Vol. 49, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1985, p. 644-6490099-2240/85/030644-06$02.00/0Copyright ©) 1985, American Society for Microbiology

Bovine Serum Eliminates Rapid Nonspecific Toxic Reactions duringBioassay of Stored Fish for Clostridium botulinum Toxint

MYRON SOLBERG,* LAURIE S. POST, DAVID FURGANG, AND CHARLES GRAHAM

Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers, The State University,New Brunswick, New Jersey 08903

Received 16 July 1984/Accepted 18 December 1984

When stored fish or some fish products were tested for the presence of Clostridium botulinum toxin,nonspecific toxic reactions in mice often occurred, rendering the bioassay inconclusive. The nonspecific toxicreactions were mediated by the gram-negative microbiota, inherent to the fish, which were the source of lethal,heat-stable endotoxins. The treatment of assay samples with bovine serum eliminated nonspecific reactionsthrough the interaction of constituent serum immunoglobulin M (IgM) with endotoxic material. Removal of1gM from bovine serum through treatment with protein A or concanavalin A resulted in a loss of protectiveactivity.

During a recent study of toxin production by Clostridiumbotulinum type E inoculated into fish fillets packaged undermodified atmospheres, nonspecific toxic reactions occurredperiodically during the mouse lethality assay for C. botuli-num toxin. The nonbotulinum-like deaths first occurred onthe day of or immediately after the disappearance of oxygenin the packages. Death was rapid, usually within 1 to 2 h.Segner and Schmidt (9) reported nonspecific deaths duringmouse botulinum assays of irradiated and nonirradiatedhaddock fillets inoculated with C. botulinum type E. Non-specific deaths occurred between 24 and 48 h after injection.Bacteria isolated from marine environments are predomi-nately gram-negative asporogenous rods (10). The outermembranes of gram-negative bacteria contain heat-stablelipopolysaccharides or endotoxins. A lethal injection of anendotoxin results in hemorrhagic shock within 1 to 2 h (1).On a cellular level, endotoxins elicit alterations of mitochon-drial function in animals (5). The gram-negative microbiotainherent to the fish may have grown in the presence ofresidual oxygen and, upon death, released endotoxic mate-rial.Serum is known to possess bactericidal and bacteriolytic

activity against susceptible representatives of almost everygram-negative genus (13). The predominant natural antibod-ies to these species are contained in the immunoglobulin M(IgM) fraction of human plasma (6). Michael and Rosen (6)have reported that IgM is 140-fold more active againstSalmonella typhi and 560-fold more active against Esche-richia coli than is IgG on a weight basis. Robbins et al. (8)have found that IgM is 18 times more active than is IgG insera of rabbits immunized with Salmonella typhimurium.

This study was undertaken to investigate the use of bovineserum to protect mice from nonspecific toxic reactionsduring assays of fish fillets for C. botulinum toxin, todetermine the mechanism of the protection, and to deter-mine whether the lethal reaction is mediated by the microbi-ota inherent to the fish. Tro this end, constituent IgM wasremoved from bovine serum with protein A or concanavalinA (ConA), and the effect on the protective activity of thebovine serum was evaluated during botulinum assays of

* Corresponding author.t Publication no. D-10201-1-84 of the New Jersey Experiment

Station.

stored fish samples and microbial cultures isolated fromstored fish samples.

MATERIALS AND METHODSFish samples. As a part of an ongoing investigation of C.

botulinum toxin production and organoleptic deterioration infresh fish fillets subjected to modified atmosphere storage,samples were selected which gave a nonspecific toxic reac-tion during the mouse lethality assay for C. botulinum toxin.Fresh whole cod had been purchased from the Fulton FishMarket (New York City) and filleted by a local retailer. Thefillets were cut into 100-g portions and inoculated (50 sporesper g; 10 10-,ul drops) with a mixture of five strains of C.botulinum type E or equal portions of sterile distilled water.The inoculated and uninoculated fillets were packaged inindividual barrier nylon ethylene vinyl acetate bags (02 trans-mission, 2.6 ± 0.1 ml/100 in2 (111.76 cm2) per atm of pressureper 24 h; H20 permeability, 0.003 ml/100 in2 per 24 h at 95%relative humidity), evacuated and flushed with 100% CO2.Samples selected for study were representative of thosewhich produced nonspecific toxic reactions in mice. Inocu-lated packages stored at 4°C for 21 days and uninoculatedpackages stored at 4°C for 60 days were examined.

