Immunization of sheep against experimental Clostridiurn perfringens type A enteritis

L. NIILO, A. H. W. HAUSCHILD, AND W. J. DORWARD Carzada Department of Agricr~ltrrre, Animal Diseases Research Znstitrrle (Western), Lethbridge, Alberta

and Research Laboratories of the Food and Drug Directorate, Department of Natior~al Health and

Welfare, Ottawa, Canada

Received October 21, 1970

NIILO, L., A. H. W. HAUSCHILD, and W. J. DORWARD. 1971. Immunization of sheep against experimental Clostridirrm perfringens type A enteritis. Can. J. Microbial. 17: 391-395.

Lambs were injected parenterally with formalized cell extract, or bacterin, prepared from sporulating cultures of an enteropathogenic strain of Clostridiurnperfringens type A. This procedure induced an anti- body in the serum which neutralized the erythema1 and enteropathogenic activities of the cell extract. Immunodiffusion of the serum against the toxic cell extract produced a single, specific band associated with enteropathogenic activity. Protective immunity was not evident when the immunized lambs were challenged by the intraduodenal and the ligated intestinal loop methods with toxic cell extract or with whole cells of C. perfringens. Non-immunized lambs, challenged nine times by the intraduodenal method over a period of 70 days, had no measurable antibody and remained susceptible to experimental enteritis.

Introduction Clostridiutn perfringens type A may cause

enteritis in man (12) and animals (8). The disease has been reproduced experimentally in sheep and rabbits by intraintestinal administration of live cells in a suitable medium, cells grown in a spor- ulation medium, and extracts from sporulated cells (4, 8, 10). These preparations also produce fluid accumulation in ligated intestinal loops of lambs and rabbits (3, 5, 9, 11). The enteropatho- genic factor of the extract from sporulated cells also produces erythema in the skin of rabbits and guinea pigs; this factor can be assayed quanti- tatively by the skin test (7).

Our earlier work (7, 11) showed that in vitro incubation of C. perfringens cell extract with antiserum prepared in rabbits completely neu- tralized the activity of the enteropathogenic fac- tor, both in the ligated intestinal loops of lambs and in the guinea pig skin. This work was con- ducted to ascertain whether or not parenterally- induced circulating antibody was protective against Cperfringens type A enteritis and whether one or more attacks of such enteritis would confer a degree of immunity against subsequent challenges.

the same breed. Both breeds appeared to respond equally to the treatments described below.

Immuniza tion Clostridiutn perfringens type A, strain 8239 (NCTC),

was grown in sporulation medium of Duncan and Strong (2) for 5 h at 37C. A formalized extract was prepared from sonically disrupted cells as described previously (10, 11). For the preparation of bacterin, the 5-h cells were washed, suspended to about 3% concentration in 0.85% NaC1, and treated with formalin (1 1).

The preparations were mixed 1 :1 (v:v) with Freund's complete adjuvant (Difco), and 6.0-ml volumes of the mixtures injected at weekly intervals over a period of 6 weeks. The first four injections were made at multiple subcutaneous sites; the last two doses were given intra- muscularly. A 2-week interval was allowed between the last injection and the first challenge.

The effect of repeated attacks of experimental enteritis on possible development of immunity was tested on four lambs by the intraduodenal administration of alternating doses of cell extract (10) and vegetative (whole) cells in CP-2VS medium (9). Each of these animals received a total of nine doses over a period of 70 days.

Challenge The surgical procedures for the intraduodenal intuba-

tion and the ligation of the small intestine were described previously (8, 9). Thirty milliliters of 5-h culture of strain 8239 in CP-2V medium was carefully mixed with 220.0 ml of deaerated CP-2VS medium and administered as a challenge via the intraduodenal tube (9). The number of cells introduced oer animal was about 2.7 X 1010. Enteropathogenic cell extract of the same strain grown

Materials and Methods in sporulation medium was administered in a diluted Esperirrzerztal Arzirnals form: 8.0 ml of the extract in 250.0 ml of 0.85% NaCl

Seven-month-old Suffolk lambs, weighing 30 to 35 kg per animal. Animals that were challenged repeatedly were and 6-month-old Cheviot lambs, weighing 20 to 25 kg, allowed to rest for at least 3 days between the challenges, nere used. In each experimental group, the animals were and for 5 days between the last challenge and the terminal matched by a co~nparable number of control aninlals of experiment by the ligated intestinal loop method.

