Separation of botulinum-positive and -negative fish samples by means of a pattern recognition method applied to headspace gas chromatograms.

A gas chromatographic headspace technique was used to analyze the gas produced during putrefaction of pond-raised, degutted trout, incubated in evacuated plastic pouches. The following samples were analyzed; 10 samples which, due to natural contamination with Clostridium botulinum, were toxic when injected into mice, 10 samples which were nontoxic when injected, and 9 samples inoculated with one strain of C. botulinum type E. The gas chromatograms showed the presence of 118 compounds in most samples. Quantitative differences among most chromatograms could be observed, but no compound was unique to any of the three groups. By means of a specific pattern recognition method, all negative samples were shown to fall into one group and were distinctly separated from the toxic samples. No differences could be observed between the two groups of inoculated and naturally contaminated trout samples. The results suggest that headspace analysis combined with pattern recognition analysis might prove to be a valuable method for screening studies of foods containing living cells of C. botulinum.     

Growth potential of Clostridium botulinum in fresh mushrooms packaged in semipermeable plastic film.

Fresh mushrooms (Agaricus bisporus) were inoculated in the stem, gill, or cap with Clostridium botulinum spores. They were placed with uninoculated mushrooms in paper board trays, which were then covered and sealed in a polyvinyl chloride stretch film to simulate prepackaged mushrooms available at retail stores. When incubated at 20 C, botulinum toxin could be detected as early as day 3, or 4, when the mushrooms still appear edible. Mushrooms inoculated in the stem with 1,000 type A spores frequently became botulinogenic; higher spore levels were needed if gills or caps were inoculation sites. Type B spores were less apt to produce toxic mushrooms. Respiration of the fresh mushrooms used up O2 more rapidly than could enter through the semipermeable wrapping film, so that the equilibrium O2 concentration became low enough for growth of C. botulinum. Inoculated mushrooms did not become botulinogenic when held at 4 C.     

Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat

A cocktail of washed spores from six psychrotrophic Clostridium strains isolated from blown vacuum-packed meats was inoculated onto lamb chumps. A second washed spore cocktail of four toxigenic reference Cl. botulinum strains, types A, B (two strains) and E, and a Cl. butyricum type E strain, was similarly inoculated onto lamb chumps. All inoculated lamb chumps were individually vacuum-packed and placed into storage at various temperatures typical of good to grossly abusive chilled storage (-1 degree C to 15 degrees C). All packs were observed for gas production (pack-'blowing') over a 12 week storage period. On gas production, or after 12 weeks of storage, packs were examined by mouse bioassay for botulinum toxin production. The packs inoculated with the meat isolate cocktail showed evidence of gas production earlier than packs inoculated with reference strains. No botulinum toxin was recovered from the meat isolate inoculated packs, while botulinal toxin was detected in reference strain inoculated packs down to a nominal storage temperature of 2 degrees C.     

Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.     

Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media.

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.     

Analysis of Tobacco Headspace Volatiles Using Tenax GC or Active Carbon†

Two simple methods, the Tenax GC method and the active carbon method, have been developed to analyze the headspace volatiles of tobacco. The headspace volatiles swept by helium from cut tobacco were collected by either Tenax GC or active carbon and were analyzed by gas chromatography after desorption by heating the Tenax GC or by extracting the active carbon with dichloromethane. The gas chromatograms of the headspace volatiles of flue-cured tobacco obtained by these two methods differed. Both methods showed good reproducibility and were applied to the analysis of the headspace volatiles of American and Japanese flue-cured tobacco samples. By principal component analysis, three or four principal components were. extracted from the 15 selected peaks of the headspace volatiles of these samples that had been obtained from both the Tenax GC and the active carbon methods.     

