Use of lectin-magnetic separation (LMS) for detecting Toxoplasma gondii oocysts in environmental water samples.

Proof-of-principle of lectin-magnetic separation (LMS) for isolating Toxoplasma oocysts (pre-treated with 0.5% acidified pepsin (AP)) from water for subsequent detection by microscopy or molecular methods has been shown. However, application of this technique in the routine water-analysis laboratory requires that the method is tested, modified, and optimized. The current study describes attempts to apply the LMS technique on supernatants from water samples previously analyzed for contamination with Cryptosporidium and Giardia using standard methods, and the supernatant following immunomagnetic separation (IMS) retained. Experiments on AP-treatment of Toxoplasma oocysts in situ in such samples demonstrated that overnight incubation at 37 °C was adequate, but excess AP had to be removed before continuing to LMS; neutralization in sodium hydroxide and a single wash step was found to be suitable. Mucilaginous material in post-IMS samples that had been stored at room temperature without washing, which was found to be probably an exudate from bacterial and fungal overgrowth, hampered the isolation of T. gondii oocysts by LMS beads. For detection, microscopy was successful only for clean samples, as debris occluded viewing in dirtier samples. Although qPCR was successful, for some samples non-specific inhibition occurred, as demonstrated by inhibition of an internal amplification control in the qPCR reaction. For some, but not all, samples this could be addressed by dilution. Finally, the optimized methodology was used for a pilot project in which 23 post-IMS water sample concentrates were analyzed. Of these, only 20 provided interpretable results (without qPCR inhibition) of which one sample was positive, and confirmed by sequencing of PCR product, indicating that Toxoplasma oocysts occur in Norwegian drinking water samples. In conclusion, we suggest that post-IMS samples may be suitable for analysis for Toxoplasma oocysts using LMS, only if freshly processed or washed before being refrigerated. In addition, application of AP treatment requires a neutralization step before proceeding to LMS. For detection, qPCR, rather than microscopy, is the most appropriate approach, although some inhibition may still occur, and therefore inclusion of an internal amplification control is important. Our study indicates that, despite some limitations, this approach would be appropriate for further large-scale analysis of samples of raw and treated drinking water.

Lectin-magnetic separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR.

Although standard methods for analyzing water samples for the protozoan parasites Cryptosporidium spp. and Giardia duodenalis are available and widely used, equivalent methods for analyzing water samples for Toxoplasma gondii oocysts are lacking. This is partly due to the lack of a readily available, reliable immunomagnetic separation technique (IMS). Here we investigated the use of lectin-magnetic separation (LMS) for isolating T. gondii oocysts from water sample concentrates, with subsequent detection by microscopy or molecular methods. Four different types of magnetic beads coated with wheat germ agglutinin (WGA) were tested for capture of oocysts from clean or dirty water samples. Dynabeads (Myone T1 and M-280) consistently provided mean capture efficiencies from 1 ml clean water in excess of 97%. High recoveries were also found with Tamavidin beads (in excess of 90%) when LMS was used for capture from a small (1 ml) volume. Dissociation (required for detection by microscopy) using 0.1N hydrochloric acid (HCl), as standard in IMS, was not successful, but could be achieved using a combination of acidified pepsin (AP) and N-acetyl d-glucosamine. Although simple centrifugation was as effective as LMS when concentrating high numbers of oocysts from clean water, LMS provided superior results when oocysts numbers were low or the water sample was dirty. Application of LMS integrated with qPCR enabled detection of 10 oocysts per 10 ml dirty water sample concentrate. These findings indicate that LMS with WGA coupled to magnetic beads could be an efficient isolation step in the analysis of water sample concentrates for T. gondii oocysts, with detection either by microscopy or by qPCR.

Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts.

The surfaces of aged (10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labelled with fluorescein isothiocyanate while aged oocysts did not. In contrast, the walls of aged oocysts bound a lectin (wheat germ agglutinin, WGA), but not the walls of fresh oocysts. Exposure of oocysts to detergent solutions or trypsin did not affect the binding properties of the walls of the oocysts. However, exposure of fresh oocysts to acidified pepsin enabled labelling of the walls with WGA, presumably due to the relevant moieties on the oocyst walls becoming exposed. WGA binding, but not mAb binding, was partially abrogated with periodate exposure. These findings reveal a significant difference in the binding properties of oocyst walls from "aged" and "fresh" oocysts. The results are of relevance when considering technologies for isolating or detecting T. gondii oocysts in environmental samples based on oocyst surface properties, as used for other protozoan parasites. Our results suggest the possibility of developing a WGA-based separation procedure for isolating Toxoplasma oocysts from environmental matrices, in which pepsin pre-treatment would be included to ensure that both fresh and aged oocysts were isolated.