title: Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa.
abstract: In this study, the use of skimmed milk flocculation (SMF) to simultaneously concentrate viruses, bacteria and protozoa was evaluated. We selected strains of faecal indicator bacteria and pathogens, such as Escherichia coli and Helicobacter pylori. The viruses selected were adenovirus (HAdV 35), rotavirus (RoV SA-11), the bacteriophage MS2 and bovine viral diarrhoea virus (BVDV). The protozoa tested were Acanthamoeba, Giardia and Cryptosporidium. The mean recoveries with q(RT)PCR were 66% (HAdV 35), 24% (MS2), 28% (RoV SA-11), 15% (BVDV), 60% (E. coli), 30% (H. pylori) and 21% (Acanthamoeba castellanii). When testing the infectivity, the mean recoveries were 59% (HAdV 35), 12% (MS2), 26% (RoV SA-11) and 0.7% (BVDV). The protozoa Giardia lamblia and Cryptosporidium parvum were studied by immunofluorescence with recoveries of 18% and 13%, respectively. Although q(RT)PCR consistently showed higher quantification values (as expected), q(RT)PCR and the infectivity assays showed similar recoveries for HAdV 35 and RoV SA-11. Additionally, we investigated modelling the variability and uncertainty of the recovery with this method to extrapolate the quantification obtained by q(RT)PCR and estimate the real concentration. The 95% prediction intervals of the real concentration of the microorganisms inoculated were calculated using a general non-parametric bootstrap procedure adapted in our context to estimate the technical error of the measurements. SMF shows recoveries with a low variability that permits the use of a mathematical approximation to predict the concentration of the pathogen and indicator with acceptable low intervals. The values of uncertainty may be used for a quantitative microbial risk analysis or diagnostic purposes.
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain.
authors.forename: Eloy
authors.initials: E
authors.lastname: Gonzales-Gustavson
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain.
authors.forename: Yexenia
authors.initials: Y
authors.lastname: Cárdenas-Youngs
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Calvo
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain.
authors.forename: Marcelle Figueira Marques
authors.initials: MF
authors.lastname: da Silva
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain.
authors.forename: Ayalkibet
authors.initials: A
authors.lastname: Hundesa
authors.affiliation: Instituto de Ingeniería del Agua y Medio Ambiente, Ciudad Politécnica de la Innovación, Ed. 8G, Acceso D, planta 2, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Amorós
authors.affiliation: Instituto de Ingeniería del Agua y Medio Ambiente, Ciudad Politécnica de la Innovación, Ed. 8G, Acceso D, planta 2, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Moreno
authors.affiliation: Instituto de Ingeniería del Agua y Medio Ambiente, Ciudad Politécnica de la Innovación, Ed. 8G, Acceso D, planta 2, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Moreno-Mesonero
authors.affiliation: IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Rosell
authors.affiliation: IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
authors.forename: Llilianne
authors.initials: L
authors.lastname: Ganges
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Araujo
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalonia, Spain. Electronic address: rgirones@ub.edu.
authors.forename: Rosina
authors.initials: R
authors.lastname: Girones
mesh_terms: D000818:Animals; D001419:Bacteria / Q000302:isolation & purification*; D002417:Cattle; D003458:Cryptosporidium / Q000302:isolation & purification; D016785:Cryptosporidium parvum / Q000302:isolation & purification; D004926:Escherichia coli / Q000302:isolation & purification; D005427:Flocculation; D005872:Giardia / Q000302:isolation & purification; D008828:Microbiological Techniques; D008892:Milk; D060888:Real-Time Polymerase Chain Reaction; D035501:Uncertainty; D014780:Viruses / Q000302:isolation & purification*; D014867:Water / Q000469:parasitology*; D014871:Water Microbiology
chemical_list: D014867:Water
keywords: Microorganisms; Real concentration; Recovery; Skimmed milk flocculation; Water contamination; q(RT)PCR