Fresh cod fillets were purchased for use in the identifica-tion of those bacterial species responsible for the nonspecifictoxic response. Portions (100 g) were packaged in barrierbags, flushed with a gas mixture containing 90% C02-8%N2-2% 02, and incubated at 8°C for 8 days, after whichnonspecific toxic reactions were recorded.

Analyses for C. botulinum and nonspecific toxins. A 100-gsample of fish was blended with 100 ml of gel-phosphatebuffer at pH 6.2 (3) for 2 min in a Stomacher 400 (DynatechLaboratories, Inc., Alexandria, Va.). A 25-g portion of theslurry was centrifuged at 17,400 x g for 30 min at 5°C. Thesupernatant was decanted. A 1.8-ml sample of the superna-tant was treated with 0.2 ml of a fresh 10% trypsin solution(3) and incubated for 1 h at 37°C. C. botulinum toxin wasdetected by a modified Bacteriological Analytical Manual(3) mouse lethality assay. A 0.5-ml portion of the treatedsample was injected intraperitoneally into a single femaleSwiss Webster mouse weighing 18 to 22 g. If the mouse diedduring the initial screen, the screen assay was repeated, andanother mouse was injected with a heated (80°C, 15 min),trypsin-treated sample of supernatant. To confirm the pres-

644

ELIMINATION OF NONSPECIFIC TOXIC REACTIONS 645

ence of C. botulinum toxin, a mouse protected with type Eantiserum (Centers for Disease Control, Atlanta, Ga.) waschallenged with the suspect toxin-containing sample alongwith another screen assay. Nonspecific toxic reactions wereindicated when both heated and unheated samples werelethal and the mice were not protected by the monovalentantitoxin. Symptoms were also quite different from thoseproduced by botulinum toxin.

Treatment of samples with bovine serum. A 1.8-ml sampleof supernatant implicated in nonspecific death was incubatedfor 1 h at 37°C with 1 ml of Pentex bovine serum (stored at-20°C and defrosted as needed; Miles Laboratories, Inc.,Elkhart, Ind.), after which the sample was subjected totrypsinization procedures. Gel-phosphate buffer (pH 7.8)was used to dilute a portion of the inoculum to the sameextent to assure that clearing of the nonspecific reaction wasdue to the bovine serum and was not the effect of dilution.Bovine serum-treated and gel phosphate-diluted sampleswere carried through the complete assay for the presence ofbotulinum toxin. Samples were treated with bovine serumafter the heat-inactivation procedure.Treatment of bovine serum with protein A. Protein A

(Sigma Chemical Co., St. Louis, Mo.) was obtained as asalt-free lyophilized powder and was reconstituted withgel-phosphate buffer (pH 7.8) to a concentration of 1 mg/ml.Protein A was complexed with bovine serum in a 1:20(vol/vol) ratio and immediately spectrophotographed with aGilford model 2600 spectrophotometer (Oberlin, Ohio)equipped with a Hewlett Packard plotter (Palo Alto, Calif.).Wavelengths were scanned between 200 and 343 nm tovisually monitor changes in characteristic protein peaks as aresult of sample treatment. The following incubation proce-dure was derived from Sjoquist et al. (11). The complex wasincubated for 2 h at 37°C and scanned spectrophotometri-cally. The samples were further incubated for 22 h at 4°C andagain scanned before storage at -20°C. In preparation forthe bioassay procedures, the complex was scanned, centri-fuged at 17,400 x g for 30 min (5°C), and rescanned. Toevaluate the potential loss of serum activity during the 2 h ofincubation at 37°C and the 22 h at 4°C, samples of bovineserum and samples of protein A were carried through theincubation regimen as experimental temperature controls,and spectrophotographs were prepared after each incubationand storage step.

