The role of enterotoxin in Clostridilcm perfringens type A enteritis

A. H. W. HAUSCHILD, L. NIILO, AND W. J. DORWARD Research Laboratories of the Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Canada

and Canada Departtner~t of Agriculture, Animal Diseases Research Institlrte (Western), Letlibridge, Alberta

Accepted April 2, 1971

HAUSCHILD, A. H. W., L. NIILO, and W. J. DORWARD. 1971. The role of enterotoxin in Clostridiltm perfringelis type A enteritis. Can. J. Microbiol. 17: 987-991.

Vegetative cells of three strains of Clostridiilrn perfringens type A, free of erythema1 activity, were suspended in fresh medium and injected into ligated intestinal loops of lambs. Examination of the loop contents after 6.5 h showed significant accumulation of fluid, multiplication and sporulation of C . perfringens, and erythemal activity in both the supernatant fluids and the sediments.

The erythema1 factor produced in vivo was identical with the erythemal factor of sporulated cells of C . perfringerrs grown in vitro, and again caused accunlulation of fluid when transferred into ligated intestinal loops of recipient lambs. Immune rabbit serum prepared against extracts from sporulated cells of C. perfringeris, and absorbed with extracts from vegetative cells of the same strain, completely neu- tralized the enterotoxic and erythemal activities of the in vivo-produced factor.

It is concluded that the erythemal factor is the causative agent in C . perfringens type A enteritis. The term "Clostridilim perfringens enterotoxin" is proposed to characterize the erythemal factor.

Introduction Clostridiun? perfiirzgens type A causes food

poisoning in man characterized by diarrhea and abdominal pain (16). The disease has been reproduced in human volunteers by ingestion of large numbers of bacterial cells (1, 15). Diarrhea has also been induced in rabbits (4) and lambs (1 1) by administering vegetative cells, suspended in nutrient medium, into the intestines. In- jections of similar cell suspensions into ligated intestinal loops of rabbits (6) and lambs (12) have resulted in accumulation of fluid, and dis- tention of the ligated loops. The suitability of the loop technique for the study of C. perfiingens type A enteritis has been demonstrated in a number of publications (4, 5, 12, 14). It was shown recently (13) that the causative factor in the diarrhea of lambs was not introduced as an ingredient of the vegetative cells, but was pro- duced in vivo by C. perfiingens.

Diarrhea in rabbits and lambs and fluid accumulation in ligated intestinal loops of the same species have also been produced with ex- tracts from sporulating cells of C. perfiingens grown in vitro (5, 13, 14). The causative com- ponent of the extract is heat-sensitive, pre- cipitable with ammonium sulfate, antigenic, and non-dialyzable (5, 14), and causes erythema in the skins of guinea pigs and rabbits (9). In young cultures it occurs exclusively within the cells (13, 14).

Received January 18, 1971

The work described here shows that the enteropathogenicity of C. perfritzgeizs in ligated intestinal loops of lambs is associated with the production of an erythemal factor which is identical with the cellular erythemal factor produced in vitro, and that this factor is the causative agent in C . perfringens enteritis.

For reasons stated in the Discussion, the term "Clostridium perfiingens enterotoxin" is being proposed for the erythemal factor and will be used in this paper.

Materials and Methods Experimental At~it~rals

Six-months-old Cheviot lambs weighing 22 to 25 kg were used. The animals were starved for 42 h before sur- gery. The ligated intestinal loop technique was used as described previously (12, 14), except that the length of the loops infected with vegetative cells (Table 1) was about 30 cm. The animals were killed either 6.5 h after injection of vegetative cells, or 5 h after injection of enterotoxin.

Productiotz of Etzterotoxin itz Vivo The strains of C.perfringetrs type A (8239, 80535, S-79)

and the growth conditions have been described previously (13). In each of two animals, nine ligated loops, three per test strain, were infected with mixtures of 1.0 ml of 16-h vegetative culture in medium CP-2V (12) and 6.0 ml of medium CP-2VS (12). The control loops received 1.0 ml of medium CP-2V and 6 ml of CP-2VS.

The loop contents, harvested after 6.5 h, were filtered through a double layer of cheesecloth. Total bacterial cells and viable spores in the filtrates were enumerated as described previously (13, 14).

