Type E botulism in salmonids and conditions contributing to outbreaks

Aquaculture, 41 (1984) 293-309 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

293

TYPE E BOTULISM IN SALMONIDS AND CONDITIONS CONTRIBUTING TO OUTBREAKS

M.W. EKLUND, F.T. POYSKY, M.E. PETERSON, L.W. PECK’, and W.D. BRUNSONZ Northwest and Alaska Fisheries Center, NMFS, NOAA, Utilization Research Division, 2725 Montlake Boulevard East, Seattle, WA 98112 (U.S.A.)

’ Washington State Department of Fisheries, Olympia, WA (U.S.A.) ’ Washington State Game Department, (Accepted

Olympia, WA (U.S.A.)

11 June 1984)

ABSTRACT Eklund, M.W., Poysky, F.T., Peterson, M.E., Peck, L.W. and Brunson, W.D., 1984. Type E botulism in salmonids and conditions contributing to outbreaks. Aquaculture, 41: 293-309. Type E botulism was first recognized as a major cause of fish mortality in the United States in 1979. Between 1979 and 1983, fish botulism was confirmed in 18 different outbreaks resulting in losses of over 2.3 million juvenile salmon and steelhead trout. These outbreaks occurred in both excavated earth-bottom and asphalt-lined ponds. The source of the toxin was from the growth of Clostridium botulinum type E in the dead fiih that were not removed from the rearing ponds because of pond depth and turbid water. Live fish developed botulism and died after they cannibalized the dead fish and ingested lethal concentrations of toxic flesh. Based upon laboratory and field studies, a botulism cycle is described and the conditions contributing to the length of the cycle and fish mortality are discussed.

INTRODUCTION

Clostridium bo tulinum is a spore-forming anaerobic bacterium that produces neuroparalytic toxins lethal to man and animals. Based upon the production of antigenically specific neurotoxins, this bacterial species is classified into different types designated by the letters A through G. Only the nonproteolytic types B, E, and F have the unique property of growing and producing toxin at temperatures as low as 3.3”C. Of these, type E is the most prevalent in marine and freshwater environments of the Northern Hemisphere. Type E botulism was recognized for the first time in 1979 as a major cause of fish mortality in the United States (Eklund et al., 198213). This outbreak occurred in a l/2-acre earth-bottom pond and caused the loss of over 1 million yearling coho salmon (Oncorhynchus kisutch). Similar outbreaks have been reported in trout in Britain (Cann and Taylor, 1982) 0044-8486/84/$03.00

o 1984 Elsevier Science Publishers B.V.

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and Denmark (Huss and Eskildsen, 1974) where the disease was originally called bankruptcy disease. These outbreaks all occurred in excavated earth ponds. Between 1979 and 1983, type E botulism outbreaks occurred at different fish-rearing ponds both in Washington and Oregon State, resulting in losses of over 2.3 million fish. These outbreaks affected yearling chinook (Oncorhynchus tshazuytscha) and coho salmon and steelhead trout (Salmo gairdneri). In contrast to the outbreaks reported earlier, these outbreaks occurred in both earth-bottom and asphalt-lined ponds. This report discusses these outbreaks and conditions contributing to the growth and toxin production by C. botuhum type E in dead fish that remain on the pond bottoms. It also describes the botulism cycle which starts when the live fish ingest preformed C. botulinum type E toxin as they cannibalize the toxic decomposing carcasses. MATERIALS

Botulinum

AND METHODS

toxin assay

The intestines from sacrificed moribund fish were homogenized in a tissue homogenizer or cut into small pieces, and toxin was extracted at a ratio of 2 ml of buffer to 6 g of intestines. The flesh of moribund fish or the whole carcasses of dead fish collected from the ponds were blended and extracted at a ratio of 1 ml of buffer per 8 g of flesh. After a l-h extraction, the samples were centrifuged and 0.5 ml of a 1 to 2 dilution of either trypsinized or untrypsinized (Duff et al., 1956) supernatant fluids were injected intraperitoneally (I.P.) into Swiss Webster mice weighing 18 to 26 g. Dilutions of the supernatant fluid were assayed to determine the toxin titers. Tubifex worms were homogenized with a tissue homogenizer, extracted with gelatin-phosphate, and supernatant fluids assayed. Water was collected from the outlet of the ponds, dialyzed with polyethylene glycol (Kahn, 1959) in an attempt to concentrate any botulinal toxin, and assayed for toxin. Toxin from pure cultures of C. botulinum type E, isolated from ponds, sediments, and fish, was produced in TPG medium (5% trypticase, 0.5% peptone, and 0.4% glucose) containing 0.1% sodium thioglycollate, and the bacterial cells were removed by centrifugation and filtration. Filtrates were assayed for toxin and titered before and after trypsinization. C. botulinurn type E toxin was identified in the extract using the mouse protection test and monovalent C. botulinurn antiserum (Eklund et al., 1967). Isolation and enumeration

