PATHOGENIC CLOSTRIDIA

Transcription

1 SEROLOGY OF THE SOLUBLE ANTIGENS OF THE PATHOGENIC CLOSTRIDIA PAUL D. ELLNER' AND STANLEY S. GREEN Department of Medial Mirobiology, College of Mediine, University of Vermont, Burlington, Vermont Reeived for publiation 7 June 963 ABSTRACT ELLNER, PAUL D. (University of Vermont, Burlington), AND STANLEY S. GREEN. Serology of the soluble antigens of the pathogeni lostridia. J. Bateriol. 86: Soluble antigens of 4 strains, representing nine speies of lostridia ommonly ourring in human infetions, were prepared by growing the organisms in a nonantigeni medium. Serologial studies demonstrated the ourrene of onsiderable strain variation within eah speies. Interations among the nine speies, as well as with the previously haraterized Clostridium perfringens, were also investigated. Extreme heterogeneity was observed among the speies studied, with many ross-reations due to ommon antigens, although speies-speifi antigens were also found in some ases. Oasional weak reations were also demonstrated between ertain lostridial antisera and the soluble antigens of three of the four speies of Baillus studied. The 9 or more speies omprising the genus Clostridium represent an extremely heterogeneous olletion, with the ommon harateristis of being gram-positive, anaerobi, atalase-negative, spore-forming rods. Inluded in this genus are a wide variety of saprophyti forms normally ourring in soil and in the intestinal trats of man and animals. These speies vary widely in their biohemial ativities, and inlude thermophili speies and nitrogen-fixing speies, as well as a small number of speies pathogeni for man. Identifiation of the pathogeni anaerobes has been aomplished by biohemial reations. Spray (936) set up a "Tentative Key" for the sporulating anaerobes by the use of physiologial tests and morphologial harateristis. Other lassifiations by biohemial reations have been Present address: Department of Mirobiology, College of Physiians and Surgeons of Columbia University, New York, N.Y. formulated by Duffett (93), Reed and Orr (94), Marshal, Wetzler, and Kawatomari (96), Brooks and Epps (99), and Kaufman and Weaver (96). The initial serologial grouping of lostridia was attempted by Robertson (9). In his study of Vibrion septique (Clostridium septium), Robertson found three agglutinating types. By use of washed ultures as antigens, three serologial raes were found, and toxin ould be demonstrated for all strains tested. Clark and Hall (937) demonstrated rossreations of C. sordellii and C. bifermentans, using the agglutination of ells by speifi antisera. They also observed that animals proteted with C. bifermentans antiserum resisted infetion with C. sordellii. Studies by MCoy and MClung (938) indiated that rossagglutination ours among some speies of lostridia, and serologial investigations of individual or small groups of speies of lostridia have also been done (Coleman and Gunnison, 98; Gunnison, 937, 947; Smith, 937; Miles and Miles, 947, 9; Oakley, Warrak, and Clarke, 947; Mandia and Bruner, 9). Double diffusion in agar gel was utilized by Bjorkland and Berengo (94) to study the interations of seven speies of lostridia. This was the first attempt at an overall study of the pathogeni lostridia. Ellner and Bohan (96) used the same tehnique to study the soluble antigens of C. perfringens types A-F. This study showed marked heterogeneity among the types, as well as strain variation within eah type. Walker (963), with spore antigens of the C. sordellii-c. bifermentans omplex, found ross-agglutination between the two speies. In a preliminary study with C. perfringens, C. sporogenes, and C. novyi types A and B, Ellner and Green (963a) showed strain variation and speies interations among these speies by utilizing the soluble antigens in an agar gel system. The present study represents an attempt to 84 Downloaded from on September 9, 8 by guest