Bioassays with protein A-treated bovine serum. Mice wereinjected with trypsinized samples of suspect supernatant toestablish the nonspecific toxic response. The complete bio-assay for C. botulinum toxin was performed, in which 1.8 mlof supernatant was treated with 1.0 ml of fresh bovine serumor diluted with 1.8 ml of gel-phosphate buffer. Abbreviatedbioassays were performed with portions (1.8 ml) of thesupernatants treated with 1.0 ml of incubated bovine serum(experimental temperature control) or with centrifuged pro-tein A-treated bovine serum. The concentrations of proteinA-treated bovine serum, incubated bovine serum, and gel-phosphate were doubled to determine whether any resultantlack of protection was due to the protein A treatment of thebovine serum or to the effects of temperature manipulation.

Treatment of bovine serum with ConA. ConA Sepharose(Pharmacia Fine Chemicals, Piscataway, N.J.), which con-sisted of ConA coupled to sepharose 4B beads, was pre-pared for batch affinity chromatography. Samples (5 ml) ofbeads were washed with 25 ml of starting buffer (0.02 MTris-hydrochloride, 0.5 M NaCl [pH 7.4]) and centrifuged at482 x g for 10 minutes. The procedure was repeated twice.Beads were examined microscopically for packing or com-

pression of the gel. The beads were washed from thecentrifuge tube with 10 ml of bovine serum into a sterilebeaker and reacted overnight at 8°C with gentle stirring. Thereaction mixture was centrifuged at 482 x g for 10 min toremove the beads. The supernatant (ConA-treated bovineserum) was scanned spectrophotometrically as before andstored at -20°C. The beads were eluted with 30 ml of boratebuffer (0.1 M, [pH 6.5]) for 48 h at 8°C with gentle stirring.The mixture was centrifuged at 482 x g for 10 min, and theeluent was scanned spectrophotometrically. The eluent wasdialyzed to dryness versus 30% polyethylene glycol (20 M).The dialysis tubing was rinsed with 2 ml of Tris-hydrochlo-ride buffer (concentrated eluent) and scanned spectrophoto-metrically or with 2 ml of ConA-treated bovine serum(restored bovine serum) and scanned. The samples werestored at -20°C. A sample of fresh bovine serum was carriedthrough the incubation regimen (8°C for 24 h, followed bystorage at -20°C) as a temperature control and was scannedbefore the following bioassay procedures.

Bioassays with ConA-treated bovine serum. Trypsinizedscreening assays were carried out with a suspect supernatantto establish the nonspecific toxic response. The completebioassay for C. botulinum toxin was performed, in whichsupernatants were treated with fresh bovine serum or dilutedwith Tris-hydrochloride. Abbreviated bioassays were per-formed with portions of the supernatants treated in the sameproportion with bovine serum which had been subjected tothe temperature regimen alone, with ConA-treated bovineserum, with restored bovine serum, and with concentratedeluent.

Role of trypsin treatment in nonspecific toxic reactions.Trypsinized and nontrypsinized supernatants were assayedfor nonspecific toxic reactions with and without bovineserum treatment. The order of bovine serum addition fol-lowed by trypsinization was then reversed. The sample wastrypsin treated and then incubated with bovine serum for 15min at 37°C before injection.

