susceptibility of coho salmon, oncorhynchus kisutch (walbaum), to different toxins of clostridium...

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Susceptibility of coho salmon, Oncorhynchus kisutch (Walbaum), to different toxins of Clostridium botulinum Melvin W Eklund , Frank T Poysky, Rohinee N Paranjpye, Mark E Peterson & Gretchen A Pelroy US Department of Commerce, NOAA, National Marine Fisheries Service, Northwest Fisheries Science Center, Resource Enhancement and Utilization Technologies Division, Seattle,WA, USA Correspondence: R N Paranjpye, 2725 Montlake Blvd E, Seattle,WA 98112, USA. E-mail: [email protected] Present address: M W Eklund, Mel Eklund & Associates,1872735th Avenue NE, Seattle,WA 98155, USA. Abstract Coho salmon Oncorhynchus kisutch (Walbaum), held at 15 1C were tested for their susceptibility to toxins of proteolytic and nonproteolytic Clostridium botulinum types A, B, C 1 ,C 2 , D, E, F, and Gadministered by the oral and intraperitoneal (i.p.) routes. By the oral route, the ¢sh were most susceptibile to type E neurotoxin, which was lethal at a dose equivalent to 90 mouse in- traperitoneal minimum lethal doses (MLDs). The oral lethal dose increased to 2000 MLD for nonproteolytic and proteolytic type F neurotoxins, but the toxin types A, B, and C 1 were not lethal to ¢sh at 2000MLD and type D was not lethal at 20000 MLD (highest titre tested). The ¢sh were not susceptible to 200 MLD (the highest titres tested) of type G neurotoxin or C 2 cyto- toxin. By the i.p. route, all of the toxins except type G were lethal to coho salmon. Type E neurotoxin was the most toxic at a level of one-half the mouse MLD. Coho salmon held at temperatures ranging from 1 to 20 1C were sensitive to type E neurotoxin by both the oral and i.p. routes. As the temperature decreased the ¢sh became more resistant to type E neurotoxin by the oral route, but the i.p. dose remained one-half the mouse MLD at all temperatures. Keywords: Clostridium botulinum, toxins, salmon Introduction Clostridium botulinum is a spore-forming bacterium that produces paralytic neurotoxins that are the most potent toxins known. This bacterial species in- cludes a very heterogenous group of strains that are divided into seven di¡erent toxin types (A, B, C 1 , D, E, F, and G) on the basis of the antigenic speci¢city of the neurotoxins that are produced (Smith & Sugiya- ma1988). In addition, most strains of type C and D produce a lethal C2 toxin that does not cause neuro- paralytic activity. Even though the neurotoxins have similar pharma- cological action, the bacteria producing these neuro- toxins di¡er markedly in their biochemical and physiological characteristics. On the basis of these characteristics, the species can be further divided into four groups. Group 1 includes types A, B, and F and has a minimum growth temperature of10 1C (Tanner & Oglesby 1936). Group II includes nonproteolytic types B, E, and F, and can growat temperatures as low as 3.0 1C (Schmidt, Lechowich & Folinazzo 1961; Eklund, Poysky & Wieler 1967a, b). Because of their nonproteolytic characteristic, their toxin precursors require activation with trypsin or other proteolytic enzymes to achieve full toxicity. Strains belonging to group III produce either neurotoxin types C 1 or D. Members of this group grow poorly at 15 1C and not at all at 10 1C (Smith & Sugiyama 1988). The neurot- oxins of these types are mediated by speci¢c bacterio- phage (Eklund & Poysky 1974). When these strains lose these bacteriophages, they cease to produce neu- rotoxins, but most of the strains will continue to pro- duce a C 2 toxin, which requires trypsin treatment to obtain full toxicity (Eklund & Poysky 1972). Types C and D have usually caused botulism in animals and birds. Strains belonging to group IV produce only type G neurotoxin, but the characteristics of these strains di¡er markedly from the strains of the other three groups and closely resemble C. subterminale , an or- ganism frequently found in soil. Type G was ¢rst iso- lated from vineyards in Argentina and to date has not Aquaculture Research, 2004, 35, 594^600 594 r 2004 Blackwell Publishing Ltd

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Susceptibility of coho salmon, Oncorhynchus kisutch

(Walbaum), to different toxins of Clostridium botulinum

MelvinW Eklund�, FrankT Poysky, Rohinee N Paranjpye, Mark E Peterson & Gretchen A PelroyUS Department of Commerce, NOAA, National Marine Fisheries Service, Northwest Fisheries Science Center, Resource

Enhancement and UtilizationTechnologies Division, Seattle,WA, USA

Correspondence: R N Paranjpye, 2725 Montlake Blvd E, Seattle,WA 98112, USA. E-mail: [email protected]�Present address:MW Eklund, Mel Eklund & Associates,1872735th Avenue NE, Seattle,WA 98155, USA.

