Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water

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.

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France

“…Thus, we add BSA to reduce the incidence of inhibitors in our samples, as suggested by Rochelle et al (1997). We have already demonstrated the efficiency of this procedure for environmental water samples (Villena et al 2004); however, PCR inhibitors are still present in some environmental samples (Kourenti & Karanis 2006). Here, using BSA allowed us to remove PCR inhibitors from 11 of 13 samples, with the positive detection of Toxoplasma in three.…”

“…In this study, the PCR sensitivity ranged from 1 oocyst/L up to 1000 oocysts/L, depending on the source of seeded water (the best sensitivity was obtained with deionised water). Moreover, PCR was more sensitive than the bioassay by mice inoculation (Villena et al 2004). Schwab and McDevitt (2003) have developed a PCR method capable of detecting fewer than 50 oocysts in experimental conditions.…”

“…Often, however, these methods for the detection of waterborne protozoa were reported in laboratory conditions with high inoculums in seeding experiments, but not in natural samples. We have developed a method for the detection of Toxoplasma oocysts in environmental water that can be combined with assays to detect Cryptosporidium and Giardia in the same sample, simplifying the procedure for health authorities (Villena et al 2004). The concentration-filtration step is critical, as it is marked by the loss of seeded oocysts.…”

“…We have developed a strategy for the recovery and detection of oocysts and cysts, including those of Toxoplasma gondii, Cryptosporidium spp., and Giardia, which are the main parasites associated with waterborne outbreaks (Villena et al 2004). Briefly, after the filtration of large volumes (25-100 L) of environmental samples using Envirocheck capsules (Pall Life Sciences), particulate matter was eluted from the capsule filter (1 µm absolute pore size) with an elution buffer containing detergents (Tween-80 and Laureth-12, Pall Life Sciences).…”

“…To detect PCR inhibitors, DNA from a mimetic plasmid insert (1 pg/mL) was added in a second run in all environmental samples. In case of the presence of inhibitors, 8 µg of bovine serum albumin (BSA, Boehringer, Germany) was added to a new reaction mixture before the next amplification (Villena et al 2004). …”