River and pond

of type E

sediment

samples

and contents

of fish intestines

were

295

streaked onto egg yolk agar plates before and after enrichment in TPG medium. Isolated colonies exhibiting irridescence were picked into TPG medium and assayed for botulinal toxin after 3 days at 30°C. Type E populations were enumerated in fish intestines and sediments in TPG medium using the 3-tube most probable number (MPN) procedure. The presence of type E toxin was confirmation of type E growth in the TPG medium. When spores were used in the experiments, they were enumerated by the procedures outlined by Eklund et al. (1982a). Type E toxin production in dead fish at different temperatures Healthy fish were sacrificed and the intestinal tracts were inoculated with either 10 or 100 000 type E spores. The fish were packaged and stored incubation periods. After each at 5”, lo”, 15”, and 20°C for different incubation period, three fish from each spore level were blended with gelatin-phosphate buffer and supernatant fluids were assayed for type E toxin. Cannibalism studies Healthy coho salmon were sacrificed and the intestinal tracts inoculated with 3000 type E spores and placed on the bottom of rectangular polyethylene fish tanks 4 feet (122 cm) in depth. Each tank, equipped with an aerator, was supplied with 8 l/min of dechlorinated city water at a constant temperature (15°C). Simultaneously, 12 healthy live coho salmon that had never been exposed to botulinal toxin were added to the tank. The live fish were observed twice daily for botulism symptoms and the dead fish were observed for signs of cannibalism activity. Type E growth and toxin production were determined in two of the dead fish after 2 and 6 days. Sensitivity of fish to type E toxin Coho salmon (12-15 g) were tested for sensitivity to different concentrations of type E toxin by the I.P. and oral routes. Untrypsinized and trypsinized toxin were injected I.P. with 0.1 ml of different concentrations of filtered toxin from pure cultures of type E. When the toxin was administered orally, 0.1 ml of different concentrations of filtered toxin were added to a number 5 size gelatin capsule and the capsule was force fed to the fish with the aid of a plastic rod. This procedure was superior to feeding the toxin by intubation with a syringe and plastic canula which resulted in the regurgitation of the toxin by the fish when they were returned to the fish tanks. The same toxin used for the fish experiments was also titered using the mouse I.P. assay. The lethal dose for fish was based upon the mouse I.P. lethal dose (MLD). The sensitivity of fish to type E toxin was determined Living Stream tanks at lo, 5”) lo”, 15”) and 20°C in 520-l refrigerated

296

(manufactured by Frigid Units, Inc.). The fish were acclimated from 12’C water temperature to the higher and lower temperatures in increments of 2°C per day. When the desired temperatures were obtained, the fish were held for an additional 4 days prior to testing. Fish were observed for typical botulism symptoms at least twice each day. Accumulative effect of low titers of type E toxin A modified fish pellet was prepared by mixing 15 g of ground OMP (Oregon Moist Pellet) with 50 ml of 2.4% molten agar at 60°C. This mixture was then added to a 20-ml syringe barrel fitted with an end plug and placed in a water bath at 45°C. After the agar had equilibrated to 45”C, the desired concentration of filtered toxin from a pure culture of type E was added and mixed. The plunger and barrel were assembled, removed from the water bath, and the end plug replaced with tygon tubing, 30 cm long and 3 mm inside diameter. The mixture was forced into the tygon tubing and the tubing immediately placed on ice to cool. Within 30 seconds, the agar had solidified and additional molten agar in the syringe was forced into the tubing replacing the solidified agar OMP pellet, which was extracted onto a sterile tray. The extruded material was cut into 5 mm lengths so that the concentration of toxin consumed by the fish could be measured. Representative pellets were assayed for type E toxin using the mouse assay. Coho salmon 12-15 g in weight were held at 15°C under conditions outlined under methodology for cannibalism studies, and each fish was fed two agar pellets (containing a total of 10 or 20 mouse MLD of toxin) daily. The fish were observed for botulism symptoms and death. RESULTS

Botulism outbreaks in salmon and steelhead trout Fish losses from 18 confirmed fish botulism outbreaks that occurred in rearing ponds in Washington and Oregon State between 1979 and 1983 are summarized in Table I. These outbreaks caused losses of over 1.8 million yearling coho and chinook salmon (28 tons) and 550 000 steelhead trout (12 tons). Mortality ranged from 0.8 to 50% in the salmon and from 9.6 to 79.3% in the steelhead trout, with an overall average of 16% of the fish planted in these ponds. Most of the outbreaks in salmon occurred between August and November and stopped when the water temperature decreased in late fall. The outbreaks in rearing ponds El and Ez (Table I), however, occurred later in the year because of the warmer water supplied by a large dam. Botulism outbreaks in salmon occurred in four excavated earth ponds and in five asphaltlined ponds. Several inches of sediment had accumulated in four of the lined ponds, whereas less than l/4 inch (0.6 cm) of sediment was present

