Clostridial Toxins in Strangulation Intestinal Obstructionin the Rabbit *

GEORGE H. BORNSIDE, PH.D., ISIDORE COHN, JR., M.D.

From the Department of Surgery, Louisiana State University School of Medicine,New Orleans, Louisiana

STUDY of the cause of death in strangu-lation intestinal obstruction has revealedthe importance of several factors-locationof obstruction, length of strangulated seg-ment, fluid and electrolyte balance, bloodloss, intraluminal pressure, hemorrhagicshock, and the so-called "bacterial fac-tor."8, 23 Recent studies with antibiotics inexperimental strangulation obstruction 4-

6, 9 have pointed to the need for re-evalua-tion of the contribution of intestinal bac-teria and their toxins to the cause of death.A precise understanding of the cause of

death in strangulation obstruction nowseems dependent upon a clarification ofthe nature and role of the bacterial factor.This report presents evidence that clos-tridial toxins are the major toxic factorof the peritoneal fluid in experimentalstrangulation obstruction in the rabbit.The rabbit has been used in previous

studies of strangulation obstruction. 24Wahren 22 reported that experimentalstrangulation obstruction in the rabbit pre-sented the picture of an intoxication rap-idly leading to death. He postulated thatthe toxins were formed in the strangulatedsegment of intestine, and that the toxinso formed entered the general circulationvia the peritoneum. He seems not to havegiven serious consideration to bacterialtoxins.

* Submitted for publication October 15, 1959.This investigation was supported in part by

grants from the Edward G. Schlieder EducationalFoundation, and from the National Institute ofAllergy and Infectious Diseases, United StatesPublic Health Service, Grants E-524 and E-870.

The rabbit was used in this study toprovide pools of peritoneal fluid from ani-mals strangulated by means of a simplesurgical technic. The goal of experi-rmental studies has been the characteriza-tion and identification of the toxic agentsin the peritoneal fluid produced dur-ing strangulation obstruction. Quantitativeanalysis of intestinal bacteria in the stran-gulated rabbit shows a striking increasein the number of clostridia. Moreover,neutralization of the toxicity of peritonealfluid by clostridial antitoxins emphasizesthe importance of clostridial toxins.

Materials and MethodsSurgical Procedure. Adult albino rab-

bits of both sexes weighing 1.6 to 3.4 kg.were used. Operations were performedunder 1.5 per cent sodium nembutal anes-thesia (2 ml./kg.), employing all the asep-tic precautions of a hospital operatingroom. A midline incision was used. Thelength of the small intestine was measured.A point located 75 per cent of the distancedistal to the duodeno-jejunal junction wasselected. At this site, a 15 cm. strangulatedclosed loop obstruction was created bytying the two ends of the loop togetherwith umbilical tape (Fig. 1). The vasculararcades of the mesentery were also encom-passed by the tape, which was tightenedto occlude only the venous return from thesegment. The tape was then loosened andretightened to assure that only the venousreturn was being occluded. Criteria forvenous occlusion were (1) a pronouncedcolor change in the loop, (2) pulsation in

330

Volume 152 CLOSTRIDIAL TOXINS IN STRANGULATION INTESTINAL OBSTRUCTION 331Number 2

FIG. 1. A 15-cm.closed loop strangulatedobstruction photographed5 minutes after tying to-gether the two ends ofthe loop.

the arteries of the mesenteric arcade, and(3) the appearance of petechia within thearcade (Fig. 2). The abdomen was closedin two layers.

After the operation rabbits were re-turned to cages and allowed access onlyto water. They were observed until death,which generally occurred in less than 24hours. The loop was perforated in onlyfour of 96 rabbits. In nine rabbits the loopwas not tied tightly enough to cause stran-gulation. In seven rabbits, the gangrenouscondition of the loop indicated arterialstrangulation had been produced. None ofthe preceding preparations was acceptablefor subsequent laboratory studies. In addi-tion, no peritoneal fluid was collected andstudied if the loop were not grossly dis-tended.At autopsy, the typical strangulated loop

was distended, and there was no indicationof perforation (Fig. 3). Foul smelling,dark-red fluid was generally present withinthe peritoneal cavity, and was lethal formice.At death the peritoneal fluid was aspi-

rated with sterile syringes and immediatelyfrozen. All frozen peritoneal fluids col-lected from at least 25 consecutive strangu-lated rabbits were thawed, pooled, divided

into small working aliquots of 10 ml., andimmediately refrozen and stored untilused.

FIG. 2. A 15-cm. closed loop strangulated ob-struction after the criteria for venous occlusiondescribed in the text had been fulfilled.

332 BORNSIDE AND COHN

Albino mice of eitlher sex (Rockland AllPurpose strain f ), and weighing 20 to 24Gm., were used throughout the study toassay the letlhality of peritoneal fluids. Themethod of Reed and Muenchl1 was usedto estimate the LD50 dose. Peritoneal fluidwas tested by intraperitoneal injection inmice with volumes no greater than 2 ml.

