INFECTION AND IMMUNITY, Jan. 1971, p. 171-178Copyright © 1971 American Society for Microbiology

Vol. 3, No. IPrinted in U.S.A.

Clostridium perfringens Type A Food PoisoningII. Response of the Rabbit Ileum as an Indication of Enteropathogenicity

of Strains of Clostridium perfringens in Human Beings

DOROTHY H. STRONG, CHARLES L. DUNCAN, AND GIUSEPPE PERNA

Food Research Institute, Departments ofFood Science, Bacteriology, and Medicine, and UniversityHealth Services, University of Wisconsin, Madison 53706

Received for publication 22 June 1970

The effect of feeding human beings individual strains of Clostridium perfringens or

culture filtrates thereof was examined. The strains selected for challenge includedboth those which had previously been shown to produce fluid accumulation in theligated ileum or overt diarrhea when injected into the nonligated ileum of the rabbit,or had produced both, and those which did not regularly produce these responses.

Challenge doses prepared by allowing each strain to grow in beef stew for 3 hr at 46 Cresulted in a 61% incidence of diarrhea when rabbit-positive cells were used. Nodiarrhea occurred among the subjects fed rabbit-negative strains prepared in a similarmanner. The procedures employed in preparing the challenge dose appeared to in-fluence the results obtained. When cell-free filtrates were fed, 4 of 15 persons con-

suming filtrates from rabbit-positive strains developed diarrhea. All subjects fedfiltrates from rabbit-negative strains remained free from diarrhea. Serological testswere carried out to compare the identity of the strains of C. perfringens consumedby the subjects and those excreted in the feces. Heat resistance measured as Dloovalues varied greatly among the rabbit-positive strains.

Although significant progress concerning theetiology of human disease is frequently realizedthrough the use of animal models, convincingproof of any findings must ultimately depend ondemonstrated parallelism between the animal andthe human response. In attempting to study thepossible causative agent operating in the produc-tion of Clostridium perfringens food poisoningsymptoms, various animals have been employed(11, 14, 18, 19, 23).

Recently, the use of ligated loops of intestinehas been proposed as an assay method for deter-mining the enteropathogenicity of strains of C.perfringens. Duncan et al. (8) employed this pro-cedure, utilizing young rabbits as test animals.Almost simultaneously Hauschild et al. (12) de-scribed the application of the same technique inlambs. The latter workers used a strain of C. per-fringens which had been previously shown in theirlaboratory to have produced intestinal disturb-ance in five of six cases when fed to human beings(13). Their evidence supported the propositionthat a positive response in a ligated loop of lambintestine indicated a strain of C. perfringens whichwould produce symptoms commonly developedin C. perfringens food poisoning.More recently, a correlation has been estab-

lished between the accumulation of fluid in theileum of the rabbit and a diarrheal response inmonkeys (7).The purpose of this study was to test whether a

positive response obtained after introduction of astrain of C. perfringens or its cell extract or cul-ture filtrate into the ileum of a young rabbit (5, 6)does denote a strain capable of producing thefood poisoning syndrome in human volunteers.The procedures for performing the rabbit testhave been described elsewhere (8).

In this paper, as in the preceding report (7), theterm "rabbit-positive" is used to describe strainsof C. perfringens, or products thereof, which re-peatedly have been shown to produce in youngrabbits either fluid accumulation in the ligatedileum or overt diarrhea subsequent to intrailealinjection of the nonligated gut, or both. Con-versely, "rabbit-negative" denotes the absence ofsuch response in any regular pattern.

MATERIALS AND METHODS

Cultures. Eight rabbit-positive cultures of C.perfringens type A were employed. These includedthe following, with the Hobbs serological type shownin parentheses: NCTC 8239 (H3), NCTC 8798 (H9),NCTC 10239 (H12), and NCTC 10240 (H13), allisolated and typed in the laboratory of Betty Hobbs

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and originally obtained from food or feces associatedwith human food poisoning. In addition, the followingstrains were tested: E-13 (H16) provided by H. E.Hall of the U.S. Public Health Service, 027 (serologicaltype unknown) recovered in this laboratory frombeef liver, and 68900 (serological type unknown)and 79394 (H10) isolated from foods associated withtwo food poisoning incidents in Madison, Wis.Rabbit-negative strains included NCTC 8247, alsoprocured from B. Hobbs; F42 isolated at the U.S.Public Health Service laboratory in Cincinnati, Ohio;FD1 provided by Stanley Harmon of the Food andDrug Administration in Washington, D.C.; and215b isolated in this laboratory. The original isolationsof the rabbit-negative strains NCTC 8247 (H4),F42 (Hobbs type VII), and FD1 (not typable byHobbs sera) were made from food poisoning-asso-ciated food or feces. Strain 215b (H18) was originallyrecovered from beef liver.

