waterborne toxoplasmosis outbreaks have been documented (2-4) . The first occurred in Panama in 1979 (3) . An epidemiological investigation identified the source as creek water contaminated by oocysts excreted by jungle cats . A large waterborne outbreak occurred in British Columbia (Canada) in 1995, with 110 cases of human acute Toxoplasma infection (4) . The source was municipal drinking water, probably contaminated by cougar and/or domestic cat feces (1) . The largest outbreak, with 290 human cases, was recently reported in Brazil and involved an unfiltered water reservoir (22) . Bahia-Oliveira et al . found a high T . gondii prevalence in a Brazilian community, related to drinking unfiltered water (2) . Likewise, Hall et al . identified drinking water as the probable source of infection in a community of strict vegetarians (18) .

Waterborne transmission of the intestinal parasites Giardia duodenalis and Cryptosporidium parvum is also well documented (16, 36, 37) . Over 160 waterborne outbreaks of giardiasis and cryptosporidiosis have been reported, and well-documented cases have been described in the United States and United Kingdom (26, 27, 29) . T . gondii oocysts are resistant to the usual processes used to disinfect drinking water (42) . Increase awareness of the risk of waterborne toxoplasmosis outbreaks has led to a search for methods that can efficiently detect oocysts in environmental water .

Here, we evaluated a strategy used since 2001 in our laboratory to detect T . gondii oocysts in water, along with Giardia spp . and Cryptosporidium spp . The strategy involves three basic steps: (i) concentration and filtration of the water sample to recover small numbers of Toxoplasma oocysts, (ii) elution and purification on a density gradient, and (iii) detection by PCR amplification and bioassay (mouse inoculation) to determine the presence and the infectivity of recovered oocysts . This strategy was applied to 139 water samples over a 20-month period .


   MATERIALS AND METHODS

 
Oocyst stock preparation.
Oocysts were isolated from the feces of a cat experimentally infected with 3,000 cysts of a chronic type II strain (RMS/99/GUI) . Nonsporulated T . gondii oocysts were purified and induced to sporulate as described by Dubey and Frenkel (10) . Briefly, in step 1, the feces were emulsified in water and centrifuged; the supernatant was discarded, and sediment 1 was recovered . Step 2 consisted of mixing sediment 1 with 10 volumes of sucrose solution (specific gravity, 1.15) and centrifuging (1,250 x g) . After step 2, oocysts were found in supernatant 2 . In step 3, approximately 5 ml of supernatant 2 was mixed with 45 ml of water and then centrifuged (1,250 x g); the oocysts were now in sediment 3 . Five milliliters of 2% aqueous H2SO4 was added to sediment 3, and the tube was aerated at room temperature for 7 days . Sporulated oocysts were stored at 4°C until used in seeding experiments .

Seeding.
Samples of deionized water (DW), public drinking water (PDW), and raw surface water (RSW) (100 liters each) were seeded with known numbers of oocysts . The oocyst stock suspension was counted with a hemocytometer, and the mean value obtained for 10 different samples was analyzed . The stock suspension was adjusted to 105 oocysts/ml . DW and PDW (100 liters) were seeded with 102 or 103 oocysts, and RSW (40 liters; maximal filtration capacity) was seeded with 4 x 104, 4 x 103, or 4 x 102 oocysts .

Environmental samples.
The sampling sites were selected by local public health officials in the four departments of the Champagne-Ardenne region . The survey was carried out over a 20-month period (June 2001 to January 2003) . A total of 139 samples were analyzed, consisting of RSW collected near water plant intakes (n = 45), underground water (UW) (n = 50), and PDW (n = 44) . Samples from different geographical locations exhibited a range of turbidities measured by the nephelometric procedure; 100 liters was collected in 10-liter polypropylene barrels at each source and transported to the laboratory for immediate processing . The filtered volumes were 100 liters of PDW, 25 to 100 liters of UW, and 7 to 45 liters of RSW . Some samples also contained Giardia spp . and Cryptosporidium spp., which were detected by indirect immunofluorescence after immunomagnetic separation (IMS), using method 1623 of the U.S . Environmental Protection Agency (USEPA) (15) .

