McLaughlin M R, Balaa M F, Sims J, King R
United States Department of Agriculture, Agricultural Research Service, Waste Management and Forage Research Unit, Mississippi State, MS 39762, USA.
J Environ Qual. 2006 Feb 2;35(2):522-8. doi: 10.2134/jeq2005.0080. Print 2006 Mar-Apr.
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 mum) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 degrees C, chloroform was added and samples stored at 5 degrees C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 10(8) to 2.1 x 10(9) 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 microm), 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 to 148 pfu per mL. In limited host range tests, 66 isolates recovered by the enrichment protocol produced plaques only on Enteritidis and Typhimurium strains of Salmonella and none produced plaques on lagoon isolates 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). Electron microscopy 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.
从密西西比州的九个猪粪泻湖中收集了与沙门氏菌相关的噬菌体。通过富集方案或直接从废水中分离噬菌体。对于富集，经氯仿处理的样品经过过滤（0.22微米），并通过在胰蛋白酶大豆肉汤中添加沙门氏菌菌株混合物进行选择性富集。在35℃过夜培养后，加入氯仿并将样品储存在5℃。通过双层琼脂平板（DAL）噬菌斑测定法对富集样品针对单个沙门氏菌分离株进行测试。产生了每毫升2.9×10⁸至2.1×10⁹噬菌斑形成单位（pfu）的噬菌体滴度，但无法估计泻湖中的噬菌体滴度。对于直接分离，废水通过离心澄清、过滤（0.22微米），并用于DAL噬菌斑测定法以选择针对15种沙门氏菌菌株的单噬菌斑分离株。噬菌斑计数在沙门氏菌菌株和泻湖之间有所不同。直接噬菌体回收最敏感的菌株是ATCC 13311。用ATCC 13311直接分离估计的噬菌体滴度在各泻湖之间为每毫升12至148 pfu。 在有限的宿主范围测试中，通过富集方案回收的66个分离株仅在肠炎沙门氏菌和鼠伤寒沙门氏菌菌株上产生噬菌斑，而在柠檬酸杆菌、大肠杆菌、变形杆菌、普罗威登斯菌或沙雷氏菌的泻湖分离株上均未产生噬菌斑。电子显微镜（EM）显示纯化的富集分离株具有短尾噬菌体科形态（无尾的50纳米二十面体头部带有尾刺）。澄清浓缩废水的电子显微镜显示无尾噬菌体与有尾噬菌体的比例为5.5:1。分离出的噬菌体有潜力作为沙门氏菌的分型试剂、特异性指示物和生物防治剂。