Bacteriological Protocol. All cultures wereincubated at 370 C. Anaerobic plate cultures wereincubated in Brewer jars in an atmosphere ofhydrogen. Quadrant streaking was used for quali-tative studies. The serial dilution method usingsurface inoculation was employed for quantitativestudies. Blood agar medium was composed ofTrypticase Soy Agar Base (BBL) with 4% agarand 5% human blood.

Each specimen was streaked onto a blood agarplate for anaerobic incubation. Streaked plates ofblood agar, Difco MacConkey Agar, and DifcoStaphylococcus Medium #110 were incubatedaerobically. Tubes of Difco Heart Infusion Broth,Fluid Thioglycollate Medium, and Deep MeatMedium were also incubated.

Cultures were examined after either 24 or 48hours. Gram stains of all isolates from aerobicplates were prepared and examined. Yeasts werenot identified further. Isolates from aerobic plateswere inoculated into the heart infusion broth andstreaked out on eosin-methylene blue (EMB)agar. Typical coliform bacteria (members of the

f Obtained from Rockland Farms, New City,New York.

Annals of SurgeryAugust 1960

genera Eschericliia and Aerobacter) were identi-fied by means of their characteristic colonialmorphology on EMB agar. Other Gram-negativerods were suibjected to rouitine diagnostic pro-cedures.

Gram-positive cocci were classed as entero-cocci, streptococci, and staphylococci. Heart in-fusion broth cultures of coccal isolates wereheated at 600 C. for 30 minutes. They were thenstreaked to blood agar and examined after 24hours. Hydrogen peroxide solution (30%o) wasadded to the remainder of the cultures to test forcatalase activity. Enterococci were defined ascatalase-negative cocci which survived the heattreatment; streptococci were defined as catalase-negative cocci which did not survive heating. Allcatalase-positive cocci were classed as staphylo-cocci.

Anaerobically incubated blood plates wereexamined after 48 hours. To separate facultativeanaerobes from obligate anaerobes, isolates werestreaked to blood agar for aerobic incubation andto fluid thioglycollate medium. Isolates whichgrew on the blood agar plates were considered tobe aerobes and were discarded. Gram stains of allisolates growing in the thioglycollate mediumwere prepared and examined. Clostridia were de-fined as anaerobic Gram-positive spore-formingrods. Clostridium perfringens (C. welchii) wasspecifically identified by its typical colonial mor-phology, hemolysis of blood, growth in thioglycol-late medium, "stormy" fermentation of milk, andthe microscopic appearance of cells. Bacteroidesspecies were defined as Gram-negative anaerobicrods. Additional general definitions were asfollows: Bacillus species-aerobic Gram-positive

FIG. 3. A typicalclosed loop strangulatedobstruction at autopsy ina rabbit.

Volume 152 CLOSTRIDIAL TOXINS IN STRANGULATION INTESTINAL OBSTRUCTION 333Number 233

TABLE 1. Distribution of Bacteria Recovered from Healthy Laboratory Rabbits

Generic Mid-small Tip ofIdentification Peritoneum, Intestine, Appendix, Colon, Stool,of Isolates % % % % %

Anaerobes:

Clostridium 0 7 14 7 29Bacteroides 0 71 93 100 94

Aerobes:

Bacillus 6 93 93 93 100Coliforms 0 43 100 93 53Proteus 0 0 0 0 12Bacterium 0 7 7 14 29Streptococcus-

Enterococcus 0 7 21 14 70Staphylococcuts 0 0 7 0 53Pseutdonmonas 0 0 0 0 12Yeast 0 7 0 0 0

Sterile 95 0 0 0 0

Number of rabbitsfrom whichspecimen taken 18 14 14 14 17

spore-forming rods; Bacterium species-aerobicGram-positive nonsporing rods. Isolation of Lacto-bacillus species was not achieved by means ofthe above protocol, and was not attempted.

All three initial broth cultures of a givenspecimen were maintained for 5 days to insure asufficient period of incubation. After this time,plates were streaked from all of the broths;growth served as a check on isolations made fromthe initial plates.

Use of the adjective sterile henceforthin descriptions of experimental studies withperitoneal fluids indicates that the particu-lar specimen has been subjected to, andhas passed, the following rigorous sterilitytest: inoculation into heart infusion broth,fluid thioglycollate medium, and deepmeat medium; after five days each culture,whether growth was evident or not, wasstreaked onto blood agar plates for aerobicand anaerobic incubation. Absence ofgrowth on the plate after 48 hours incu-bation allowed a diagnosis of sterile. Ifgr-owth (lid occutr at alny stage (Itirin( asterilitv test, the isolate WIas subjected tothe diagnostic bacteriological protocol, andwas not considered sterile.

