The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct plating methods, enhances efficiency in processing large numbers of samples.

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Verotoxin 1 is an Escherichia coli-derived subunit toxin that specifically binds to the glycolipid globotriosylceramide and is cytotoxic for cells that contain this plasma membrane glycolipid. Glycolipid incorporation experiments have now been performed using human lymphoid cells of the B lineage that lack this receptor, to conclusively demonstrate that globotriosylceramide alone is a functional receptor for this toxin. Globotriosylceramide incorporated into the membrane of toxin-resistant cells provides intracellular access to verotoxin by receptor-mediated endocytosis. Protein synthesis is then inhibited and globotriosylceramide-containing cells are killed.Verotoxin (VT) is an Escherichia coli elaborated subunit toxin (1, 2) highly homologous with Shiga toxin (from Shigella dysenteriae type 1) and, therefore, also referred to as Shiga-like toxin or SLT (2, 3). At least two variants of VT have been described, termed VT1 and VT2 (4) [or SLTI and SLTII (5)]. Research interest has focused on these toxins since they have been strongly implicated as the causative agent of the renal damage seen in the hemolytic uremic syndrome (HUS) (6, 7). Evidence for the presence of VT has been found in nearly 90% of patients with HUS in this hospital over the last 5 years.A4 HUS is usually preceded by hemorrhagic colitis (6) and similar gastrointestinal symptoms are obtained when purified VT is administered to rabbits (8).Most cells are entirely resistant to VT cytopathology. HeLa (9), Vero (6), and Daudi human lymphoma cells (10) have, however, been shown to be highly susceptible. It is possible that the basis of such differential sensitivity resides at the level of the cell surface toxin receptor.VT1 (11), VT2 (12), and Shiga toxin (9, 13) have been shown to bind specifically to the neutral glycolipid globotriosylceramide (galactose al-4galactose,81-4glucosylcera-mide; Gb3). We have proposed (10) MATERIALS AND METHODS Gb3 and globotetraosylceramide (Gb4) were purified from human renal tissue by a modification of the procedure as described by Strasberg et al. (14). The chloroform/methanol tissue extract was first applied on a Bio-Sil A (Bio-Rad) silica column in chloroform. The column was extensively washed with chloroform and neutral glycolipids were eluted with acetone/methanol, 9:1 (vol/vol). The neutral glycolipid fraction was then applied on a second Bio-Sil A column in chloroform/methanol, 98:2 (vol/vol). Glycolipids were then resolved with a linear solvent gradient comprising equal weights of chloroform/methanol, 15:1 (vol/vol), to chloroform/methanol, 4:1 (vol/vol). Digalactosyldiacylglycerol (DGDG) was purchased from Supelco (Ind), galactosylceramide (GC) was from Sigma (Ill), and phosphatidylethanolamine (PE) and phosphatidylserine (PS) were from Avanti. VT1 was purified as described (1). Rabbit anti-VT antibodies were kindly provided by S. Richardson (Department of Microbiology, Hospital for Sick Children). Monoclonal antibody against the B subunit of VT1 (13-C4) was a generous gift from A. O'Brien (Unifor...

Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage) wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage φEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs) are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs) and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein. FindingsBacteriophages (phages) are promising potential alternatives to antibiotics as therapeutics to reduce carriage of pathogens by food animals, thus preventing the spread of organisms such as Escherichia coli O157:H7 along the food chain. Our research has shown that a cocktail of virulent phages can eliminate E. coli O157:H7 from experimentally infected calves [1,2]. Phage V8, isolated originally from sewage [3] was renamed wV8 in our laboratory to indicate that it was obtained from the National Microbiology Laboratory (Winnipeg), and was included in the phage cocktail due to its complementary host range on common phage types (PTs) of E. coli O157:H7. Here we report on the genome and proteome of phage wV8, noting its very close similarity to the Salmonella phage Felix O1 [4][5][6][7].

A method is described for determination of the concentration of infectious phage particles by the direct plating plaque assay, which is simpler and faster than the double agar overlay plaque procedure outlined in the previous chapter.

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed using monoclonal antibodies (MAbs) as a rapid, economical alternative to culture isolation procedures for detection of Salmonella. Four MAbs previously shown to react with Salmonella strains representing 18 different serogroups were evaluated as capture antibodies and, after biotinylation, as detection antibodies. One MAb (M183) was selected for use in the ELISA to capture and detect Salmonella antigens. The detection limit of the ELISA was evaluated using Salmonella enterica subspecies enterica serovar Typhimurium and various selective and nonselective Salmonella enrichment media. The highest detection limit (ca. 10(4) CFU/ml) was achieved using an enrichment broth containing brain heart infusion, yeast extract, sodium hydrogen selenite, and sodium cholate (BYSC) after preenrichment in buffered peptone water. The ELISA detected all Salmonella serovars tested, which included representative serovars of serogroups B, C, D, and E and gave negative results for all non-Salmonella species tested. Samples (106) from various sources, including fecal samples from humans and pigeons, chicken carcass rinses, chicken parts, feed, and the environment, were used to evaluate the performance of the ELISA. The ELISA had a specificity and sensitivity of 100 and 91%, respectively, and a kappa value of 0.93 relative to the culture methods. Such an ELISA has the potential to be used in the implementation of the pathogen reduction and hazard analysis critical control point systems as well as in clinical laboratories.

Mini-Tn10luxABcam/Ptac-ATS was constructed in order to develop a luciferase-transducing bacteriophage for detecting Escherichia coli O157:H7. The transposon was designed to deliver a 3.6-kb insertion that confers n-decanal-dependent bioluminescence and resistance to chloramphenicol and was constructed using mini-Tn10cam/Ptac-ATS in the plasmid pNK2884 and luxAB from Vibrio harveyi. xV10, a temperate bacteriophage infecting common phage types of Escherichia coli O157:H7, was mutagenized as a prophage in E. coli O157:H7 strain R508. xV10: :luxABcamA1-23 was rescued from the strain by propagating it on a strain lacking the bacteriophage and the vector containing the transposon. The bacteriophage transduced n-decanal-dependent bioluminescence to E. coli O157:H7 strain R508 that was measurable approximately 1 h post infection. ß