Oocyst detection . (i) Filtration.
Each sample was concentrated using Envirochek capsules (Pall Gelman Laboratory) as specified by the manufacturer . We tested both the original Gelman Envirochek Standard (GES) (a polyethersulfonate membrane with a 1-µm absolute pore size, designed for 10 to 100-liter volumes) and Envirochek HV (GEHV) (a new 1-µm track-etched membrane for water volumes of 500 liters or more) . All the samples were concentrated using GES capsules, and some (seeded DW) were also concentrated with GEHV capsules . Particulate matter was eluted from the capsule filter with elution buffer containing detergents (Tween 80 and Laureht-12; Pall Gelman Laboratory) . Eluates were centrifuged (30 min at 1,250 x g and 4°C); distilled water was added to the pellet-eluting solution (adjusted to 10 ml), and the solution was subjected to IMS for detection of Giardia spp . and Cryptosporidium spp . After this IMS step, the eluate was centrifuged (10 min at 1,250 x g and 4°C), supernatant 1 was discarded and pellet 1 was suspended in sucrose suspension (density, 1.15; ratio: 1:3) . After centrifugation (10 min at 1,250 x g and 4°C), 2 ml of supernatant 2 (which would contain oocysts) was collected and mixed with 8 ml of DW . After last centrifugation (10 min at 1,250 x g and 4°C), supernatant 3 was discarded and sediment 3 was used for T . gondii detection .

(ii) Detection.
The sediment obtained after sucrose separation was separated into two parts . One was used for PCR, and the other was used for bioassay (mouse inoculation) after sporulation .

(a) PCR detection.
The sediment was resuspended in 1 ml of Tris-EDTA buffer and centrifuged (3 min at 1,250 x g) . Three cycles of freezing (–80°C)-thawing were done, and the pellet was subjected first to proteinase K digestion (1 h at 60°C) and then to DNA extraction with QIAamp DNA minikits (Qiagen, Courtaboeuf, France) as specified by the manufacturer . DNA was then subjected to real-time PCR for detection of the T . gondii B1 gene with the following primers described by Lin et al . (25) Toxo-F (5 µM; 5'-TCC CCT CTG CTG GCG AAA AGT-3') and Toxo-R (5 µM; 5'-AGC GTT, CGT GGT CAA CTA TCG ATT G-3) . The target DNA for real-time PCR amplification was the published sequence of the 35-fold repetitive B1 gene of the RH strain (6) . Template DNA (5 µl) was added to a reaction mixture (final volume, 25 µl) containing 0.75 U of Taq DNA polymerase (Platinum; Bio-Rad), 20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2, 200 µM each dGTP, dATP, and dCTP, 400 µM dUTP, 0.5 U of UDG, 12.5 pmol of each primer, and 5 pmol of TaqMan fluorescent probe (2 µM; 5'FAM-TCT GTG CAA CTT TGG TGT ATT CGC AG-3'TAMRA) . TaqMan PCR runs were performed in triplicate with an ICycler device (Bio-Rad) . After initial activation of Taq polymerase at 95°C for 5 min, 45 PCR cycles were run at 95°C for 15 s and 60°C for 1 min . As the standard curve constructed with 10-fold serial-dilutions of purified T . gondii tachyzoites, this PCR was considered an end-point assay and so not appropriate for oocyst quantification . The PCR amplification was considered positive when DNA was detected in the three wells . To detect PCR inhibitors, DNA from a mimetic plasmid insert (corresponding to the target on the B1 gene deleted of nucleotides) was added in a second run in all DNA environmental samples, amplified with others primers, and revealed with SYBRgreen . When inhibitors were present (plasmid insert unamplified), 8 µg of bovine serum albumin (BSA; Boehringer, Mannheim, Germany) was added to a new reaction mixture before amplification (PCR/BSA) .

(b) Bioassay.
We used a previously described procedure to induce the sporulation and infectivity of any T . gondii oocysts in the sediment (12) . After addition of 5 ml of 2% H2SO4, the sediment was aerated at room temperature for 7 days . The suspension was neutralized by adding 3.3% sodium hydroxide and then centrifuged (10 min at 1,250 x g and 4°C) . The supernatant was discarded, and the sediment was resuspended in 1.5 ml of normal saline containing antibiotics . Female Swiss-Webster mice (three mice per sample) were inoculated by gavage with well-mixed resuspended sample sediment . All mice were first confirmed to be seronegative for T . gondii . Blood was collected 6 weeks after infestation for serological analysis by high-sensitivity direct agglutination as previously described (31) . Infection was demonstrated by seroconversion (specific immunoglobulin G antibody detection) and brain examination of sacrificed mice (2 months after inoculation) .