Abstract

Coho salmonOncorhynchuskisutch (Walbaum), heldat15 1C were tested for their susceptibility to toxins ofproteolytic and nonproteolytic Clostridium botulinumtypes A, B, C1, C2, D, E, F, and G administered by theoral and intraperitoneal (i.p.) routes. By the oral route,the ¢sh were most susceptibile to type E neurotoxin,whichwas lethal at a dose equivalent to 90 mouse in-traperitoneal minimum lethal doses (MLDs). The orallethal dose increased to 2000MLD for nonproteolyticand proteolytic type Fneurotoxins, but the toxin typesA, B, and C1were not lethal to ¢sh at 2000MLD andtype D was not lethal at 20000MLD (highest titretested). The ¢sh were not susceptible to 200MLD (thehighest titres tested) of type G neurotoxin or C2 cyto-toxin. By the i.p. route, all of the toxins except type Gwere lethal to coho salmon. Type E neurotoxin wasthe most toxic at a level of one-half the mouse MLD.Coho salmon held at temperatures ranging from 1to 20 1C were sensitive to type E neurotoxin by boththe oral and i.p. routes. As the temperature decreasedthe ¢sh becamemore resistant to type E neurotoxin bythe oral route, but the i.p. dose remained one-half themouse MLD at all temperatures.

Keywords: Clostridium botulinum, toxins, salmon

Introduction

Clostridium botulinum is a spore-forming bacteriumthat produces paralytic neurotoxins that are themost potent toxins known. This bacterial species in-cludes a very heterogenous group of strains that aredivided into seven di¡erent toxin types (A, B, C1, D, E,

F, and G) on the basis of the antigenic speci¢city ofthe neurotoxins that are produced (Smith & Sugiya-ma 1988). In addition, most strains of type C and Dproduce a lethal C2 toxin that does not cause neuro-paralytic activity.Even though the neurotoxins have similar pharma-

cological action, the bacteria producing these neuro-toxins di¡er markedly in their biochemical andphysiological characteristics. On the basis of thesecharacteristics, the species can be further divided intofour groups. Group 1 includes types A, B, and F andhas aminimumgrowth temperature of10 1C (Tanner& Oglesby 1936). Group II includes nonproteolytictypes B, E, and F, and can grow at temperatures aslow as 3.0 1C (Schmidt, Lechowich & Folinazzo 1961;Eklund, Poysky & Wieler 1967a, b). Because of theirnonproteolytic characteristic, their toxin precursorsrequire activation with trypsin or other proteolyticenzymes to achieve full toxicity. Strains belonging togroup III produce either neurotoxin types C1 or D.Members of this group grow poorly at 15 1C and notat all at 10 1C (Smith & Sugiyama 1988). The neurot-oxins of these types are mediated by speci¢c bacterio-phage (Eklund & Poysky 1974). When these strainslose these bacteriophages, they cease to produce neu-rotoxins, but most of the strains will continue to pro-duce a C2 toxin, which requires trypsin treatment toobtain full toxicity (Eklund & Poysky 1972). Types Cand D have usually caused botulism in animals andbirds. Strains belonging to group IVproduce only typeG neurotoxin, but the characteristics of these strainsdi¡er markedly from the strains of the other threegroups and closely resemble C. subterminale, an or-ganism frequently found in soil. Type G was ¢rst iso-lated fromvineyards in Argentina and to date has not