Preparation of challenge dose. The cell suspensionswere prepared as previously described (7). Twodifferent carriers were used in presenting the challengedose to the subjects. In the first series of tests, foreach subject, 20 ml of cell suspension, providing asan average 3.3 X 1010 total viable cells and 2.5 X 108spores, was added to 80 ml of chocolate-flavoreddairy drink contained in a commercial-type waxcarton. The cell suspension was added just before theconsumption of the dairy drink by the subject. Thedairy drink was at refrigerator temperature.

In a second series of experiments, the carrier wascommercially canned beef stew. Cans (7.5 oz) of thestew were placed in a incubator at 46 C in sufficientnumbers so that one can was available for each sub-ject for each test. In the first experiments in thisseries, the required number of cans of beef stew wasremoved from the incubator, each was flamed withalcohol and opened, the inoculum (average, 2.5 X1010 total cells; 7.8 X 107 spores) was added to thecontents of the can, and the mixture was stirred witha sterile wooden tongue depressor. Immediatelythereafter the subject consumed the stew togetherwith buttered bread and milk or coffee, or both. Forall later experiments, the inoculum was added asep-tically to the warm stew, with subsequent incubationof the stew at 46 C for 3 hr. The average counts atthe time of feeding were 4.4 X 1010 total cells and3.0 X 108 spores.

Additional cartons of chocolate dairy drink orcans of beef stew were prepared exactly as were thoseconsumed by the subjects. These were used to deter-mine total viable cell and spore counts. For all countdeterminations, peptone water (0.1%) was used asthe diluent, and pour plates were made with TSN(BBL) agar. Spore counts were made after heat-shocking at 75 C for 20 min. Anaerobic incubationwas at 46 C, under a 901% nitrogen, 10% carbondioxide mixture.When cell-free preparations were substituted for

viable cultures as the challenge dose, for each test,each volunteer was fed 8 g of lyophilized concentratedculture filtrate prepared from a single strain of C.perfringens. To prepare the 48-hr-old culture filtrates(concentrated culture supernatant fluids) for use as

challenge feedings, the procedures described earlierwere utilized (6). For feeding, the lyophilized prepa-ration was resuspended in 100 ml of chocolate dairydrink. Submitted to the test were filtrates of rabbit-positive strains NCTC 8239, NCTC 8798, NCTC10239, and NCTC 10240, rabbit-negative strainsNCTC 8247, FD1, F42, and 215b, and uninoculatedmedium subjected to all of the procedures utilized inpreparing the culture filtrates.Management of the subjects. All subjects, both male

and female, were at least 21 years of age. Each personwas examined by the cooperating gastroenterologistwho also supervised hematological studies. Eachprospective subject presented fecal specimens at theState Hygiene Laboratory for examination for para-sites. Only when the physician had cleared the indi-vidual was he eligible to become an experimentalsubject.

Presenting the challenge dose to the subjects wasroutinely done late on Tuesday afternoon. The sub-jects were requested to eat their evening meal assoon as possible after having consumed the challengedose. In most instances, on the preceding Mondaymorning each subject submitted a preliminary fecalspecimen. A second fecal sample was obtained duringthe diarrheal episode if such developed (DS); ifdiarrhea did not develop, the subject submitted afecal sample from the first defecation to occur 24hr after consuming the challenge dose (24-hr S). Afinal fecal sample was obtained on the 6th day afterthe ingestion of the challenge dose (6-day S). Totalviable cell and spore counts for C. perfringens weremade on all fecal specimens, and the results wereexpressed as counts per gram (dry weight) of fecalmaterial. Fecal specimens were not obtained when theconcentrated culture filtrates from NCTC strains8247, 8798, 10240, and strain 215b were fed. Indi-viduals served as subjects once every 14 days.

For each feeding test, the subjects were dividedinto groups of four or five members. Each personwithin a group received an identical challenge dose.Each subject completed and returned a chart whichdescribed his response to the challenge dose. Intotal, 131 feeding tests were made.