   RESULTS

 
The results of PCR and bioassay detection of oocysts seeded in DW, PDW, and RSW are summarized in Table 1 . PCR success rates in DW were 60% for the detection of 1 oocyst/liter and 100% for 10 oocysts/liter . The detection rate was 60% for 10 oocysts/liter in PDW and 50% for 1,000 oocysts/liter in RSW . The sensitivity of the PCR assay ranged from less than 10 to more than 1,000 oocysts/liter, depending on the source of the seeded sample . PCR was more sensitive than mouse inoculation . The detection rate at the bioassay detection limit in DW (1 oocyst/liter; Table 1) with the GES capsule was 30% . GES and GEHV capsules were evaluated for the recovery of T . gondii oocysts (Table 2) . A 100-liter volume of DW seeded with 103 oocysts was filtered at 1.5 liters/min using the two capsules . Samples were concentrated as described above and tested by PCR and bioassay . PCR success rates were 100% for the detection of 10 oocysts/liter in DW with the GES capsule, and 80% with the GEHV capsule . The positivity rate was 60% (three of five) with both capsules in the bioassay; two of five GES-filtered samples were totally negative in the bioassay, while only two of three mice were positive with the other three samples .


TABLE 1 . Water samplesa and results of PCR and bioassay

 

TABLE 2 . Comparison of the ability of GES and GEHV capsules to filter 100 liters of deionized water seeded with 103 T . gondii oocysts

 
A total of 139 water samples from 80 sites were tested by PCR and mouse inoculation . The turbidity of RSW samples ranged from 0.30 to 30.1 nephelometric turbidity units, while that of PDW samples ranged from 0.03 to 1.9 units . Among the 86 samples not containing PCR inhibitors, 9 were positive for Toxoplasma DNA and 77 were negative (Table 3) . The other 53 samples contained PCR inhibitors . PCR-BSA overcame this inhibition in 39 cases (73.5%), leading to the detection of Toxoplasma DNA in one further positive sample . Inhibitors were more frequent in RSW (29 of 45; 64.4%) than in UW (18 of 50; 36%) or PDW (6 of 44; 13.6%) . After PCR-BSA, only RSW (10 of 45; 22.2%) and UW (4 of 50; 8%) samples remained uninterpretable (principally samples with high turbidity) .


TABLE 3 . DNA amplification by PCR or PCR-BSA in 139 samplesa

 
Finally, among the 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%); 14 samples (10%; 10 RSW and 4 UW) were not amplified because of PCR inhibitors . None of the samples were positive by bioassay .


   DISCUSSION

 
T . gondii oocysts can persist for long periods in the environment . Experimental ingestion of a single oocyst can infect mice and pigs (12) while no data have been reported for human toxoplasmosis . T . gondii oocysts also exhibit remarkable resistance to various inactivation procedures based on chemical reagents (8, 9) and disinfection processes used by water utilities (42) . The different methods for T . gondii oocyst recovery were recently reviewed (14) . Standard methods currently available for detection of Cryptosporidium spp . and Giardia spp . in water include concentration-filtration of large volumes, elution and clarification on density gradients or by IMS, and immunofluorescence assay (IFA) using monoclonal antibodies (32) . Here, we describe a rapid method for Toxoplasma detection in environmental water, including detection of Cryptosporidium spp . and Giardia spp . in the same sample .

The concentration-filtration step is critical and results in a marked loss of seeded oocysts (30) . Recently, two chemical flocculation procedures and one centrifugation procedure were evaluated by Kourenti et al . for T . gondii oocyst concentration on DW, with recovery rates of more than 80% (23) . While flocculation is simple and inexpensive, filtration is more robust for processing turbid water . Moreover, samples concentrated by filtration are less likely to contain PCR inhibitors, which appeared to be eliminated by using GES filters (28) . For T . gondii, since oocyst numbers in random environmental samples are probably small, we used GES capsules to filter 7 to 45 liters of RSW and 100 liters of PDW . Since GEHV capsules permit the filtration of larger volumes (up to 1,000 liters), we also tested them with T . gondii-seeded samples . No major difference was found between the two capsules, and GEHV could thus be considered interesting for filtering larger volumes potentially containing T . gondii oocysts . The concentration-filtration process leads to the accumulation of debris in the filter, and oocyst detection requires clarification procedures to separate oocysts from debris (34) . We used sucrose flotation which is commonly used for T . gondii oocyst purification (9, 12), since no monoclonal antibodies against the T . gondii oocyst outer wall are currently available (ruling out the use of IMS for concentration and IFA for quantitative detection) .

