title: Application of PCR to a clinical and environmental investigation of a case of equine botulism.
abstract: PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples.
authors.affiliation: Department of Microbiology, University of Queensland, St. Lucia, Australia.
authors.forename: E A
authors.initials: EA
authors.lastname: Szabo
authors.forename: J M
authors.initials: JM
authors.lastname: Pemberton
authors.forename: A M
authors.initials: AM
authors.lastname: Gibson
authors.forename: R J
authors.initials: RJ
authors.lastname: Thomas
authors.forename: R R
authors.initials: RR
authors.lastname: Pascoe
authors.forename: P M
authors.initials: PM
authors.lastname: Desmarchelier
mesh_terms: D000818:Animals; D001109:Archives; D001483:Base Sequence; D001681:Biological Assay; D001905:Botulinum Toxins / Q000096:biosynthesis / Q000235:genetics*; D001906:Botulism / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D003013:Clostridium / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D005798:Genes, Bacterial / Q000235:genetics*; D006734:Horse Diseases / Q000453:epidemiology / Q000382:microbiology*; D006736:Horses; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D013048:Specimen Handling; D006113:United Kingdom / Q000453:epidemiology
chemical_list: D017931:DNA Primers; D009498:Neurotoxins; D001905:Botulinum Toxins