Identification of fish microbiota and their role in nonspecifictoxic reactions. Two 100-g samples of cod packaged in a 90%C02-8% N2-2% 02 atmosphere and stored at 8°C for 8 dayswere blended as previously described with 100 ml of peptone(0.1%). Samples were assayed for the presence of botulinumtoxin or nonspecific deaths with and without trypsinization.After serial dilutions, the slurry was plated in duplicate onCETCH agar for the enumeration of pseudomonads (12),tryptic soy agar (TS) (Difco) for total aerobic counts, andviolet red bile glucose agar (Oxoid) for the enumeration oftotal members of the family Enterobacteriaceae. The plateswere incubated for 48 h at 26°C. Colonies of several mor-phological types were selected from plates of CETCH andTS agar (25 colonies per agar plate) and were streaked ontoTS agar plates. After 48 h of incubation at 26°C, one colonyfrom each plate was transferred to a piece of Whatman no. 1filter paper and tested for the presence of oxidase (2).Additional colonies from the same plate were Gram stainedand examined morphologically. Those colonies which wereoxidase-positive gram-negative rods were inoculated intoOxiferm tubes (Roche Diagnostics, Nutley, N.J.) for rapididentification through nine biochemical assays. Those colo-nies which were oxidase-negative, gram-negative rods orcoccobacilli were inoculated into Enterotubes (Roche Diag-nostics) for rapid identification by biochemical means.Genus and species as well as the relative proportion of

each bacterial type isolated from the TS agar plates weredetermined. Typical colonies of each organism were se-lected and maintained on TS agar slants stored at 4°C.

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Strains were transferred from stock slants to 10 ml of TSbroth and incubated for 24 h at 26°C. After one consecutivetransfer to fresh broth (1% inoculum), plate counts weremade of each broth culture on TS agar. Culture supernatantswere prepared by centrifugation of 48-h broth cultures at17,400 x g for 30 min. The supernatants were assayed fortoxin, with and without bovine serum treatment, individu-ally and in a mixture representative of the relative propor-tion to which each strain developed on the fish. Trypsinizedand nontrypsinized samples were assayed. The supernatantswere diluted to the population range which had developed inthe sample package and were assayed for toxin by a bioassayscheme which included treatment with bovine serum, dilu-ent, ConA-treated bovine serum, and restored bovine serumin proportions as described previously.

RESULTSTreatment of bovine serum with protein A. Table 1 sum-

marizes the results of bioassays with protein A-treatedbovine serum. Uninoculated samples of cod packaged in avacuum pack flushed with a 100%'o CO2 atmosphere andstored for 60 days at 4°C demonstrated a typical nonspecifictoxic response. Injection of the untreated supernatant or theheated supernatant and the injection of supernatant into amouse protected with antiserum all resulted in lethalitiesusually within 0.5 to 2 h. Death was preceded by laboredbreathing, prostration, and necrosis of the tissue around theeyes and occasionally at the site of injection. Treatment ofthe sample with bovine serum eliminated the nonspecifictoxic response in all but 1 of 13 mouse screens and in allheat-treated and antiserum-protected samples. The clearingof the lethal effect was not the result of dilution, sincesamples diluted with 1.0 ml gel-phosphate buffer continuedto elicit a nonspecific toxic response. The experimentallytreated bovine serum which had been subjected to the 2-hincubation at 37°C and the 22-h incubation at 4°C failed toprovide protection when supernatants were treated with 1.0ml. When the amount of the described serum, which wasserving as a temperature treatment control, was increased to2.0 ml, protection was restored. An equivalent increase inthe amount of diluent added to a control sample asstired that

TABLE 1. Summary of bioassays with protein A-treated bovineserum

Survival ratiob

Supernatant treatmenta SupernatantSupernaantatretmentaHeated in an anti-Supernatant supernatant E-protected

mouse

Untreatedc 0/12 0/9 0/9BSd 12/13 3/3 3/3Diluente

1.0 ml 0/12 0/3 0/32.0 ml 0/1

Protein A-treated BS1.0 ml 0/32.0 ml 0/1a Sample size, 1.8 ml of supernatant trypsinized with 0.2 ml of fresh 10%

trypsin solution. Supernatants were taken from a composite of three 100-gsamples of uninoculated cod which were vacuum packaged, flushed with a100o CO, atmosphere, and stored for 60 days at 4°C.

b Survival ratio, number of surviving mice/number of mice injected.c Survival ratio was 4/4 for 0.2 ml of trypsin plus 1.8 ml of gel-phosphate

without supernatant.d BS, Bovine serum.Gel-phosphate buffer (pH 7.8).