Results

Bacteriology of Normal LaboratoryRabbits. The intestinal bacteriology of agroup of laboratory rabbits was studied toobtain baseline data as a standard of com-parison for bacteriological studies of stran-gulated rabbits. Specimens were takenaseptically from the lumen of the colon 15cm. distal to the ileocecal valve, from thetip of the appendix, and from the mid-small intestine. Swabs of the peritonealcavity were made before any of thesespecimens were taken. Stool specimenswere also obtained, and diluted 1/10 withsaline.The results in Table 1 show Bacteroides

species were the most common anaerobicintestinal flora in normal rabbits. Membersof the genus Bacillus and coliforms werethe most frequent aerobic isolates through-ouit the latter hallf of the gastro-intestinaltract, anid in the stools. Of the 18 inormalrabbits fromii which peritoineal swab speci-mens were obtained, all but one specimenwere sterile. The sole organism was a Bacil-

334 BORNSIDE A

lus species, and was probably a contami-nant. A sterile peritoneal cavity in therabbit is in agreement with the findings ofSchweinberg and Sylvester.19

Bacteriology of Strangulated Rabbits.Rabbits used in experiments were autop-sied immediately at death. Specimens were

taken of peritoneal fluid, fluid from withinthe strangulated loop, intestinal contentsproximal and distal to the closed strangu-lated loop (Table 2), and blood. Clostridiawere the predominant anaerobic florawithin the intestine of the strangulatedrabbits. This was in sharp contrast to thepredominance of Bacteroides species innormal laboratory rabbits. Among theaerobes, coliforms and members of thegenus Bacillus, respectively, were againthe most frequent isolates. Clostridia andcoliforms were recovered most frequentlyfrom the peritoneal fluids although theperitoneal fluid produced by five out of 35strangulated rabbits ( 147c ) was sterile.Blood from eight rabbits was cultured. Sixspecimens were sterile. Only clostridiawere isolated from the remaining speci-

LND COHN Annals of SurgeryAugust 1960

mens, but were present at a concentrationtoo low for quantitative study. Strangu-lated rabbits did not die with a bacteremia.The species constituting the most pre-

dominant intestinal flora of the normal rab-bit and strangulated rabbit are comparedgraphically in Figure 4. It would appear

that if a toxin of bacterial origin were pro-duced in the strangulated loop of intestine,it might possibly be either a clostridialexotoxin or a lipopolysacchardic endotoxinof the Gram-negative coliforms.Toxic Properties of Pooled Peritoneal

Fluid. Pooled rabbit peritoneal fluid was

centrifuged at 00 to 5° C. using an Inter-national Refrigerated Centrifuge (modelPR2). For low speed separation, peritonealfluid was subjected to 600 x G for 20 min-utes. The conditions for high speed cen-

trifugation were 24,000 x G for 30 minutes.Exploratory studies of the toxicity of

centrifuged aliquots of pooled peritonealfluid (PPF) indicated that after either lowspeed or high speed centrifugation thesupernatant fraction was lethal for assay

mice in 24 hours. The unwashed residue,

TABLE 2. Distribution of Bacteria Recovered from Rabbits with Venous Strangulation Intestinal Obstruction

Distally ProximallyAdjacent to Adjacent to

Generic Peritoneal Strangulated Strangulated StrangulatedIdentification Fluid, Loop, Loop, Loop,of Isolates % % % %

Anaerobes:

Clostridiumni 74 100 100 100Bacteroides 17 27 30 35

Aerobes:Bacilliis 11 33 5; 47Coliforms 52 74 79 91Proteuis 6 20 12 24Bacteriu'u 11 27 15 29Streptococclus 3 7 0 6Enterococcus 9 13 3 18Staphvlococcuis 0 0 3 0

Sterile 14 0 0 0

Number of rabbitsfrom which speci-men was taken 35 15 33 17

Volume 152 CLOSTRIDIAL TOXINS IN STRANGULATION INTESTINAL OBSTRUCTION 335Number 2

Quantitative Bacteriology of Normal and StrangulatedRabbits

Normal S5 jghed Normal StrongultedIt g . V

FIG. 4. Quantitativebacteriology of speci-mens from normal andstrangulated rabbits. Lineacross bar indicates me-dian count. Numeral inbar indicates number ofspecimens exhibiting nogrowth.

At0

EaC,.toS%

a

CE0

Clostridiumm Coliforms

i0121010 1ios

Io2104 lJS102

growth I

Bocteroides Bacillus

1012~ ~~

I°B.|S0 i

Af growth

a lo-I f1. t one . tasshod olb (an fuid bom nb

bassO

resuspended to original volume with saline,was not lethal. It was subsequently shownthat supernates could be sterilized and stillbe lethal. After high speed centrifugation,supernates were sterilized by passagethrough either ultra-fine sintered glass fil-ters, or Millipore filter membranes, or Seitzfilter pads. Sterilization of supernates couldalso be accomplished by incubation withKanamycin (10 mg./ml. of supernate) forthree hours, or exposure of a thin layerof superrate to ultra-violet radiation (2537A) for 15 minutes at a distance of fourinches from the source. In addition, bothsupernatant and residue fractions after low

Omfluid tone

(a fluidbad

speed centrifugation could be sterilized byshaking with glass beads. For this proce-dure a special Shaker Head 20 was attachedto the shaft of the centrifuge, and bacteriain the fraction were disintegrated by shak-ing with glass beads (0.2 mm. diameter)for 30 minutes with the shaft of the centri-fuge at 1,400 RPM.The significance of these studies was the

consistent result that (1) lethality of cen-trifuged aliquots of pooled peritoneal fluidrested in the supernate; and (2) sterilizedsupernates retained their lethality. There-fore, centrifugation followed by steriliza-tion of the supernate provided an efficient

