A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

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.     

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.     

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

Differentiation of Clostridium botulinum Types A, B, and E by Pyrolysis-Gas-Liquid Chromatography

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.     

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.     

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.     

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.     

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.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.     

The Inhibition of Clostridium Botulinum Type B and E in Salami Sausage

Vegetative cells and spores of Clostridium botulinum type B and E were inoculated into salami sausages with and without the preservatives sodium nitrite and sodium benzoate. The growth and toxin production of Clostridium botulinum type B and E were inhibited in this type of salami sausages, even without any addition of preservatives. The use of a starter culture with pH-lowering components has both technological and hygienic advantages.     

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.     

Examination of Prepared Foods in Plastic Packages for Clostridium botulinum

The incidence of Clostridium botulinum organisms was determined in a variety of plastic-packaged "vulnerable" foods (food requiring little or no heating prior to consumption). A total of 113 foods were examined by use of an enrichment recovery procedure followed by toxin testing in animals. Results of the survey indicate that the incidence of C. botulinum organisms in these vulnerable foods is extremely low. The ability of inoculated food products to support growth and toxigenesis of C. botulinum type E was then tested. The 64 packaged foods were inoculated with type E spores and incubated anaerobically at 30 C for 11 days. A slurry of each food was prepared, smears for fluorescent-antibody testing were made, and animal tests were performed for toxin. If the animal tests were negative, enrichment cultures were prepared from the slurry and incubated at 30 C. On direct examination of the slurries for toxin, only samples of turkey roll and soybean cake supported growth and toxigenesis by C. botulinum type E. However, the enrichment culture method was able to induce growth and toxin production in 60 of the remaining 62 samples.     

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.     

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.     

Metabiotic effect of Bacillus licheniformis on Clostridium botulinum: implications for home-canned tomatoes.

The metabiotic effect of Bacillus licheniformis on Clostridium botulinum was examined. B. licheniformis elevated the pH of a model system with an initial pH of 4.4 so that C. botulinum grew and produced toxin. Toxin production was observed when spores from both species were coinoculated at levels as low as 10 spores per ml. When pint jars of tomatoes were used, canner size contributed to a 10,000-fold difference in the lethality of a boiling water bath process on B. licheniformis spores. Botulinal toxin was not detected in pH-elevated jars of tomatoes containing C. botulinum spores.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.