2 VOL. 86, 963 eluidate the serologial relationships among the ten lostridial speies most frequently involved in human disease. MATERIALS AND METHODS Cultures. Cultures were obtained from various soures as indiated in Table. All strains were streaked for purity on plates of latose-egg yolk-milk agar (Willis and Hobbs, 99) and blood agar. Surose, maltose, and saliin fermentations were determined by the rapid method of Kaufman and Weaver (96). Tryptiase Latose Iron Agar (BBL) was used to determine HS prodution. Indole prodution was determined with Kovak's reagent in Indole-Nitrite Medium (BBL). The gluose-gelatin medium of Willis and Hobbs (99) was used to measure fermentation and liquefation. Urea hydrolysis was tested for in the medium of Brooks and Epps (99), with phenol red substituted for the universal indiator. All tubed media were boiled for min and ooled prior to use, and all ultures exept Baillus speies were inubated anaerobially, either by the addition of a reduing substane or by the inubation of plates in a Brewer jar. Pathogeniity of C. bifermentans and C. sordellii was assayed by the method of Brooks and Epps (99). Produttion of antigen. The medium employed was that of Ellner and Bohan (96), modified by the use of dialyzed portions of NZ Amine type A (Sheffield Chemial, Norwih, N.Y.) and Thiotone (BBL) rather than the peptones themselves. A -ml portion of antigen medium was inoulated from the appropriate stok ulture and inubated at 37 C until ative growth was evidenit. This served as the inoulum for the -liter bathes used for antigen prodution. The time of inubation and the gluose onentration were varied with the speies (Table ). Cells were removed by entrifugation at, X g for min, and the supernatant fluid was filtered under positive pressure through a type G filter (Gelman Instrument Co., Chelsea, Mih.) to insure the removal of all baterial ells. The soluble antigens were preipitated from the ell-free supernatant fluid by saturation with ammonium sulfate to 7%O. The material that preipitated was olleted by entrifugation at, X g for min, and the preipitate was dissolved and dialyzed against.8% NaCl. SEROLOGY OF PATHOGENIC CLOSTRIDIA TABLE. Soures of Clostridium and Baillus ultures Speies No. Strain Soure* C. perfringens type A C. sporogenes C. novyi type A C. novi typeb C. histolytium C. bifermentans C. sordellii C.7'septium C. fallax C. tetani B. subtilis B. ereus B. ereus var. myoides B. anthrais 6 strains R UVM CN 733 (P)t CN 73 (P) 3 CN 694 (P) 4 6 (P) Weaver (NP) 6 64 (NP) 7 CN 69 (NP) 8 CN 6 (NP) 9 CN 6 (NP) * (a) L. S. MClung, Indiana University; (b) H. Noyes, Walter Reed Army Institute of Researh, Washington, D.C.; () M. Sterne, Wellome Researh Laboratories, Kent, England; (d) R. H. Weaver, University of Kentuky, Lexington; (e) University of Vermont, Burlington; (f) Amerian Type Culture Colletion, Washington, D.C.; (g) ited in Ellner and Bohan (96). t P = pathogeni; NP = nonpathogeni. g a b C d d d b a a e b a d a b a f f f f f f f Downloaded from on September 9, 8 by guest

3 86 TABLE. Period of inubation and gluose onentration employed for antigen prodution Organism Time Gluose hr % Clostridium histolytium 4 C. septium... 4 C. sporogenes... 7 C. fallax... 7 Baillus anthrais... 7 B. ereus... 7 B. ereus var. myoides 7 B. subtilis... 7 C. novyi type A C. novyi type B C. tetani C. bifermentans C. sordellii The protein onentration was estimated by the method of Warburg and Christian (94). Thimerosal was added to a final onentration of :,, and the antigens were stored at C. Immunization of animals. The soluble antigeni preparations harateristi of eah strain within a speies were pooled, and the protein onentration of eah pool was adjusted to to mg of protein per ml. A l)ortion of eah pool was treated with.%, formalin (Fulthorpe and Thomson, 96), inubated at 37 C for 7 hr, and stored at C for at least days to destroy toxiity. Rabbits were immunized as previously desribed by Ellner and Bohan (96). Three rabbits were employed for eah antigen pool. The sera of the three rabbits were pooled, and an equal volume of saturated ammonium sulfate was added. The resulting globulin preipitate was olleted by entrifugation, dissolved in a volume of.8% NaCl equivalent to half the original volume of the pooled sera, and dialyzed against.8% NaCl. Thimerosal was added to a final onentration of :,, and the pooled globulin was stored at C until used. Agar gel diffusion. The tehnique employed was that of Ellner and Bohan (96) exept that plates were inubated at C. RESULTS Biohemial reations. All strains gave typial reations, with the following exeptions: strain of C. novyi type B fermented surose and saliin, and gave a positive proteolyti reation on latose-egg yolk-milk agar; strains 3 and 4 of ELLNER AND GREEN J. BACTERIOL. C. septium failed to ferment saliin or liquefy gelatin. The identity of strain of C. novyi type B was onfirmed by animal protetion tests with guinea pigs and an antiserum speifi for this organism; the two aberrant strains of C. septium were identified by their mirosopi appearane in infeted guinea pigs and their olonial morphology. Strain variation within speies. The strain variation among the 6 type A strains of C. perfringens was desribed in a previous report (Ellner and Bohan, 96). The soluble antigens of the five strains of C. sporogenes were tested individually with the pooled globulin produed against the C. sporogenes antigen pool. The number of lines obtained varied from two to four, depending on the strain (Fig. ). The total number of antigens represented by the pool was five. Of these, two were ommon to all strains, one was unique to strains and 4, another was unique to strains and 4, and a third antigen was present only in strain. By the same proedure, a total of seven antigens were found among the six strains of C. novyi type A. Six of these lines were shared by all strains, and the seventh was present only in strains 4 and (Fig. ). With the two strains of C. novyi type B, a total of four antigens were found. Three of these antigens were ommon to both strains, and an additional antigen was unique to strain (Fig. 3). With the four strains of C. bifermentans, a total of seven lines were observed. Of these, five were ommon to all strains, an additional antigen was shared by strains by strains and 3, and another antigen was unique to strain 4 (Fig. 4). Among the nine strains of C. sordellii, a total of seven antigens ould be demonstrated. Five of these were ommon to all strains, one was unique to strain 3, and one was unique to strain 4 (Fig. and 6). No antigeni differenes ould be observed between pathogeni and nonpathogeni strains. A total of six antigens were found to be present among the four strains of C. histolytium. Four of these were ommon to all strains, and an additional two were shared by strains, 3, and 4 (Fig. 7). With the six strains of C. tetani, no fewer than antigens were found. None of these antigens was ommon to all strains. Two antigens were Downloaded from on September 9, 8 by guest