17 3 I

m

-1

BORNSIDE AND COHN Annals of SurgeryAugust 1960

TAXBLE 3. Pr-operties and Bacteriology of Pooled Peritonacl Flutids

Properties PPF #1 PPF #2

# Rabbits 26 28

Av. vol. PF/rabbit 14.8 ml. 9.4 ml.Total vol. PF collected 385 ml. 262 ml.Av. survival time of ea. rabbit 20.8 hrs. 22.9 hrs.Av. weight of each rabbit 2.6 kg. 2.7 kg.Mouse LD5o of crude PPF 0.45 ml. 0.038 ml.

Bacteriology

Anaerobes:

Clostridium 1,300 X 103/ml. 900 X 103/ml.Bacteroides 2 X 103/ml. None

Aerobes:

Bacillus None 16 X 103/ml.Coliforms 160 X 103/ml. 77 X 103/ml.Streptococcus 39 X 103/ml. NoneEnterococcus 27 X 103/ml. NoneStaphylococcus 60/ml. None

Total bacteria 1,528 X 103/ml. 993 X 103/ml.

means of dissociating any toxic and lethal tion to clostridial exotoxins as the toxiceffects due to bacterial infection from the agent in strangulation intestinal obstruc-contribution of pre-existing toxin. This was tion in rabbits.evidence that the lethality of peritoneal Bacteriological analysis of the two poolsfluid was attibutable in part to a toxin, of rabbit peritoneal fluid used in this studyprobably of bacterial origin. (Table 3) shows the clostridial count andWhen toxic supernates were heated at the total bacterial count to be of the same

56° C. for as little as 15 minutes, the super- magnitude. The LD50 dosage of PPF #2nate, although not sterile, was no longer for mice was approximately 12 times lesslethal. This temperature does not inacti- than PPF #1, and allowed much morevate endotoxins, and it was presumed that work to be done with PPF #2 than wasthe lethality of sterile supernate was not possible with PPF #1.due to bacterial endotoxins. The relatively The total nitrogen content of PPF #2constant clostridial content of pooled peri- was determined by means of the titrimetrictoneal fluids (Table 3) directed our atten- micro-Keldahl procedure, from which data

TABLE 4. Protein Content of Pooled Rabbit Peritoneal Fluid #2

Nonprotein ProteinTotal Nitrogen, Nitrogen, Nitrogen, Protein,

Pooled Peritoneal Fluid mg/ml mg/ml mg/ml mg/ml

Crude 12.915 i 0.134* 1.272 i 0.004 11.643 i 0.130 72.885 i 0.814High-speed centrifugation:

Supernate 12.630 i 0.071 1.514 4 0.004 11.116 i 0.067 69.586 i 0.419Filtered supemate 12.200 i 0.218 1.268 i 0.007 10.932 ± 0.211 68.435 ± 1.321

Residue 0.475 + 0.033 0.004 + 0.002 0.471 i 0.031 2.948 ± 0.194

* Mean of determination from three aliquots ± standard error of mean.

336

Volume 152 CLOSTRIDIAL TOXINS IN STRANGULATION INTESTINAL OBSTRUCTION 337Number 2

TABLE 5. Toxicity of PPF #2 for AMice in 24 Hours

LD5o Dose, Toxicity ofml. 1 ml., I.P.t

Crude 0.038

High speed centrifugation:Supernate 0.057Supernate (sterile) 0.057*, (0.014)**

Residue: 1st wash saline 2/3 (3/3)**2nd wash saline 0/33rd wash saline 0/3Residue, resuspended to original volume with saline 0/3 (2/3)

* Eight ml. (1.4 LD5o) injected I.P. with 12 ml. of saline brought about the death of a 2 kg. rabbit in 19 hours.** All values in parentheses refer to data based upon death in 48 hours.t Number of mice dead/number of mice challenged.

the protein content was calculated (Table4). The lethal sterile high speed supernateaccounted for 94 per cent of the proteincontent of crude pooled PF.Assay mice died within 24 hours when

challenged with PPF #1, or else survivedthe injection. Death within 24 hours was

therefore selected as a parameter for assays

of lethality. However, with the more toxicPPF #2, assay mice not dead in 24 hoursmight die by 48 hours, but not after thatperiod. It is possible that more than one

toxin is active-one killing within 24 hoursif present in high enough concentration,the other recognized only when the fasteracting toxin is not present in sufficient con-

centration.Although exploratory studies indicated

that the residue obtained from high speedcentrifugation was not lethal for assay

mice, the following study was pursued toobtain quantitative data regarding this ob-

servation. The residue from high speedcentrifugation of PPF #2 was resuspendedto original volume with saline and recentri-fuged at high speed. This procedure wasrepeated three times. The washings as wellas the washed residue were tested for tox-icity (Table 5). The activity of the firstwash was attributed to toxin adhering tothe residue and the wall of the centrifugetube. This interpretation is borne out bythe innocuousness of the two subsequentwashings. The ultimate toxicity of thewashed residue is most likely the result ofan overwhelming infection from the resi-due. However, challenge with one ml. ofthe thrice washed and resuspended residuecorresponds to 26.3 LD50 doses of the ini-tial crude PPF #2 on a volume basis. Thusthe lethality of the washed high speedresidue from PPF #2 is significantly lessthan that of the high speed supernate.

