Incidence of Clostridium botulinum Type E in Salmon and Other Marine Fish in the Pacific Northwest

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1 APPLIED MICROBIOLOGY, Apr. 1968, p Copyright 1968 American Society for Microbiology Vol. 16, No. 4 Printed in U.S.A. Incidence of Clostridium botulinum Type E in Salmon and Other Marine Fish in the Pacific Northwest JAMES M. CRAIG,' SIDNEY HAYES, AND K. S. PILCHER Department of Microbiology, Oregon State University, Corvallis, Oregon 971 Received for publication 5 December 1967 Salmon, sole, cod, oysters, clams, and crabs from ocean waters along the coast of Oregon and Washington were examined for the presence of Clostridium botulinum type E. The organism was detected by identification of the type E toxin in enrichment cultures of the viscera of individual fish. Of 69 salmon specimens, 48 yielded cultures containing toxin lethal to mice, and almost half of the toxic cultures were shown to contain botulinal toxin, chiefly type E. Eighteen of 11 sole and cod specimens, 4 of 22 Dungeness crab specimens, 5 of 16 oyster specimens, and 27 of 115 clam specimens gave rise to cultures containing botulinal toxin which was usually type E, although types A and B were occasionally encountered. The 196 outbreaks of botulinum food poisoning resulting from the ingestion of type E botulinal toxin in smoked whitefish and smoked whitefish chubs from the Great Lakes (1, 1), as well as earlier outbreaks, aroused interest in the incidence of Clostridium botulinum in other marine species such as salmon, tuna, shrimp, and crab. The distribution of this organism in fish from the Great Lakes was reported by Bott et al. (2). Recently, C. botulinum has been identified in marine sediments from the Gulf of Mexico and the Pacific coast (7, 16); it has also been found in several species of fish (5, 9, 10) and shrimp (). Eklund (8) reported the presence of the organism in a large percentage of Dungeness crabs collected in the Pacific Northwest. The survey reported here was undertaken to study the incidence of C. botulinum type E in the most important species of fish and shellfish of the Pacific Northwest. MATERIALS AND METHODS Gills and viscera of fish were removed with sterile instruments and were placed in sterile polyethylene bags with 70 ml of TPG medium ( 14). Air was expelled from the bags, which were then closed with rubber bands. All samples were kept on ice until delivered to the laboratory and, if not to be tested within 24 hr, were frozen and stored at -20 C. Samples were incubated at 28 C for 5 days. A portion of the fluid was removed from the plastic bag, centrifuged at about 1,000 X g for 0 min, and then filtered through a 0.2,u membrane filter in a Swinney adapter. The sterile filtrate was adjusted to ph 6.2 with 1 N HCI and was activated with I '; trypsin for I On leave from San Jose State College. 1 hr at 7 C (6). Tenfold and 100-fold dilutions of the filtrate were prepared in gelatin phosphate buffer (0.2% gelatin and 1% Na2HPO4, adjusted to ph 6.2 with 1 N HCl), and 0.5 ml of each dilution was injected intraperitoneally into each of two Swiss mice (18 to 22 g). During the latter part of this investigation, filtration of the incubated samples was omitted to expedite the testing of larger numbers of specimens, and dilutions of 1:5 and 1: 10 were used for injection. This method seemed to give satisfactory results with the fish and shellfish specimens. Only sterile filtered samples, however, were used in typing toxic cultures with monovalent antisera. In all mouse tests, the animals were observed closely for 48 hr for typical symptoms of botulism and for death; surviving mice were discarded after 4 days. The shellfish tested were collected, washed free of sediment in salt water, and placed in plastic bags in groups of three to six specimens for most species. Within 6 hr, the shells were scrubbed with a brush and tap water, dried, and opened with a sterile knife. The tissue and liquor from a group of six specimens were removed aseptically and were pooled in a sterile plastic bag with 20 to 40 ml of TPG medium. The bag was then incubated at 28 C for 5 days, and the mixed culture was tested for toxicity as described above. Toxic samples were serologically typed by repeating the injections of trypsin-activated, diluted sterile filtrate into mice that had been individually protected with C. botulinum types A, B, C, D, or E antisera. The antisera were supplied by the Division of Microbiology, Food and Drug Administration. After typing the toxin, recovery of the organism was attempted. A 2 ml amount of the material containing the sediment from the centrifuged culture, or 2 ml of the uncentrifuged culture (both had been stored at -20 C), was treated for 1 hr with enough 954, ethyl alcohol to give a 50%, alcohol concentra- 55

