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B Billcliffe

Publications and source records attributed to B Billcliffe.

6 recordsLinked to original sources

Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization.

The intracellular localization of Legionella pneumophila serogroup 1 within Acanthamoeba castellanii rendered the bacteria non-culturable on supplemented BCYE agar. DNA amplification, using two 19-mer primers, and hybridization using a 25-mer oligonucleotide probe, permitted detection of Leg. pneumophila in approximately 81% (29/36) of samples where the bacteria could not be detected using culture. A combination of co-cultivation of samples with Leg. pneumophila-naive A. polyphaga or Hartmannella vermiformis, incubation in a defined liquid medium or use of catalase indicated that approximately 31% (9/29) of the samples contained Leg. pneumophila which were viable although not culturable.

Acanthamoeba↗

A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectin-stimulated spreading of BHK21 cells.

When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.

Adenylate Cyclase Toxin↗

Qualitative and quantitative methods for detecting staphylococcal epidermolytic toxin.

Concentrated supernates of cultures of 98 strains of Staphylococcus aureus were screened for the production of epidermolytic toxin by (1) biological tests in 3-day-old mice, (2) double-diffusion precipitation tests against specific antiserum, and (3) the appearance of characteristic protein bands on thin-layer-gel isoelectric focusing. Positive results were obtained in all three of these tests with supernates from 11 of these cultures; the same 11 strains, and no others, produced epidermal splitting when newborn mice were challenged with viable organisms. Of the 14 phage-group II strains included in the survey, eight (58%) produced epidermolytic toxin. Three toxinogenic strains belonged to phage groups other than group II. A radial-immunodiffusion test employing antiserum to purified epidermolytic toxin proved satisfactory for measuring amounts of epidermolytic toxin in excess of 200 mug per ml. The results of immunodiffusion tests indicated that six of the 11 positive strains produced two serologically distinct forms of epidermolytic toxin and that the remainder produced only one of these. A striking correlation was observed between the presence of toxin of serotypes i and ii and the occurrence of protein bands i and ii in thin-layer isoelectric-focusing gels. These tests should facilitate the qualitative and quantitative assessment of the production of different serotypes of epidermolytic toxin by S. aureus in future surveys of strains isolated from toxic epidermal necrolysis of Ritter's type and impetigo. The two forms of epidermolytic toxin previously designated ETA (pI=7-0) and ETB (pI=6-0) were detected by preparative isoelectric focusing of sero-type-i toxin. Evidence suggests that studies of the effect of heat should provide a means of investigating the relationship between the different molecular forms of epidermolytic toxin.

Animals↗