Phylogeographical lineages in brown trout (Salmo trutta): investigating microgeographical differentiation between native populations from Northern Spain

SUMMARY 1. The large microgeographical differentiation revealed by allozyme studies in brown trout ( Salmo trutta) populations is one of the most striking features of this species. Additionally, allozymes showed great genetic differences between Atlantic and Mediterranean populations on a macrogeographical scale.
2. This study was carried out in order to assess whether the great differences observed between Atlantic and Mediterranean populations persisted where the two are geographically close (the 'microgeographical scale'). Sixteen populations of brown trout, S. trutta , were screened for genetic variation at 25 allozyme loci. The sampling sites, which occupied a relatively small geographical area, were distributed across Cantabrian (Atlantic) and Mediterranean drainages in Northern Spain.
3. The neighbour-joining tree, inferred from Nei's genetic distance, showed that brown trout populations clustered into two different groups. These groups corresponded to the Cantabrian and the Mediterranean groups of populations, although no clear geographical pattern emerged within each of the groups. This geographical pattern is basically caused by significant differences in the frequency distribution of the CK-A1 * locus, with a higher frequency of * 115 in Cantabrian samples (0.586 ± 0.091) while allele * 100 was more frequent in Mediterranean samples (0.931 ± 0.038). In addition, this study revealed alleles exclusive to the Mediterranean and Cantabrian populations, agreeing with previous findings.
4. Genetic differentiation between Cantabrian and Mediterranean regions (14.19%) was similar to that estimated in Spain at a larger scale (13%), showing that most of the differences between the regions can be observed even in a small geographical area.     

Demonstration and Isolation of Clostridium botulinum Types from Whitefish Chubs Collected at Fish Smoking Plants of the Milwaukee Area

A total of 1,071 whitefish chub samples were examined at eight stages of processing, including sampling aboard ship, various processing steps in the smoking plant, and display in retail cases. The frequency of Clostridium botulinum contamination of freshly caught and eviscerated chubs was approximately 13 to 14%. The highest percentage of contamination (20%) was noted among chubs sampled at the brining step of processing. The prevalence of contamination among chubs sampled at other processing stages prior to the smoking operation ranged from 6 to 14%. Of 858 freshly smoked chubs that had been processed at 180 F for 30 min (internal temperature of loin muscle), 10 were contaminated with C. botulinum (1 Type B and 9 Type E). The use of heat-shocked (60 C for 15 min) and nonheat-shocked enrichment cultures in combination yielded a greater number of positive samples than either method yielded when used alone. Each toxic enrichment culture obtained was subcultured to obtain isolation of the toxigenic organism. Toxigenic pure cultures of C. botulinum were obtained from 80% of the fish samples observed to produce toxic enrichment cultures.     

Nonspecific Toxicities in the Mouse Assay Test for Botulinum Toxin

In inoculated pack experiments on Clostridium botulinum type E, unirradiated and 0.1-Mrad irradiated haddock fillets often gave nonspecific toxicities by the mouse assay test for botulinum toxin. Samples given 0.2-Mrad radiation failed to produce nonspecific reactions. Nonspecific deaths sometimes occurred within 24 hr after injection, although deaths between 24 and 48 hr were more common. The symptoms and the pattern of these deaths suggested a septicemia. Heart-blood cultured from mice showing nonspecific symptoms indicated an infectious process. Among 23 isolates from the blood, eight were identified as Proteus vulgaris, two P. morganii, one P. rettgeri, one Providence subgroup B, two Aerobacter aerogenes, one Actinobacillus, three enterococci, one Alcaligenes marshalli, and four Erysipelothrix insidiosa. The E. insidiosa, Aerobacter, Providence group, and most of the Proteus isolates were infectious for mice when injected by the intraperitoneal route. But the enterococci, Alcaligenes, and Actinobacillus isolates were not infectious and probably represent secondary invaders. The cultural characteristics of the E. insidiosa isolates conform to those described in the literature, with the exception that the four strains grew in the temperature range 50 F (10 C) to 40 F (4.4 C). Nonspecific toxicities were avoided in assays for botulinum toxin by the protection of mice with chloramphenicol and oxytetracycline.     

Use of Immunofluorescence and Animal Tests to Detect Growth and Toxin Production by Clostridium botulinum Type E in Food

The appearance of Clostridium botulinum type E organisms and of toxin in experimentally inoculated packages of turkey roll was followed to study the time relationship between the presence of vegetative cells and the demonstration of toxin. The presence of vegetative cells was determined by immunofluorescence, and animal tests were used to assay toxin production. Growth initiated from detoxified spores of C. botulinum type E resulted in toxin formation within 24 hr. Presence of fluorescing vegetative cells and of toxin coincided from 1 to 14 days of incubation. Beginning with the next testing date, day 21, differences were observed. Toxin could be detected for a longer time than vegetative cells. Neither toxin nor organisms could be found after 56 days of incubation. The mouse lethal dose tests (MLD per gram of turkey roll) showed fluctuations in the amount of toxin present throughout the period of testing. Maximal amounts of toxin were present during the period when fluorescing organisms were also more numerous. The applications of immunofluorescence in the study and in the diagnosis of botulism is discussed.     