TABLE 2. Summary of bioassays with ConA-treated bovineserum

Survival ratio'Supernatant

Supernatant treatmenta Heated in an anti-Supernatant supernatant E-protected

mouse

UninoculatedUntreatedc 0/12 0/9 0/9

BS (1.0 ml)d 12/13 3/3 3/3

Diluent (1.0 ml)e 0/12 0/13 0/3

ConA-treated BS (1.0 ml) 0/6

Restored BS (1.0 ml) 4/4

InoculatedUntreatedc 0/3 0/3 0/4

BS1.0 ml 0/4 1/3 4/41.5 ml 1/1

Diluent1.0 ml 0/3 0/1 0/11.5 ml 0/1

ConA-treated BS (1.0 ml) 0/3 0/3

Restored BS (1.0 ml) 0/2 2/2

Eluent (1.0 ml) 0/1 1/1

a Sample size, 1.8 ml of supernatant trypsinized with 0.2 ml of fresh 10%trypsin solution. Supernatants were taken from a composite of three 100-gsamples of uninoculated cod as described in footnote a of Table 1 or from a100-g sample of inoculated cod that was vacuum packaged, flushed with a10%/0 C02 atmosphere, and stored for 21 days at 4°C. The inoculated samplecontained 50 spores of C. botulinum type E per g at the initiation of theexperiment.bAs described in footnote b of Table 1.' Survival ratio was 5/5 for 0.2 ml of trypsin plus 1.8 ml of gel-phosphate or

Tris-hydrochloride without supernatant.d BS, Bovine serum.e Gel-phosphate buffer (pH 7.8) or Tris-hydrochloride buffer (pH 7.4).

protection was not due to dilution effects. The nonspecifictoxic response was not eliminated when samples werecombined with 1.0 or 2.0 ml of protein A-treated bovineserum. The lack of protection was not due to experimentaltreatment or dilution, since protection was afforded by 2.0ml of the temperature control and samples diluted with 2.0ml of diluent continued to elicit a nonspecific toxic response.

Spectrographs of the bovine serum showed two peaks ofabsorption at 206 and 278 nm. The temperature-treatedcontrol showed moderate decreases in absorption at thesewavelengths when compared with fresh bovine serum. Bo-vine serum treated with protein A and subjected to centri-fugation showed greater decreases in absorbance readingswhen compared with a fresh bovine serum control. A sampleof protein A carried through the incubation regimen as atemperature control, maintained a relatively stable spectrumdespite the various temperature manipulations.Treatment of bovine serum with ConA. Table 2 summarizes

the results of bioassays with ConA-treated bovine serum.Uninoculated samples of cod demonstrated a typical non-specific toxic response, as described previously, which was

646 SOLBERG ET AL.

ELIMINATION OF NONSPECIFIC TOXIC REACTIONS 647

TABLE 3. Distribution of microbiota inherent to cod fillets'

o% of populationMeim No. of TotalMedium isolates CFU/ml P. vesic- P. P. P. fluo- Pseudomo- E. K. S. P.

ularis putida cepacia rescens nas spp. coli ozaerae rubidea ureae

CETCH 20 1.5 x 107 70 15 10 0 0 5 0 0 0VRBGb 25 1.0 x 106 52 4 8 8 4 0 20 4 0TS 30 2.6 x 107 43 0 53 0 0 0 0 0 4

a Fillets were vacuum packaged, flushed with 90% C0O-8% N2-2% 02, and incubated at 8°C for 8 days.b VRBG, Violet red bile glucose.

eliminated with bovine serum treatment. The addition ofConA-treated bovine serum afforded no protection, whereastreatment with restored bovine serum eliminated the lethaleffect of the supernatant. Dilution of the lethal effect wasdiscounted, since the lethality of the supernatant was main-tained despite the addition of 1.0 ml of diluent. A bovineserum temperature control continued to provide protectiondespite temperature manipulations.Samples of cod inoculated with 50 spores of C. botulinum