297 TABLE I Fish losses from type E botulism in 1979-1983 Rearing* facility

Year

Duration of outbreak

Water temperature (ranee) “C

Total fish population

Total fish losses

Mortality (%I

A A B B C D

1979 1980 1980 1983 1981 1981 1981 1981 1982 1980 1980 1981 1980 1980 1981 1981 1980 1981

Aug.-Nov. Aug.-Sept. Sept.--Ott. Aug.-&t. Aug.-Sept. Aug.-Sept. Sept.-Dee. Sept.-Dee. July-Sept. Sept.-&t. Sept.-Feb. Sept.-Dec. Aug.-Feb. Aug.-Feb. Aug.-Jan. Aug.-Jan. July-Jan. Jan.-Mar.

10.5-14.5 6.0-18.3 10.0-15.6 8.9-20.0 12.8-17.8 15.6-23.3 12.2-15.6 12.2-15.6 13.9-17.2 5.0-11.7 5.0-11.7 7.8-10.6 5.0-10.6 5.0-10.6 -

2 1 8.7 1.5 2 1.5 3.5 5 9.3 2.8 2.9 2.5 7.1 3.0 7.6 3.1 1.9 1.5

1 5.3 2.6 1.9 3.5 3.8 7.1 7.2 2.8 1.1 2.3 1.1 1.0 1.0 7.3 3.8 4.7 2.4

50.0 5.3 29.9 1.3 0.8 2.5 2.0 14.4 30.1 39.3 79.3 52.0 26.7 33.3 9.6 12.3 24.7 16.0

Elb E*b F G, G* G, H, Hz H, HZ I JC

5.5-13.3 8.9-12.8

x x x x x x x x x x x x x x x x x x

lo6 lo6 10s lo6 lo6 lo6 lo6 lo5 lo5 lo5 lo5 lo5 lo4 lo4 lo* lo4 10s 105

x x x x x x x x x x x x x x x x x x

lo6 lo4 lo5 lo4 lo4 lo4 lo4 lo4 10’ lo5 lo5 lo5 lo4 lo4 lo3 10’ lo4 lo4

aFaciiities A through F involved coho salmon and Facility D involved coho and chinook salmon and a few incidental rainbow trout. Facilities G through J involved steelhead trout. Two separate ponds were involved at Facilities E, G, and H. bWater for rearing ponds was from a large dam and temperatures remained higher into late fall. CWater for rearing ponds was cooling water from an electrical plant.

in the fifth pond at the time of the outbreak. Dead fish, however, were found on the bottom of all ponds. When steelhead trout were involved, the fish losses usually started in July and continued into January and February. The outbreak in Facility J was an exception in that it occurred between January and February. Water for this facility was from an electrical plant cooling tower which enabled the manager to maintain higher water temperatures in the ponds during the winter months (8.9” and 12.8%). All of the outbreaks in steelhead occurred in excavated earth ponds.

Signs of botulism Shortly before the development of botulism signs, the fish became hypersensitive and nervous. As the disease progressed, the muscles controlling the pectoral, pelvic, anal, adipose, and dorsal fins were paralyzed by the C. botulinum type E neurotoxin. This began with the pectoral fin, and the

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paralysis rapidly continued toward the tail. The caudal fin (tail), however, remained active which permitted the fish to propel itself, but its inability to control the other fins resulted in a loss of equilibrium and direction. As a result, moribund fish would often swim head-on into the sides of fish tanks or they were unable to swim against the water current of the fish ponds and were often forced onto outlet screens. Some fish escaped the water current and swam toward the shoreline of the ponds where they would lie on their sides. When disturbed, they swam toward deeper water, again losing their equilibrium and occasionally surfacing on their backs. After resting for a brief period of time, the fish would again swim for a short distance and stop. Because of the paralysis of the fin muscles, they were unable to maintain a horizontal balance and would sink to the bottom tail first. This behavior continued and death occurred within hours at water temperatures between 10” and 20°C to weeks at temperatures below 5°C. Many of the fish remained in deeper waters and were not observed. After death, the gill covers of the fish were extended and the bodies often curved. The last lo-15 mm of the intestinal tract contained very viscous ambercolored fecal material suggesting a state of constipation, a condition frequently associated with animal and human botulism. Confirmation of botulism C. botulinum type E toxin was demonstrated in the stomach and intestines and occasionally in the blood and flesh of fish exhibiting botulism symptoms. When the botulinal toxin was extracted from the moribund fish and the supernatant fluids were injected intraperitoneally into mice or other healthy fish, the latter developed signs of botulism and died. Botulism was also produced in fish that were fed toxin extracted from dead fish collected from the bottom of rearing ponds, in which botulism outbreaks were occurring. In addition, toxins from pure cultures of C. botulinurn type E isolated from pond sediments produced the same botulism symptoms in fish by both the oral and I.P. routes. When aliquots of these same toxic supernatant fluids were mixed with C. botulinum type E antitoxin, the toxin was neutralized and the supernatants were no longer toxic to either mice or fish. This specificity of neutralization confirmed the presence of C. botulinum type E toxin in the fish flesh or intestines and type E botulism. In some of the outbreaks, type E toxin was confirmed in the intestinal contents of each moribund fish, and toxin titers ranged from 4 to 200 MLD/ g of intestines. In these same fish, type E toxin was frequently detected in the flesh, blood, liver, and kidney. In other outbreaks, especially when the water temperatures in the rearing ponds were below 10% and the time between ingestion of toxin and botulism symptoms was extended, type E toxin was detected less frequently in the moribund fish. When attempts were made to increase the sensitivity of the assay by using less buffer to extract the toxin from the fish flesh or intestines, the