Serological typing of C. perfringens isolated frompostchallenge fecal specimens. To assess whether theC. perfringens cells detected in the fecal specimenscollected after oral ingestion of the challenge doseby the subject (i.e., the DS or 24-hr-S fecal samples)belonged to the strain which had been recently con-sumed, a limited number of serological typing reac-tions were carried out. Specific antisera were em-ployed to confirm the identity of the C. perfringensisolates. In addition to using the antisera of the strainmost recently fed, antisera for strains consumed inearlier feedings were employed, to test for possible"carry-over" of the strains. For controls, similarserological testing was carried out on strains isolatedfrom feces of subjects before initial challenge. Theserological identification of the strains was determinedby the agglutination test (9). r'Heat resistance determinations. High-heat resistance

of spores of C. perfringens has, in some instances,appeared to be a characteristic associated with the

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food poisoning strains. However, it has been reportedthat both heat-sensitive and heat-resistant strainsmay cause food poisoning (22). It was, therefore, ofinterest to determine whether the rabbit-positive andrabbit-negative strains, which were fed to the sub-jects during the course of these experiments, differedin this regard.The Dloo value (time in minutes for inactivation of

90% of the spores at 100 C) of spores of each strainwas determined. Methods previously described (4)were used, except that TSN (BBL) instead of SPS(Difco) agar was used to determine viable counts.In the present experiments, the time required for thecontents of the thermal death time tubes to reach100 C ("come-up time") was 42 sec.

RESULTS AND DISCUSSIONThe results presented in Table 1 indicated

that, of the persons challenged by rabbit-positiveviable cells of C. perfringens, 57% reported one

or more of the symptoms commonly associatedwith C. perfringens food poisoning. Similarly,21% of persons fed rabbit-negative cultures re-ported such symptoms.

Closer inspection of the data suggests that theresponses of the human subjects, when fed rabbit-positive or rabbit-negative strains of C. perfrin-gens, were somewhat more divergent than first ap-peared (Tables 1 and 2). Occurrence of diarrheais probably the most objective of the symptoms.Based upon this criterion, 26 or 45% of the per-sons tested showed signs of C. perfringens foodpoisoning when fed rabbit-positive strains,whereas only 1 of 29 or 3.4% of those tested re-sponded similarly when rabbit-negative strainswere fed. In the latter instance, the one personreporting diarrhea indicated that the symptomdid not appear until 31 hr after the culture hadbeen consumed. In known C. perfringens food

TABLE 1. Relationi of response inl the rabbit ileum to response in human subjects subsequenzt to inigestionof challenzge doses of viable cells of Clostridium perfringens

Rabbitreaction' Carrier for cellsb

CDDBS-GCDDBS-1BS-GCDDBS-IBS-GCDDBS-GBS-GBS-GBS-GBS-GCDDBS-GBS-GCDDBS-GCDDBS-GCDD

BS + 0. 1%0 pep-tone water

No. of cells fed

Total count

6.6 X 1095.8 X 1010

1.1 X 10103.2 X 1094.1 X 1018

1.6 X 10114.7 X 10103.6 X 10101.3 X 10101.8 X 1094.5 X 10123.0 X 1010

3.2 X 10117.9 X 10101.2 X 1072.2 X 1010

7.9 X 10103.0 X 10107.1 X 10101.2 X 10103.0 X 10°

Spores

7.8 X 1081.6 X l0

1.5 X 10

1.5 X 1082.1 X 1084.2 X 1075.4 X 1066.4 X 1083.4 X 1072.7 X 1061.6 X 1083.2 X 1073.2 X 1085.2 X 105

1.9 X 1027.1 X 1084.6 X 1081.9 X 1081.9 X 1091.2 X 109

Human subjects

No. posi-tiveC/no.at test

2/53/31/51/42/44/51/41/41/43/43/44/43/44/43/50/40/41/51/41/40/31/50/4

No. diar-rhea/no.at test

2/53/30/51/42/43/51/41/40/42/43/42/42/44/41/50/40/40/50/40/40/30/50/4

a R+ (rabbit-positive) denotes strains of C. perfrinzgens, and products thereof, which have repeatedlybeen shown to produce fluid accumulation in the ligated ileum of young rabbits or overt diarrhea sub-sequent to intraileal injection of the nonligated gut, or both. Conversely, R- (rabbit-negative) indicatesthe absence of these responses in any regular pattern.

b Abbreviations: CDD, Chocolate dairy drink inoculated immediately before being fed; BS-G, previ-ous to feeding, cells grown for 3 hr in canned beef stew; BS-I, canned beef stew inoculated immediatelybefore being fed.