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been implicated in any botulism outbreaks. It is pro-teolytic, produces low titres of neurotoxin and willnot growat10 1C (Smith & Sugiyama1988).The characteristics of members of the di¡erent

groups are very important in the survival, growthand toxin production by the organisms under di¡er-ent conditions and their ability to cause botulism. Allof the neurotoxins produced by the di¡erent C. botu-linum types are lethal, but the sensitivity of man,animals, birds and ¢shvaries markedly with the neu-rotoxin type.The distributionof these toxin typesvar-ies also with the geographical area, but type E isusually the most prevalent in marine and freshwaterenvironments of the Northern Hemisphere (Smith &Sugiyama 1988). Food-borne, wound and infant (of-ten referred as toxicoinfection) botulism are thethree clinical forms that are currently recognized.Food-borne botulism is caused by the ingestion ofpreformed toxin during the growth of C. botulinumin a food or feed. Botulism from wound or toxico-infectionare caused by the growth and toxin produc-tion by C. botulinum in damaged tissue or in theintestines of the victim respectively.Of these clinical forms, the ingestion of preformed

neurotoxin, or food-borne botulism, has been respon-sible for ¢sh botulism outbreaks. Type E botulism hasbeen recognized as a major cause of ¢sh mortality inDanish (Huss & Eskildsen 1974) and British troutfarms (Cann & Taylor1982), and in salmon and steel-head trout in hatchery rearing ponds in the UnitedStates (Eklund, Peterson, Poysky, Peck & Conrad1982). In these outbreaks, the source of the botulinalneurotoxin was from cannibalism of dead ¢sh thathad accumulated on the bottom of rearing ponds. Asecond source of the neurotoxin in Danish troutfarmswas fromgrowthand toxin production byC. bo-tulinum type E in minced marine trash ¢sh stored atnonrefrigerated temperatures before it reached thefarms (Huss & Eskildsen1974). In one of the botulismoutbreaks from the minced ¢sh, the intestines of oneof the ¢sh contained both type B and E neurotoxinsand the feed also contained 50MLD (minimum lethaldose) of type B neurotoxin per gram. The role thattype B toxin played in the death of the trout was notdetermined. In recent years, botulinal toxin type Cwas demonstrated in the laboratory to cause botulismin tilapia, Oreochromis mossambicus (Peters) when ad-ministered both orally and via i.p. inoculation (Dr T.Rocke, pers. comm.). In another study, C. botulinumtypesA, B, C, D, and Ewere shownto be toxic to tilapiawhen injected intraperitoneally, with the ¢sh beingmost sensitive to type E (Lalitha & Gopakumar 2001).

Even though type E has been shown as the majorcause of botulism in salmon, the potential of botu-lism outbreaks from the other types of C. botulinumhas not been studied. This report discusses the sensi-tivity of coho salmon Oncorhynchus kisutch (Wal-baum) to the di¡erent toxins of C. botulinum types Athrough G via the intraperitoneal and oral routes. Italso discusses the sensitivity of coho salmon to type Eneurotoxin in ¢sh held at water temperaturesranging from1to 20 1C, the sources of toxins in out-breaks, and procedures used in the detection of neu-rotoxin in ¢shwith signs of botulism.

Materials and methods

Cultures

The C. botulinum strains used to prepare the di¡erenttoxins and their sources were as follows: Type Astrain B1G4, nonproteolytic type B strain 2B, non-proteolytic type F strain 70F, type E strain EF4, andtype C strain 468C-AO28 cured of bacteriophageand producing only C2 toxin (Eklund, NorthwestFisheries Science Center, Seattle,WA, USA); proteoly-tic type B strain 7169 (W. P. Segner, Continental Can,Chicago, IL, USA); type C stain162 producing C1 neu-rotoxin (A. Baillie, Unilever Research Laboratories,Bedford, UK); type D strain 1873 (H Iida, HokkaidoInstitute of Public Health, Sopporo, Japan); proteoly-tic type F strain Langeland (L.V. Holdeman,VI Poly-technic Institute, Blacksburg, VA, USA); and type Gstrain 89G (D. A. Kautter, Food and DrugAdministra-tion,Washington, DC, USA).