299

extracts also included higher concentrations of fish proteins and solubles which caused death when introduced I.P. into mice. This effect, however, was not neutralized by botulinal antitoxins. Thus, the effect of the concentrated proteins limited the sensitivity of the

other toxic toxic assay.

Source of type E toxin The prerequisite of any botulism outbreak is that victims must be exposed to the botulinal toxin. This is usually from the ingestion of a food in which C. botulinurn has grown and produced its deadly toxin. In some cases, such as infant botulism in humans, the C. botulinum bacteria can grow and produce toxin in the intestinal tract, but this has seldom resulted in death. Tubifex worms, sediments, water, feed, and dead fish were therefore collected from the rearing ponds in which the outbreaks were occurring to identify the toxin source for fish. Toxin was not detectable in any of the water or feed samples. Low titers of type E toxin (2 MLD/g) were detected in one of the 35 tubifex worm samples collected under a dead fish and in one of the 149 sediment samples containing fragments of decomposed fish. The major source of toxin (4 to 20 000 MLD/g) was from dead fish that accumulated on the pond bottoms. Ingestion of type E toxin during cannibalism In most outbreaks, the water in the ponds was either too deep or too turbid for adequate studies to be made on site. Laboratory studies were therefore started to determine the effects of cannibalism on botulism outbreaks. Healthy fish were sacrificed and inoculated (via the intestinal tract) with 3000 type E spores and placed in a fish tank at 15°C along with 12 live fish. Within 2 days, the type E bacteria grew in the dead fish and produced over 200 MLD of toxin per gram of flesh. At this time, the live fish began to cannibalize the soft belly wall of the dead fish (Fig. 1). This continued until only the skeletons remained (Fig. 2). By the 8th day, seven of the live fish developed signs of botulism and died. The dead fish were left in the fish tank and within several additional days, they became toxic and were cannibalized by the surviving fish. On the 26th day after the start of the experiment, the five remaining live fish developed botulism and died. Similar results were obtained by Huss and Eskildsen (1974) in their studies with trout. In rearing ponds, live fish were occasionally observed cannibalizing carcasses of dead fish, and fish skeletons were also found during fish botulism outbreaks. In several outbreaks, fragments of fish flesh and small fish bones and type E toxin also were found in the stomachs of the moribund fish. Furthermore, when live fish were removed from ponds containing toxic dead fish and placed in shallower ponds, which enabled the workers to continually remove any subsequent dead fish, the outbreaks stopped within a 4-7 day period.

300

Fig. 1. Beginning of cannibalism on toxic dead fish.

Fig. 2. Effect of 7 days of cannibalism on toxic dead fish.

The results from these laboratory experiments and observations at the rearing ponds indicate that the fish must ingest the preformed type E toxin, which in these outbreaks was from dead fish, before a botulism outbreak could be induced. Studies to determine

whether type E would grow in intestines

To determine whether type E could colonize in the fish intestine and produce toxin, sediments containing 24 000 type E organisms per gram were returned to the laboratory and placed in fish tanks with water at 15°C. After 1 month and the daily addition of OMP pellets, the type E population increased to over 100 000/g. There was also a simultaneous increase in the

