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Title: ISOLATION OF SALMONELLA BACTERIOPHAGES FROM SWINE EFFLUENT LAGOONS

Author
item McLaughlin, Michael
item El Balaa, Mohamad
item SIMS, J - WESTERN KENTUCKY UNIV
item ANDERSLAND, J - WESTERN KENTUCKY UNIV
item KING, R - WESTERN KENTUCKY UNIV

Submitted to: Journal of Environmental Quality
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/12/2005
Publication Date: 2/2/2006
Citation: McLaughlin, M.R., Balaa, M.F., Sims, J., King, R. 2006. Isolation of Salmonella bacteriophages from swine effluent lagoons. Journal of Environmental Quality. 35:522-528.

Interpretive Summary: Bacteriophages, or phages as they are called, are viruses that infect bacteria. Phages occur widely in the natural environment wherever bacteria occur. Phage infection of a bacterial cell often causes the bacterial cell to produce new phages at the expense of shutting down its own functions. The end result of such phage infection is death of the bacterial cell. The infected bacterial cell is ruptured and multiple copies of new phage particles are released into the environment, where they may come in contact with other bacterial cells and continue the infection cycle. Phages often have very specific host requirements and can only infect and attack certain strains of bacteria. Knowledge of this specificity is used by medical and food safety personnel, enabling them to use specific phages to help identify, track and even treat bacterial infections. In research reported here, Agricultural Research Service scientists at Mississippi State, MS worked jointly with scientists from Western Kentucky University at Bowling Green, KY to make a collection of phages from swine lagoons. Methods were adapted to isolate and identify phages which attacked and killed only strains of Salmonella. In addition to determining the bacterial host specificity of the phages, the phages were also classified scientifically according to their unique size and shape. Information from the phage study will enable scientists to better understand the bacterial ecology of swine manure lagoons. The Salmonella phages will be useful in future research to develop new methods of identification and control for Salmonella.

Technical Abstract: Bacteriophages (phages) associated with Salmonella were collected from nine swine manure lagoons in Mississippi. Phages were isolated by an enrichment protocol or directly from effluent. For enrichment, chloroform-treated samples were filtered (0.22 µm) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 C, chloroform was added and samples stored at 5°C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 108 to 2.1 x 109 plaque forming units (pfu) per ml were produced, but estimation of phage titers in lagoons was not possible. For direct isolation, effluent was clarified by centrifugation, filtered (0.22 µm) and used in DAL plaque assays to select single-plaque isolates for 15 Salmonella strains. Plaque counts varied among Salmonella strains and lagoons. The most sensitive strain for direct phage recovery was ATCC 13311. Phage titers estimated by direct isolation with ATCC 13311 ranged among lagoons from 12-148 pfu per ml. Host range tests of 66 isolates recovered by the enrichment protocol, showed all were specific for Salmonella serotypes Enteritidis and Typhimurium and none produced plaques on lagoon isolates of species of Citrobacter, Escherichia, Proteus, Providencia or Serratia. Electron microscopy (EM) showed purified enrichment isolates had Podoviridae morphology (tailless 50 nm icosahedral heads with tail spikes). EM of clarified concentrated effluent showed 5.5:1 tailless to tailed phages. The isolated phages have potential as typing reagents, specific indicators and biocontrol agents of Salmonella.