toxin type E per g demonstrated a typical nonspecific toxicresponse (Table 2). The presence of botulinal toxin wasmasked throughout the assay procedure due to the lethaleffects of the heat-stable nonspecific toxin. The addition of1.0 ml of bovine serum alleviated the lethal effect of thenonspecific toxin, and the results were more typical of anassay positive for botulinum toxin. The bovine serum-treated supernatant was lethal. Heat treatment of the super-natant and subsequent treatment with 1.0 ml of bovineserum allowed for the survival of one of three mice. Themice were completely protected against bovine serum-treated supernatant by injections of anti-E serum. Thevariable protection provided by heat treatment indicates thatin the absence of the heat-labile botulinal toxin, any residualnonspecific toxic factor was able to exert a lethal effect.Increasing the bovine serum treatment to 1.5 ml providedcomplete protection and allowed for a typical assay responseto heat treatment. Supernatants treated with 1.0 or 1.5 ml ofdiluent were lethal in each phase of the assay. Samplesmixed with 1.0 ml of ConA-treated bovine serum continuedto elicit a nonspecific toxic reaction, and the mice were notprotected by antiserum to C. botulinum type E. Superna-

2.4

WU 1.8Uz

1.2

o d.40.6 a

0.0o 0 0 0 0 o o 0o N t 0 0 0 N ItN N Ncm N 0 e 0

WAVELENGTH (nm)FIG. 1. Spectrographs depicting relative absorbances of the fol-

lowing: a, concentrated eluent (IgM eluted from ConA-coatedbeads); b, ConA-treated bovine serum (IgM-depleted serum); c,untreated fresh bovine serum; d, restored bovine serum (treatedbovine serum plus eluent).

tants treated with restored bovine serum or eluent werelethal but the protection conferred by anti-E serum indicatedthat lethality was due to C. botulinum toxin and was not dueto a nonspecific toxic response.

IgM, present in the bovine serum, was bound to theConA-coated beads and was removed with the centrifuga-tion of the beads from solution. IgM, when eluted from thebeads, was able to confer protection alone or when recom-bined with bovine serum deficient in the immunoglobulin(Table 2). Figure 1 is a spectrograph which depicts therelative absorbances of restored bovine serum, fresh bovineserum, ConA-treated bovine serum, and the concentratedmaterial eluted from beads reacted with bovine serum (elu-ent), assumed to be IgM. A decrease in absorbance isevident between fresh bovine serum and ConA-treated bo-vine serum. Absorbance was increased above that of freshbovine serum when the concentrated eluent was added backto the ConA-treated, IgM-depleted bovine serum.

Effect of trypsinization on bioassay results. Nontrypsinizedextracts of uninoculated fish did not elicit a nonspecific toxicresponse. Fresh 10% trypsin solution was injected into aseries of mice, and all survived, eliminating trypsin as thelethal agent. The elimination of the nonspecific toxic reac-tion was not the result of bovine serum inactivation of thetrypsin, since reversal of the treatment procedure (trypsin-ization followed by the addition of bovine serum) alsoresulted in complete protection of the mice.

Identification of fish microbiota. Total counts on CETCHagar reached 1.5 x 107 CFU/ml and were composed ofPseudomonas vesicularis (70%), Pseudomonas putida (15%),Pseudomonas cepacia (10%), and E. coli (5%) (Table 3). Thetotal population on violet red bile glucose agar was 106CFU/ml which included both typical oxidase-negative pur-plish-red subsurface colonies and oxidase-positive colonieswhich were pigmented pink, white, brown, and gray. P.vesicularis accounted for a large proportion of the totalpopulation, 52%, although colonies were easily distinguish-able from the members of the Enterobacteriaceae. Addi-tional pseudomonad species formed another 24% of thepopulation. Of the Enterobacteriaceae family, only Klebsi-ella ozaerae (Enterobacter agglomerans) and Serratiarubidea were isolated, and they constituted 24% of the totalpopulation (Table 3). Total aerobic counts on TS agarreached 2.6 x 107 CFU/ml. The microbial population wascomposed of P. cepacia (53%), P. vesicularis (43%) andPasteurella ureae (4%). Those species chosen for continuedinvestigation were cultured in TS broth. Total counts (CFUper milliliter) for isolates in broth culture were as follows: P.vesicularis, 3.7 x 109; P. putida, 1.1 x 109; E. coli, 4.2 x 109;P. cepacia, 2.3 x 108; P. ureae, 7.2 x 108.