301

tubifex worm population. Live fish were then added to the tanks and OMP pellets were fed in excess daily. Even though the fish were seen picking at the sediments for food, no ill effects were observed during the Z-month study. Similar experiments were made by placing 50 fish in wire holding pens placed on the bottom of rearing ponds in which a botulism outbreak was occurring. The sediments in the bottom of the pens exceeded 150 000 type E organisms per gram and also contained tubifex worms. No ill effects were observ_edin these fish during the 5-week study. In a second set of experiments, healthy fish were fed OMP pellets containing sulfamethazine for 3 days in order to free the intestines of competing bacterial flora and then fed modified OMP pellets containing 500 000 type E spores each day for 5 days. In addition, the same concentration of type E spores was fed to fish that had not been previously exposed to any antibiotics. Several of the fish were sacrificed during the experiment, but type E toxin was not demonstrated in the fish flesh or intestines. All of the remaining fish continued to be healthy during the 4-week study. Based upon these experiments, it is unlikely that type E could grow and produce toxin in the intestines of healthy fish. It is possible, however, that under certain conditions following the ingestion of preformed toxin and the onset of constipation, type E bacteria could grow and produce toxin in the intestines. At this stage of botulism, however, the major damage from the ingested preformed toxin (from dead fish) would have already occurred and any additional effect from subsequent toxin production by type E cells in the intestines would be negligible. On the other hand, if sublethal concentrations of toxin were ingested, then the subsequent growth and toxin production of type E cells could be more important. Even though the possibility exists that under specific conditions type E could grow in the intestines of moribund fish, we believe that this would have played a minor role in the outbreaks reported in this paper. Conditions that contribute to botulism outbreaks and fish mortality C. botulinurn type E populations from sediments and fish. The water supply for each of the rearing ponds discussed in this paper was from rivers. Sediments collected from several of these rivers above the intake for these rearing ponds were tested and found to be contaminated with C. botulinurn type E. These water sources therefore furnish the rearing ponds with a low but continuous source of type E spores. Sediments and moribund fish were also collected from the different ponds in which botulism outbreaks were occurring, and populations of C. botulinurn type E were enumerated by the MPN procedure. Type E was demonstrated in all of the 210 sediments collected from the different fish rearing ponds and the type E populations were as low as 23 and as high as 240 000 per gram. Type E was concentrated in sediments near the out-

302

let of some ponds, whereas in other ponds the type E populations varied according to the water currents. The intestines of all fish with botulism symptoms contained type E bacteria ranging from 75 to 110 000 per gram of intestines. The main source of these organisms was from sediments and the ingestion of dead fish flesh in which type E had grown and produced toxin. Location of type E organisms in sediments. During one of the larger botulism outbreaks, the surviving fish were removed and the excavated earth pond was drained slowly. Sediments were collected from different areas within a radius of 10 m from the pond outlet and type E populations were determined at the different depths. The largest population of type E, ranging from 28 000 to 150 000 per gram, was concentrated in the top 1.5 cm of the sediments. The second (3.5 cm) and third (10 cm) layers both contained between 4600 and 46 000 type E organisms per gram. Large populations of tubifex worms were located in the second layer whereas the third layer contained mainly gravel. The top 1.5 cm of sediment contained waste feed, fecal material, and dead fish, which supported the growth and accumulation of type E spores and vegetative cells. In addition, the tubifex worms continually moved between the first and second layers of the sediments, undoubtedly transporting type E organisms. The worms and other bacteria also utilize the oxygen, thus contributing to the anaerobic conditions needed for type E growth. The concentration of type E bacteria in the top 1.5 cm of the sediments therefore increases the changes that both live and dead fish will be contaminated. Nonsymptomatic fish were collected from this pond prior to drainage and from other ponds where type E occurred in large numbers. All of the 76 healthy fish collected were shown to be contaminated with the type E organisms. Since type E must grow and produce toxin in order to induce botulism, this contamination was of no immediate danger to live fish. If any of the fish die, however, type E could grow and produce toxin in the decomposing carcasses, initiating a botulism outbreak. Effect of spore concentration, time, and temperature on toxin production in dead fish. In laboratory studies, toxin was produced after 24 h of incubation at 20% in dead fish that received an inoculum of 100 000 spores (Fig. 3). When the inoculum was reduced to 10 spores, toxin was not detected until after 48 h of incubation and the toxin titers remained consistently lower throughout the incubation period. Even though the spores were inoculated into the intestinal tract, the toxin was evenly distributed throughout the flesh after 48 h of incubation at 20°C. As the incubation temperature decreased from 20°C to lO”C, longer periods of time were required for toxin production. The lo-spore inoculum required twice as long to produce toxin between 10” and 20°C as did the 100 OOO-spore inoculum. At 10°C toxin titers from the lo-spore inoculum

303

n

n

10 spores IO5 spores

A

Nontoxic with 10 spores

B

Nontoxic with both 10 and IO5 spores

Incubation

temperature

IO0 c

1246

2 4

5”

10

8

c

16243236

1

Incubation time (days) Fig. 3. Effect of spore inoculum, incubation time, and temperature on toxin production by C. bo tulinum type E in dead fish.