To test both the supernatant fluids and the clostridial cells of the filtrates for enterotoxin, the filtrates were centrifuged at 12 000 relative centrifugal force (r.c.f.) for

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988 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17. 1971

10 min, and the supernates (designated loop supernatant fluids) were collected individually. For each animal, the sediments from each group of three loops infected with the same strain were pooled, suspended in 0.85% NaCI, ruptured with a sonifier (13), and centrifuged at 12 000 r.c.f. for 10 rnin. The supernatant fluids (designated sedi- ment extracts) were collected, and the sediments dis- carded. The erythemal activities of the loop supernatant fluids and of the sediment extracts were assayed in guinea pigs (9).

The loop supernatant fluids contained < 0.5 to 8 ery- thema units per milliliter. Samples with activities of 6 to 8 units/ml were concentrated by dialysis against poly- ethylene glycol (PEG) 20 000 to contain 32 ? 16 units/ ml. The sediment extracts, except for one with no detect- able erythemal activity (Table I), were diluted with 0.85% NaCl to contain 32 + 16 erythema units/ml. Preparations containing less than 2 erythema units/ml (sediment extract 80535 and supernatant fluid 8239) were used as controls (see Fig. 1 and Table 3).

Antisern The sporulation of C. perfi.inigeris in DS medium (3):

and the methods for the preparation of cell extracts and of rabbit iinmune sera against the cell extracts have been described (13).

Immune serum prepared against extracts from sporu- lated cells of strain 8239 was absorbed with extracts from vegetative cells of C. perfringens grown in medium CP-2V by mixing the serum 3 :1 with a cell extract of strain 8239 (Table 3) or 1 :l with a cell extract of strain 80535 (Fig. 1 ; b, 4. The serum -cell extract mixtures were kept at 22°C for 2 h and centrifuged at 12 000 r.c.f. for 10 rnin. The sediments were discarded. The treatment eliminated all the major precipitin bands found in immunodiffusion against extracts from sporulated cells of strain 8239, with the exception of the enterotoxin-specific band (Fig. 1). The immunodiffusion technique has been described pre- viously (17).

The rabbit immune serum to be used for neutralization of enterotoxin produced in vivo (Table 3) was concen-

trated by dialysis against PEG 20 000 to about 50% of its original volume. The concentrated serum contained 50 to 60 antierythema units/ml. Normal serum, concentrated in the same way, was used as control.

Activity of it1 Vivo Produced Enrerotoxin in Ligated Zniesti- ~zal Loops

Concentrated supernatant fluids from infected loops. and sediment extracts were mixed 1 :1 either with absorbed and concentrated immune serum, or with concentrated normal serum. The mixtures were kept at 22°C for 30 rnin and injected into ligated intestinal loops in volumes of 2.0 rnl per loop. The loop fluid volumes were measured 5 h later.

Identificatiorz of Enrerotoxi~z Prodrlced in Vivo An extract from the pooled sediments of three ligated

loops infected with vegetative cells of strain 8239 was compared with an extract from sporulated cells of strain 8239 grown in DS medium (13). Samples of both extracts were subjected to the treatments listed in Table 2. Dia- lysis was carried out at 5OC for 24 h against 0.85% NaCI. The procedures for ammonium sulfate precipitation and neutralization with iinmune serum prepared against cell extracts of sporulated strain 8239 were as described pre- viously (14), except that 400 mg of (NH4)2S04 per milli- liter of extract was used. Samples of extract were also mixed 1 :1 with C. perfringens type A diagnostic anti- serum (Wellcome Laboratories, Beckenham, England) and kept at 22OC for 30 min.

Results

Production of Eiiterotoxitz In Vivo The cells of the three strains of C. petfringens

injected into ligated intestinal loops had no spores and contained no erythemal activity. Table 1 shows that after 6.5 h each of the strains had grown and sporulated, and significant

TABLE 1 Growth and sporulation of C. perfringenls, and production of erythemal activity in ligated intestinal

loops of lambs

Mean values per loop after 6.5 h

Total erythemal activ., units

Inoculum, C. per%. cells Lamb Fluid vol., Total Total viable Sup. Sedim.

straln no. No. ml* cells* spores* fluid* extr.**

8239 4.4X108 1 33 3.4XlOlo 2.3X109 11 0 24 2 48 n.d. 2.9X109 10 43

80535 1.4X109 1 29 1 . 8 ~ 1 0 1 0 2.6x109 < 10 153 2 29 n.d. 9.0X107 30 < 6

S-79 6.2X108 1 34 1.2X1010 4.0X109 < 10 86 2 38 n.d. 2 . 0 ~ 1 0 9 10 89

Medium 0 1 8 3.2X109 4.8X107 < 2 n.d. only 2 3 n.d. 2.0X106 < 1 n.d.

