PLATE ISINCE Defalle (1902) first reported the antigenicity of the spores in Bacillus species, opinion has been divided regarding the value of spore antigens in the differentiation of serological types among spore-forming bacteria. Defalle (1902) and Mellon and Anderson (1919) were of the view that spores possessed specific spore antigens distinct from those of the vegetative forms of the same species. The later report of Starin and Dack (1923) that spores were not antigenic and of Krauskopf and McCoy (1937) that spores did not produce antibodies to themselves but gave rise to antibodies specific for the H and 0 antigens of the bacillary forms, perhaps discouraged further study, but Howie and Cruickshank (1940) were able to prove beyond reasonable doubt that specific spore antibodies could be produced that were distinct from H and 0 antibodies produced by vegetative cells.The work of Moussa (1956) established the potential of spore antigens in the taxonomy of the clostridia. Meisel and Rymkiewicz (1959) studied a limited number of strains in each of seven species of clostridia and found more than one type in most of these species on the basis of their spore agglutinogens. The present investigation covers the immunological reactivity of the spores of Clostridium sporogenes ; their relationship to the cultural and biochemical characters of this organism has already been reported (Princewill, 1978). MATERIALS AND METHODSCultures. The 84 cultures of C. sporogenes used in this investigation, and the sources from which they originated, have been described elsewhere (Princewill, 1978). Cultures were maintained in cooked-meat medium (CMM, Southern Group Laboratory, Hither Green Hospital, London, SE 13).Production of spores. The stock cultures were inoculated into fresh CMM and incubated at 37°C for 24 h. Subcultures were made in two transfers into reinforced clostridial medium (RCM, Hirsch and Grinsted, 1954), from which 2-ml inocula were made on RCM agar slopes in 1 14-ml (4-oz) screw-capped bottles. The cultures were incubated anaerobically at 37°C for 7 days and the growth from 9-10 bottles harvested by washing with chilled 30-40 nll of distilled water. The pooled washings were allowed to stand for 15-20 min. for coarse debris to settle out, and the supernatant was decanted and centrifuged at 400g for 10 min. The sedimented spores were resuspended and washed once more in chilled water. The turbidity of the final suspension was adjusted against Brown's opacity tube no. 10 (Burroughs Wellcome) and distributed in 12-ml amounts into 28-m1(1 oz) bottles and stood in ice ready for sonication in a 150-W ultrasonic disintegrator (MSE) with a 9.5-mm (# in.) titanium probe at an amplitude of 18 flpm (peak to peak) for 5 min. To prevent germination of spores
All the 28 strains of Clostridium sporogenes type I tested produced thiaminase. Only 2 of the 16 strains of Cl. sporogenes type II tested were positive for the enzyme; these gave a weak positive reaction. The single strain of Cl. sporogenes type III behaved in a manner similar to the strains of type I, giving a strong positive thiaminase reaction. Thiaminase production amongst the strains of Cl. sporogenes does in the main support the cultural, biochemical and immunological properties described earlier.
ALTHOUGH Clostridium sporogenes and C. histolyticum show obvious cultural and biochemical differences, several workers have adduced evidence for the sharing of vegetative and spore antigens by these two species (Smith, 1937;Mandia, 1955; Meisel and Rymkiewicz, 1959;Lynt, Solomon and Kautter, 1972). In their attempts to classify the proteolytic clostridia by means of vegetative antigens, Mandia (1951;1952a; 19526;1955) and Mandia and Bruner (195 1 ) did not include C. bifermentans which is an important proteolytic species. Meisel and Rymkiewicz (1959) showed that the spore antigens of this organism did not cross-react with spore antigens of the other proteolytic species they studied. However, cross reactions were reported by Walker (1 963) between the spore antigens of C. bifermentans and C. sordelli, which are closely related species (Brooks and Epps, 1959). Princewill (1979) used the spore antigens to serotype strains of C. sporogenes. That study has now been extended to investigate the taxonomic relationship between C. sporogenes, C. histolyticum, C. bifermentans and C. butyricum. MATERIALS AND METHODS Strains of clostridiu.The 84 strains of C. sporogenes used in this study and their origin have been described elsewhere (Princewill, 1978). The 69 strains of C. histolyticum were: nos. 119, 294,646 from Dr H. Meisel, Serum Research Institute, Warsaw, Poland; CN nos. 919,920,948, 949, 950, 1693, 3056, 3252, 3254, 3255, 3256, 3257, 3258, 3259, 3260, 3261, 3265,3266,3267, 3268,3269,3270,3271,3272,3273,3274,3276,3277,3278,3279,3280,3281,3282,3283,3284, 3285,3286,3287,3288,3289,3290,3292,3293,3294,3295,3296,3297,3298,3300,3301,3302, 3303, 3304, 3305, 3306, 3307, 3308, 3309, 3310, 331 1, 3312, 3313, 3786, 4760, 6304 from the Wellcome Research Laboratories, Langley Court, Beckenham, Kent; and from The National Collection of Industrial Bacteria no. NCIB503, Torrey Research Station (NCIB), Aberdeen. Other species were: C. bifermentans NCIB strains nos. 506,2912,2929,6800,6928 from NCIB; C. beijerinckii no. NCIB9362 from NCIB; C. tyrobutyricum no. CNRZSOO and C. succharobutyricum no. CNRZ555 from Dr J. L. Bergere, INRA, Jouy-en-Josas, France; C. butyricum no. CNRZ5328 from Dr Bergere, and C. butyricum no. SA I1 from Dr J. W. Classens, Bloemfontein, South Africa.Lyophilised cultures were reconstituted, grown in cooked-meat medium (CMM from Southern Group Laboratory, Hither Green Hospital, London) and cultured on fresh horse-blood agar. After incubation at 37°C for 48 h, single colonies were isolated and their identity was checked by routine methods (Princewill, 1978).
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