2 554 CRAIG, HAYES, AND PILCHER APPL. MICROBIOL. tion, as described by Johnston, Harmon, and Kautter (11). Dilutions of 1:10, 1:100, and 1:1,000 were prepared in gelatin phosphate buffer, and each of these dilutions, as well as the undiluted mixture, was streaked on plates of Liver Veal Agar (Difco) with 4%S egg yolk added and on Blood Agar Base (Difco) with 5%c human blood (outdated bank blood). One loopful was streaked on each plate. All plates were incubated in a nitrogen atmosphere in a Case anaerobic jar for 6 hr at 28 C. Colonies resembling C. botulinum were transferred to TPG medium, incubated for 5 days at 28 C, and tested for toxicity in mice. If toxic, the toxin was typed by the method described above. Pure cultures were most easily isolated and identified either from the I :100 or 1:1,000 dilutions after alcohol treatment. RESULTS Cultural examination of considerable numbers of the chief marine fishes and shellfish of the Pacific Northwest was carried out to determine the relative frequency with which C. botulinum type E or its specific toxin could be identified in cultures from freshly caught specimens. The results obtained with three species of salnon, as well as with steelhead trout, sturgeon, sole, and cod, are presented in Table 1. A significant percentage of the specimens from each of the three salmon species gave rise to culture filtrates which were toxic to mice by intraperitoneal injection. This percentage varied from about 8% for all groups of coho salmon to about 24% for the sockeye. The time of death of injected mice varied from to 20 hr in most cases, which is considered characteristic of type E botulinum toxin. Of the toxic specimens from salmon, 22 were identified immunologically by retesting in mice protected by botulinum antitoxin of a particular type, as well as in unprotected mice. We could not type 26 cultures because toxicity had been lost during storage at -15 C. The majority of specimens successfully typed were shown to contain type E botulinum toxin, indicating the presence of the organism in the mixed culture. Two specimens of sockeye salmon produced cultures containing type A botulinum toxin, and one produced the rare type F toxin (4). Among the steelhead trout specimens, which were fewer in number, about 2 % yielded toxic culture filtrates. Five out of seven contained type E toxin, whereas the other two were found to contain type B. About 11 % of the sturgeon examined produced toxic filtrates, and all contained type E toxin. These data indicated quite clearly that C. botulinum, usually type E, was present in a significant percentage of all of these fish. We did not make extensive efforts to determine exactly the organs or tissues in which the organism was present. However, individual specimens of liver and muscle from 66 salmon and 9 steelhead trout were tested without obtaining any toxic cultures. It seems probable, in view of the TABLE 1. Comparative incidence of Clostridium botulinum type E in fish taken at different sites aloig the Washing-ton and Oregon Coast Identity of specimens Sockeye salmon... Chinook salmon... Chinook salmon... Coho salmon... Steelhead trout... Sole and cod... Sole and cod... Sturgeon... Source of specimens Pacific Ocean, Westport, Wash. Straight of Juan de Fuca, Port Angeles, Wash. Pacific Ocean, LaPush, Wash. Pacific Ocean, Westport, Wash. Pacific Ocean, Depoe Bay, Ore. Alsea River (Oregon coast) Pacific Ocean, 5 to 8 miles offshore, 5 miles north of mouth of Columbia river Pacific Ocean, 15 to 20 miles west of Coos Bay, Ore. No. of specimens tested Toxigenic specimens' No. of toxic specimens' proven to I containbotulinum No. Per cent toxin ; type E 2; type A I; type F 0 0 2; type E 2; type E I; type E 2; type B 1; type E I; type B ; type E ; type E a Serological typing of all toxic cultures was attempted but was often unsuccessful because toxicity had been lost during storage at -15 C. Most of the cultures successfully typed contained type E toxin.