4 Downloaded from FIG.. Antigens of the five individual strains of Clostridium sporogenes against pooled homologous globulin. FIG.. Antigens of the six individual strains of Clostridium novyi type A against pooled homologous globulin. FIG. 3. Antigens of the two individual strains of Clostridium novyi type B and the antigen pool (NovB) against pooled homologous globulin. FIG. 4. Antigens of the four individual strains of Clostridium bifermentans and uninoulated medium (M) against pooled homologous globulin. FIG.. Antigens of strains,, 3,, 6, and 7 of Clostridium sordellii against pooled homologous globulin ḞIG. 6. Antigens of Clostridium sordellii strains 4, 8, and 9, pooled P strains (P), pooled NP strains (NP), and total pool (Sor) against pooled homologous globulin. 87 on September 9, 8 by guest

5 IL,..I's Iet 3 f.ai *: X P.a i. ral m S..efp 3 Downloaded from I I 8) FIG. 7. Antigens of the four individual strains of Clostridium histolytium and uninoulated medium (M) against pooled homologous globulin. FIG. 8. Antigens of the six individual strains of Clostridium tetani against pooled homologous globulin. FIG. 9. Antigens of the two individual strains of Clostridium fallax and the antigen pool (Fal) and uninoulated medium (M) against pooled homologous globulin. FIG.. Antigens of the four individual strains of Clostridium septium and the antigen pool (Sep) and uninoulated medium (M) against pooled homologous globulin. FIG.. Antigen pools of Clostridium novyi type A (NovA), C. novyi type B (NovB), C. sporogenes (Spo), and C. perfringens type A (Per) against pooled C. perfringens globulin (Per). FIG.. Antigen pools of Clostridium histolytium (His), C. bifermentans (Bif), C. sordellii (Sor), and C. perfringens (Per) against pooled C. perfringens globulin (Per). 88 ;or on September 9, 8 by guest