Toxin production and assay

Pure cultures of C. botulinum were grown anaer-obically inTYG broth (Eklund & Poysky1974) at 30 1Cfor 4 days. After incubation, the bacterial cells were re-moved by centrifugation and ¢ltration of culturesthrough 0.45-mm ¢lters (Millipore, Bedford, MA, USA).Culture ¢ltrates were assayed before and after treat-ment with a ¢nal concentration of 0.25% trypsin (Difco1:250, Detroit, MI, USA) following procedures of Du¡,Wright & Yarinsky (1956) and Eklund & Poysky (1972).Neurotoxins from nonproteolytic types B, E, and Fandthe C2 toxin from type C strain 468C-AO28 were theonly toxins treated with trypsin. In some experimentswith type E, both trypsinized anduntrypsinized neuro-toxins were used in the experiments. Higher titres of C2toxin were obtained by culturing type C strain 468C-

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AO28 in intussuscepted ‘cellophane tubes’ followingprocedures of Eklund & Poysky (1972).Toxin titres weretested using the mouse bioassay after diluting ¢lteredculture supernatants ingelatin^phosphate bu¡er.C. bo-tulinum toxins were con¢rmed with monovalent anti-serum (obtained from Centers for Disease Control andPrevention, Atlanta, GA, USA) and C2 antiserum pro-duced at our laboratory (Eklund & Poysky1972).The ¢l-tered toxins were held at 1 1C and titered using themouse bioassay, the same day that the ¢shwere tested.

Fish

Coho salmonwere obtained fromone of theWashing-ton State Department of Fish andWildlife hatcheries.Fish were held in 3 ft � 1ft � 4 ft deep polyethylenetanks equipped with aerators and supplied with8 Lmin�1 of dechlorinated city water.When the ¢shwere tested for sensitivity to type E strain EF4, neu-rotoxin at 20, 15, 10, 5, and 1 1C, they were held in520-l refrigerated tanks (Living Stream, Frigid Units,Toledo, OH, USA).The ¢shwere acclimated from10 1Cto the higher or lower temperatures in incrementsof 2 1C day�1. After the desired temperature wasreached, the ¢shwere held an additional 4 days priorto testing.

Susceptibility of ¢sh to C. botulinum toxins

Coho salmon (weighing12^15 g) were tested for sus-ceptibility to the di¡erent dilutions of toxins of C. bo-tulinum by the intraperitoneal (i.p.) and oral routes at15 1C. Untrypsinized and trypsinized toxins wereinjected i.p. with 0.1mL of di¡erent dilutions and con-centrations of toxin. When the toxin was adminis-tered orally, 0.1mL of di¡erent concentrations oftoxin was added to a number 5-size gelatin capsuleand force fed to the ¢sh. This procedure was superiorto feeding by intubationwith a syringe and plastic ca-nula, which often resulted in the regurgitation of thetoxin. If a capsule was regurgitated, it could immedi-ately be observed £oating in thewater.The lethal dosefor ¢shwas based upon the mouse i.p. lethal dose.

Results

Mortality of coho salmon exposed toC. botulinum toxins by the oral route

The oral lethal doses of di¡erent types of C. botulinumtoxins for juvenile coho salmon held at 15 1C are

summarized in Table 1. Of the di¡erent toxin types(A^G), type E was the most toxic to the coho salmonat a lethal dose as lowas 90MLD.When theneurotox-ins of both nonproteolytic and proteolytic type Fwere tested, the lethal oral dose increased to2000MLD. For C. botulinum proteolytic types A andB, nonproteolytic B, and C1,2000MLD of neurotoxin,the highest titre used in these experiments, werenontoxic to the ¢sh. The culture of type D producedthe highest neurotoxin titre, and 20000MLD wasnontoxic. Type G strains do not produce very high ti-tres of neurotoxin, and 200MLD, the highest titretested, had no e¡ect on the ¢sh. The C2 toxin is pro-duced at low titres by many strains of types C and D.Coho salmon were not sensitive to C2 toxin at a levelof 200MLD.

Mortality of coho salmon exposed toC. botulinum toxins by i.p. route

The i.p. lethal doses of toxins from pure cultures of C.botulinum types A through G are also summarized inTable1.When inoculated by the i.p. route, type E neu-rotoxin was also the most lethal to coho salmon. Thelethal dose for the ¢sh was one-half of the minimumi.p. lethal dose for mice. The i.p. lethal dose increasedto 20MLD for the nonproteolytic type F and the C2

toxins, and to 200 MLD for types A, proteolytic andnonproteolytic type B, type D, and proteolytic type F.With the C1neurotoxin, a dose of 2000MLD was re-quired to produce mortality. Type G neurotoxin wasnot lethal via the I.P. route at a titre of 200MLD, thehighest titre tested.