never exceeded 200 MLD/g. In comparison, 100 000 MLD of toxin was produced with the higher spore inoculum. When the temperature was lowered to 5”C, toxin was not detected during a 40-day incubation period with the lo-spore inoculum. Titers of 200 MLD/g, however, were produced at 5°C after 40 days by the 100 OOO-spore inoculum. Based upon the softness of the dead fish after 40 days at 5°C and the probability that the carcasses would be cannibalized earlier by the living fish, it is unlikely that the carcasses would remain intact in a fish pond for sufficient time at 5°C for toxin production. The concentration of type E spores and time and temperature of incubation therefore govern the growth rate and amount of toxin produced in dead fish, which in turn have a marked effect on the fish mortality in rearing ponds. Sensitivity of coho salmon to type E toxin at different temperatures. The lethal oral dose of toxin for fish increased as the water temperature decreased. When the toxin from a pure culture of type E was force fed in

304 TABLE II Sensitivity of coho salmon to C. botulinum

type E toxin

Water temperature (“C)

Mouse lethal dosesa Oral route

Mouse lethal dose@ I.P. route

20 15 10 5 1

90 90 180 180 450

0.5 0.5 0.5 0.5 0.5

aMouse intraperitoneal lethal doses per 12-15

at different temperatures

g fish, based upon trypsinized toxin titer.

TABLE III Effect of water temperature on botulism signs in coho salmon Temperature (“Cl

20 15 10 5 1

Oral route

I.P. route

Hours prior to botulism symptomsa

Hours prior to botulism symptomsb

< 19 < 19 26-44 96-142 176-432

24-72 44-126 90-256 200-344 278-344

aFish force fed 450 MLD of toxin in a gelatin capsule; hours between toxin ingestion and symptoms. bFish injected intraperitoneally with 0.5 MLD of toxin; hours between injection and symptoms.

gelatin capsules to fish, only 90 MLD (based upon trypsinized mouse I.P. lethal dose) was lethal to fish held at 20°C. If the fish were held in water at 1°C the lethal dose was increased to 450 MLD of toxin (Table II). Similar results were observed when steelhead trout were used. Lower water temperature not only increased the lethal dose of toxin, but it also increased the time between ingestion of toxin and the onset of botulism (Table III). Fish fed 450 MLD of toxin developed signs of botulism within 19 h at temperatures above 15°C and death occurred within 29 h. This time frame increased as the water temperature decreased and at 1°C botulism was delayed until 176-432 h and death occurred after an additional 456 h (19 days). The minimum intraperitoneal lethal dose of toxin from pure cultures of type E for fish was one-half of the minimum lethal dose for mice when the fish were in the temperature range of 1°C to 20°C (Table II). This was true for both trypsinized and untrypsinized toxin. When the minimum

305

lethal dose of toxin was administered intraperitoneally, symptoms within 24-72 h at 20°C. This time period 5°C reduction in temperature.

fish developed doubled for each

Accumulutiue

effect of type E toxin. In two of the coho salmon botulism outbreaks in ponds with water temperatures of 12°C to 15°C the dead fish collected from the pond bottoms contained only 20 MLD of type E toxin per gram and only a small percentage of the moribund fish contained detectable levels of toxin in their intestines. Based upon a minimum lethal oral dose of 90 MLD of toxin at 15°C and 180 MLD at 10°C from a single exposure of toxin, a 20-g live fish would be required to ingest at least 5 to 9 g of dead fish flesh containing 20 MLD of toxin per gram in one day to initiate botulism. Since this markedly exceeds the daily capacity of a yearling salmon, they therefore must have cannibalized the dead fish over an extended period of time before sufficient toxin was ingested to initiate botulism. To determine the cumulative effect of toxin ingestion, consecutive daily oral doses of low titer toxin were fed to fish held at 15°C (Table IV). Fish fed only 10 or 20 MLD of toxin daily developed botulism symptoms and died during a lo-25-day period. Because of the long period of time between toxin ingestion and symptoms, type E toxin was not detectable in any of the intestines of these fish that were analyzed within several hours after death. TABLE IV Effect

of multiple daily feedings of low titer type E toxin on fiih botulism at 15°C

MLD of toxin fed per daya

Number of days fish fed type E toxin 10

15

21

22

23

25

Number of fish dying from botulismb 10 20

1-212ll---3

aDose in mouse lethal doses based upon trypsinized toxin produced by pure cultures of type E. bSix fish used in 10 MLD experiment, Five fish used in 20 MLD experiment.