TABLE 6. Toxicity of Ultracentrifuged Sterile High-speed Supernate of PPF #2 in 24 Hours

LD50 Dose, Toxicity of 1 ml.Ultracentrifuged ml. I.P.t

Supernate 0.036 (0.015)**Residue*, resuspended to original volume with saline 0/3 (2/3)**

* The residlue was a sterile sediiment; its toxicity is attributed to contamination of wall of centrifuge tube withsupernate.

** Figures in parentheses refer to 48-hour dlata.t Number of mice (lea(l/numb)er of mice challenge(l.

338 BORNSIDEUltracentrifugation. A Model L Spinco

Ultracentrifuge was used with a number40 rotor. Sterile high speed supernate inultraviolet-sterilized plastic tubes was sub-jected to a mean force of 105,400 x G for1 hour at 50 C. After decanting the darkred supernate, there was but a slightamount of like-colored sediment at the bot-tom of the tube. This pellet, when resus-pended to the original volume with saline,was not as lethal as the supernatant frac-tion (Table 6). The ultracentrifuged super-nate was, however, more lethal than thehigh-speed supernate (Table 5) fromwhich it was prepared. As the toxin wasnot sedimented under conditions whichwould have removed serum globulin, it ispossible that the molecular weight of thetoxin was less than 100,000. More work is,however, necessary before this point canbe verified.The toxicity data for the sterile residue

from ultracentrifugation is comparable tothat for washed high speed residue. How-ever, the lethal result in 48 hours can mostlikely be attributed to adsorption of sometoxin onto the sedimented material.Immunogenicity. Repeated inoculation

of sublethal doses of sterile high speedsupernates of PPF #2 increased the re-sistance of mice to challenge with toxicPPF. Mice injected intraperitoneally witheither three or four repeated sublethaldoses of sterile supernate during a twoweek period survived challenge with 3-4LD50 doses of the same preparation.The heat lability of PPF was studied

further by heating aliquots of sterile highspeed supernate for 15 minutes and 30minutes at 570 C. and for ten minutes at1000 C. After heat treatment each aliquotwas centrifuged at 600 x G for 15 minutesto remove coagulated protein. Groups ofthree mice were challenged with 0.02 ml.,0.04 ml., and 0.08 ml of each heated prepa-ration. None of these 27 mice died. Thesemice were challenged seven days laterwitlh 0.08 ml. (1.4 L,D.;,,) of active sterile

,ND COHN Annals of SurgeryAugust 1960

high speed supernate. There were 22deaths in 26 mice challenged (85%) mor-tality). Accordingly, prior challenge withheated nonlethal preparation conferred noactive immunity to mice.

Clostridial Antitoxins. Exploratory stud-ies with PPF #1 had shown that poly-valent gas gangrene antitoxin protectedmice challenged with approximately 4LD50 doses of sterile high speed supernate.The protection afforded mice by commer-cial gas gangrene antitoxins * was investi-gated further. A controlled experiment wasdesigned to assay the protection againstchallenge 24 hours later with sterile highspeed supernate of PPF #2 (Table 7).The pentavalent antitoxin conferred a

striking measure of protection against thelethal activity of high speed supernate.No protection was given by the trivalentantitoxin, the normal control serum, or noserum therapy. These data indicate a majorrole for clostridial toxins in the lethal ac-tivity of the peritoneal fluid produced inrabbits during experimental strangulationobstruction. They also point to the possi-bility of identifying the toxins contributedby particular species of the genus Clos-tridium.

Accordingly, the protection afforded bymonovalent clostridial antitoxin ** was as-

* Antitoxins are polyvalent products of equineorigin, and were kindly furnished by Dr. I. S.Danielson of Lederle Laboratories and Dr. M. Z.Bierly, Jr., of Wyeth Laboratories. The Lederleproduct is pentavalent and has the following mini-mal composition (15 ml.): C. perfringens anti-toxin 10,000 units; C. septicum antitoxin 10,000units; C. histolyticum antitoxin 3,000 units; C.novyi antitoxin 1,500; C. sordelli antitoxin 1,500units. The Wyeth product is trivalent, and 15 ml.are composed of the same minimal unitage ofC. perfringens, C. septicum, and C. novyi anti-toxins. The control serum was prepared byLederle Laboratories from slaughterhouse horseserum.

** Small volumes of unrefined monovalentantitoxins against toxins of the several clostridialspecies contributing to the pentavalent gas gan-grene antitoxin were kindly supplied by Dr. I. S.Danielson of Lederle Laboratories.

w- -v - T v,Al

Volume 152 CLOSTRIDIAL TOXINS IN STRANGULATION INTESTINAL OBSTRUCTIONNumber 2

TABLE 7. Protection Afforded Mice by Gas Gangrene Antitoxin (0.2 ml., I.P.) Against Challenge24 Hours Later with Sterile Supernate from PPF #2

Volume of Sterile High Speed Supernate of PPF #2Injected I.P.; LD50 Dose = 0.057 ml.