6 VOL. 86, 963 SEROLOGY OF PATHOGENIC CLOSTRIDIA 89 unique to strain ; four antigens to strains and ; another four antigens were shared by strains 3, 4,, and 6; a single antigen was shared by strains 4,, and 6; and an additional antigen was shared by strains, 3, 4,, and 6 (Fig. 8). With the two strains of C. fallax, a total of seven antigens were found, all of whih were ommon to both strains (Fig. 9). Among the four strains of C. septium, a total of nine antigens were deteted, three of whih were ommon to all strains. Two additional antigens were shared by strains and ; another antigen was shared by strains,, and 3; two antigens were unique to strain 3; and one antigen was unique to strain 4 (Fig. ). Relationship between speies. Globulin prepared against pooled C. perfringens antigens reated with the antigens of all the speies tested (Fig.,, and 3). However, C. perfringens appeared to have one antigen unique to this speies. C. sporogenes globulin reated only with the antigens of C. novyi types A and B (Fig. 4,, and 6). One of the antigens of C. sporogenes was found to be speies-speifi. Globulin prepared against C. novyi type A reated only with the antigens of C. sporogenes, C. novyi type B, C. bifermentans, and C. sordellii (Fig. 7, 8, 9, and ). At least one speiesspeifi antigen was deteted. Globulin prepared against C. novyi type B reated with the pooled antigens of all speies tested with the exeption of C. perfringens (Fig.,, and 3). No antigens were found in C. novyi type B that were not also present in one or more other speies. C. bifermentans globulin reated with the antigens of all speies tested exept C. perfringens and C. sporogenes (Fig. 4 and ). Although no antigens were found to be unique to C. bifermentans, this speies an be distinguished from C. sordellii by the use of absorbed sera (Ellner and Green, 963b). C. sordellii globulin reated with the antigens of all speies tested exept C. novyi types A and B (Fig. 6 and 7). One unique antigen was present in this speies. C. histolytium globulin reated with the antigens of all speies tested (Fig. 8 and 9), as did globulin prepared against C. fallax (Fig. 3 and 3). Neither of these speies was found to ontain unique antigens. Although C. tetani globulin dislosed lines when tested against individual strains of this speies, the pooled antigens dislosed only 6 lines. C. tetani globulin reated with the antigens of all speies tested exept C. sporogenes and C. novyi type A (Fig. 3 and 33). Only one of the C. tetani antigens appeared to be speies-speifi. C. septwum globulin reated with the antigens of all speies tested exept C. perfringens and C. sporogenes (Fig. 34 and 3). Two antigens are unique to this speies. The ross-reations of all speies are summarized in Table 3. Reations with Baillus antigens. The soluble antigens prepared with the four speies of Baillus were eah tested individually against the globulins prepared against the ten lostridial speies. B. anthrais reated with the globulins of C. bifermentans, C. sordellii, C. fallax, and C. septium. B. ereus var. myoides ross-reated with the same globulins as B. anthrais plus the globulin prepared against C. novyi type A. B. ereus only reated with C. septium globulin, and B. subtilis failed to reat with any of the globulins. All reations between Baillus antigens and lostridial globulins were extremely faint. The results of these interations are summarized in Table 4. Control studies. Resuilts obtained when uninoulated medium was tested against various pooled globulins indiated that the medium itself was nonantigeni (Fig. 4, 7, 9, and ). DISCUSSION It is apparent from these studies that the soluble antigens of the lostridial speies examined are extremely heterogeneous. C. perfringens, C. sporogenes, C. tetani, and C. septium strains varied onsiderably, while the strains of the remaining speies were remarkably onstant. Speies-unique antigens were found in C. perfringens, C. sporogenes, C. novyi type A, C. sordellii, C. tetani, and C. septium. Although no attempt has been made to examine the soluble antigeni preparations for toxigeniity or other biologial ativity, it seems reasonable to assume, in view of the method of preparation, that many suh materials are present. Furthermore, while efforts were made to exlude ellular antigens, the possibility exists that materials suh as lipopolysaharides, flagellar antigens, or other antigeni materials normally assoiated with strutural omponents of the ell may have beome solubilized through autolysis and ould also be present in our preparations. Downloaded from on September 9, 8 by guest

7 Downloaded from FIG. 3. Antigen pools of Clostridium septium (Sep), C. novyi type A (NovA), C. novyi type B (NovB), C. tetani (Tet), C. fallax (Fal), and C. perfringens (Per) against pooled C. perfringens globulin (Per). FIG. 4. Antigen pools of Clostridiun tetani (Tet), C. fallax (Fal), C. bifermentans (Bif), C. sordellii (Sor), C. histolytium (His), and C. sporogenes (Spo) against pooled C. sporogenes globulin (Spo). FIG(.. Antigen pools of Clostridium novyi type A (NovA), C. novyi type B (NovB), C. sporogenes, (Spo), and C. perfringens (Per) against pooled C. sporogenes globulin (Spo). FIG. 6. Antigen pools of Clostridium septium (Sep), C. bifermentans (Bif), C. sordellii (Sor), C. histolytium (His), C. tetani (Tet), and C. sporogenes against pooled C. sporogenes globulin (Spo). FIG. 7. Antigen pools of Clostridium histolytium (His), C. bifermentans (Bif), C. sordellii (Sor), and C. novyi type A (NovA) against C. novyi type A globulin (NovA). FIG. 8. Antigen pools of Clostridium tetani (Tet), C. fallax (Fal), C. novyi type A (NovA), C. histolytium (His), and C. perfringens (Per) against pooled C. novyi type A globulin (NovA). 9 on September 9, 8 by guest