¢ Positive here defined as any person reporting symptoms of any type, after ingestion of challenge dose.

Strain no.

NCTC 8239NCTC 8239NCTC 8798NCTC 8798NCTC 8798NCTC 10239NCTC 10239NCTC 10239NCTC 10240NCTC 10240E136890002779394NCTC 8247NCTC 8247FD1F42F42215b215b

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poisoning outbreaks, the symptoms have beendescribed as appearing 6 to 24 hr after consump-tion of the peccant food (1). Therefore, the ques-tion arises as to whether the diarrhea experiencedby the person consuming the rabbit-negativestrain was associated with the experimental pro-cedure.

Table 2 also shows that the subjects fed rabbit-positive strains suffered more frequently and moreseverely from abdominal pain and reported othersymptoms in more instances than did those whoconsumed rabbit-negative strains, thus supportingthe ligated ileal loop test in rabbits as a means ofdistinguishing human food poisoning strains ofC. perfringens. In all cases, with the one exceptionnoted above, symptoms appeared in the subjectswithin 24 hr of administration of the test dose.Abdominal pain persisted for an average of 4.9 hrafter onset and diarrhea persisted for 5.7 hr.Another factor which apparently entered into

the response of human beings to ingestion of largenumbers of C. perfringens was the procedure em-ployed in preparing the challenge culture. The

most effective procedure appeared to be that ofplacing the viable cells in beef stew and allowingthem to grow for 3 hr at 46 C. Seventy-four percent of the persons fed rabbit-positive strains re-ported food poisoning symptoms, whereas noneof those fed rabbit-negative strains experiencedany untoward effects. Furthermore, 19 of the 31persons (61 %) showing positive responses haddiarrhea in degrees varying from mild to severe.No one in the group fed rabbit-negative strainsgrown in the beef stew exhibited diarrhea.The results obtained when cell-free concen-

trated filtrates were fed to the subjects are shownin Table 3. Four of 15 subjects developed diarrheawhen culture filtrates from rabbit-positive strainswere fed, whereas no diarrhea was reported by the16 persons who ingested filtrates prepared fromrabbit-negative strains. One subject fed filtratefrom a rabbit-positive strain vomited. These re-sults support the concept of an enteropathogenicfactor being responsible for C. perfringens foodp g symptoms (3, 6).A consideration of total viable cell and spore

TABLE 2. Symptoms reported by subjects subsequenit to consuiminlg viable cells of ClostridiuJm perfrinigents

Strain no. and carrier'

NCTC 8239, CDDNCTC 8239, BS-GNCTC 8798, CDDNCTC 8798, BS-INCTC 8798, BS-GNCTC 10239, CDDNCTC 10239, BS-INCTC 10239, BS-GNCTC 10240, CDDNCTC 10240, BS-GE13, BS-G68900, BS-G027, BS-G79394, BS-GNCTC 8247, CDDNCTC 8247, BS-GFD1, BS-GF42, CDDF42, BS-G215b, CDD215b, BS-G

CDDBS

Rabbitreac-tionb

R+R+R+R+R+R+R+R+R+R+R+R+R+R+R-R-R-R-R-R-R-

Symptomsc

Abdominal pain

Mild Moderate Severe

2, 42, 7.5

1, 62, 9

1, 61, 16

1, 63, 91, 41, 14.52, 5.5

1,21, 31, 5

1,91, 131, 20

2, 14.51, 11

1,121, 3.5

1, 9

Diarrhea

Mild jModeratel Severe

2, 2.51,12.5

1,192, 153, 161, 221,6

1,31,62, 61, 161, 31

1, 16.51 l, 13

2, 101, 11

3, 12

1, 41, 9.5

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Nausea

1, 6

2, 81,

1, 12

Headache

1, 221,2

1, 16

1, 14.5

Flatus

1

2

22

a See footnote b of Table 1.I See footnotes a of Table 1.c First value records number of cases; second value is the average number of hours after consumption

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TABLE 3. Response of human subjects to culturefiltrates prepared from selected strains of

Clostridium perfrinigensa

No. ofRabbit1 subjects

Source strain for filtrate responseb diarrheal/total no.challenged

NCTC 8239 R+ 2/4NCTC 8798 R+ 1/3NCTC 10239 R+ 1/4NCTC 10240 R+ 0/4NCTC 8247 R- 0/4FD1 R- 0/4F42 R- 0/4215b R- 0/4Uninoculated medium 0/4

a Challenge doses presented in chocolate dairydrink.

