THE ISOLATION OF CLOSTRIDIUM PERFRINGENS TYPE C FROM NECROTIC ENTERITIS OF MAN IN PAPUA-NEW GUINEA

J. R. EGERTON AND P. D. WALKER Department of Agriculture, Konedobu, Papua, and The Wellcome

Research Laboratories, Beckenham, Kent

Clostridium perfringen (Cl. welchii) is divided into types A, B, C, D, E and F (Wilsdon, 1931; Glenny et al., 1933; Bosworth, 194043; Oakley, 1949 ; Zeissler and Rassfeld-Sternberg, 1949).

Of these, the classical type A, the food poisoning variety of type A (Hobbs et al., 1953), type D (Gleeson-White and Bullen, 1955; Kohn and Warrack, 1955) and type F (Zeissler and Rassfeld-Sternberg) have previously been recorded from man. Types B, C, D and E have been regarded as specific pathogens of animals, each type having a limited host range. Thus, type B is associated chiefly with lamb dysentery (Dalling, Mason and Gordon, 1928), type C with enterotoxaemias of sheep, calves and piglets (McEwen, 1930; Griner and Johnson, 1954; Field and Gibson, 1959, type D with pulpy kidney disease of sheep (Bennetts, 1932) and type E is occasionally found as a saprophyte in the intestines of calves (Bosworth, 1940-43).

In the course of bacteriological investigations of further cases of necrotic enteritis in man such as those described by Murrell and Roth (1963), a number of strains of CI. perfringens were isolated from resec- tions of affected small intestine. Since a number of these produced the lethal 8-toxin characteristic of types B, C and F, a study of their toxico- logical and immunological properties and their heat resistance was undertaken.

MATERIALS AND METHODS

Material from the lumen of the affected portion of the resected bowel was inoculated into cooked meat broth. This was placed in a boiling waterbath for 2 min. and then incubated at 37" C for 24 hr. The resulting cultures were inoculated on to 10 per cent. sheep blood agar plates which were then incubated anaerobically for 24 hr. Organisms from suspect colonies were stained by Gram's method and subcultured on blood agar prior to being inoculated into media for biochemical tests.

Media. The media used in identification were Loeffler's inspissated serum, gelatin stabs, litmus milk, and peptone water sugar base with bromcresol purple indicator, to which one of the following carbohydrates had been added at 1 per cent. concentration: glucose, maltose, sucrose, lactose, glycerol or salicin.

Heat resistance. Tests for heat resistance were carried out exactly as described by Brooks et al. (1957).

Pathogenicity. 0.5 ml. of a 24-hr cooked meat broth culture was injected intramuscularly into the thigh of a guinea-pig.

Toxicological examination. Filtrates were examined for various toxins and antigens according to the methods described by Oakley and Warrack (1953) as modified by Brooks et a/. (1957).

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

-

RESULTS Organisms that were morphologically, culturally and biochemically

characteristic of CI. perfringens were isolated from resected bowels and from fatal cases of necrotic enteritis in man, and from pigs sus- pected of being concerned in the initiation of outbreaks of enteritis. The results of a toxicological and immunological study of the p-pro- ducing human strains are given in the table.

TABLE Typing of /l-producing strains of CI. perfringens isolated during outbreaks

of necrotic enteritis of man in Papua-New Guinea

S B

- + - + - + - + - + - + - + - +

-1

- + - +

__

Strai

F 2113

F 4853

F 5701

F 5702

263

331

924

926 934 A

934 B 986

Patient and age

F 41

M 8

M 42

F 8

M 22

M 8

F 7

F 32 M 7

M l F l

Origin

Resected jejunum

Resected ileum

Resected ileum

Resected jejunum

Resected jejunum

Resected ileum

Duodenal aspirate

Faeces Bowel contents

Jejunum Jejunum ~-

* tr = Trace

-

a - + 4

+ + + + 4-

+ + + + ._

t H.R. = Heat resistance

-

P -

+ + + + + - + + + + +

The majority of strains isolated from human cases produced a- and /?-toxins. However, in four cases (F 4852, 972, 973, 998/63) only a-producing strains were found; this does not exclude the possi- bility of /?-producing strains having been present. Since the pathological appearances in the human cases from which type-C strains were isolated were completely consistent with those seen elsewhere in human and animal infections with CI. perfringens types that produce p-toxin, there can be little doubt that these strains were responsible for the necrotic enteritis. None of the strains was heat-resistant. Spore suspensions obtained with some difficulty from two strains (263, 4853) failed to withstand heating at 100" C for 5 min.

Six strains (F 2108, F 2109, F 2111, 874/32, 843/56, 843/43) isolated from patients not proved to have the enteritis-necroticans syndrome belonged to CI. perfringens type A. Similarly, sixteen cultures (F 21 10, F 2112, F 2114, F 2115, 969; Ogonel 5, 1 1 , 12; Korfena 5, 9; Kwongi 6, 19; Miramur 1,4, 12, 16; Lunaby 8) isolated from pigs in two small surveys carried out subsequently belonged to CI. perfringens type A.

CL. PERFRINGENS FROM NECROTIC ENTERITIS 217

DISCUSSION Although enterotoxaemias caused by various types of CZ. per-

fringens are common amongst domestic animals, they rarely occur in man. The outbreaks of necrotic enteritis described in Germany in the immediate post-war period seem to have been somewhat excep- tional incidents. It is, therefore, most interesting to encounter a similar condition in Papua-New Guinea, especially since the strains respons- ible were, like Zeissler and Rassfeld-S ternberg’s isolates in Germany, producers of 8-toxin.

Murrell and Roth (1963) are of the opinion that some of their cases may have been due to gorging on contaminated pig meat at feasts. The negative findings in the series of pigs examined by us do not exclude this possibility. The similarity of the antigenic spectrum of the Papua- New Guinea strains and those isolated by Field and Gibson (1955) from piglets in Britain (Brooks et al., 1957) is perhaps worth noting. An alternative hypothesis is that over-indulgence at feasts might predispose to a rapid multiplication of 8-producing organisms already present in the intestinal tract. This type of pathogenesis is not un- common in the case of enterotoxaemias in animals.

The German cases of necrotic enteritis were caused by CI. per- fringens type F, which differs from type C only in its heat resistance. Since the strains responsible for Murrell and Roth’s cases produced u- and /3-toxins, but were not heat resistant, they must be assigned to type C. The fact that strains as similar epidemiologically, patho- logically and toxicologically as Zeissler and Rassfeld-Sternberg’s and Murrell and Roth’s are assigned to different types strengthens the view expressed by Brooks et al. (1957) and Sterne and van Heyningen (1958) that type F should be abandoned and the strains included in it should be transferred to type C (see also p. 282 in this issue).

SUMMARY An immunological and toxicological examination has been made of

a number of Clostridium perfringens strains isolated from cases of necrotic enteritis of man in Papua-New Guinea. Since they produced CI. perfringens u- and 6-toxins and were not heat resistant they have been assigned to type C .

We should like to thank Dr Anne White of the Public Health Laboratories, Colindale, for letting us have filtrates of cultures grown at Colindale from strains sent originally by one of us (J.R.E.). We are also very grateful to Dr G . Harriet Warrack who carried out the original typing and detected the presence of 8-toxin in Dr White’s filtrates.

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