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3 92 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

Ligated intestinal loops were injected with the same materials as used for the intraduodenal challenges. Meas- ured amounts of the cell extract were injected with 3.0 ml of 0.85% NaCl per loop. The volumes of whole cell: medium mixtures were 3.0 to 3.5 ml per loop and con- tained 1.8-4.5 X 108 cells. Both preparations were tested in the same animal, randomizing the injection sites. There were at least three control loops per animal; these were injected with 5.0 ml of 0.85% NaCI, 3.0 ml of medium CP-2VS (negative controls) and a minimum of 0.2 ml of the cell extract in 3.0 ml of saline (positive contro!). The loops were examined 6.5 h after inoculation. A loop reac- tion was termed positive when it contained at least 10.0 rnl of fluid, and when the adjacent interloops were empty.

Serum Antibody Assays Serum antibody against the enteropathogenic factor

was assayed by (i) immunodiffusion and (ii) neutraliza- tion of the erythema1 activity (7).

The immunodiffusion method is described below; the precipitin titer was expressed as the reciprocal of the highest dilution of serum which produced a specific precipitin band against undiluted cell extract.

Mixtures of cell extract containing 160 erythema units/ ml with varying amounts of serum were kept at about 22C for 45 min, diluted with 0.85% NaCl to two erythema units/ml and injected intradermally into guinea pigs in volumes of 0.05 ml per site (7). One antierythema unit was arbitrarily assigned to the amount of antibody that completely neutralized one erythema unit.

Neutralization of the Enteropathogenic Factor with Serrrm Enteropathogenic cell extract and serum (S4) from a

lamb immunized with formalized cell extract were mixed at various ratios (Table 2) and incubated at about 22C for 40 min before injection into ligated intestinal loops, or injected simultaneously without prior incubation.

Immunodiffrrsion Double diffusion precipitation tests of Ouchterlony

(15) were performed in 1% Noble agar (Difco). The wells were 6 mm in diameter and 6 mm apart. The diffusion reaction was allowed to develop at about 22C and it was checked periodically for up to 72 h. In most cases, satis- factory readings were obtained in 48 h. For titration of antibody, the serum samples were tested in twofold dilu- tions up to 1 :64 against undiluted cell extract.

Immune serum S4 was used to test the identity of precipitinogen in formalized and in toxic extracts from enteropathogenic cells of strains 8239 and S-79 (10, 11) as well as in extracts from non-enteropathogenic cells of several strains of C.perfringens. The latter strains were not

reacting in ovine intestinal loops or in guinea pig skin. For greater specificity to the enteropathogenic factor, the S4 serum was treated with the non-enteropathogenic cell extract of C. perfringens, strain 80535 (10, 11) grown in medium CP-2V by mixing equal volumes of the serum with this extract, incubation of the mixture at 37C for 2 h and centrifugation at 12 000 rcf for 15 min. The resulting supernatant fluid (S4T) eliminated a number of undesir- able precipitation bands.

The enteropathogenic factor of C. perfringens, strain 8239, purified by gel and ion exchange chromatography, was also tested in immunodiffusion. The detailed pro- cedure for the purification of the antigen will be published elsewhere.

Results Serum Antibody Response

Figure 1 shows the precipitation bands of untreated and formalized cell extract of C. perfringens, strain 8239, against serum S4. Al- though slightly different in position and sharp- ness, the bands were essentially the same. The precipitation bands obtained with sera from animals immunized with bacterin were similar but less intense and less distinct.