*Means from individual assays of three loops per group. ?Sediments from three loops per group pooled before extraction and assay. n.d. Not determined.

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FIG. 1. Precipitation bands in agar gel. (S4) Ininit~ne serum against extract fro111 cells of C. per.h.itrgetrs, strain 8239, in vitro; (S4T) as S4, but absorbed with extract from vegetative cells, strain 80535; (39), (35), (79) strains 8239, 80535, and S-79, respectively; (VIT) extract from in vitro-grown cells; (SED) extract frol-11 loop sediment; (SUP) loop supernatant fluid; (f) containing 32 + 16 erythema units/ml; (-) containing < 2 erythema unitsjml.

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HAUSCHILD ET AL.: CLOSTRIDIUM PERFRINGENS ENTERITIS 989

amounts of fluid had accumulated. The cells and able erythemal activity. The supernatant fluids spores recovered from the infected loops and the showed considerable variation in erythemal viable spores recovered from the control loops activity, not only between different animals were essentially all C. pefiin-pens, but the total (Table I), but also between corresponding loops cells from the control loops consisted mainly of within the same animal. non-clostridial forms. cblonies derived f;om spores from the control loops were almost all beta-hemolytic on sheep blood agar, while colonies derived from the cells and spores from the infected loops were partially hemolytic. The three strains used for infection of the loops also produced partial hemolysis only (14). It is un- likely, therefore, that the cell and spore counts of the infected loops were significantly affected by the normal gut flora.

With one exception, each group of three loops had a mean viable spore count of > 109 per loop, and their sediments contained 24 to 153 ery- thema units per loop (Table I). The exception was a group infected with strain 80535 which had a relatively low spore count and no measur-

TABLE 2

Common characteristics of enterotoxin from cells of C. perfringet~s grown in sporulation medium, and in ligated

intestinal loops of lambs

Erythema1 activity % of original

Treatment of cell extract activity

None 100 Heat; 60°C, 10 min < 2 Dialysis (non-dialyzable) 8@100 (NH&S04 precipitation 80-100 Imr~iune seruni against extract

from sporul. cells < 5 C. perfringens type A

diagnostic antiserum 8fL100

Identity of Enterotoxins Produced In Vitro and It? Vivo

Table 2 shows a number of characteristics common to both the enterotoxin extracted from sporulated cells of C. perfringens, strain 8239 grown in vitro, and the enterotoxin extracted from sediments of ligated intestinal loops that had been infected with vegetative cells of the same strain.

The enterotoxin of sporulated cells of C. per- $ringens grown in vitro is characterized by a specific precipitin band in agar gel (17). The pre- cipitin patterns of supernatant fluids from ligated intestinal loops infected with the three different strains, and of extracts from the corresponding sediments, were compared with the pattern of the extract from in vitro-grown cells (Fig. I ) . Each preparation contained either 32 k 16 (+) or < 2.0 (-) erythema units per milliliter. Figure 1 shows a specific precipitin band common to each of the preparations with erythemal activity. This band was not produced by any of the prepara- tions with little or no erythemal activity, i.e. ex- tracts from non-sporulated cells of strain 80535 grown in vitro (35 VIT), concentrated super- natant loop fluid (39 SUP), and sediment extract (35 SED). Supernatant fluids from loops infected with strain S-79 were not tested because none of these contained more than 2.0 erythema units/ ml.

TABLE 3

Neutralrzation of In vivo-produced enterotoxin of C. perfringens type A

Serum in mixture

Normal Immune* Preparation of Erythema No. loops per

enterotoxin Strain units/ml toxin-serum mix Mean fluid vol., ml

Loop sup. fluid 8239 32+16 2 30 0 < 2 4 3

80535 32+ 16 2 27 3 Loop sedim.

extract 8239 32k 16 3 30 1 80535 32+ 16 3 25 3

< 2 3 0 S-79 32+ 16 3 23 2

NOTE: Mixtures of enterotoxin and serum ( I : I ) were kept at 22°C for 30 min and injected in volumes of 2.0 ml per loop. Fluid accumulation was measured after 5 h.