8 Downloaded from FIG. 9. Antigen pools of Clostridium septium (Sep), C. histolytium (His), C. tetani (Tet), and C. novyi type A (NovA) against pooled C. novyi type A globulin (NovA). FIG.. Antigen pools of Clostridium novyi type A (NovA), C. novyi type B (NovB), C. sporogenes (Spo), and C. perfringens (Per) against pooled C. novyi type A globulin (NovA). FIG.. Antigen pools of Clostridium novyi type A (NovA), C. novyi type B (NovB), C. sporogenes (Spo), and C. perfringens (Per) against pooled C. novyi type B globulin (NovB). FIG.. Antigen pools of Clostridium septium (Sep), C. tetani (Tet), C. fallax (Fal), and C. novyi type B (NovB) against pooled C. novyi type B globulin (NovB). FIG. 3. Antigen pools of Clostridium tetani (Tet), C. fallax (Fal), C. novyi type B (NovB), C. bifermentans (Bif), C. sordellii (Sor), and C. histolytium (His) against pooled C. novyi type B globulin (NovB). FIG. 4. Antigen pools of Clostridium septium (Sep), C. perfringens (Per), C. sporogenes (Spo), C. tetani (Tet), C. fallax (Fal), and C. bifermentans (Bif) against pooled C. bifermentans globulin (Bif). 9 on September 9, 8 by guest

9 Downloaded from FIG.. Antigen pools of Clostridium perfringens (Per), C. novyi type A (NovA), C. novyi type B (NovB), C. sporogenes (Spo), C. sordellii (Sor), and C. histolytium (His) against pooled C. bifermentans globulin (Bif). FIG. 6. Antigen pools of Clostridium perfringens (Per), C. novyi type A (NovA), C. novyi type B (NovB), C. sporogenes (Spo), C. bifermentans (Bif), and C. histolytium (His) against pooled C. sordellii globulin (Sor). FIG. 7. Antigen pools of Clostridium septium (Sep), C. novyi type A (NovA), C. novyi type B (NovB), C. tetani (Tet), C. fallax (Fal), and C. sordellii (Sor) against pooled C. sordellii globulin (Sor). FIG. 8. Antigen pools of Clostridium perfringens (Per), C. novyi type A (NovA), C. novyi type B (NovB), C. sporogenes (Spo), C. bifermentans (Bif), and C. sordellii (Sor) against pooled C. histolytium globulin (His). FIG. 9. Antigen pools of Clostridium septium (Sep), C. perfringens (Per), C. tetani (Tet), C. fallax (Fal), and C. histolytium (His) against pooled C. histolytium globulin (His). FIG. 3. Antigen pools of Clostridium sordellii (Sor), C. histolytium (His), C. tetani (Tet), C. fallax (Fal), and C. septiumn (Sep) against pooled C. fallax globulin (Fal). 9 on September 9, 8 by guest

10 pe r B If 'al Pal..ar ova Tet ~jvb.:n 7B [ iep Vet f iep ler Spo is Sep Fal. FIG. 3. Antigen pools of Clostridium perfringens (Per), C. sporogenes (Spo), C. novyi type A (NovA), C. novyi type B (NovB), C. bifermentans (Bif), and C. fallax (Fal) against pooled C. fallax globulin (Fal). FIG. 3. Antigen pools of Clostridium bifermentans (Bif), C. sordellii (Sor), C. histolytium (His), C. fallax (Fal), and C. tetani (Tet) against pooled C. tetani globulin (Tet). FIG. 33. Antigen pools of Clostridium septium (Sep), C. perfringens (Per), C. sporogenes (Spo), C. novyi type A (NovA), C. novyi type B (NovB), and C. tetani (Tet) against pooled C. tetani globulin (Tet). FIG. 34. Antigen pools of Clostridium sporogenes (Spo), C. novyi type A (NovA), C. novyi type B (NovB), C. perfringens (Per), C. histolytium (His), and C. septium (Sep) against pooled C. septium globulin (Sep). FIG. 3. Antigen pools of Clostridium bifermentans (Bif), C. sordellii (Sor), C. tetani (Tet), C. fallax (Fal), and C. septium (Sep) against pooled C. septium globulin (Sep). 93 So-r Tet )ep ova Downloaded from on September 9, 8 by guest

