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

Abstract

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.