I See footnote a of Table 1 for explanation ofsymbols.

counts of C. perfringens in the feces of the experi-mental subjects reveals little which is definitive.Figure 1 illustrates the average and the range ofthe viable cell and spore counts which were en-countered. When the total cell and spore countsfor the preliminary fecal sample (which was ob-tained previous to the feeding of each challengedose of viable cells of C. perfringens) were takenas a base line, in 19 of 21 tests there was some in-crease in average total counts for the samples (DSor 24-hr S) secured subsequent to the feeding.Compared in the same way, the spore counts werealways somewhat higher in the time shortly afterthe subjects had consumed the suspensions ofcells. Six days after the feeding of the respectivechallenge doses to each group of subjects, thefecal total cell and spore counts for C. perfringens(6-day S) presented no regular picture when thecounts were compared with those of the prelimi-nary sample. There was no comparable increasein viable cell or spore counts after the feeding offiltrates as challenge doses. When the counts oftotal viable cells and spores of C. perfringens pergram (dry weight) of feces for those subjectswithin a group who developed diarrhea weresingled out and compared with the average for allmembers of the group, again no regular patternappeared.The tests carried out with Hobbs antisera for

comparing the identity of strains of C. perfringensfed with those recovered from the postchallengefecal specimens of the subjects indicated the fol-lowing. (i) When strain NCTC 8239 (H3) was fed,the strain was detected in two of three of the fecalsamples (DS or 24-hr S) obtained from the threesubjects postchallenge; detections for the other

D2 TOTAL CELL COUNT[j] SPORE COUNTH AVERAGE VALUE

(0_ -F-z

0106~~~~~~~~1105-~-

0) k o VI( )- ( u '

V rWt IJ Z I

_3 _c v_m' .I O NJO :M

PS R+ STr^AINS R- 6-DAY CONTRO(.SSTRAINS -

FIG. 1. Ranige antd average offecal counits of Clos-tridium perfrinigenis per gram of dry weight of feces.Abbreviationis: PS, sample obtainied previous to chal-lenige feedinig; DS, diarrhleal sample; 24-hir S, sampleobtainied subsequentt to conisumptiont of challenge dosewheni no diarrhlea occurred; 6-day S, sample obtainzed6 days after conzsumptiont of challenge dose. All chal-lenige doses were C. perfrintgenis viable cells exceptwhere nioted to be cell filtrates. See footniote a of TableI for additionzal defintitionl of symbols.

strains were, respectively, NCTC 8247 (H4) 0/4,NCTC 8798 (H9) 3/4, NCTC 10239 (H12) 3/4,and NCTC 10240 (H13) 4/4. (ii) At the outset ofthe feeding experiments, before the subjects hadconsumed any of the test cultures, strains sero-logically identical with NCTC 8247 and NCTC8798 were each detected in the feces of one person;subsequently, when these strains were fed, thefeces of these two persons were negative for therespective strains. (iii) In only one case did achallenge strain (NCTC 8798) apparently persistin the digestive tract for 2 weeks, to appear subse-quently after the feeding of another strain. (iv) Allcross-agglutinations with specific strains werenegative. (v) In some cases (NCTC 8239, oneinstance; NCTC 10239, 2 instances; NCTC 10240,one instance), isolates picked from plates inocu-lated with samples of feces heated at 75 C for 20min yielded positive agglutination tests, whereasthose isolates obtained from unheated feces werenegative, thus suggesting good sporulation in theintestines. The failure to detect strain NCTC 8247in the feces of subjects subsequent to challengemay be explained by the fact that, although viablecells were fed in the magnitude of 1010 cells, only

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102 spores were present. Since vegetative cells ap-pear to be more affected by acidity than spores(8a), it may be that this strain did not survivepassage through the stomach.Table 4 indicates the results obtained when D,oo

values were determined as a measure of heat re-sistance for the rabbit-positive and the rabbit-negative strains of C. perfringens. For rabbit-positive strains, the D1oo values varied from 0.70to 38.27. These results point out that strains pro-ducing a response in the ileum of the rabbit maybe of varied heat resistance. Fewer observationswere made on rabbit-negative strains, but the dataavailable suggest that such strains are charac-terized by a low degree of heat tolerance. How-ever, evidence has been presented by Sutton andHobbs (22), among others, that C. perfringensfood poisoning may be caused by heat-sensitiveas well as heat-stable strains of C. perfringens.During 1968 and for the period of January to

