title: Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels.
abstract: Giardia duodenalis, Cryptosporidium parvum and Toxoplasma gondii are important parasitic protists linked to water- and food-borne diseases. The accurate detection of these pathogens is central to the diagnosis, tracking, monitoring and surveillance of these protists in humans, animals and the environment. In this study, we established a multiplex real-time PCR (qPCR), coupled to high resolution melting (HRM) analysis, for the specific detection and quantification of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). Once optimised, this assay was applied to the testing of samples (n = 232) of treated wastewater and mussels (Mytilus galloprovincialis). Of 119 water samples, 28.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens; of 113 mussel samples, 66.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens, and 13.2% were test-positive for only T. gondii. The specificity of all amplicons produced was verified by direct sequencing. The oo/cysts numbers (per 5 μl of DNA sample) ranged from 10 to 64. The present multiplex assay achieved an efficiency of 100% and a R(2) value of >0.99. Current evidence indicates that this assay provides a promising tool for the simultaneous detection and quantitation of three key protist taxa.
authors.affiliation: Department of Science of Agriculture, Food and Environment, University of Foggia, 71121 Foggia, Italy; Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia. Electronic address: marianna.marangi@unifg.it.
authors.forename: Marianna
authors.initials: M
authors.lastname: Marangi
authors.affiliation: Department of Science of Agriculture, Food and Environment, University of Foggia, 71121 Foggia, Italy.
authors.forename: Annunziata
authors.initials: A
authors.lastname: Giangaspero
authors.affiliation: Department of Science of Agriculture, Food and Environment, University of Foggia, 71121 Foggia, Italy.
authors.forename: Vita
authors.initials: V
authors.lastname: Lacasella
authors.affiliation: Department of Agricultural and Environmental Science, University of Bari, 70126 Bari, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Lonigro
authors.affiliation: Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia. Electronic address: robinbg@unimelb.edu.au.
authors.forename: Robin B
authors.initials: RB
authors.lastname: Gasser
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000382:microbiology*; D003458:Cryptosporidium / Q000737:chemistry*; D005872:Giardia / Q000737:chemistry*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D014122:Toxoplasma / Q000737:chemistry*; D062065:Wastewater / Q000382:microbiology*
chemical_list: D062065:Wastewater
keywords: Cryptosporidium; Giardia; Multiplex PCR; Protists; Toxoplasma