Role of fish microbiota in nonspecific toxic reactions. Trypsi-nized supernatants from centrifuged broth cultures of eachisolated species elicited a nonspecific toxic reaction duringthe mouse bioassay individually and in a combination repre-

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APPL. ENVIRON. MICROBIOL.

sentative of the total fish microbial population isolated on TSagar. Undiluted culture supernatants from P. vesicularis, P.cepacia, and P. ureae were combined in relative percentagesof 43, 53, and 4%, respectively. Nontrypsinized sampleswere not toxic. Table 4 summarizes the results of bioassayswith trypsinized supernatants diluted to the population rangewithin which each species developed on the fish. All culturesupernatants were lethal at each stage of the assay (screen,heat, protect) except that of E. coli, which did not elicit atoxic response in the diluted state, although undiluted sam-ples were toxic. The addition of bovine serum eliminated thenonspecific toxic reaction, whereas the protective effect ofbovine serum was removed with ConA treatment. Theaddition of restored bovine serum to culture supernatantsonce again conferred protection.

DISCUSSION

The nonspecific toxic reaction occurred periodically dur-ing the botulinum assay of fish packaged and stored undermodified atmospheres. The reaction in mice ranged in sever-ity from temporary respiratory distress and eye damage to

TABLE 4. Summary of bioassays in the determination of thosespecies responsible for the nonspecific toxic reaction in mice

Survival ratiob

Supernatant Supernatanttreatmenta Supematant Heated in an anti-

supernatant E-protectedmouse

P. vesicularisDiluentc 0/2 0/2 0/2Fresh BSd 2/2 2/2 2/2ConA-treated BS 0/2 0/2 0/2Restored BS 2/2 2/2 2/2

P. putidaDiluent 0/2 0/2 0/2Fresh BS 2/2 2/2 2/2ConA-treated BS 0/2 0/2 0/2Restored BS 2/2 2/2 2/2

P. cepaciaDiluent 0/2 0/2 0/2Fresh BS 2/2 2/2 2/2ConA-treated BS 0/2 0/2 0/2Restored BS 2/2 2/2 2/2

E. colieDiluent 2/2 2/2 2/2Fresh BS 2/2 2/2 2/2ConA-treated BS 2/2 1/2 2/2Restored BS 2/2 2/2 2/2

P. ureaeDiluent 0/2 0/2 0/2Fresh BS 2/2 2/2 2/2ConA-treated BS 0/2 0/2 0/2Restored BS 2/2 2/2 2/2a Sample size, 1.8 ml of supernatant. Supernatants were taken from 48-h TS

broth cultures and diluted to population ranges which developed on fish: P.vesicularis, 1:100; P. putida, 1:100; P. cepacia, 1:10; E. coli, 1:1,000; P.ureae, 1:100. All samples were trypsinized with 0.2 ml of a fresh 10% trypsinsolution. All supernatant treatments were 1.0 ml.

b As described in footnote b of Table 1.c Diluent was gel-phosphate buffer (pH 7.8).d BS, Bovine serum.eE. coli at higher concentrations caused nonspecific toxic reactions.

rapid death. The reaction appeared to be more acute inheated samples and appeared to be activated by the additionof trypsin. Nontrypsinized samples of both fish and culturesupernatants did not elicit a nonspecific toxic response.Trypsin itself was eliminated as the causative agent. Trypsintreatment of fragmented fish flesh or cellular debris in thesupernatants may have exposed additional quantities ofendotoxic materials necessary to reach some threshold con-centration for biological activity, or the trypsin may havealtered the size and conformation of a proteinaceous endo-toxin interacting factor, thus releasing the endotoxin. Thetrypsinization procedure is essential when samples are eval-uated for toxin production by nonproteolytic strains of C.botulinum and is recommended when proteolytic strains areassayed under acidic conditions (7). Elimination of trypsintreatment is not a feasible means for preventing nonspecifictoxic reactions.Blended fish fillets and the culture supernatants of organ-