11 94 ELLNER AND GREEN J. BACTERIOL. Globulin prepared against C. perfringens... C. sporogenes... C. novyi type A.. C. novyi type B... C. bifermnlentans... C. sordellii. C. histolytiunt... C. tetani... C. fallax... C. septium. () 4 (7) 4 6 Antigens (4) 4 4 (7) 7 6 (7) (6) 4 4 X lz t3 U al (6) (7) * Parentheses indiate homologous reations. TABLE 4. TABLE 3. Cross-reations between lostridial speies *. 6 -;~ (3)*l Reations between Baillus antigens and lostridial globulins Globulin prepared against 3 Antigens CZ >, a: a.*-_ C. perf?ringens C. sporogenes C. novyi type A C. novyi type B. C. bifermitentans C. sordellii C. histolytiun C. tetani.tani... C. fallax C. septium The ross-reations observed between C. perfringens, C. sordellii, C. histolytium, C. tetani, and C. fallax are reiproal. C. perfringens globulin also reated with the soluble antigens of C. sporogenes, C. novyi types A and B, C. bifermentans, and C. septium, although the globulins of these speies failed to reat with C. perfringens antigen. Although we have not attempted to identify lines of preipitation ourring in these ross-reations, it should be noted that the theta toxin of C. perfringens is serologially related to the oxygen-labile hemolysin of C. tetani (Malennan, 96) and the epsilon toxin of C. histolytium (Howard, 93). Miles and Miles (947, 9) showed that the leithinase of C. perfringens was serologially related to the leithinase of C. bifermentans, and we assume that the strains examined by these workers inluded strains that some urrent workers would lass as C. sordellii. The studies of Bjorklund and Berengo (94) onfirmed the reiproal ross-reation of C. perfringens and C. tetani and the nonreiproal reations between C. perfringens and C. septium. The globulin prepared against the soluble antigens of C. sporogenes ross-reated with the soluble antigens of C. novyi types A and B. Mandia and Bruner (9) were able to identify four heat-labile somati antigens assoiated with this speies and lassified them in group of the proteolyti lostridia. Smith (937) found that rough variants of C. histolytium agglutinated with sera prepared against C. sporogenes. Globulin prepared against the soluble antigens of C. novyi type A ross-reated with soluble antigens of C. novyi type B, C. sporogenes, C. bifermentans, and C. sordellii. All these rossreations were reiproal exept C. sordellii, sine C. sordellii globulin failed to reat with the antigens of C. novyi type A. It should be noted that Oakley et al. (947) found the alpha toxin of C. novyi type A to be serologially homogeneous with the alpha toxin of type B of this speies. Furthermore, the gamma toxin of type A appears to be a leithinase, and one an only speulate whether this fator is responsible in part for the ross-reations with the C. bifermentans-c. sordelli group. C. novyi type B globulin ross-reated with the soluble antigens of C novyi type A, C. histolytium, C. sporogenes, C. tetani, C. fallax, C. septium, C. bifermentans, and C. sordellii. As with C. novyi type A, all reations were reiproal, exept that with C. sordellii globulin whih failed to reat with the antigens of C. novyi type B. Again, as in the ase of C. novyi type A, it should be noted that the alpha toxin of the two types is homogeneous, and one might also speulate on the signifiane of the beta-leithinase of type B with respet to the ross-reation with the C. bifermentans-c. sordellii group. Downloaded from on September 9, 8 by guest