DISCUSSION

Based upon earlier records from the salmonid rearing facilities in Washington and Oregon State, in which 18 recent botulism outbreaks occurred, and discussions with managers and fish pathologists, botulism has undoubtedly plagued the salmonid industry for at least 25 years, resulting in estimated

306

losses of 10 to 20 million fish. Records from one facility alone indicate that over 2 million fish died from a mysterious disease between 1960 and 1979. Similar sporadic unidentified outbreaks also occurred at other rearing ponds during this same period of time. According to fish pathologists studying these earlier losses, the moribund fish displayed symptoms exhibited by fish in recent botulism outbreaks. Based upon laboratory and field studies and observations at the 18 different botulism outbreaks, the contributing conditions are defined and summarized in Fig. 4. Epidemiological studies of botulism outbreaks in humans, birds, etc., show that the victims of any botulism outbreak must have a source of botulinal toxin. In the fish botulism outbreaks reported in this study, the fish ingested C. botulinum type E toxin during cannibalization of toxic decomposing dead fish that accumulated on the pond bottoms. When the fish were separated from the toxin source (dead fish), the outbreaks were stopped. Similar results were obtained by Huss and Eskildsen (1974), Eklund et al. (1982b), and Cann and Taylor (1982) during earlier studies of fish botulism outbreaks in Denmark, United States, and United Kingdom.

Dead fish remain on pond bottom

Live fish get botulism 1 - 25 days later and die

Type E grows and produces toxin in dead fish

Live fish consume type E cells and toxin by cannibalizing decomposing fish

Fig. 4. Botulism cycle in fish-rearing ponds.

These studies show that C. botulinum type E is ubiquitous in most rivers and streams which serve as water supplies for rearing ponds. In addition, the type E organisms can be introduced into ponds by fish transported from a contaminated area or from feed, birds, etc. Waste feed, fecal material,

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and carcasses of dead fish accumulating in the ponds serve as excellent growth media for the type E organisms at temperatures above 3°C. During the growth cycle of type E bacteria, numerous spores are produced which can remain dormant in the sediments for many years. The majority of live fish reared in these environments become contaminated with either type E vegetative cells or spores as they come in contact with the pond sediments. If these fish die from any cause and the carcasses accumulate in the sediments at water temperatures above 5”C, a botulism outbreak can occur. In most of the outbreaks investigated in this study, fish dying from furunculosis or other fish diseases or even the death of a few fish during their transfer from one rearing area to another have been sufficient to start the botulism cycle. Because of the water depth and turbidity, many of these fish often remain in the sediments where they become contaminated with additional type E spores. At elevated temperatures of 15” to 2O”C, a few type E spores can germinate in the dead fish, grow, and produce millions of additional type E cells and high titers of toxin within several days. Shortly after toxin production and the softening of the carcass flesh, the live fish begin to cannibalize the dead fish, consuming lethal concentrations of toxin and also large populations of type E bacteria. When these fish die, their intestines and flesh can contain over 200 MLD of toxin and over 100 000 type E bacteria per gram. This large population of type E bacteria grows rapidly and produces larger amounts of toxin in the new carcasses. If these carcasses contain 20 000 MLD of type E toxin/g, one dead fish could theoretically cause botulism in over 100 live fish. Initially, only a few fish are involved in an outbreak, but losses increase rapidly as the type E population and toxin titers increase in the dead fish that accumulate in the ponds. These conditions account for the doubling in the daily losses which occurred in many of the outbreaks. For example, in one outbreak, furunculosis caused losses of several thousand fish out of a total population of 2 million fish. Some of these dead fish remained in the deep turbid water of the ponds, and within one week botulism symptoms were observed in the live fish and daily losses continued at a doubling rate until they exceeded 70 000 fish per day. In another outbreak, a few fish died shortly after being transferred from a shallow concrete-lined pond into an excavated earth pond. These dead fish remained in deep water and C. botulinurn type E grew and produced toxin in the decomposing flesh. Within several days, the water temperatures increased to 23°C and live fish began displaying botulism symptoms. After an additional 7 days, 38 000 fish had died from botulism. When the fish were transferred to the shallower concrete-lined ponds and the dead fish were removed daily, the outbreak stopped within 5 days. In general, the daily losses during a salmon botulism outbreak exceeded those in steelhead outbreaks. This in part is attributed to the higher density of salmon in the ponds and also the warmer temperatures encountered during the outbreaks.