Mortality of coho salmon exposed toC. botulinum type E neurotoxin atdi¡erent temperatures

The sensitivity of coho salmon to type E neurotoxinby the oral and i.p. routes and at di¡erent water tem-peratures is summarized inTable 2. At 20 1C, the oraltoxicity for untrypsinized toxin precursor was83MLD. In comparison, when the toxin precursorwas activated with trypsin prior to testing, the doserequired to cause mortality increased to 125MLD.When the ¢sh were held in water at temperaturesof 15 and 10 1C, the lethal doses were 100 and 83MLD respectively. Decreasing the water temperaturefurther to 5 1C increased the lethal dose of type Eneurotoxin to 250MLD. Coho salmon were the mostresistant to type E neurotoxin when exposed at

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awater temperature of1 1C. At this temperature, thelethal dose was 500MLD for both the untrypsinizedand trypsinized supernatant £uids. By the i.p. route,coho salmon were more sensitive to the toxin thanmice, and a dose of 0.5MLD was lethal to the ¢sh atall of the temperatures studied. These data with typeE strain EF4, which produces very high toxin titres,were comparable to results reported by Eklund,Poysky, Peterson, Peck & Brunson (1984) with otherstrains of type E.

Signs of botulism in ¢sh

Before the onset of signs of botulism, the ¢sh becamevery nervous. As the botulismprogressed, themusclescontrolling the ¢sh ¢ns were paralysed by theneurotoxins. This began with the pectoral ¢n and

the paralysis continued toward the tail. The caudal¢n continued to be active after the other ¢ns were af-fected. The caudal ¢n enabled the ¢sh to move, butthe ¢sh was unable to control the other ¢ns, whichresulted in a loss of equilibrium. As a result of thisparalysis, the ¢sh would swim without directionalcontrol into the sides of the ¢sh tank. Because ofthe paralysis of the ¢n muscles, they were unable tomaintain a horizontal balance and would sink to thebottom of the tank tail ¢rst, rising occasionally to thesurface only to repeat the cycle again. After death,the gills were extended and the carcasses were oftentwisted sideways.When C2 toxin was administered by the i.p. route,

the ¢sh became very nervous, but did not show neu-roparalytic signs. The C2 toxin caused the gills to be-come very bright red in colour and the ¢sh generally

Table 1 Lethal doses of di¡erent types of C. botulinum toxin for juvenile coho salmon held at15 1C

Toxin type

Oral route Intraperitoneal route

MLD� testedNumber fish dead/number fish tested MLD tested

Number fish dead/number fish tested

A 2000 0/2 2000 2/2

1000 0/2 200 2/2

400 0/2 40 0/2

B 2000 0/2 200 2/2

1000 0/2 40 0/2

400 0/2

B n.p.w z 2000 0/2 200 2/2

1000 0/2 40 0/2

400 0/2

C1 2000 0/2 2000 2/2

1000 0/2 200 0/2

400 0/2

C2 200 0/2 200 2/2

40 0/2 20 2/2

10 0/2

D 20 000 0/2 2000 2/2

4000 0/2 200 2/2

40 0/2

Ez 180 3/3 1.0 3/3

90 3/3 0.5 3/3

45 0/3 0.25 0/3

F 2000 2/2 200 2/2

1000 0/2 40 0/2

F n.p.w z 2000 1/2 200 2/2

1000 0/2 20 2/2

10 0/2

G 200 0/2 200 0/2

Control ¢sh fed gelatin capsules with gelatin phosphate bu¡er without toxin or injected with gelatin phosphate without toxin survivedthe duration of the experiments.�Toxic dose based upon mouse intraperitoneal minimum lethal dose (MLD).wTypes B n.p. and F n.p. are nonproteolytic strains of types B and F.zNeurotoxins of nonproteolytic types B n.p., E, and F n.p. and C2 toxin were treated with trypsin before testing.

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remained on the bottom of the tanks in a twistedposition.The C2 toxin is not a neurotoxin, but a toxinthat exhibits lethal, increased movement of £uidsacross membranes and enterotoxic activity (Ohishi& Dasgupta1987).