Polyvalent Gas-Gangrene Antitoxin 0.06 ml. 0.10 ml.

Pentavalent (Lederle) 0/4* 1/19Trivalent (Wyeth) 13/15

Normal serum 3/4 (4/4)** 19/19No serum therapy 2/3 (3/3) 14/17 (16/17)

* Number of mice dead/number of mice challenged.** Figures in parentheses refer to 48-hour data.

sayed in a similar experimental procedure(Table 8).The results indicated that a striking level

of protection against the lethal activity ofPPF #2 was conferred by the C. novyiand C. sordelli antitoxins. The C. septicumantitoxin protected during the first 24hours after challenge with PPF #2, butnot after 48 hours. A possible explanationfor the short term protection afforded bysepticum antitoxin may be that sterilePPF #2 contains some C. septicum toxin,neutralization of which provides a short-term measure of protection against theeventually lethal effect of the slower actingmixture of C. novyi and C. sordelli toxins.These latter two toxins may be present inlower concentrations or they may be lesspotent than the septicum toxin. Quantita-tive evaluation of these speculations is notyet possible. However, the data in Table 8

do show that the toxins of neither C. per-fringens nor C. histolyticum exert a role inthe lethal activity of peritoneal fluid pro-duced in rabbits. These toxins may verywell be absent.There is presently no explanation for the

fact that the trivalent gas gangrene anti-toxin (containing perfringens, septicum,and novyi antitoxins) did not protect miceagainst challenge with lethal peritonealfluid.

DiscussionThe following conclusions are in har-

mony with the results of this study. (1)Massive numbers of clostridia are presentin the fluid within the strangulated closedloop, and considerable, but lesser, num-

bers are found in the peritoneal fluid.(2) The lethality of pooled peritonealfluids results primarily from toxins rather

TABLE 8. Protection Afforded Alice by Specific M1onovalent Clostridial Antitoxins (0.2 ml., I.P.) AgainstChallenge 24 Hours Later wit/h Sterile Supernate from PPF #2

Antitoxin Against Mouse Mortality after ChallengeToxins of Clostridial MLD, x%ith 0.1 ml. Sterile High-

Species units/ml.* speed Supernate PPF #2

C. perfringens Type A 115 9/10 (10/10)**C. septicunm 450 2/9 (9/9)C. novvi 9 0/9C. sordelli 100 0/10C. histolyticllm 35 9/10 (10/10)

* Potencv of each antitoxin as assayed by Lederle Laboratories.** Mortality: Number mice (lea(d in 24 hours/number mice challenged; ratio in l)arentheses is mortality in 48

hours in those instances where there was a change.

339

340 BORNSIDE AND COHN

than from bacterial infection. (3) Cen-trifugation offers an efficient means ofseparating pre-formed toxin from infectiousbacteria. (4) Centrifugation followed bypassage of the supernate through bacterialfilters yields sterile fluids which are toxic.

Bacteriological studies of various fluidsin the strangulated rabbit point to the pos-sibility that the toxic activity of peritonealfluids is due to either intracellular endo-toxins of Gram-negative bacteria or toextracellular exotoxins of Gram-positiveclostridia. In view of current interest inthe role of endotoxins as the bacterial fac-tor causing the irreversibility of prolongedhemorrhagic shock, it was of importanceto evaluate the relative contribution ofendotoxins to the lethality of our prepara-tion. Fine 11 has proposed "that when deathoccurs in traumatic shock in spite of propertherapy, the probable cause is a bacterialtoxin which produces irretrievable collapseof the circulation," and that "if there is nosystemic or local infection to account forthe toxin, an endotoxin derived from theintraintestinal bacteria is the most likelyoffending agent."

It has been shown in this report that atemperature of 560 C. for 15 minutes in-activates the lethality and immunogenicityof sterile preparations of peritoneal fluid.Endotoxins, being lipopolysacchardic com-plexes, withstand boiling. Accordingly, thecontribution of endotoxin to the biologicalactivity of the peritoneal fluid produced instrangulation obstruction of the rabbit ap-pears to be minor. The results of investiga-tions of the effect of irreversible hemor-rhagic shock upon the bacteriology ofdogs10' '4 also tend to minimize the like-lihood of any obvious contribution of endo-toxin to hemorrhagic shock associated withstrangulation obstruction. Furthermore, thestudies of Sanford and Noyes 18 employingCr51-labeled endotoxin do not suggest thatendotoxin is absorbed from the gastro-intestinal tract of dogs committed to ir-reversible hemorrhagic shock.

