title: 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.
authors.affiliation: Parasitology, Department of Food Safety and Infection Biology, Norwegian University of Life Sciences - Faculty of Veterinary Medicine, Adamstuen Campus, P.O. Box 8146 Dep, N-0033 Oslo, Norway; College of Veterinary Medicine, Hawassa University, P.O. Box 1337, Hawassa, Ethiopia.
authors.forename: Jemere Bekele
authors.initials: JB
authors.lastname: Harito
authors.affiliation: Alere Technologies A.S., Oslo, Norway.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Campbell
authors.affiliation: Parasitology, Department of Food Safety and Infection Biology, Norwegian University of Life Sciences - Faculty of Veterinary Medicine, Adamstuen Campus, P.O. Box 8146 Dep, N-0033 Oslo, Norway.
authors.forename: Kristoffer Relling
authors.initials: KR
authors.lastname: Tysnes
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, BARC-East, Beltsville, MD 20705-2350, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Dubey
authors.affiliation: Parasitology, Department of Food Safety and Infection Biology, Norwegian University of Life Sciences - Faculty of Veterinary Medicine, Adamstuen Campus, P.O. Box 8146 Dep, N-0033 Oslo, Norway. Electronic address: lucy.robertson@nmbu.no.
authors.forename: Lucy J
authors.initials: LJ
authors.lastname: Robertson
mesh_terms: D000818:Animals; D018189:Immunomagnetic Separation; D037102:Lectins; D008853:Microscopy; D033661:Oocysts; D014122:Toxoplasma / Q000302:isolation & purification*; D014867:Water / Q000469:parasitology
chemical_list: D037102:Lectins; D014867:Water
keywords: Detection; Lectin-magnetic-separation; Recovery efficiency; Toxoplasma gondii oocysts; Water; Wheat germ agglutinin