Discussion

The data presented in this paper demonstrates thewide variation in the susceptibility of coho salmonto di¡erent botulinal toxins. Coho salmon were mostsusceptible (0.5MLD) to type E neurotoxin by the i.p.route.When the neurotoxins, except types E and F,were tested by the i.p. route, the lethal doses rangedfrom 20 to over 2000MLD. In contrast, only type E,nonproteoltyic F and proteolytic type F neurotoxinswere shown to be lethal to coho salmon by the oralroute. Earlier studies have shown that there is across-neutralization between the neurotoxin of typeF strains and type E antiserum (Eklund et al. 1967a)and may explain why they produce similar toxicityin ¢sh. Of the other toxin types, titres greater than2000MLD (highest titres tested) would have to beingested to cause botulism. The lethal dose for type

G was not identi¢ed, because these strains producelow titres of toxin and 200MLD was the highest titretested.Our results also demonstrate the di¡erences in the

susceptibility of ¢sh to botulinum toxins by the oraland i.p. routes. Botulinum toxins administered bythe oral route can be inactivated by the digestive en-zymes and/or not be absorbed into the bloodstream.In comparison, toxin administered by the i.p. routeis not exposed to these inactivating e¡ects (Smith &Sugiyama1988).Temperature along with trypsin activation also

plays an important role in the susceptibility of the¢sh to type E neurotoxin.When coho salmon weretested for susceptibility to type E neurotoxin at di¡er-ent water temperatures, the lethal oral dose at 20 1Cwas 125MLD when the toxin precursor was treatedwith trypsin and 83MLD when the toxin precursorwas not treated with trypsin. Since C. botulinum typeE does not produce proteolytic enzymes, its toxin pre-cursor must be activated with trypsin or other pro-teolytic enzymes to obtain full toxicity. However,once the toxin precursor has been activated, con-tinued enzyme activity can decrease toxin titres or

Table 2 Lethal doses of C. botulinum type E toxin for juvenile coho salmon at di¡erent temperatures

Water temperature (1C)

Oral route Intraperitoneal route

MLD� tested

Number fish dead/number fish tested

MLD tested

Number fish dead/number fish tested

Untry Try Try

20 125 3/3 3/3 1.0 3/3

100 3/3 0/3 0.5 2/3

83 1/3 0/3 0.25 0/3

50 0/3 0/3

15 125 3/3 3/3 1.0 3/3

100 3/3 2/3 0.5 2/3

83 0/3 0/3 0.25 0/3

50 0/3 0/3

10 125 3/3 3/3 1.0 3/3

100 3/3 3/3 0.5 3/3

83 3/3 3/3 0.25 0/3

50 0/3 0/3

5 1000 3/3 NT 1.0 3/3

500 2/3 NT 0.5 3/3

250 1/3 NT 0.25 0/3

125 0/3 NT

1 1000 3/3 3/3 1.0 3/3

500 1/3 3/3 0.5 3/3

250 0/3 0/3 0.25 0/3

Control ¢sh that were fed gelatin capsules with gelatin phosphate bu¡er without toxin, or injected with gelatin phosphate withouttoxin survived the duration of the experiments.�Toxin titers based on mouse intraperitoneal minimum lethal dose (MLD) for trypsinized toxin.Untry, untrypsinized toxin; try, trypsinized toxin; NT, not tested.

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completely inactivate the neurotoxin. This appearedto happen in the 20 1C experiment, where pretreat-ment of the toxin precursor with trypsin and furtherexposure of the activated toxin by the ¢sh intestinalenzymes began to inactivate the neurotoxin, result-ing in a decreased lethal dose.When the toxin (with-out trypsin treatment) was fed, the ¢sh intestinalenzymes were able to activate the toxin precursorand the highest lethal dose was obtained with thistoxin form.In contrast, when ¢sh were held at 1 1C the lethal

dosewas 500MLD of type E neurotoxinwith or with-out trypsin treatment.The number of ¢sh susceptiblewas slightly higher when the toxin was activatedwith trypsin before feeding and lower when the toxinwas administered without trypsin treatment. In thiscase, the activity of the intestinal enzymes at1 1C ap-peared to be slower and prior trypsin treatment wasrequired for the increased lethal dose. A similar phe-nomenon has been observed with seafood productsthat have been inoculated with spores of type E. Theenzymes of spoilage bacteriawill oftenactivate type Etoxin precursor and if the activated toxin is then trea-ted with trypsin, the neurotoxin is either destroyedor the titre is markedly reduced (Pelroy, Eklund, Para-njpye, Suzuki & Peterson1982). In other cases, in theabsence of other proteolytic activity, trypsin treat-ment is necessary to activate the toxin.Water temperatures are also important in deter-