12 VOL. 86j 963 SEROLOGY OF PATHOGENIC CLOSTRIDIA 9 Globulins prepared against C. bifermentans and C. sordellii both ross-reated with soluble antigens of C. histolytium, C. tetani, C. fallax, and C. septiumz. In addition, C. bifermentans globulin ross-reated with the antigens of C. novyi types A and B as well as C. sordellii, while C. sordellii globulin reated with the antigens of C. perfringens, C. bifermentans, and C. sporogenes. C. sporogenes globulin, however, failed to reat with the antigens of C. sordellii. Cross-reations ourred between C. histolytium globulin and the soluble antigens of C. perfringens, C. novyi types A and B, C. bifermentans, C. sordellii, C. tetani, C. fallax, C. septium, and C. sporogenes. The globulins of C. sporogenes and C. novyi type A failed to reat with C. histolytium antigen. Of the five toxins asribed to C. histolytium, the lethal toxin has been found to ross-reat with the alpha toxin of C. septium (Sterne and Warrak, 96), and, as mentioned previously, the oxygen-labile epsilon hemolysin appears to be related to hemolysins of C. perfringens, C. novyi, and C. septium. Mandia (9) lassified C. histolytium in group III of the proteolyti lostridia and observed some rossreations with C. sporogenes. Cross-reations between rough strains of this organism and C. sporogenes were also observed by Smith (937). Bjorklund and Berengo (94) found that C. tetani antitoxin gave 8 to lines when reated with the homologous toxin. This finding orrelates very well with the lines we observed among the various strains of C. tetani and the 6 lines produed by the C. tetani pool when reated with the homologous globulin. Ten types of C. tetani have been desribed (Coleman and Gunnison, 98; Gunnison, 937, 947) based upon heat-stable somati antigens. One suh antigen was found to be ommon to all strains, and an additional heat-stable antigen was ommon to types II, IV, V, and IX. Type-speifi reations, however, were due to flagellar antigens and were present in types, III, VII, and VIII. Type VI (a nonmotile strain) was found to be devoid of flagellar antigens. Of the six strains of C. tetani employed in our studies, strain was type II, strain 3 was type VI, and strain was type V. The type designation of the remaining three strains was unknown. Our studies onfirmed Bjorklund and Berengo's (94) findings that C. tetani serum ross-reats with the antigens of C. perfringens, C. histolytium, and C. septium. However, we also found that C. tetani globulin ross-reats with the soluble antigens of C. novyi type B, C. bifermentans, C. sordellii, and C. septium. Cross-reations were observed between the soluble antigens of C. perfringens, C. novyi types A and B, C. bifermentans, C. sordellii, C. histolytium, C. tetani, C. septium, and C. sporogenes with the globulin prepared against C. fallax. Globulins prepared against C. novyi type A and C. sporogenes failed to reat with antigens of C. fallax. The unusual urved lines obtained with C. fallax globulin and homologous antigen is suggestive of a disparity between the moleular weights of the antigen and of the antibody (Kabat and Mayer, 96). C. septium globulin ross-reated with the soluble antigens of C. novyi types A and B, C. bifermentans, C. sordellii, C. histolytium, C. tetani, and C. fallax. C. novyi type A globulin failed to reat with C. septium antigens. Of the four toxins assoiated with this organism, the alpha toxin has been found to ross-reat with the alpha toxin of C. histolytium (Sterne and Warrak, 96). Bjorklund and Berengo (94) failed to observe any ross-reations between C. septium and C. histolytium, but Sterne and Warrak's finding of suh a ross-reation onfirms our observations of a reation between these two speies. Reations between globulins of C. septium, C. fallax, C. sordellii, C. bifermentans, and C. novyi type A and antigens of one or more speies of Baillus were observed. Most reations ourred with B. ereus var. myoides, some with B. anthrais and B. ereus, and none with B. subtilis. It should be emphasized that these reations were all extremely faint and only appeared after prolonged inubation. Sine omparative studies were not done, it is not known whether these reations indiate antigens ommon to both genera, or distantly related antigens. Two phenomena deserve omment, namely, the ourrene of nonreiproal reations, and the oasional observation that heterologous reations may give rise to a greater number of lines than homologous reations. Indeed, both observations may well be manifestations of the same phenomenon. Nonreiproal reations were also observed by Bjorklund and Berengo (94), who suggested that they might be due to differenes in immune responses of individual animals. We must, however, rejet this postulated explanation, sine our studies employed globulin pools. Downloaded from on September 9, 8 by guest