Annals of SurgeryAugust 1960

Studies of the immunogenicity and heatlability of sterile pooled rabbit peritonealfluid indicate the presence of a toxic pro-tein, and oriented attention to clostridialexotoxins. Neutralization of the lethal ac-tivity of peritoneal fluid for assay mice bygas gangrene antitoxin verified the impor-tance of clostridial exotoxins in the rabbitpreparation. Analysis of the protection con-ferred by monovalent clostridial antitoxinsdemonstrates that the toxins of C. novyi,C. sordelli, and C. septicum exert a role inlethal activity. However, no evidence forthe presence of the toxins of C. perfringenswas obtained although this species wasregularly isolated from the strangulatedrabbit, but not from the normal rabbit.

Earlier studies of strangulation obstruc-tion employing dogs have suggested amajor role for the toxins of C. perfrin-gens.'s6 21, 25 However, fundamental physio-logical differences between herbivores andcarnivores may parallel differences in ex-perimental results using rabbits and dogs.It would be anticipated that there mightbe differences not only in the normal in-testinal flora, but also in the flora subse-quent to experimental strangulation ob-struction. Comparison of the flora of thenormal rabbit with that of the strangulatedrabbit (Fig. 4) bears out this anticipation.Increase in the permeability of the trauma-tized intestinal wall and seepage of bloodinto the peritoneal cavity probably accountfor the presence of bacteria and of clos-tridial toxins in peritoneal fluid. Althoughcontamination of peritoneal fluid couldoccur as a result of perforation of theclosed loop, this possibility is unlikely inthis study as only peritoneal fluids fromrabbits having distended loops showing nogross perforation were harvested.The production of bacterial toxins in

strangulation obstruction is supported bythe studies of Blain et al.5' " and Cohn etal.9 The work of Barnett2 has placed freshemphasis upon the role of infectious bac-teria in experimental strangulation obstruc-

Volume 152 CLOSTRIDIAL TOXINS IN STRANGULATION INTESTINAL OBSTRUCTION 34Number 234tion in the dog. Barnett and Neely3 re-ported that intraperitoneal injection of alethal dose of raw peritoneal fluid, pro-duced by strangulated donor dogs, broughtabout a severe leukopenia. However, in-jection of C. perfringens toxin into dogswas followed by a leukocytosis. Theseauthors concluded that the alpha-toxin(lecithinase) of C. perfringens does notcontribute to the marked leukopenia asso-ciated with the death of dogs receivingstrangulation obstruction fluid. It is note-worthy that there is no decisive evidencefor the presence of alpha toxin (lecithi-nase) in either experimental intestinal ob-struction 12, 21 or experimental or clinicalgangrenous myositis.1 13 Investigation ofthe physiological effects of exotoxins pro-duced by clostridial species other than C.perfringens may help to explain some ofthe effects of raw strangulation fluid inrecipient animals.

Extrapolation of results obtained fromstudy of strangulation obstruction in rab-bits to either dogs or humans requiresextreme caution. Noer15 has reported thatthe anatomy of the small-intestinal circula-tion of the rabbit is similar to that of manwhereas that of the dog differs greatly fromman. Further insight into the characteriza-tion of the toxemia of strangulation ob-struction may be correlated with majoranatomical or physiological differencesamong vertebrate species. Alternatively,the characteristics of this toxemia amongthe several species may reveal only minorvariations on the same theme.

Summary1. Fifteen-cm. lengths of rabbit small in-

testine were strangulated and obstructedby tying the ends together with umbilicaltape to form a closed loop. Peritoneal fluidwas collected and studied from animalswith a strangulation obstruction showinggross distention and presenting no evi-dence of perforation.

2. Clostridia were the predominating an-

aerobic organisms within the strangulatedintestine whereas Bacteroides species pre-dominated as the anaerobic intestinal floraof the normal rabbit. Coliforms and Bacil-lus species were the most frequent andnumerous aerobes in both the strangulatedand the normal rabbit.

3. Pooled peritoneal fluid (PPF) fromrabbits with strangulation intestinal ob-struction was lethal for assay mice. Follow-ing centrifugation of PPF, the supernatantfraction was lethal; the residue was notlethal. Supernates sterilized by filtrationwere lethal. Ultracentrifugation of thesterile supernate from centrifuged PPF didnot remove the toxin(s).

4. Repeated sublethal doses of sterilesupernate protected assay mice againstchallenge with the same preparation. Thelethality of sterile supernate was inacti-vated by heating. Heated preparationswere no longer immunogenetic. Thesefindings emphasized the protein nature ofthe toxic agent(s).

5. Gas gangrene antitoxin protected as-say mice against the lethal result of chal-lenge with sterile PPF. Antitoxins preparedfrom Clostridium novyi, C. sordelli, andC. septicum conferred protection to assaymice challenged with sterile PPF. Anti-toxins prepared from C. perfringens(welchii) and C. histolyticum did not pro-tect assay mice.

6. The relation of these results to otherstudies of the bacterial factor in experi-mental strangulation intestinal obstructionis discussed.

7. It is concluded that clostridial toxinsare the major lethal factor of the peritonealfluid in intestinal strangulation obstructionin the rabbit.

AcknowledgmentsThe authors wish to thank Drs. M. Atik, A. M.

Cotlar, and T. L. Hudson for their assistance withthe surgical phases of this work. The able assist-ance of Miss Bette M. Beauclair, Miss DorisDePaoli, and Mr. A. T. Knecht, Jr. is gratefullyacknowledged.

