Location: Animal Parasitic Diseases LaboratoryTitle: Lectin-Magnetic Separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR
|HARITO, JEMERE - Hawassa University|
|TYSNER, KRISTOFFER - Norwegian University Of Life Sciences|
|CAMPBELL, ANDREW - Alere As|
|ROBERTSON, LUCY - Norwegian University Of Life Sciences|
Submitted to: Water Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2017
Publication Date: 2/21/2017
Citation: Harito, J., Tysner, K., Campbell, A., Dubey, J.P., Robertson, L. 2017. Lectin-Magnetic Separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR. Water Research. 114:228-236.
Interpretive Summary: Toxoplasmosis, caused by the single celled parasite, Toxoplasma gondii, continues to be a public health problem worldwide. This parasite infects all warm-blooded hosts, including humans. It causes mental retardation and loss of vision in children, and abortion in livestock. The ingestion of food and water contaminated with resistant stage of the parasite, the oocyst, is a major mode of transmission of this parasite. Of all the hosts infected, only cats are known to excrete oocysts in feces. Cats can excrete millions of oocysts after eating an infected prey, such as a mouse or a bird. Oocysts can survive outdoors for months. The number of oocysts in municipal waters is low and difficult to detect by microscopy of centrifuged water samples. In the present paper the authors tested 4 types of magnetic beads coated with wheat germ agglutinin for their efficacy to separate oocysts from water. Dynabeads (Myone T1 and M-280) in conjuction with a molecular biology assay of genetic material (PCR) provided the best results to detect T. gondii DNA in water. These results will be of interest to biologists, parasitologists, and epidemiologists.
Technical 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 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 Toxoplasma 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 turbid 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 acidified pepsin (AP) solution and 0.5 M solutions of 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 a single oocyst/ml in 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.