June 1969, the attack rate for persons exposed tothe possibility of contracting C. perfringens foodpoisoning was stated to be, overall, 33.9 and56.7%, respectively (16, 17). In the present ex-periments, attack rates obtained for the selectedpopulation which was fed rabbit-positive strainswere essentially identical (57%) with the 1969figure. Under conditions most closely resemblinga "natural" outbreak (i.e., viable cells allowed togrow for 3 hr in beef stew held at 46 C), the attackrate when the criterion for illness was diarrhea was61.0% for subjects fed rabbit-positive cells and0% for those fed rabbit-negative strains.When diarrhea occurred in members of the test

groups fed viable cells, the range of time of ap-

TABLE 4. Heat resistance (Dloo value) of strains ofClostridium perfringens which have demonstrated

different responses in the ileum ofthe rabbit

Strain no. Rabbit D1oo valuereactiona (min)

NCTC 8239 R+ 20.61NCTC 8798 R+ 27.45NCTC 10239 R+ 12.74NCTC 10240 R+ 1.59E13 R+ 38.2768900 R+ 0.70027 R+ 1.4879394 R+ 8.93NCTC 8247 R- NTbFD1 R- 0.58F42 R- 1.16215b R- 0.97

a See footnote a of Table 1 for explanation ofsymbols.

b Not tested. Strain did not produce spores insufficient numbers to permit test.

pearance was 2.5 to 22 hr, with one exception,noted above, in which the period of incubationwas 31 hr. Despaul (1) stated that the time for ap-pearance of symptoms after consumption of foodcontaminated with large numbers of viable C. per-fringens cells is 6 to 24 hr. Hauschild and Thatcher(13) reported diarrhea to occur between 4 and 9hr after cell ingestion and to continue for periodsof 4 to 21 hr. The four subjects demonstratingdiarrhea subsequent to the ingestion of concen-trated culture filtrates reported the appearance ofthe symptoms in 2 to 2.5 hr after consumption ofthe challenge.

In the laboratory study done by Hauschild andThatcher (13), certain food poisoning symptoms,typical of those induced by the presence of largenumbers of C. perfringens in food, were inducedin five of six human volunteers subsequent tofeeding 4 X 109 to 6 X 109 vegetative cells ofstrain S-79. For feeding, the cells were suspendedin cooked milk and the subjects consumed anadditional 100 ml of milk, apparently followedimmediately by their eating lunch. In our experi-ments, the effects produced by feeding the viablecultures to the subjects seemed to be enhanced ifconsiderable amounts of food were eaten at thetime the cultures were presented. A logical ex-planation for this observation would appear to bethe buffering action of food on the acidity of thestomach. Strains of C. perfringens have beenshown to be susceptible in some degree to theaction of acid in the range of thepH of the stom-ach (8a, 10). In our laboratory, it has not beenpossible to induce diarrhea in rabbits, a speciesknown to maintain low pH value in the emptystomach, by oral challenge (5). Obviously, thefate of the cells in the stomach is closely relatedto the sequential effect in the gut.

Interpretation of data relative to the number oftotal viable cells or spores of C. perfringens, orboth, in the feces of "normal" people and of per-sons who have experienced diarrhea as a conse-quence of consumption of large numbers of theorganisms in food is difficult. Variation exists be-tween individuals and for a single individual atdifferent times. The average viable counts we ob-tained for total cells and for spores of C. perfrin-gens in feces during the prechallenge dose periodwere the same or higher than those that have beenreported for the general population by other in-vestigators. The range of average total cell countsfor groups for all experiments was 1.5 X 105 to9.2 X 107 and for spores was 1.1 x 104 to 2.3 x107 per g of dry feces. These values should be con-sidered approximate since the effect of heat shock,and hence the relationship between total countsand spore counts, cannot be definitively estab-

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lished; nevertheless, the figures presumably re-flect, with some accuracy, the concentration ofviable organisms which existed.