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Botulism outbreaks usually occurred during the summer and fall when the water temperatures were higher and decreased when the water temperatures dropped below 10°C and stopped at temperatures below 5°C. As the water temperature decreased, the growth rate of type E bacteria also decreased (growth stops at 3°C) and less toxin was produced in the dead carcasses. In addition, more toxin was required to initiate botulism and the time between ingestion of the toxin and onset of symptoms was greatly extended. As a result, the botulism cycle was slower and fewer fish were involved. At higher water temperatures (above 15”C), the dead fish often contained many lethal doses of type E toxin per gram of flesh. When the live fish ingested the toxic dead fish, they generally developed botulism symptoms within a 24-48-hour period and botulism was readily confirmed following the demonstration of type E toxin in the moribund fish intestines and flesh. In two salmon botulism outbreaks which occurred at temperatures below 15”C, the flesh of the dead fish collected from the pond bottoms frequently contained insufficient toxin to initiate botulism in live fish from a single day’s ingestion of the toxic dead flesh. Multiple ingestions of the toxic dead flesh were therefore required in order to obtain lethal doses of toxin. This was confirmed in laboratory studies which showed that lethal doses of toxin could be acquired by a fish when it ingested as little as 10 mouse intraperitoneal lethal doses daily during a lo-23-day period. Because of this increased time period between ingestion of the toxin and the onset of botulism symptoms, it is more difficult to demonstrate type E toxin in the moribund fish. In these cases, when botulism is suspected, strong presumptive evidence can be obtained by demonstrating the toxin in dead fish from the pond bottoms and by observing moribund fish for signs of botulism. Numerous moribund fish may have to be examined for confirmation. We have observed that dead fish with fungus growth on their surfaces generally have remained in the pond for sufficient periods of time for type E to grow and produce toxin in the flesh. Even under these conditions, however, all dead fish will not contain detectable levels of type E toxin. If one attempts to increase the sensitivity of the botulinal assay by extracting the toxin from a number of fish intestines or flesh with smaller volumes of saline or gelatin-phosphate, soluble fish proteins are also concentrated which produce botulism-like symptoms and death between 2 and 24 h when introduced I.P. into mice. This lethal effect from high concentrations of fish proteins, however, is not neutralized by C. botulinurn type E or other botulinal antitoxins. One is therefore limited to the sensitivity of the mouse assay in confirming low levels of botulinal toxin in moribund fish. These studies also show that salmon and steelhead are equally sensitive to type E toxin by either the oral or I.P. route. When the toxin is administered by the I.P. route, fish are twice as sensitive as the mouse. Based upon the sensitivity of fish to C. botulinum toxin by the I.P. route, it has been suggested by Skulberg and Grande (1967) and Crisley (1960) that fish may

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be an alternative to mice in the botulinal toxin assay. The main disadvantage of using fish is the longer period of time required to obtain results. During botulism outbreaks, extreme precaution should be taken to prevent the spread of the disease to other animals or to humans. If marketable size fish are involved, the fish should not be marketed during the outbreak; otherwise, there is a possibility of introducing food-borne botulism in humans. Further precautions to be followed during an outbreak have been outlined by Eklund et al. (1982b). The botulism outbreaks described in this paper and by Cann and Taylor (1982) and Huss and Eskildsen (1974) were caused by C. botuIinum type E. Nonproteolytic strains of C. botulinurntypes B and F also share some of the same characteristics as type E (Eklund et al., 1967), especially the ability to grow at temperatures as low as 38°F (3.3’(Z), and under certain conditions they could also be involved in fish botulism outbreaks. ACKNOWLEDGEMENTS

We gratefully acknowledge the personnel and managers of the Washington State Department of Fisheries, Washington State Game Department, Oregon Department of Fish and Wildlife Hatcheries, and Kevin Amos, James W. Wood, Richard L. Westgard, Lew Atkins, Chuck Johnson, Richard Holt, and J.F. Conrad for their very cooperative efforts in these studies. We also acknowledge Lamia Mseitif and Gretchen Pelroy for their assistance during parts of these studies.

REFERENCES Cann, D.C. and Taylor, L.Y., 1982. An outbreak of botulism in rainbow trout, Salmo gairdneri Richardson, farmed in Britain. J. Fish. Dis., 5 : 393-399. Crisley, F.D., 1960. Routine method of goldfish assay of the toxin in crude culture centrifugates of Clostridium botulinum type A. Appl. Microbial., 8: 282-286. Duff, J.T., Wright, G.G. and Yarinsky, A., 1966. Activation of Clostridium botulinum type E toxin by trypsin. J. Bacterial., 12: 455-460. Eklund, M.W., Poysky, F.T. and Wieler, D.I., 1967. Characteristics of Clostridium botuhum type F isolated from Pacific Coast of the United States. Appl. Microbial., 15: 1316-1323. Eklund, M.W., Pelroy, G-A., Paranjpye, R., Peterson, M.E. and Teeny, F-M., 1982a. Inhibition of Clostridium botulinum types A and E toxin production by liquid smoke and NaCl in hot-process smoke-flavored fish. J. Food Prot., 45: 935-941. Eklund, M.W., Peterson, M.E., Poysky, F.T., Peck, L.W. and Conrad, J.F., 1982b. Botulism in juvenile coho salmon (Oncorhynchus kisutch) in the United States. Aquaculture, 27: l-11. Huss, H.H. and Eskildsen, U., 1974. Botulism in farmed trout caused by Clostridium botulinurn type E. Vet. Med., 26: 733-738. Kahn, J., 1959. A simple method for concentration of fluids containing proteins. Nature (London), 1813: 1055. Skulberg, A. and Grande, M., 1967. Susceptibility of rainbow trout (Salmo gairdneri Richardson) to Clostridium botulinum toxin. Trans. Am. Fish. Sot., 96: 67-70.