Figures 2 to 7 show precipitation patterns of various cell extracts tested against the absorbed (S4T) and the original serum (S4). The ente- ropathogenic cell extract of strain 8239 produced one major and two minor bands that were not produced by the non-enteropathogenic extract of strain 80535 (Figs. 2 and 3). These bands were also absent in the precipitation pattern of four other strains of C. perfringens not possessing enteropathogenic activity (Figs. 4 and 5), but the major band was produced by the enteropath- ogenic extract of strain S-79 (Figs. 6 and 7). The purified enteropathogenic factor from strain 8239 produced a single line which was identical with the major specific band of the crude extracts of strains 8239 and S-79 (Figs. 6 and 7).

Immunodiffusion precipitin titers, based on the highest dilution of serum producing detect- able reaction against undiluted cell extract, are

FIG. 1. Precipitation bands in agar gel. (39EX) C. perfringens, strain 8239, cell extract; (39TOX) the same preparation, toxoided; (S4) immune serum. FIG. 2. Strain 8239 extract (39EX) compared with extract from nonsporulated cells of strain 80535 (35EX). FIG. 3. Antigens as in Fig. 2, but serum (S4T) absorbed with strain 80535 extract. FIG. 4. Cell extract antigens of various strains of C. perfringens. (75) "classical" strain, bovine origin; (84) "classical" strain, human origin; (18) "classical" strain, bovine origin; (Nl) normal bovine isolate; (35) strain 80535; (39) strain 8239. FIG. 5. Antigens as in Fig. 4, but serum (S4T) absorbed with strain 80535 extract. FIG. 6. Strain 8239 extract (39) compared with purified extract of the same strain (39P) and with strain S-79 extract. FIG. 7. Antigens as in Fig. 6 tested against absorbed serum (S4T). FIG. 8. Part of the small intestinal tract of a lamb showing positive (distended with fluid) and negative loops inoculated with (a) CP-2VS medium; (b) cell extract + immune serum, incubated; (c) whole cells + CP-2VS medium; (d) 0.85% NaCl; (e) ce!l extract; (f) cell extract + immune serum, incubated; (g) and (h) whole cells + med~um; (i) cell extract + Immune serum, not incubated. The dots indicate ligatures.

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NULO ET AL.: IMMUNIZATION OF SHEEP 393

shown in Table 1. No precipitin reaction oc- repeatedly over the 70-day period by intra- curred with preimmunization sera, sera from the duodenal administration of cells and cell extract control animals, or sera from lambs treated to produce experimental enteritis.

TABLE 1 Postimmunization serum antibody in experimental sheep

Serum antibody

Immunizing Precipitin Antierythema inoculum titera titerb

Formalized cell extract, parenteral 45+16 47+12

Formalized bacterin, parenteral 20+7 15+5

Cells: medium and cell extract, intraduodenal 0 < 4

None 0 NDc

OReciprocal of highest rcacting senun dilution in agar gel, mean + standard deviation.

bAnti-erythema units /ml, mean + standard deviation. not done.

Serum Neutralization Neutralization of the erythema factor by

postimmunization sera is shown in Table 1. The data are consistent with those of the precipitin test. However, since the data are expressed in arbitrary antierythema units (7), they are not directly comparable with the precipitin titers.

The results of the trials to neutralize the ente- ropathogenic factor with immune serum S4 in ligated intestinal loops are shown in Table 2. When the cell extract was mixed with the immune serum and incubated for 40 min at 22C before injection into the intestinal loops, the ente- ropathogenic factor was completely neutralized. Without incubation, the serum was less effective.

TABLE 2 Effect of immune serum on the activity of cell extract of C. perfringens, strain 8239,

in ligated intestinal loops of lambs

No. positive' loops/No. loops tested

Extract, ml Loop

inoculum Serum, ml 0.3 0.2 0.1 0.05

Extract 0 NDb 414 414 518 Extract + serum,

incubated 2.0 0110 016 ND ND Extract + serum, not

incubated 2.0 415 215 ND ND 3 .o ND 115 ND ND

distended with at least 10.0 ml of fluid. bNot done.