Rapid and sensitive pathogen detection methods are essential for the public-water industry . PCR was described as more rapid, sensitive, and specific for Cryptosporidium species detection in environmental water samples (17, 19, 21); the sensitivity was found to be comparable for TaqMan PCR and conventional IFA (7) . For DNA extraction, Sluter et al . (38) showed that three cycles of freezing-thawing were sufficient to expose Cryptosporidium oocyst DNA, resulting in higher sensitivity than proteinase K digestion or sonication . For T . gondii DNA extraction, we chose freezing-thawing for its speed, low cost, and simplicity . Nevertheless, PCR inhibitors may not be completely removed by flotation procedures and can compromise the sensitivity of molecular detection (5) . Kreader (24) and Rochelle et al . (33) showed that BSA can be added to the PCR mix to avoid inhibition in samples containing humic acids (mixtures of complex polyphenolics produced during the decomposition of organic matter) (41) . In our study, the use of PCR-BSA reduced the incidence of inhibitors from 38 to 10% of samples . The sensitivity of the PCR assay was reduced by up to 100-fold in oocyst-seeded RSW compared with DW and PDW . This is consistent with results from Johnson et al . (21) for PCR-based Cryptosporidium oocyst detection in water .

Finally, among the 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%) . Three cases involved RSW, whose environmental matrices may be contaminated by soil washing after peaks in rainfall (4) . This could also be the case for the UW samples (six positive samples), which were chosen by local public health officials because of frequent pathogen recovery (including Giardia spp . and Cryptosporidium spp.) . The detection of Toxoplasma DNA in PDW was more surprising, since none of the samples were positive by bioassay . In a previous study (20), T . gondii identification after filtration was based on mouse inoculation . Mouse bioassay is still the reference method to detect viable oocysts, but 7 days is required for sporulation, before mouse inoculation, and an additional 4 weeks is required to obtain the immunological results (20) . As previously reported by Kourenti et al . (23), our seeding data suggest that Toxoplasma oocysts remain infective for mice . However, our bioassay results with environmental water samples were disappointing and showed that the efficiency of the bioassay for 103 oocyst-seeded, & filtered, and eluted water samples was poor . Dubey et al . (12) and Isaac-Renton et al . (20), using the same method, failed to detect Toxoplasma oocysts in PDW . Previously, it was reported that the different isolation steps and vigorous shaking necessary for consistent oocyst elution may cause a loss of infectivity (35, 39) . All these results show that while the mouse bioassay is still the reference test for viable oocysts, its efficiency is poor when it is applied to filtered environmental water samples . This could explain why none of our T . gondii PCR-positive samples were positive by bioassay . Another cause could be DNA contamination or detection of DNA from noninfective oocysts present in the environment . To avoid contamination, DNA extraction, PCR mix preparation, and PCR amplification were done in separated rooms . TaqMan PCR minimizes the possibility of cross-contamination because it involves a closed-vessel system and because of the presence of UDG in the mix (7) . To distinguish between DNA from living and from dead oocysts, reverse transcription-PCR could be used as an important indirect technique for oocyst viability determination while it selectively detects viable organisms .

In conclusion, we describe here a strategy for the detection of multiple waterborne parasites, including Toxoplasma oocysts, in environmental water samples . PCR amplification was able to detect Toxoplasma DNA, even when the mouse bioassay was negative . These results suffer from some limitations, mainly owing to the flotation step . The development of monoclonal antibodies against the Toxoplasma oocyst wall would permit the use of IMS and also microscopic quantification by IFA . Moreover, efforts are under way in our laboratory to develop a quantitative reverse transcription-PCR method to differentiate between viable and nonviable oocysts detected in environmental samples since the mouse bioassay takes too long for public health sentinel purposes .

 


   ACKNOWLEDGMENTS

 
This work was supported by Ministère de l'Aménagement du Territoire et de l'Environnement (convention d'aide ENOOC12) .

We thank the Communauté de Communes de l'Agglomération de Reims, the Conseil Regional and DRASS (Champagne-Ardenne), and the DDASS (Marne, Haute-Marne, Ardennes, Aube) for their cooperation in the collection of environmental water samples, and we thank Pall Life Science for providing GEHV capsules . We thank D . Young for editing the manuscript and M . L . Dardé for helpful discussions .


   FOOTNOTES

 
* Corresponding author . Mailing address: Laboratoire de Parasitologie-Mycologie, CHU Maison Blanche, 45 rue Cognacq-Jay, 51092 Reims, France . Phone: 00(0)3-26-78-42-20 . Fax: 00(0)3-26-78-73-28 . E-mail: ivillena{at}chu-reims.fr .


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