THE TYPES OF CLOSTRIDIUM PEWRINGENS

M. STERNE AND G. HARRIET WARRACK Wellcome Research Laboratories, Beckenham, Kent

As in the case of most pathogenic bacteria, the subdivision of Clos- tridium perfringens (Cl. welchii) has been based on medical rather than botanical criteria. Wilsdon (1 931) divided the species into four types, A, B, C and D, depending on the ability of their antisera to neutralise the lethal effects of strains isolated from various conditions in man and animals. The types distinguished by Wilsdon corresponded to well- defined epizootiological groups, in that type A included the organisms responsible for human gas-gangrene, type B those responsible for lamb dysentery, and types C and D those respectively responsible for " struck" and " pulpy kidney ", two clinically distinct forms of enterotoxaemia of sheep. Glenny et al. (1933) showed that the specific characters of the four types depended on their ability to elaborate one or more lethal toxins which they named, a, 8 and C. Thus, type A produced a-toxin, type B produced a, 8 and E, type C produced a and p, and type D produced a and E. In addition, a lethal toxin (designated 7) was detected in filtrates of type-B and type-C cultures, and a haemolysin (designated 6) in type-C filtrates. Oakley (1943) prepared a detailed review of the work done on the distribution of antigens amongst the different types. By this time, 8 * and 7 -toxins had been named (Prigge, 1936; Ipsen and Davoli, 1939), in addition to the " major '' toxins, which defined the types. Bosworth (19-3) isolated from a calf a strain of CI. perfringens that produced a lethal toxin not neutralisable by existing antisera. He named the toxin iota (I) and proposed a new type (E) to include the strains producing iota toxin. Thus, five types were now generally recognised, each based on the production of dis- tinctive lethal toxins. Oakley and his co-workers isolated and char- acterised further antigens ( K , A, p, v), which proved useful as ancillary criteria for type differentiation and brought the total of distinguishable antigens to twelve. However, division into types remained dependent on the distribution of the four " major " toxins a, 8, E and z (Oakley and Warrack, 1953). The term " major " antigens is used as a con- venience to describe those that define the type, since they are responsible for the gross pathological changes seen in the diseases.

In 1949, Zeissler and Rassfeld-Sternberg isolated strains of CI. perfringens from fatal cases of necrotic enteritis of man in West Ger- many. These were shown (Oakley, 1949) to produce a- and 8-toxins, a finding that justified assigning them to type C. However, they were found to be unusually heat-resistant and to lack ancillary antigens

* Prigge named this a-toxin, but since a was already pre-empted for the type-A Ieci- thinase, 0 was substituted (Anonymous, 1942).

1. PATH. 8ACT.-VOL. 88 (1964) 279

280 M. STERNE AND C. HARRlET WARRACK

such as 6, 8 and K, which had come to be regarded as characteristic although not definitive of type C. Therefore, despite the possession of the major antigens characteristic of type C, Zeissler and Rassfeld- Sternberg’s isolates were assigned to a new type, F.

Brooks, Sterne and Warrack (1957) examined a large number of CI. perfringens strains, including many new isolates, and showed that ecologically significant subtypes existed within the major divisions and that these could be distinguished by differences in the minor antigens. Thus, Hobbs et a/. (1953) described a group, heat-resistant CI. perfringens type A, that lacked the ability to produce &toxin and were responsible for outbreaks of food poisoning in man. Type-B strains from the Middle East produced K-toxin (collagenase) in place of the A-toxin (proteinase) hitherto regarded as characteristic, and also failed to produce hyaluronidase, which was produced in quantity by all classical strains of type B (Brooks and Entessar, 1957). In North America, extensive outbreaks of enterotoxaemia in calves and in neonatal lambs were found to be caused by type C (Griner and Bracken, 1953; Griner and Johnson, 1954). These strains did not produce &toxin, which is invariably produced by the classical type-C strains. Field and Gibson (1955) identified CI. perfringens type C as the cause of a neonatal enterotoxaemia of piglets in Britain. These strains too differed from the classical type C in being unable to produce &toxin, and were other- wise distinguishable from Griner’s strains (Brooks et al., 1957). Thus, four “ subtypes ” of CI. perfringens type C had now been demonstrated, or five if type F could be regarded as belonging to type C. These “ subtypes ” showed a consistent association of certain serological properties with epizootiological and other ecological differences.

Recently, Murrell and Roth (1963) in Papua-New Guinea described cases of necrotic enteritis in man that closely resembled the enteritis necroticans of man in Germany from which Zeissler and Rassfeld- Sternberg (1949) isolated the CI. perfringens strains subsequently assigned to type F. From Murrell and Roth’s material, Egerton isolated and Warrack and Walker typed (Egerton and Walker, 1964) strains of CI. perfringens that resembled Zeissler and Rassfeld-Sternberg’s isolates in producing a- and /?-toxins, but differed in producing 8-haemolysin, hyaluronidase, and traces of K-collagenase. Epidemiological and pathological considerations strongly favoured grouping the Papua-New Guinea and the German strains together. However, the Papua-New Guinea strains lacked heat- resistance, the definitive character of type F, so had perforce to be separated from it. Therefore, since the Papua-New Guinea strains produced /?-toxin, they were assigned to type C.