mining which type of C. botulinum will be involvedin a botulism outbreak. Of the di¡erent types of C. bo-tulinum, only type E has been implicated in botulismoutbreaks in steelhead trout and salmon when thewater temperaturewas between 5 and 25 1C (Eklundet al.1982). This in part is related to the ability of typeE to growand produce neurotoxin at temperatures aslow as 3 1C and the higher incidence of type E inponds where outbreaks have occurred. In most ofthese outbreaks, the source of the neurotoxin wasfrom growth and toxin production by C. botulinumtype E in dead ¢sh that remained on the bottoms ofthe rearing ponds and the cannibalizing of thesedead ¢sh by live ¢sh (Eklund et al.1982,1984).The nonproteolytic C. botulinum strains of types B

and F from Group II can also grow and produce neu-rotoxin at temperatures as low as 3 1C (Eklund et al.1967a, b). Under certain conditions, they could alsobe involved in ¢sh botulismoutbreaks. However, theyhave not been reported in botulismoutbreaks in cohosalmon or trout. This in part may be related to thelower incidence of these types in freshwater environ-ments of rearing ponds. In addition, coho salmon

were more resistant to the toxins of nonproteolytictypes B and F than they were for type E neurotoxinat water temperatures of15 1C.With the exception of type G, the other types of C.

botulinum have been shown to be present in terres-trial, freshwater and marine environments of theNorthern Hemisphere. Proteolytic types A, B and Ffrom Group I and types C and D from Group III havenot been involved in botulism outbreaks involvingtrout or salmon. This can possibly be attributed tothe lower incidence of members of Groups I and IIIand their inability to grow at or below10 1C and therelative resistance of coho salmon to these toxins bythe oral route.Recent studies suggest that C. botulinum toxins

other than type E can be involved in botulism out-breaks when the water temperatures are higher thanthose observed in salmon and trout ponds. For exam-ple, in an outbreak in 1996 in the Salton Sea, tilapiawere suspected to have died from neurotoxin of C.botulinum type C when the water temperature was35^37 1C (Dr T. Rocke, pers. comm.). This tempera-ture is optimum for the growth and toxin productionby C. botulinum type C. In addition, it is also possiblethat the sensitivity of tilapia to botulinal neurotoxinsincreases at higher temperatures as it does in sal-mon. If the water temperature had been lower than15 1C, C. botulinum type C organismwould grow veryslowly and probably be less likely to be involved in abotulism outbreak. In comparison, C. botulinum typeE would probably be less involved at temperaturesof 35^37 1C because these temperatures limit itsgrowth and toxin production.Another source of botulinal toxins for ¢sh are un-

refrigerated feed or feed ingredients that support thegrowth of C. botulinum. During a botulism outbreakin trout in Denmark, both type B and E neurotoxinwas detected in the intestines of ¢sh dying frombotulism and these neurotoxins were traced back tothe unrefrigerated minced trash ¢sh used as feed(Huss & Eskildsen 1974). Type B neurotoxin waspresent in the feed at 50MLD g�1. The role thattype B neurotoxin played in the outbreak was notdetermined.C. botulinum types other than type E could also

produce neurotoxin in feeds or feed ingredients thatare not adequately refrigerated, but salmon are rela-tively resistant to these other neurotoxin types. Therelatively high resistance of trout to the neurotoxinsof types A, B, C, and D and the sensitivity to type Eneurotoxin via the oral route has also been reportedby Skulberg & Grande (1967). However, the neuro-

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toxins of the nonproteolytic types B and F and pro-teolytic types Fand Gwere not studied.The ability of the di¡erent types of C. botulinum to

grow and produce neurotoxin at di¡erent tempera-tures, and the susceptibility of the ¢sh to these di¡er-ent neurotoxins are very important in determiningwhether a botulism outbreak will occur and whatC. botulinum type will be involved.

Acknowledgments

We gratefully acknowledge theWashington State De-partment of Fish and Wildlife for the coho salmonand the US Center for Disease Control and Preven-tion, Atlanta, GA, for the C. botulinum antitoxins.Wealso acknowledge Lamia Mseitif for her assistanceduring parts of these studies.

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