isms isolated from the fish flesh produced the same charac-teristic toxic reaction. The gram-negative endotoxin-produc-ing organisms identified were established as the cause of thenonspecific toxic response. The organisms constituting thelargest proportion were oxidase-positive pseudomonads. E.coli, a common endotoxin-producing organism, induced apotent toxic effect in mice when present in sufficient num-bers. The various Pseudomonas species isolated from thefish were probably the principal endotoxin-containing agents,since they were present in the greatest numbers and brothculture supernatants were lethal at a dilution level represent-ative of the actual microbial population in the fish. Treat-ment of fish or culture supernatants with bovine serumeliminated the nonspecific toxic reaction in mice.Bovine serum protection was thought to be mediated by

the constituent IgM content. IgM is an effective inactivatorof gram-negative endotoxins. The role of IgM in bovineserum treatment was elucidated by a series of procedures.The serum was first treated with protein A, a cell wallconstituent of most Staphylococcus aureus strains known toprecipitate normal human and guinea pig immunoglobulinsprincipally by binding with the Fc fragment of IgG and, tosome extent, that of IgM (4). A loss of protection wasobserved with protein A treatment. Since protein A binds toboth IgG and IgM, a second procedure with the morespecific ConA was carried out. ConA, a lectin, binds specifi-cally to the polysaccharide portion of IgM. Removal of theIgM from the bovine serum through binding with the ConA-coated beads resulted in a loss of protection which wasrestored when the beads were eluted and the eluent wasadded back to the depleted bovine serum. IgM was, there-fore, specifically identified as the protective factor in bovineserum. The amount of bovine serum required for eliminationof endotoxic reactions may depend on the IgM content of theserum and the endotoxin concentration. In the heat treat-ment step of the botulinal toxin assay, greater amounts ofbovine serum may be needed for complete protection. Acontrol diluted to the same extent would be required.When fish or fish products are tested for the presence of C.

botulinum toxin, nonspecific toxic reactions in mice may beexpected, especially if there is any oxygen in the storageenvironment, as a result of the gram-negative bacterialspecies inherent to a marine environment. The bioassay isrendered inconclusive due to the presence of heat-stableendotoxins. The treatment of assay samples with bovineserum eliminates the nonspecific toxic reaction, principally,through the action of serum IgM on endotoxic material.As a result of these studies, it is recommended that food

sample extracts which are subjected to trypsinization before

648 SOLBERG ET AL.

ELIMINATION OF NONSPECIFIC TOXIC REACTIONS 649

mouse bioassays for botulinum toxin be mixed with bovineserum rich in IgM at a 1.8:1.5 (vol/vol) ratio and incubated at37°C for 1 h before injection of mice to eliminate nonspecifictoxic reactions which often result in mouse death andmasking of the botulinum toxin response. In most of thestudies, 1.8-ml samples treated with 1.0 ml of bovine serumwere used. This resulted in an occasional nonspecific toxicresponse which was eliminated by increasing the volume ofbovine serum. Therefore, in the recommended procedure,an increased volume of bovine serum is used.

ACKNOWLEDGMENTSThis work was supported by state funds, by U.S. Hatch Act funds,

and by Booz, Allen and Hamilton, prime contractor to the U.S.Food and Drug Administration.

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media and reagents, p. 296. Difco Laboratories, Detroit.3. Food and Drug Administration. 1978. Bacteriological analytical

manual, p. XIV 1-16. Food and Drug Administration, Washing-ton, D.C.

4. Forsgren, A., and J. Sjoquist. 1967. "Protein A" from Staphylo-coccus aureus III. Reaction with rabbit y-globulin. J. Immunol.99:19-24.

5. McGivney, A., and S. G. Bradley. 1979. Action of bacterialendotoxin and Lipid A on mitochondrial enzyme activities ofcells in culture and subcellular fractions. Infect. Immun.25:664-671.

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VOL. 49, 1985