TABLE 3

Response of immunized and control sheep to intraduodenal challenge with cell extract and whole cells of C. perfringens, strain 8239

Challenge inoculum

Extract, 8.0 ml Cells: medium, 30.0 : 220.0 ml

Diarrhea (No.)

Immunizing Positive/ Positive/ inoculum total +++a -tC total +++ ++ 4-

Formalized cell extract, parentera1 313 3 o o 619 o 4 2

Formalized bacterin, parentera1 313 1 2 o 518 o 3 2

Cells: medium and cell extract, intraduodenald 15/16 4 6 5 14/20 0 9 5

None 6 I6 2 3 1 416 0 3 1 - .--.

.Profuse diarrhea. bModerate diarrhea. ;Pasty unformed feces. q o t a i of nine challenges over a period of 70 days.

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CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

TABLE 4

Response of ligated intestinal loops of immunized and control sheep to cell extract and whole cells of C. perfringens, strain 8239

- No. positivea loops/No. loops tested

Loop inoculum

Extract, ml Cells: medium, ml Immunizing

inoculum 0.2 0.1 0.05 0.2:2.8 0.5:3.0

Formalized cell extract, parenteral 10/10 8/10 518 518 7/10

Formalized bacterin, parentera] 414 7/10 418 216 516

Cells: medium and cell extract, intraduodenal ND 112 114 212

None 518 2 15 415 .Distended with at least 10.0 ml of fluid. bNot done.

Challenge Table 3 shows the response of the immunized

and control sheep to challenge with cell extract and whole cells of C. perfringens, strain 8239. The most uniform response was obtained with the cell extract. Although the use of whole cell : medium mixtures gave some variation, there was no significant difference in the results between the immunized or repeatedly challenged animals, and the control animals.

No significant difference was found in intes- tinal loop response between the four groups of animals (Table 4). About 75y0 of the positive loops contained 20.0 ml to 40.0 ml of fluid. Although the fluid volumes were always meas- ured, it was possible to judge the reactions on appearance as negative or positive (Fig. 8).

Discussion

Hauschild (7) has shown that the enteropath- ogenic factor of C. perfringens causes erythema in the skin of guinea pigs and rabbits, and that both the antigen and its antibody can be assayed quantitatively by the skin test. The immuno- diffusion test described here showed that the antigen precipitated with the antibody in a single specific band which allowed quantitative assay of the antibody in vitro. Presumably, the antigen could be measured by the same method.

The complete inhibition of fluid accumulation in the intestinal loops by the ovine immune serum, when incubated with the antigen before injec- tion, conforms with our earlier findings (11)

when rabbit antisera were used. The incomplete inhibition of the fluid accumulation without preliminary incubation may suggest either a rapid binding of the enteropathogenic factor to effec- tive tissue sites, or elective affinity of the factor for such tissues. Similar observations were made by Mosley and Ahmed (14) with the choleragenic toxin of Vibrio clzolerae in the intestinal loops of rabbits. They suggested that the cholera toxin was rapidly bound to the active site, presumably the intestinal epithelial cells (14).

The circulating antibody in the immunized lambs was not protective either against experi- mental C. perfrillgens enteritis or against fluid accumulation in the infected intestinal loops. These results suggest that the circulating anti- body either does not have sufficient access to the sites affected by the enteropathogenic factor, or the same mechanism exists as observed in the intestinal loops with intraluminally introduced antiserum. Mosley and Ahmed (14) reported that circulating antibody to the choleragenic toxin in rabbits provided only limited protection against ileal loop challenge with the antigen. In contrast, Finkelstein and Atthasampunna (6) found significant protection against the cholera- genic toxin in the ileal loop of parenterally im- munized rabbits.