3 VOL. 16, 1968 C. BOTULINUM TYPE E IN MARINE FISH 555 findings of other investigators with other fish (2, 10), that the intestinal tract was the chief source of the clostridia. As the data for salmon collected from different locations along the coast and from the Columbia River became available, it became apparent that the proportion of fish of a given species harboring toxigenic organisms varied with the location at which the fish were collected. Table 1 shows the percentage of toxigenic specimens of chinook and coho salmon caught in the and in ocean waters at various points along the Washington and Oregon coast. It is evident that, with both species of salmon, the proportion of the fish found to yield toxic cultures was higher among those caught in the than among those caught in ocean waters. The differences between the coho salmon caught in the river and those caught in the ocean were found to be statistically significant. (For example, when the coho caught in the Columbia were compared with those caught in the ocean near Westport, the value of chi square was The probability of this value being exceeded by chance is less than 0.02.) This seems to indicate that the river is a more abundant source of the toxigenic bacteria than is the ocean, and that a large number of these fish probably acquired the organisms only after entering the river. From the results of the serological typing of the toxic cultures (Table 1), it seems probable that the toxin-forming organisms were chiefly C. botulinum type E. The group of 0 steelhead trout studied were taken from the Alsea River, which is another coastal stream flowing into the Pacific. It is interesting that the percentage of these fish found to produce toxic cultures was also relatively high and was similar to the comparable figures for the salmon. In addition to the anadromous fish described above, various species of sole and cod are of importance in the northwest. Through the cooperation of commercial fishermen and packers in Coos Bay and Astoria, Ore., specimens of these fish were made available for examination. The methods employed were the same as those used for salmon. These species are often referred to as "bottom fish" because they are found in ocean waters at relatively great depths. The presence of C. botulinum type E was demonstrated in most of these fish species (Table 2). The proportion of specimens of the various species harboring the organism was quite variable. In some cases, such as the English sole, Red Rock cod, and Black cod, it was relatively high, whereas in others it was similar to the proportion found in oceancaught salmon. For the purposes of this study, it is sufficient to note that the organism was often present in these fish. Table 1 also shows comparative data for sole and cod varieties caught in two different areas along the Oregon coast. One group of 55 fish, caught in the ocean west of Coos Bay, about 200 miles south of the mouth of the Columbia, yielded 6% toxigenic specimens, whereas the second group of 58, caught a few miles offshore and about 5 miles north of the mouth of the river, gave 28 % toxic cultures. This difference was also found to be significant (Chi square value was 9.86; P = less than 0.01). It is possible that the difference may also reflect the influence of the river as a source of the organism. Of 19 toxic cultures from these groups of fish, 18 were successfully typed; TABLE 2. Incidence of Clostridium botulinum type E in sole, cod, and perch from coastal waters of Oregon No. of No. of No. of toxic specimens Identity of specimens Source of specimens specimens specimens proven to contain tested' toxigenic botulinum toxin Dover sole... Coos Bay English sole... Coos Bay and Astoria 19 6 Petroli sole... Coos Bay and Astoria ; type E Rex sole... Coos Bay Ling cod... Coos Bay and Astoria ; type E True cod... Coos Bay and Astoria ; type E Red Rock cod... Astoria and Coos Bay 1 6 6; type E Green cod... Coos Bay Black cod... Astoria 6 2 1; type E 1; type B Grouper... Coos Bay 9 1 Not tested Perch... Coos Bay a All specimens were gills and viscera from individual fish and were tested by the same method used for salmon.