The table gives a conspectus of the species CI. perfringens and its subdivisions. The scheme is, basically, that of Oakley and Warrack (1953), as modified by Brooks et al. (1957) to accommodate new isolates, and is republished here with the addition of the new Papua-New Guinea variety and with the elimination of type F by its transfer to type C.

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.

282 M. STERNE AND G. HARRIET WARRACK

DISCUSSION The desirability of maintaining the type status of Zeissler and

Rassfeld-Sternberg’s isolates has been questioned (Brooks et al., 1957 ; Sterne and van Heyningen, 1958) on the grounds that the differences between ecologically important strains within types A, B and C are as great as that considered sufficient to distinguish type F from type C. For example Hobbs et al.’s heat-resistant type A might equally well have been considered as type G. However, no serious irrationality resulted from this separation until the present time, when the separation of type F on the basis of heat resistance parts Murrell’s from Zeissler’s strains, although both cause virtually the identical disease in man and possess similar antigenic patterns. This indicates that the selection of heat resistance as a criterion for distinguishing between types F and C was unsound, and reinforces the recommendation that type F should be abandoned and the strains included in it transferred to type C. This would not only rationalise existing contradictions, but would reinstate Wilsdon’s original criteria for type differentiation, which were neglected when type F was established.

We have considered it inadvisable to distinguish the varieties within the types by means of letters or numbers, since this might restrict possibilities of future regrouping as further information becomes available. If such distinctions were, however, considered expedient, then the varieties could be noted by suffixes as Al, A2; B1, Bz; C1, CZ, C3, Cq, Cg, according to the order of their accession to the type.

SUMMARY The scheme proposed by Oakley and Warrack (1953) for the classi-

fication of Clostridium perfringens and modified by Brooks et al. (1957) to include more recent findings, has now been elaborated to include the type-C strains isolated from cases of necrotic enteritis of man in Papua-New Guinea.

It is recommended that type F be abandoned and the strains included in it transferred to type C.

REFERENCES ANONYMOUS . . . . . . . BOGWORTH, T. J. . . . . . BROOKS, M. ELIZABETH, AND EN-

TESSAR, F. . . . . . . . BROOKS. M. ELIZABETH, STERNE, M.,

EGERTON, J. R., AND WALKER, P. D.

GLENNY, A. T., BARR, MOLLIE, LLEWELLYN-JONES, MONA, DALL- ING, T., AND Ross, HELEN E.

GRINER, L. A., AND BRACKEN, F. K.

AND WARRACK, G. HARRIET

FIELD, H. I., AND GIBSON, E. A. .

1942. Nature (Lond.), 149, 56. 1940-43. J. Comp. Path., 53, 245. 1957. Brit. Vet. J., 113, 506.

1957. This Journal, 74, 185.

1964. This Journal, 88, 275. 1955. Vet. Rec., 67, 31. 1933. This Journal, 37, 53.

1953. J. Amer. Vet. Med. ASSOC., 122, 99.

THE TYPES OF CLOSTRIDIUM PERFRINGENS 283

GRINER, L. A., AND JOHNSON, H. W. 1954.

HOBBS, BETTY C., SMITH, MURIEL E., OAKLEY, C. L., WARRACK, G . HARRIET, AND CRUICKSHANK, J. C.

IPSEN, J., AND DAVOLI, R. . . . MURRELL, T. G. C., AND ROTH, L. . OAKLEY, C. L. . . . . . .

OAKLEY, C. L., AND WARRACK, G.

PRIGGE, R. . . . . . . . STERNE, M., AND VAN HEYNINGEN,

. . . . . . 7 7

HARRIET

W. E.

1953.

1939. 1963. 1943. 1949. 1953.

1936. 1958.

WILSDON, A. J. . . . . . . 1931.

ZEISSLER, J., AND RASSFELD-STERN- 1949. BERG, L.

J. Amer. Vet. Med. Assoc., 125,

J . Hyg. (Cumb.), 51, 75. 125.

Bull. Hlth Org. L.o.N., 8, 833. Med. J . Austral., 1, 61. Bull. Hyg., 18, 781. Brit. Med. J., 1, 269. J . Hyg. (Cut&.), 51, 102.

Z. Imrnun.-Forsch., 89, 417. In Bacterial and mycotic infections

of man, 3rd ed., edited by R. J. Dubos, Philadelphia, p. 343.

Rep. Inst. h i m . Path., Univ. Camb., 2nd Rep., p. 53.

Brit. Med. J., 1, 267.