13 96 ELLNER AND GREEN J. BACTERIOL. On the other hand, while no ready evidene is available, we do onur with Bjorklund's alternate postulate that some antigens might our in low onentrations in some preparations insuffiient to ause visible preipitation. Despite the extreme heterogeneity and the great number of ross-reations observed, preliminary experiments with absorbed sera suggest that it may be possible in some ases to produe group-speifi or even speies-speifi sera. Should it be possible to produe suh sera, the impliations are that they ould prove extremely useful as an additional adjunt in the identifiation of the pathogeni lostridia, perhaps by means of the fluoresent-antibody tehnique. The utility of suh proedures, however, must be determined by additional studies. ACKNOWLEDGMENT The authors are indebted to Louis DS. Smith for his generous gift of C. novyi type B antiserum. LITERATURE CITED BJORKLUND, B., AND A. BERENGO. 94. Studies on the antigeni omposition of some lostridia toxins with the aid of gel diffusion. Ata Pathol. Mirobiol. Sand. 34: BROOKS, M. E., AND H. B. G. EPPS. 99. Taxonomi studies of the genus Clostridium: Clostridium bifermentans and C. sordellii. J. Gen. Mirobiol. :44-. CLARK, F. E., AND I. C. HALL A omparative study of Baillus bifermentans (Tissier and Martelly), Baillus entrosporogenes (Hall) and ertain losely related proteolyti Xanaerobes. J. Bateriol. 33:3-4. COLEMAN, G. E., AND J. B. GUNNISON. 98. Serologi types of Clostridium tetani. J. Infet. Diseases 43: DUFFETT, N. D. 93. The differentiation of Baillus fallax (Weinberg and S6guin) from Baillus arnis (Klein). J. Bateriol. 9:73-8. ELLNER, P. D., AND C. D. BOHAN. 96. Serology of the soluble antigens of Clostridium perfringens types A-F by agar-gel diffusion. J. Bateriol. 83: ELLNER, P. D., AND S. S. GREEN. 963a. Serologi relationships between lostridia based on soluble antigens. Bateriol. Pro., p. 64. ELLNER, P. D., AND S. S. GREEN. 963b. Serologial grouping of the pathogeni lostridia. J. Bateriol. 86:98-. FULTHORPE, A. J., AND R.. THOMSON. 96. Antigeni effiieny of tetanus toxoids modified by exess formalin or by heat and phenol. Immunology 3:6-34. GUNNISON, J. B Agglutination reations of the heat stable antigens of Clostridium tetani. J. Immunol. : GUNNISON, J. B Complement fixation reations of the heat labile and the heat stable antigens of Clostridium tetani. J. Immunol. 7:67-7. HOWARD, J. G. 93. The hemolysin of Clostridium histolytium. Brit. J. Exptl. Pathol. 34: KABAT, E. A., AND M. M. MAYER. 96. Experimental immunohemistry. Charles C Thomas, Publisher, Springfield, Ill. KAUFMAN, L., AND R. H. WEAVER. 96. Rapid methods for the identifiation of lostridia. J. Bateriol. 79:9-. MCoy, E., AND L. S. MCCLUNG Serologial relations among spore-forming anaerobi bateria. Bateriol. Rev. : MALENNAN, J. D. 96. The histotoxi lostridial infetions of man. Bateriol. Rev. 6: MANDIA, J. W. 9. Serologial identifiation of ultures of Clostridium histolytium as group III of the proteolyti lostridia. J. Infet. Diseases 9:48-. MANDIA, J. W., AND D. W. BRUNER, 9. The serologial identifiation of ultures of C. sporogenes. J. Immunol. 66:497-. MARSHAL, J. D., T. WETZLER, AND T. KAWA- TOMARI. 96. Differentiation of the Clostridia assoiated with medial speimens. U.S. Armed Fores Med. J. 7:44-4. MILES, E. M., AND A. A. MILES The leithinase of C. bifermentans and its relationship to the alpha toxin of C. welhii. J. Gen. Mirobiol. : MILES, E. M., AND A. A. MILES. 9. The relation of toxiity and enzyme ativity in the leithinases of C. bifermentans and C. welhii. J. Gen. Mirobiol. 4:-3. OAKLEY, C. L., G. H. WARRACK, AND P. H. CLARKE The toxins of Clostridium oedemations (C. novyi). J. Gen. Mirobiol., :9-7. REED, G. B., AND J. H. ORR. 94. Rapid identifiation of gas gangrene anaerobes. War Med. :493-. ROBERTSON, M. 9. Serologial grouping of Vibrion septique and their relation to the prodution of toxin. J. Pathol. Bateriol. 3:3-7. SMITH, L Some serologial aspets of the S-R hange in Clostridium histolytium. J. Bateriol. 34: SPRAY, R. S Semisolid media for ultivation and identifiation of the sporulating anaerobes. J. Bateriol. 3:3-. STERNE, M., AND H. G. WARRACK. 96. The inter- Downloaded from on September 9, 8 by guest

14 VOL. 86, 963 SEROLOGY OF PATHOGENIC CLOSTRIDIA 97 ations between C. septium and C. histoly- WARBURG, O., AND W. CHRISTIAN. 94. Isolierung tium toxins and antitoxins. J. Pathol. Ba- und Krystallisation des Garunsferments teriol. 84: Enolase. Naturwissenshaften 9:89-9. WALKER, P. D The spore antigens of Clos- WILLIS, A. T., AND G. HOBBS. 99. Some new tridium sporogenes, C. bifermentans and C. media for the isolation and identifiation of sordellii. J. Pathol. Bateriol. 8:4-49. Clostridia. J. Pathol. Bateriol. 77:-. Downloaded from on September 9, 8 by guest