Wednesday, November 15, 2006

EPA Worst Case Water Microcosms for Testing Phage Biocontrol of Salmonella.

Michael McLaughlin and John Brooks. USDA-ARS, USDA-ARS, PO Box 5367 810 Hwy 12 E, Mississippi State, MS 39762-5367

A method using laboratory microcosms is proposed for evaluating the effectiveness of selected phages for biocontrol of Salmonella within swine effluent and manure impacted environments.  The method uses EPA (U.S. Environmental Protection Agency) worst case water as a high organic matter matrix to simulate lagoon effluent.  EPA worst case water has been typically defined to simulate high turbidity and organic matter in water involved in challenges of point of use water filtration devices. Using this matrix, these laboratory microcosms provide a similar, but simpler and more defined environment than the complex and malodorous lagoon effluent from which the phages were originally isolated.  Physical and chemical parameters (organic content, total dissolved solids, temperature, pH and turbidity) are controlled, evaluated and defined to allow more precise evaluation of microbiological parameters (Salmonella and phage strains).  The microcosm matrix is loaded with one or more selected Salmonella isolates and treated with Salmonella-specific phages alone and in combinations.  Survival of the Salmonella and persistence of the phage(s) are monitored by conventional culture plate and plaque assay methods, respectively.  Concurrent extraction of test samples for RNA and subsequent use of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for specific amplification of phage transcription genes is proposed as a supplement to conventional microbiological phage assays.  Inactivation rates and log reductions of Salmonella and phage are analyzed to assess effects of altering physicochemical and microbiological parameters.  The method is intended for initial use in selecting phages for subsequent ‘real world’ testing in lagoon effluents and other more complex manure impacted environmental matrices.