The failure of nine repeated challenges with cells or cell extracts of C. perfiil7gens to induce clinical immunity in sheep agrees with the find- ing of Duncan and Strong (4), who noted no protective immunity in rabbits challenged for a second time 16 days after the first experimental

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NIILO ET AL.: IMMUNIZATION OF SHEEP 395

infection. The lack of clinical immunity to re- peated attacks of C. perfringem food poisoning has been observed in humans (1). In the sheep, the circulating antibody also failed to develop. Animals having enteric infections with certain pathogenic protozoa develop immunity against subsequent reinfection. Such protective immu- nity appears to be associated with local immune mechanisms in the intestinal mucosa rather than with circulating antibody (13). In the present experiments, apparently little or no protective local antibody was formed, probably because the intestinal mucosa is not invaded by the or- ganism (8), and because the contact between the immunologically competent cells and the enter- opathogenic factor may be too short to initiate an immune response.

Acknowledgments

The authors gratefully acknowledge the valua- ble technical assistance of Mr. R. Hilsheimer and Mr. L. H. Dow.

1. DISCHE, F. E., and S. D. ELEK. 1957. Experimental food poisoning by Clostridirrnr welchii. Lancet, 2: 71-74.

2. DUNCAN, C. L., and D. H. STRONG. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16: 82-89.

3. DUNCAN, C. L., and D. H. STRONG. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridir~m perfring- ens. J . Bacteriol. 100: 86-94.

4. DUNCAN, C. L., and D. H. STRONG. 1969. Experi- mental production of diarrhea in rabbits with Clostridium perfringens. Can. J . Microbiol. 15: 765- 770.

5. DUNCAN, C. L., H. SUGIYAMA, and D. H. STRONG. 1968. Rabbit ileal loop response to strains of Clostri- dium perfringens. J . Bacteriol. 95: 1560-1566.

6. FINKELSTEIN, R. A., and P. A~HASAMPUNNA. 1967. Immunity against experimental cholera. Proc. Soc. Exp. Biol. Med. 125: 465-468.

7. HAUSCHILD, A. H. W. 1970. Erythema1 activity of the cellular enteropathogenic factor of Clostridilinl perfringens type A. Can. J. Microbiol. 16: 651-654.

8. HAUSCHILD, A. H. W., L. NIILO, and W. J. DORWARD. 1967. Experimental enteritis with food poisoning and classical strains of Clostridirrrn perfringerrs type A in lambs. J. Infect. Dis. 117: 379-386.

9. HAUSCHILD, A. H. W., L. NULO, and W. J. DORWARD. 1968. Clostridiurn perfringerrs type A infection of ligated intestinal loops in lambs. Appl. Microbiol. 16: 1235-1239.

10. HAUSCHILD, A. H. W., L. NIILO, and W. J. DORWARD. 1970. Enteropathogenic factors of food poisoning Clostridirrrn perfringens type A. Can. J. Microbiol. 16: 331-338.

11. HAUSCHILD, A. H. W., L. NIILO, and W. J. DORWARD. 1970. Response of ligated intestinal loops in lambs to an enteropathogenic factor of Clostridiurn perfringens type A. Can. J. Microbiol. 16: 339-343.

12. HOBBS, B. C. 1965. Clostridium ~velchii as a food poisoning organism. J. Appl. Bacteriol. 28: 71C82.

13. HORTON-SMITH, C., P. L. LONG, A. E. PIERCE, and M. E. ROSE. 1963. Immunity to coccidia in domestic animals. In Immunity to protozoa. Edited by P. C. C. Garnham, A. E. Pierce, and I. Roitt. Blackwell Scientific Publications, Oxford.

14. MOSLEY, W. H., and A. AHMED. 1969. Active and passive immunization in the adult rabbit ileal loop model as an assay for production of anti-toxin immunity by cholera vaccines. J. Bacteriol. 100: 547-549.

15. OUCHTERLONY, 0. 1958. Diffusion-in-gel methods for immunological analysis. Progr. Allergy, 5: 1-78.

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