Interrelationship of Heat and Relative Humidity in the Destruction of Clostridium botulinum Type E Spores on Whitefish Chubs

Heat destruction of types B and E Clostridium botulinum spores on whitefish chubs was observed to be dependent upon the relative humidity (RH) in the chamber in which fish were heated. Experimental conditions were designed to simulate those attainable in commercial fish-smoking plants. Low numbers of type E spores were destroyed with regularity, within 30 min, on fish which were held at an internal temperature of 77 C (170.6 F) in an atmosphere of at least 70% RH. However, an internal temperature of 82 C (179.6 F) and a minimum RH of 70% were required to destroy several hundred thousand type E spores. Quantitative estimates of spore destruction were arrived at with a modified most probable number procedure. Type E spore populations were reduced by 2 to 4 logarithms at 77 C (170.6 F), by 5 to 6 logarithms at 82 C (179.6 F), and by more than 6 logarithms at 88 C (190.4 F) when fish were heated in an atmosphere of 70% RH. A 5 to 6 logarithm reduction of spores was also observed when fish inoculated with type B spores were processed at 82 C (179.6 F) in an atmosphere of 70% RH.     

Sensitivity of an Enrichment Culture Procedure for Detection of Clostridium botulinum Type E in Raw and Smoked Whitefish Chubs

The sensitivity of an enrichment culture procedure for detecting Clostridium botulinum type E in whitefish chubs (Leucichthys sp.) was assayed. Data demonstrated that fish inoculated with 10 or more viable C. botulinum spores regularly develop specifically neutralizable enrichment cultures. Mild heat treatment (60 C, 15 min) substantially reduced the sensitivity of enrichment culturing. This effect was particularly noticeable in the culturing of fish which harbored fewer than 10 spores each. Evidence is presented which indicates that sensitivity of enrichment, without heat, approaches the level of one spore per fish. Smoked whitefish chubs, containing from one to several hundred spores each, were examined for toxin content after storage at 5, 10, 15, and 28 C for as long as 32 days. The lowest temperature at which detectable toxin was produced was 15 C. This occurred in 1 of 10 fish incubated for 14 days. C. botulinum was regularly recovered, by enrichment culture, from fish inoculated with small numbers of spores, even though toxin was not detected by direct extraction of incubated fish. Persistence of C. botulinum type E spores was observed to decline with an increase in the temperature and time at which inoculated fish were stored.     

Isolation of Clostridium botulinum from tropical fish

Electroporation was used to facilitate transformation of Listeria species with plasmid DNA. Optimal conditions for transformation of L. monocytogenes were a field strength of 8.5 kV/cm, 200 Ohms resistance, 25 microF capacitor with a time constant of 5 ms. With these conditions, 3.9 x 10(6) transformants/micrograms DNA were obtained. Under the same conditions, L. innocua and L. ivanovii exhibited a frequency of transformation similar to that of L. monocytogenes but a somewhat lower level was obtained with L. seeligeri.     

Biodiversity of Clostridium botulinum Type E Strains Isolated from Fish and Fishery Products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Demonstration and characterization of the cell wall carbohydrate and protein antigens from Clostridium botulinum type E Saroma

Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100. The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine. Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer. Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.     

Isolation and Role of Nonheme Iron Protein in Clostridium botulinum

A type of iron-bound protein was isolated from Clostridium botulinum by a modification of the method used for isolating ferredoxin from C. pasteurianum. This method involved acetone and diethylaminoethyl cellulose treatments followed by ammonium sulfate fractionation. The protein exhibited maximal absorption in the ultraviolet region near 260 mμ. Portions of the isolated iron protein were separated by disc electrophoresis and, following specific iron-bound protein staining, showed a positive reaction in the same position on the gel column as was first demonstrated by use of cell-free extract. Evidence accumulated by use of a cell-free extract of C. botulinum suggests that pyruvate is metabolized through a phosphoroclastic system as demonstrated in other clostridia. It is probable that ferredoxin is an electron mediator between pyruvic oxidase and hydrogenase for hydrogen evolution and acetyl phosphate formation.     

Survival and Outgrowth of Clostridium botulinum Type E Spores in Smoked Fish

Chub injected in the loin muscle with 10(6)Clostridium botulinum type E spores were smoked to an internal temperature of 180 F (82.2 C) for 30 min, sealed in plastic bags, and incubated at room temperature (20 to 25 C) for 7 days. Viable type E spores were found in practically all such fish. Toxin formation by the survivors in the smoked fish was dependent on the brine concentration of the smoked fish. A brine concentration of 3% or higher, as measured in the loin muscle, inhibited toxin formation. Six different type E strains gave similar results. Only a few hundred of the million spores in the inoculum survived the smoking. Moisture in the atmosphere during smoking did not reduce the incidence of fish with type E survivors.     

甘草内生真菌的分离及鉴定

植物体内普遍存在着内生菌,为了探寻传统药材甘草的质量与其内生菌之间可能存在的关系。对采自新疆的甘草根部进行了内生真菌的分离及鉴定。选用CYM真菌培养基,对其内生真菌进行分离及纯化,选择其中最占优势的两种菌落分别进行了菌落形态观察及菌丝形态(棉兰染色)观察,以及基因组DNA的18S rDNA和ITS rDNA序列鉴定和系统进化分析。本研究结果表明从甘草根部主要分离到两种内生真菌,分别属于Fusarium镰刀菌属和Gibberella赤霉菌属。     

Recovery of Clostridium botulinum from mud samples incubated at different temperatures

Summary In mud samples naturally contaminated with Clostridium botulinum type C, other types cannot be recovered when incubated at 37°C. At incubation temperatures equal to or lower than 30°C and in the presence of type E, Clostridium botulinum type C cannot always be detected. The use of two incubation temperatures to increase the probability of detecting all types of Clostridium botulinum can therefore be recommended.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Fine Structure of Protoplasts and L-Forms of Clostridium botulinum Types A and E

Spherical bodies, that are obtained by adding penicillin and lysozyme to Clostridium botulinum types E and A cultures which are growing in an osmotically stabilized medium, are shown to be protoplasts by electron microscopy. The L-forms of these two culture types have morphologically different inclusion bodies.