Distribution of Clostridium botulinum type E in fish, shellfish and the marine environment of the Pacific Northwest, and protein patterns of the toxigenic and non-toxigenic stains Public Deposited

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  • Interest in the distribution of Clostridium botulinum type E was heightened by the sudden outbreak of human botulism from smoked whitefish chubs and canned tuna fish in 1963. The question arose as to how widely the organism is distributed among fish and shellfish in the Northwest and what potential hazard exists for the consumer of fish products. This sporeforming anaerobic orgnism is heat sensitive and had eluded detection in other surveys where heat shock had been used to eliminate non sporeforming contaminants. More recent study using other techniques than heat to facilitate recovery has shown this organism to be widespread, especially in the marine environment. This study was undertaken to find the incidence and distribution of C. botulinum in the marine organisms and environment of the Pacific Northwest and the food products derived therefrom. All species of fish were examined by incubating the gills and viscera individually in tryptone-peptone-glucose medium anaerobically at 28 C for four days and testing the culture filtrate for mouse toxicity by intraperitoneal injection. Toxic filtrates were typed by retesting them in mice protected by specific botulinal antitoxin of type A, B, E, or F. Among salmonid fish the proportions of specimens of each species yielding toxic filtrates were as follows. Sockeye salmon from the Columbia River, 14 of 59 (23.7%); Chinook salmon from the Columbia River, 19 of 106 (18.0%); Chinook salmon from the Pacific Ocean, 1 of 18 (5,6%); Coho salmon from the Columbia River, 10 of 19 (34.4%); Coho salmon from the Pacific Ocean, 13 of 186 (7.0%); Steelhead trout from the Alsea River, 7 of 37 (19.0%). About one-third of the toxic cultural filtrates were successfully typed and proven to contain botulinal toxin. Most of them proved to be type E toxin but 3 were type A, 3 were type B and one, a comparatively new type, type F, was isolated from a Sockeye salmon in the Columbia River. Pure cultures of Clostridium botulinum type E were isolated from 18 specimens and one specimen yielded a pure culture of type F from a sockeye salmon. This was the second time this type had been isolated. In all of the experimental groups the proportion of fish producing toxigenic cultures was significantly higher in those taken in the two rivers than those of the same species taken from the ocean waters. "Bottom fish" represented by Cod, Sole, Grouper and members of the Sebastodes group were also tested in the manner described above. The number of specimens yielding toxic filtrates were 28 of 157 (17.8%). When grouped according to location at which the fish were caught, those near the mouth of the Columbia River produced a greater percentage of toxic filtrates than did those caught off the open shore line. The results were as follows: Bottom fish from Astoria, 23 of 70 (32.8%), Botton fish from Coos Bay, 5 of 87 (5.6%). Sturgeon specimens produced 3 of 24 (12.5%) toxic filtrates. Most of the species contained type E; however, one type A and one type B were found on typing, with about one-third of the toxic filtrates being successfully typed. Environmental swab samples from the "deep sea" fillet processing plants produced 3 of 39 (7.7%) toxic filtrates. None of the 53 samples taken in the salmon processing plants produced toxic filtrates. Shellfish were collected along the ocean beach and in the estuaries. Three to five shellfish were combined into a single specimen and treated as described. All shellfish obtained from the estuaries demonstrated a higher percentage of toxic filtrates than those obtained from the ocean beach. The results were as follows: Razor clams, 11 of 75 (14.6%), Cockle clams, 12 of 15 (80.0%); Softshell clams, 8 of 12 (66.4%); Littleneck clams, 4 of 11(36.2%); Horseneck clams, 1 of 3 (33.3%); Oysters, 6 of 19 (31.6%); Dungeness crabs, 17 of 24 (71%). Only the razor clams were collected exclusively from the ocean beach. Loss of toxicity on holding mixed cultures at -15 C while awaiting typing was a continual problem. This accounts for only one-third of toxic filtrates being successfully typed. Electrophoretic analysis of the total bacterial proteins was carried out on cell sonicates and cell free culture filtrates by first growing cells for four days at 28 C anaerobically. The cells were separated, washed and disrupted with ultrasonic energy. The cell free culture filtrate was concentrated 10 fold by dialysis against polyethylene glycol 4000. Both the toxigenic organisms and the toxic filtrate demonstrated an extra common protein band in the upper third of the electrophoretic pattern not present in the nontoxic spectra. This band might represent the type E toxin. Differences could also be noted in the number of protein bands in the lower third of the patterns in different nontoxigenic strains and also when the toxigenic and nontoxigenic strains were compared. This could suggest an association with a phage in the toxigenic cultures.
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