342 BORNSIDE AND COHN Annals of Surgery342 ~~~~~~~~~~~~~~~~~~~~~~~~~August 1960

Bibliography

1. Balch, H. H. and 0. H. Ganley: Observationson the Pathogenesis of Clostridium welchiiMyonecrosis. Ann. Surg., 146:86, 1957.

2. Bamett, W. O.: Strangulation Obstruction:Pathophysiology and Management. Missis-sippi Doctor, 36:72, 1958.

3. Bamett, W. 0. and W. A. Neely: A Com-parison of In Vivo Changes Following Ad-ministration of Strangulation ObstructionFluid and Clostridium welchii Toxin. Surg.Gynec. & Obst., 106:667, 1958.

4. Barnett, W. 0. and T. V. Stanley: Treatmentof Experimental Strangulation Obstruction.Arch Surg., 77:196, 1958.

5. Blain, A., III and J. D. Kennedy: The Effectof Penicillin in Experimental Intestinal Ob-struction: Studies on Strangulated Low IlealObstructions. Bull. Johns Hopkins Hosp.,79:1, 1946.

6. Blain, A., III, J. D. Kennedy, R. J. Calihanand H. N. Harkins: Effect of Penicillin inExperimental Intestinal Obstruction. Cure ofStrangulated Ileal Obstructions Treated withPenicillin Prior to Late Resection. Arch.Surg., 53:378, 1946.

7. Bunting, C. H. and A. P. Jones: IntestinalObstruction in the Rabbit. J. Exp. Med.,17:192, 1913.

8. Cohn, I., Jr.: Strangulation Obstruction. In-ternat. Abstr. Surg., 103:105, 1956; in Surg.,Gynec. & Obst., Aug., 1956.

9. Cohn, I., Jr., A. Gelb and H. R. Hawthorne:Strangulation Obstruction: Effect of Pre-and Post-operative Antibacterial Agents.Ann. Surg., 138:748, 1953.

10. Culbertson, W. R., W. Elstum, W. Cole andW. A. Altmeier: Bacterial Studies in Irre-versible Hemorrhagic Shock. Arch. Surg.,79:185, 1959.

11. Fine, J.: The Role of the Intestine in Trau-matic Shock. Am. J. Gastroent., 29:596,1958.

12. Furr, W. E., R. V. Bourdeau, H. D. Rochand H. Laufman: In Vivo Effects of Clos-tridium welchii Lecithinase. Suirg., Gynec.& Obst., 95:456, 1952.

13. MacLennan, J. D. and R. G. Macfarlane:

Toxin andl Antitoxin Stud(lies of Gas Gan-grene in MIan. Lancet, 2:301, 1945.

14. Nelson, R. XI. and H. E. Noyes: Blood Cul-tuire Stutdies in Normal Dogs and in Dogsin Hemorrhagic Shock. Surgery, 35:782,1954.

15. Noer, R. J.: The Blood Vessels of the Jejunumand Ileum: A Comparative Study of Manand Certain Laboratory Animals. Am. J.Anat., 73:293, 1943.

16. Oughertson, A. W. and J. H. Powers: TheRelationship of the Toxin of Bacillus welchiito the Toxemia of Intestinal Obstriction.Arch. Surg., 18:2019, 1929.

17. Reed, L. J. and H. Muench: A SimpleMethod of Estimating Fifty Percent End-points. Am. J. Hyg., 27:493, 1938.

18. Sanford, J. P. and H. E. Noyes: Studies onthe Absorption of Escherichia coli Endo-toxin from the Gastrointestinal Tract ofDogs in the Pathogenesis of "Irreversible"Hemorrhagic Shock. J. Clin. Invest., 37:1425, 1958.

19. Schweinburg, F. B. and E. M. Sylvester:Bacteriology of the Healthy ExperimentalAnimal. Proc. Soc. Exp. Biol. & Med., 82:527, 1953.

20. Shockman, G. D., J. J. Kolb and G. Toennies:A High Speed Shaker for the Disruption ofCells at Low Temperature. Biochim. Bio-phys. Acta, 24:203, 1957.

21. Tanturi, C. A., R. E. Anderson and J. F.Canepa: Lecithinase and Hyaluronidase inExperimental Intestinal Obstruction. Surg.Gynee. & Obst., 90:171, 1950.

22. Wahren, H.: The Intoxication in IntestinalStrangulation: An Experimental Study. ActaChir. Scand., 78:121, 1936.

23. Wangensteen, 0. H.: Intestinal Obstructions.3rd Edition. Chas. C Thomas, Springfield,Illinois, 1955.

24. Wangensteen, 0. H. and S. S. Chunn: Studiesin Intestinal Obstruction. I. A comparisonof the Toxicity of Normal and ObstructedIntestinal Content. Arch. Surg., 16:606,1928.

25. Williams, B. W.: Importance of ToxaemiaDue to Anaerobic Organisms in IntestinalObstruction and Peritonitis. Brit. J. Surg.,14:295, 1926.