Application of PCR to a clinical and environmental investigation of a case of equine botulism

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

Analysis of fumigant residues - A critical review

This review concentrates on the main fumigants currently applied to commodities: phosphine (PH3) and methyl bromide (CH3Br). It discusses possible new fumigants, such as carbonyl sulfide (COS) and ethyl formate (EtF), that may be able to be applied in such a way that "residues" after fumigation do not exceed the range of natural levels. The review aims to raise some general issues and to suggest some general conclusions from a synthesis of studies on analytical chemistry and on fumigant behavior

Release of fumigant residues from grain by microwave irradiation

Multiresidue analysis of fumigants is important because of their widespread use on staple foodstuffs, such as grain. Fumigants are usually extracted from grain either by solvent extraction or by purge-and-trap techniques. In this paper, fumigant residues in wheat were "extracted" by a microwave procedure. Wheat, in gas-tight Erlenmeyer flasks, was placed in a domestic microwave oven, and fumigants were released into the headspace by microwave irradiation. Power settings for maximum release of fumigants were determined for CH3Br, PH3, CS2, and COS. Recoveries of fortified samples were >90%. Completeness of extraction was assessed from the amount of fumigant retained by the microwave-irradiated wheat. This amount, determined from both solvent extraction and from further microwave irradiation, was always small (<5% of the amount obtained from the initial procedure). Limits of quantitation were <1 ng/g for CH3Br, PH3, and CS2. These low limits were essentially due to the absence of interference from solvents. The microwave method is rapid and solvent-free. However, care is required in selecting the appropriate power setting. The safety implications of heating sealed flasks in microwave ovens should be noted

Comparison of six methods for determining aged phosphine residues in wheat

Phosphine (PH3) residues on wheat were determined by gas chromatography (GC) in a blind experiment using 6 methods to assess whether results from different methods are comparable. For one method, 2 different chromatographic conditions also were used. Samples were fumigated for 7 days and aired for 2 or 7 days to obtain "aged" residues. The methods involved determination of PH3 in the headspace over aqueous acidified acetone plus wheat (method a), over ground wheat (method b), over ground wheat plus headspace concentrations over ground wheat in acetone after transfer to another flask (method c), over ground wheat in acetone (method d), over whole wheat after microwave heating (method e), and over water after dilution of 0.5 mL aqueous acetone leachate with 10 mL buffered sodium sulfate solution (method f). For each method, gas-tight flasks and syringes were used. Data to support new methods c, d, and f are discussed. Each procedure correctly identified the order of PH3 residues as highest, medium, and absent. Quantitative results from all methods were similar, except that results from method b were on average 44% lower than the average results from other procedures. Thus each method, except method b, is suitable for determination of PH3 residues. The relative advantages of each procedure are discussed

Evaluation of Headspace Solid-Phase Microextraction for Analysis of Phosphine Residues in Wheat

In headspace (HS) analysis, a fumigant is released from a commodity into a gas-tight container by grinding, heating, or microwaves. A new technique uses HS-solid-phase microextraction (SPME) for additional preconcentration of fumigant. HS-SPME was tested for detection of phosphine (PH3), chosen for examination because of its wide use on stored commodities. PH3 was applied to 50 g wheat in separate 250 mL sealed flasks, which were equipped either with a septum for conventional HS analysis or with one of four HS-SPME fibers [100 mu m polydimethylsiloxane (PDMS), 85 mu m carboxen (CAR)/PDMS, 75 mu m CAR/PDMS, and 65 mu m PDMS/divinylbenzene (DVB)]. The wheat was heated at 45 degrees C for 20 min. In conventional HS analysis, a gaseous aliquot (80 mu L) was taken from the HS and injected into the GC instrument. In the HS-SPME procedure, the fiber was removed from the HS and exposed in the heated injection port of the GC instrument. In all cases, PH3 was determined under the same chromatographic conditions with a GC pulsed flame photometric detector. In a comparison of the efficacy of the fibers, the bipolar fibers (CAR/PDMS and PDMS/DVB) contained more PH3 than the aliquot in the conventional HS analysis; larger size bipolar fibers extracted PH3 more efficiently than smaller fibers (e.g., 85 > 75 > 65 mu m). The nonpolar fiber (PDMS) contained no PH3. Four fortification levels of PH3 on wheat were tested: 0.01, 0.05, 0.1, and 0.3 mu g/g. The response of each bipolar fiber increased with the fortification levels, but the conventional HS analysis detected no fumigant at the lowest fortification level of 0.01 mg/g. Under the conditions of the validation study, the LOD was in the range of 0.005-0.01 ng PH3/g wheat

Improved methodology for studying diffusion, sorption and desorption in timber fumigation

Methods to study movement of fumigant through timber were improved, and procedures were developed that obviated the need for sampling lines. Movement for two fumigants (methyl bromide and carbonyl sulphide) through, and sorption on, heartwood of softwood and hardwood were studied. Each fumigant was sorbed less on softwood than on hardwood and penetrated softwood better than hardwood. Neither fumigant penetrated across the grain. Carbonyl sulphide penetrated timber better than did methyl bromide, and was less sorbed on timber. A rapid method of solvent extraction was developed to enable, rapid estimation of the amount of intact fumigant sorbed in wood. This procedure enabled excellent recovery of methyl bromide as either intact fumigant or as bromide ion

Modifications of a method for determining multifumigant residues

Phosphine (PH3) and methyl bromide (CH3Br) are the main fumigants used on stored grains. Published multiresidue methods, including those based on solvent extraction and on purge-and-trap techniques, give poor recoveries of CH3Br and, in our hands, close to zero recovery of PH3 and carbonyl sulfide (COS), a potential fumigant. We examined factors influencing fumigant analysis, including stability of chemicals in leachates and partitioning of fumigant between leachate and air. The partition ratio, defined as the ratio of fumigant concentration in leachate to that in the headspace, varied between 1.1 for PH3 and >100 for ethylene dibromide (EDB). An official procedure involving solvent extraction followed by partitioning was modified by being performed in sealed flasks. This change raised the recovery of CH3Br from 28 to 85%. Volatile fumigants, including PH3 and COS, were determined from concentrations in the headspace over the leachate (aqueous acidified acetone). Recoveries were nearly quantitative at levels down to 3 ng PH3/g and 16 ng COS/g, provided that fortified samples were used as analytical standards. Thus an existing multifumigant procedure was adapted to enable determination of the main fumigants used on staple foodstuffs