'Prepared against in vitro-produced enterotoxin.

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990 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

Activity of In Vivo Produced Enterotoxin Trans- ferred itzto Ligated Intestinal Loops, and Its Nezitralization rvitlz Itllrnwze Serlun

Table 3 shows that each preparation contain- ing about 32 erythema ~inits/ml, whether con- centrated supernatant loop fluid or sediment extract, again caused significant acc~im~~lation of fluid in the ligated loops. This enterotoxic activ- ity war completely neutralized with immune serum prepared against the in vitro produced enterotoxin. The preparations with less than 2 erythema units/ml caused no fluid accumulation.

Discussion The results have shown that C. perjiingetzs

grows, sporulates, and produces an erythemal factor in the ligated intestinal loop. The question is whether or not this factor is significantly in- volved in the observed accumulation of fluid in the infected loop. That it is likely to contribute to the accumulation of fluid is indicated by our finding that the properties of the erythemal factor produced in vivo are identical with those of the factor produced in vitro. The latter has been shown to cause fluid accumulation in the ligated intestinal loop (5, 14). In addition, the loop supernatant fluids and sediment extracts with erythemal activity again produced fluid accumu- lation in recipient loops, and their enterotoxic activity was completely neutralized with immune serum against the in vitro produced toxin. There is no doubt, therefore, that the in vivo produced toxin is significantly involved in enteropatho- genicity.

There was considerable variation in the entero- toxin contents of the infected ligated loops, particularly in the supernatant fluids. However, it is probable that the enterotoxin content at a given time is largely dependent upon the develop- mental stages of C. perfringens, and these are likely to vary considerably between equally treated loops as indicated by the differences in sporulation. One cause of variation in the entero- toxin content may be the outgrowth of newly forined spores, res~ilting in the breakage of the sporangium and rapid release of enterotoxin into the intestinal fluid.

Clostridizrm perjringetzs, strain 80535, was in- criminated in a food poisoning outbreak (7) and was later used to reproduce food poisoning in human vol~inteers (10) and diarrhea in lambs (I I). However, in contrast to strains 8239 and

S-79, this strain failed to produce any enterotoxin in vitro (13, 14). The failure was explained by the inability of strain 80535 to sporulate in DS medi- Lim (13). Previous work (1 1, 14) and the data in Table 1 have shown that strain 80535 readily sporulates in the intestine. This work now shows that the conditions of the ligated intestinal loop allow the strain to produce enterotoxin as well as spores.

Enterotoxin has been defined as "a cytotoxin specific for the cells of the mucous membrane of tlie intestine" (Stedman's Medical Dictionary, 21st ed., 1966), "a toxin arising in the intestine" (Dorland's Illustrated Medical Dictionary, 24th ed., 1965), and "a toxic substance produced by nlicroorganisms that is responsible for the gas- trointestinal symptoms of some forms of food poisoning" (Webster's 3rd New International Dictionary, 1968). The toxin described here is produced in the intestine by C. perfringens and causes gastrointestinal symptoms. Its enterotoxic activity indicates that the natural site of action is either the intestinal mucosa or associated tissue. Furthermore, its gross enterotoxic action is sim- ilar to the gross actions of the enterotoxins of Escherichia coli (8, 18), and Vibrio cholerae (2). Therefore, we propose the term "Clostridiurn per- fringens enterotoxin" to characterize the eryth- emal toxin.

Acknowledgments The authors are indebted to Drs. S. E. Mag-

wood and H. Pivnick for their cooperation, to Mr. L. H. Dow. Mr. R. Hilsheimer. and Mr. W. V. Krampl for ;aluable technical assistance, and to Dr. H. W. Smith for suggesting the term "en- terotoxin" to characterize the erythemal toxin of

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HAUSCHILD ET AL.: CLOSTRIDIUM PERFRINGENS ENTERITIS 99 1

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18. SMITH, H. W., and S. HALLS. 1967. Studies on Escherichin coli enterotoxin. J . Pathol. Bacteriol. 93: 531-543.

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