Sutton (21) reported for an assorted humanpopulation a median of 3.7 X 104 and a mean of1.40 X 105 as the viable count for C. welchii (per-fringens) per g of feces. Earlier (20) he had foundthat participation in communal feeding and poorhygienic conditions influenced the carrier-rate forheat-resistant C. perfringens in human feces. In alater study, Sutton and Hobbs (22) stated thatfecal median counts for C. welchii (perJringens)of 103 per g of feces are within normal limits. In aseries of 57 samples of feces from normal humanintestines, Nakagawa and Nishida (15) found avariation of from 103 or less to 106 or more cells ofC. perfringens per ml of feces. Hauschild andThatcher (13) reported a concentration of 104 to3 X 104 spores of C. perfringens per g of feces ofsubjects before cell ingestion. Presumably, thevalues reported in the literature were based onmoist fecal material in contrast to our valueswhich are expressed in terms of dry fecal material.

Questions which might arise concerning otheraspects of our study include the possible develop-ment of immunity in our subjects, many of whomwere tested eight times, and the psychologicaleffect of suggesting to the patients symptomswhich they might encounter. Dische and Elek (2)observed earlier that repeated feedings of the samesubject with viable cultures of C. welchii (perfrin-gens) did not mitigate the symptoms occurring atthe time of the second or third challenge dose.Therefore, they concluded that ". . . there was noindication that clinical immunity had developed"(reference 2, p. 73). Our findings supported thoseof Dische and Elek.

It is difficult to assess the effect of suggestibilityin regard to the symptoms reported by the sub-jects after consuming challenge doses of viable C.perfringens. It is possible that subjective symp-toms reported were not associated with the inges-tion of the test cultures. For this reason, thoseinstances in which diarrhea occurred are regardedas especially significant.

Finally, the correlation of results obtained inthese human feeding studies with those acquiredthroughout the series of studies in which rabbitsand monkeys have been utilized deserves com-ment. A summary of the findings resulting fromthe injection of the ligated loops and the non-ligated ilea of rabbits with viable cells, cell ex-tracts, or culture filtrates for selected strains ofC. perfringens was presented in an earlier publica-tion (6). Suffice it to state here that both viablecells and cell-free products of strains NCTC 8239,NCTC 8798, NCTC 10239, NCTC 10240, E13,68900, 79394 and 027 produced, with significant

regularity, an accumulation of fluid in ileal loops,or overt diarrhea, or both, subsequent to chal-lenge in young rabbits. Viable cells of the samestrains produced diarrhea in 57% of the 35 mon-keys tested (strain 68900 not tested; 7) and in 45%of 58 persons in the current study. Culture filtratesprepared for each of four strains (NCTC 8793,NCTC 10239, NCTC 10240, and NCTC 8239)produced, overall, diarrhea in 8 of 20 monkeysand vomiting in 11 of 20 monkeys (7). The con-centrated filtrates obtained from the same fourstrains produced diarrhea in 4 of 15 human sub-jects and vomiting in 1.Of the rabbit-negative strains, NCTC 8247,

F42, and FD1 also were first isolated fromsources (i.e., food or feces) associated with C. per-fringens food poisoning. Neither viable cultures ofthese strains nor, in a limited number of trials,culture filtrates produced a response in rabbittests or in any of 20 tests in monkeys (7). Inhuman beings, only 1 somewhat doubtful positiveresponse was recorded in 22 challenges with viablecells, and an additional 12 challenges made withculture filtrates prepared from the rabbit-negativestrains were also negative. Viable cells or filtratesof strain 215b, which was originally isolated frombeef liver, did not induce a positive response inrabbits, monkeys, or human beings.The observations concerning the rabbit-nega-

tive strains of C. perfringens suggest that thefactor responsible for food poisoning has, in somecases, apparently been permanently lost duringrepeated subculturing in the laboratory. Resultsobtained with other strains indicate that the pro-duction of the diarrhea-inducing factor may occurirregularly and thus these strains would seem to beintermediate between the rabbit-negative and therabbit-positive groups.

ACKNOWLEDGMENTS

Published with the permission of the Director of the ResearchDivision of the College of Agricultural and Life Sciences, Uni-versity of Wisconsin, Madison.

This study was supported by Public Health Service grant 5-R01-FD-00068-9 from the Consumer Protection and Environ-mental Health Service and by contributions from memnber in-dustries of the Food Research Institute.

The antisera were provided through the generous cooperationof Betty Hobbs. She further cooperated with us by determining theserological type of certain of our strains. We gratefully acknowl-edge this assistance.

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