Effect of Low Temperatures on Growth of Nonproteolytic Clostridium botulinum Types B and F and Proteolytic Type G in Crabmeat and Broth 1.

The ability of unheated and heated spores of nonproteolytic Clostridium botulinum types B and F, and of the weakly proteolytic type G, to grow and produce toxin in crabmeat and broth at low temperatures was investigated. Sterilized crabmeat or broth was inoculated with 103 spores/g or ml and incubated anaerobically at 4, 8, 12 and 26 C for 180 days. Both heated and unheated spores of all three types grew and produced toxin at 26 C in broth and crabmeat. Types B and F grew in broth at 12, 8 and 4 C when unheated but only at 12 and 8 when heated; they did not grow in crabmeat at any of these temperatures, heated or not. Heated and unheated type G grew at 12 C in both broth and crabmeat but not at lower temperatures.

Evaluation of the Botulism Hazard from Nitrogen-Packed Sandwiches.

Clostridium botulinum was inoculated into hamburger, sausage and turkey sandwiches, which were subsequently placed in a nitrogen atmosphere. Growth of the bacterium was studied to assess the botulism hazard. Hamburgers inoculated with C. botulinum types A and B and incubated at room temperature became toxic on day 4 while remaining fully acceptable organoleptically. Sausages became toxic on day 7 while appearing marginally acceptable. In air at room temperature, all sandwiches were obviously decomposed before toxin was produced. Refrigeration under nitrogen prevented toxin production by types A and B; however, hamburgers inoculated with type E were toxic at 12 C in 30 days while appearing fully acceptable. All refrigerated sandwiches were either fully or marginally acceptable organoleptically throughout the 60-day observation period; none were obviously decomposed. Turkey sandwiches did not become toxic at any temperature or incubation time studied.

Toxin Production by Clostridium botulinum in Shelf-Stable Pasteurized Process Cheese Spreads.

Five non-refrigerated, pasteurized process cheese spreads, considered shelf-stable, were studied for their ability to support growth and toxin production by spores of Clostridium botulinum types A and B. Based on pH and water activity (aw) Cheese with Bacon, Limburger, Cheese Whiz, Old English, and Roka Blue cheese spreads were selected for the study. The pH ranged from 5.05 to 6.32 and the aw from 0.930 to 0.953. Fifty jars of each cheese spread were inoculated with 24,000 spores each; an additional 50 jars of the Cheese with Bacon spread received 460 spores each. The inoculum consisted of five type A and five type B strains in 0.1 ml of 0.85% NaCl. At 35 C, 46 jars of Limburger and 48 jars of Cheese with Bacon spread, which received the greater inoculum, became toxic starting at 83 and 50 days, respectively. One jar of Cheese with Bacon spread which received 460 spores became toxic. The average toxicity of the Limburger was 3000 MLD/ml of extract as compared with 54 MLD/ml for the Cheese with Bacon spread. Results of this study will be considered in determining whether these cheese spread products should be treated as low-acid canned foods under the Good Manufacturing Practice Regulations of the Food and Drug Administration.

Evaluation of the Botulism Hazard in Fresh Mushrooms Wrapped in Commercial Polyvinylchloride Film.

The botulism hazard in fresh mushrooms wrapped in commercial polyvinylchloride (PVC) film appears to be minimal. At the end of their normal shelf-life, 1,078 packages of PVC-wrapped mushrooms were all free of botulinum toxin. Since inoculated mushrooms were occasionally found to be toxic (14 in 250 packages) when only one 1/8-inch hole was punched in the wrapper, and none became toxic when two holes were present, it seems prudent to recommend that PVC-wrapped tills of mushrooms have two holes in the wrapper.

Effect of Low Temperatures on Growth of Clostridium botulinum Spores in Meat of the Blue Crab.

The ability of unheated and heated spores of Clostridium botulinum types B. E, and F to grow and produce toxin in crabmeat from the blue crab at low temperatures was investigated. Sterilized crabmeat was seeded with 103 unheated spores/g or 104 heated spores/g and incubated anaerobically at 4, 8, 12, and 26 C. Broth cultures served as controls. Both unheated and heated spores of the three strains grew and produced toxin in crabmeat at 26 C in 3 and 6 days, respectively. In addition, unheated spores of the nonproteolytic type E strain grew and produced toxin in crabmeat at 12 C in 14 days. Neither heated spores of type E nor heated or unheated spores of types B and F grew in crabmeat at any refrigerated temperature within 180 days.

Incidence of Clostridium botulinum in crabmeat from the blue crab.

The incidence of Clostridium botulinum in fresh crabmeat of blue crab was six out of 986 samples; in pasteurized crabmeat one sample out of 1,000 contained the organism.

Antigenic relationships among the proteolytic and nonproteolytic strains of Clostridium botulinum.