First isolation of Clostridium tetani from soils of Meseta Central, Costa Rica]

Clinical evidence has long pointed to the existence of Clostridium tetani in Costa Rica. Thirty soil samples were studied for clostridia, and two yielded six strains of C. tetani, four of which proved to be toxigenic when mice were inoculated intraperitoneally with the culture supernates. These four isolates could be neutralized when their toxic supernates were admixed with tetanus antitoxin.     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.     

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.     

Use of the Anaerobic Pouch in Isolating Clostridium botulinum Spores from Fresh Meats

The anaerobic film pouch was demonstrated to be an effective device for the primary isolation of Clostridium botulinum types A and B spores from raw pork, beef, and chicken. Optimal pasteurization of these meats (for reduction of nonspore microflora without affecting indigenous putrefactive anaerobic spore levels) was 50 min at 60 C. C. botulinum spores were recovered with good precision from meat samples inoculated with mixtures of C. botulinum and Putrefactive Anaerobe 3679 at 1:1 and at 1:99 ratios. Verification of C. botulinum isolates was accomplished by protection testing of subcultures in mice.     

Storage Stability of Clostridium botulinum Toxin and Spores in Processed Cheese

Growth initiated from detoxified spores of Clostridium botulinum 62A resulted in toxin production of 50 to 10,000 mouse lethal doses (MLD) per gram of processed soft surface-ripened cheese. Regular assays during subsequent storage of toxic samples at 2 to 4 C revealed a characteristic two- to fivefold increase in toxin titer during the initial 1 week to 12 months of storage. Thereafter, the toxin titer remained constant for 2 to 4 years, after which the toxicity declined rapidly. At the end of 6 years of storage at 2 to 4 C, the samples still contained 20 to 5,000 MLD of toxin per gram, with the usual toxin level at 200 to 500 MLD. Toxic culture filtrates of C. botulinum incorporated into cheese and stored at 30 C for 60 days showed no decline in toxin in processed type I cheese, but toxin decreased slightly in processed type II and type III cheese. The surface flora of these cheeses did not attack but, on the contrary, protected C. botulinum toxin during storage at 30 C. Initial difficulties in recovering C. botulinum organisms from type I cheese were traced to growth inhibitory activity which could be removed by washing with distilled water in a centrifuge. Viable spores or vegetative cells could be recovered from all samples after 4 to 5 years of storage at 2 to 4 C. After 6 years, organisms were recovered from all except three samples of type I cheese. Two other samples showed a large decrease in viable organisms. In type III cheese, spores remained remarkably stable for 6 years at the level of the initial inoculum, i.e., approximately 10(5) spores per gram.     

Contamination routes of Clostridium botulinum in the honey production environment

Factors influencing Clostridium botulinum contamination in the honey production environment were evaluated in a 3-year survey. A number of 1,168 samples from 100 apiaries and related facilities were analysed for the presence of C. botulinum types A, B, E and F, using multiplex polymerase chain reaction targeted to botA, botB, botE and botF genes. Production methods and environmental factors were registered using a questionnaire and by personal observation. Clostridium botulinum was shown to be a common finding throughout the whole honey production chain, and the type most frequently detected was group I type B. In a pulsed-field gel electrophoresis (PFGE) analysis of 202 group I type B isolates, only six different PFGE profiles were observed, of which two clearly distinct profiles predominated. This may indicate the existence of at least two different genetic lineages. The high prevalence of C. botulinum in soil and in samples associated with beeswax suggests the accumulation of soil-derived botulinal spores in wax. Additionally, according to Spearman's rank order correlation and multivariate analysis, production hygiene-dependent factors have a significant influence on the contamination, and thus the number and frequency of C. botulinum spores in honey could possibly be diminished by increasing hygienic level in honey production.