4 556 CRAIG, HAYES, AND PILCHER APPL. MICROBIOL. TABLE. Incidence of Clostrium botulinum type E in shellfish from Oregon coastal waters No. of No. of No. of toxic samples Identity of specimens Source of specimens samplesa samples proven to contain tested toxigenic botulinum toxin Clams, razor... Winchester Bay, Ore.; and an ; type E ocean beach, Seaside, Ore. 1; type B Clams, cockle... Yaquina Bay; and Yachats, ; type E Ore. Clams, softshell... Yaquina Bay, Ore.; Florence, ; type E Ore., Reedsport, Ore. Clams, littleneck... Yaquina Bay, Ore., Florence, 11 4 ; type E Ore.; Nahcotta, Wash. Clams, horseneck... Florence, Ore. 1 1 ; type E Oysters, Pacific... Yaquina Bay, Ore.; Tillamook, 16 6 Ore.; and Bay City, Ore. Crabs, Dungeness Yaquina Bay, Ore.; Winchester ; type E Bay, Ore.; Chinook, Wash. 1; type B a All clams and oysters samples consisted of to 5 shellfish; for littleneck clams, 8 to 10 were used. 16 contained type E toxin, 1 type A, and 1 type B. Crabs, oysters, and several species of clams were included in this study. They were obtained from various sites along the Oregon coast. The methods used for examination were similar to those employed with the fish viscera and have been described in the previous section. C. botulinum type E was found in all species of clams examined, and in two species 66% or more of the specimens produced toxic cultures (Table ). The lowest proportion of toxic specimens was found in the razor clams, many of which were collected from the open beach at Seaside, Oregon. The other species of clams were obtained chiefly from various bays along the coast at the mouths of coastal rivers. The type E toxin was found in most of the toxic specimens examined immunologically. More than % of the oyster specimens tested produced toxic cultures, as did more than 50% of the crabs. Type E toxin was identified in both cases. It seems clear that the organism is very commonly present in clams, oysters, and crabs found along the Oregon coast. Pure cultures of C. botulinum type E were isolated from 18 of the specimens examined in this survey. The sources of these cultures were as follows: 2 were derived from cockle clams, 1 from a softshell clam, 1 from a horseneck clam, 2 from the Pacific oysters, and 1 from a Dungeness crab; 4 from Red Rock cod, 1 from a Black cod, 1 from a coho salmon, from sturgeon, 1 from a ling cod, and 1 from a steelhead trout. One culture of C. botulinum type F was isolated from a sockeye salmon. DISCUSSION In view of the prevalence of C. botulinum type E in salmon, shellfish, and other important fish in the Pacific Northwest, the potential exists for the occurrence of cases of human botulism from consumption of marine food products which have not been processed adequately to destroy the organisms or the toxin. Such food products would presumably consist chiefly of smoked salmon, sturgeon and other fish products, and raw oysters. Thus far, however, there have been no reports of type E botulism in Oregon or Washington. It is possible that the moisture and salt content of these products and the physical conditions under which they are usually held, such as the degree of exposure to air, are not favorable for growth and toxin production by C. botulinum type E. ACKNOWLEDGMENT This research was performed under contract FDA 66-2(Neg) with the Food and Drug Administration, Department of Health, Education, and Welfare. LITERATURE CITED 1. ANONYMOUS Botulism outbreak from smoked whitefish. Food Technol. 18: Borr, T. L., J. S. DEFFNER, E. McCoy, AND E. M. FOSTER Clostridium botulinum type E in fish from the Great Lakes. J. Bacteriol. 91: CARROLL, B. J., E. S. GARRETT, G. B. REESE, AND B. Q. WARD Presence of Clostridium botulinum in the Gulf of Venezuela and the Gulf of Darien. Appl. Microbiol. 14: CRAIG, J. M., AND K. S. PILCHER Clostri-

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