Relationships of the somatic antigens among Clostridium botulinum strains have been investigated by tube agglutination and agglutinin absorption tests. Results revealed a relationship by which strains of C. botulinum are grouped by their proteolytic capacity rather than by the type of specific toxin produced. Thus, C. botulinum type E and its nontoxigenic variants, which are nonproteolytic, share common somatic antigens with the nonproteolytic strains of types B and F. Absorption of antiserum of a strain of any one type with antigen of any of the others removes the antibody to all three types. In the same manner, C. botulinum type A shares somatic antigens with the proteolytic strains of types B and F, and absorption of any one antiserum with an antigen of either of the other two types removes the antibody to all three types. Partial cross-agglutination of C. sporogenes, C. tetani, and C. histolyticum with the somatic antisera of the proteolytic group was also observed.

Serological studies of Clostridium botulinum type E and related organisms. II. Serology of spores.

Pure spore antigens for the immunization of rabbits were prepared by enzymic digestion of vegetative components and separation of the cleaned spores in polyethylene glycol. Spore antisera were prepared to strains representative of toxigenic Clostridium botulinum type E; nontoxigenic boticin E-producing variants; nontoxigenic nonproducers of boticin E; nontoxigenic "atypical" strains, which differ somewhat from C. botulinum type E in their physiology; C. botulinum types A and B; and C. bifermentans. They were tested against these and additional strains representative of the above groups, other types of C. botulinum, and other Clostridium species. There was no evidence of agglutination of flagellar or somatic antigens of vegetative cells by these antisera. Agglutination and agglutinin absorption tests showed common antigens among toxigenic type E strains and nontoxigenic variants, both producers and nonproducers of boticin E. Some nontoxigenic "atypical" strains varied in their ability to be agglutinated by type E antisera, and others did not agglutinate at all. Of those atypical strains that were not agglutinated, one was agglutinated by C. bifermentans antiserum. Antisera prepared against C. botulinum types A and B and C. bifermentans did not agglutinate the spores of type E or its variants nor share antigens common to each other. Similarly, antisera to type E, its nontoxigenic variants, and nontoxigenic atypical strains did not agglutinate other C. botulinum types or any other Clostridium species investigated.

Serological studies of Clostridium botulinum type E and related organisms.

Clostridium botulinum type E antigens prepared from washed cells by either Formalin treatment or heating at 100 C were used for immunizing rabbits. Agglutination tests showed that high levels of antibody were produced by both types of preparations. Flagellar antigens were highly strain-specific, whereas the somatic antigens were sufficiently similar to produce complete cross-agglutination. One toxigenic strain produced toxigenic and nontoxigenic progeny which were physiologically and antigenically identical in all other respects. Other nontoxigenic strains whose growth, physiological, and morphological characters were identical to type E and strains which had some physiological differences completely cross-agglutinated with type E strains via the somatic antigen. Neither type of antiserum agglutinated other clostridia against which they were tested except for C. acetobutylicum. This reaction seems to be due to a nonspecific anamnestic response and does not appear to be related to the immunizing strains. The nontoxigenic strains studied seem to have no greater antigenic differences from type E strains than the type E strains have from each other.

Antagonistic effect on Clostridium botulinum type E by organisms resembling it.

A bacteriocin-like substance, active against strains of Clostridium botulinum type E, is produced by certain nontoxic organisms whose biochemical properties and morphological characteristics are similar to type E. The substance, for which the name "boticin E" is proposed, is bacteriolytic for vegetative cells and bacteriostatic for spores of type E. Its spectrum of activity is somewhat strain-specific. Of the clostridial species tested, only C. botulinum type E and, to a lesser extent, C. perfringens and C. acetobutylicum, but not C. botulinum types A, B, or F, are sensitive. Irreversibly resistant variants originating from both vegetative cells and spores of certain strains were obtained. The active substance is heat-stable and dialyzable, and is not inactivated by chloroform but is digested by trypsin. Ethyl alcohol and acetone precipitates are fully active, whereas trichloroacetic acid precipitates are only partially active. Other nontoxic organisms producing similar antagonistic substances are discussed.

Survival and recovery of enterovirus from foods.

Type 1 poliovirus and type B1 and B6 coxsackieviruses added to eight commercial frozen or convenience foods before storage at room temperature, 10 C, and -20 C were still viable after various intervals of time up to 1 week, 1 month, and 5 months, respectively. Infectivity titers were determined in monkey kidney tissue culture. Decomposition which took place in food stored at room temperature did not affect the survival of virus, and antibiotics controlled bacterial growth during assay. A rapid, significant reduction in type B6 coxsackievirus was observed in cole slaw. Preliminary data indicate that sodium bisulfite could be the responsible ingredient. A less rapid reduction of type 1 poliovirus and type B6 coxsackievirus was found in hashed brown potatoes when stored at 10 C.