(Bornstein et al., 1941; Saphra and Silberberg, 1942; Wheeler et al., 1943; Edwards,

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1 TWO PARACOLON CULTURES RELATED ANTIGENICALLY TO SHIGELLA PARADYSENTERIAE1 W. W. FERGUSON AND WARREN E. WHEELER Bureau of Laboratories, Michigan Department of Health, Lansing, Michigan, and the Children's Hospital of Michigan, and the Department of Pediatrics, Wayne University College of Medicine, Detroit, Michigan Received for publication September 13, 195 In recent years numerous reports have been made on the antigenic interrelationship of organisms of the Salmonella, Escherichia, and Shigella genera. (Bornstein et al., 191; Saphra and Silberberg, 192; Wheeler et al., 193; Edwards, Cherry, Bruner, 193). Stuart and coworkers (193) have described the antigenic factors shared in common by Shigella alkalescens, paracolon, and coliform organisms isolated from a small population in a restricted area. Recently Saphra and Wassermann (195) have demonstrated that SalmoneUa onderstepoort and Escherichia coli 391 have an identical "O" antigen, while Shigella parady8enteriae (Flexner "V") is related to the other two by an antigen fraction. The present report deals with two paracolon strains, isolated from the same patient, which are related to various Flexner and Boyd types of Shigella parady8enteriae. Isolation of the paracolon strains was made by one of ue from the stools of a 7-month-old infant with a past history of eczema. Immediately following the addition of egg yolk to his diet, the child began to vomit and a few hours later to have a diarrhea. There was no previous history of diarrhea in the patient, nor among contacts. The diarrhea subsided within a few hours. The paracolon denoted 339 in the following description was obtained from a diarrheal specimen; the second paracolon strain, 88, was obtained from a formed specimen approximately 1 days after the original isolation. In blood serum taken shortly after the second paracolon was isolated, no agglutinins were present for either organism. Both strains upon initial isolation gave a dysenterylike reaction on Kligler's iron agar slants because of delayed gas production. It is customary in the laboratory in which the isolations were made to test all dysenterylike organisms by the slide agglutination technique with absorbed sera covering the genus Shigella and the specific types of the S. paradysenteriae complex. The sera are of commercial origin. A marked agglutination reaction was obtained in only one of the sera, Boyd P 13. Both 339 and 88 organisms were also agglutinated strongly in an unabsorbed polyvalent Flexner antiserum used for tube agglutination tests. From the preliminary evidence it was natural to conclude that the organisms were Shigella paradysenteriae. Biochemical tests revealed, however, that both organisms were of the paracolon group with nearly identical characteristics. The biochemical characteristics are listed in table 1. An antiserum was developed in a rabbit against paracolon 339 that agglutinated both 339 and 88 organisms in a dilution of 1:20,80. Absorption of this 107

2 108 W. W. FERGUSON AND WARREN E. WHEELER antiserum with 88 organisms revealed that agglutinins for both strains were removed completely. An antiserum was also prepared against 88 paracolon that agglutinated the two strains in a 1:20,80 dilution. Agglutinins for both 339 and 88 organisms were completely removed by absorption with 339 organisms. The paracolon strains were, therefore, antigenically similar. In tables 2 and 3 are recorded the results of agglutination tests with 339 and 88 sera and various Shigella cultures. Slightly different results were obtained with the two sera; both, however, agglutinated suspensions of Flexner "V" "X," and "Y" organisms in moderate titer. Neither serum in low dilution agglutinated suspensions of P 13 organisms. The reaction of 339 and 88 organisms with absorbed P 13 antiserum was interesting from a diagnostic standpoint. In order to discover whether antigenic Glucose... Lactose... Sucrose... Maltose... Mannitol... Sorbitol... Salicin... Dulcitol... Indole... Urea... Acetylmethylcarbinol. H2S. Citrate... Motility... AG = acid and gas. A = acid. TABLE 1 Characteristics of paracolon 8trains STRAIN 339 AG 1 hours AG 72 hours AG 96 hours produced utilized 72 hours sluggish at 37 C STRAIN 88 A 120 hours AG 72 hours A 168 hours produced utilized 72 hours none at 37- C or 22 C relationships existed with other enteric organisms, suspensions of both cultures were tested with a number of unabsorbed Shigella antisera and with 25 Salmonella "O" antisera. No agglutinations were obtained with the Salmonella sera. In table are recorded the reactions of 339 and 88 paracolon strains with unabsorbed Shigella antisera. Both strains were agglutinated in practically the same dilutions of serum; the results are, therefore, incorporated in one table. Flexner "Y" antiserum agglutinated 339 and 88 in a dilution of 1:1,280, indicating a fairly strong antigenic bond. Others of the Flexner and Boyd antisera, including Boyd P 13, agglutinated the organisms in low dilutions ranging from 1:80 to 1:320. When the paracolon strains were tested with type-specific paradysentery antisera of our manufacture, prepared according to Wheeler (19), s occurred in Flexner "V," "W," "Z," Boyd 103, Boyd P 119, or Boyd 88 antisera.

3 TABLE 2 Reaction of Shigella strains with paracolon 889 antiserum LIVE ANTIGENS S. paradysenteriae Flexner "V"... Flexner "W"... Flexner "X". Flexner "Z"... Flexner "Y"... Boyd Boyd P Boyd Boyd Boyd P Boyd D 1... Boyd D Boyd P Boyd P S. alkalescens... S. sonnei... S. ambigua... S. dysenteriae... SERUM DILuTION 1:0 1:80 11:160 1:320 1:60 11:1,28011:2,560 Incubation, 52 C, water bath, 18 hours. = complete agglutination TABLE 3 Reaction of Shigella strains with paracolon 88 antiserum :.,120 SALINE CON- TROL SERMt D1ALUTION SALINE LIVE. ANTIGENS _ CON- 1:0 1:80 1:160 1:320 1:60 1:1,280 1:2,560 1:5,120 TROL S. paradysenteriae Flexner "V. 2 _ Flexner"W" _ Flexner "X. 2 Flexner "Z Flexner "Y". 2 t - Boyd Boyd P _ - Boyd Boyd170. _._._ Boyd P Boyd D 1... Boyd D Boyd P27.._... 1)oyd P13.- _. _ - S. alkalescens.. S.sonnei.- S.ambigua.. S. dysenteriae.. Incubation, 52 C, water bath, 18 hours. - complete agglutination I

4 110 W. W. FEGUSON AND WARREN E. WHEELER Since strains of Flexner "X" and "Y" are regarded by Boyd (1936, 1938) and Wheeler (19) as possesing no type-specific antigens, it is evident that the relationship of the paracolon strains 339 and 88 with the Flexner group' is a sharing of minor antigens. During the preparation of a new absorbed P 13 antiserum for use in slide agglutination tests, it was discovered that agglutinins for the paracolon strains 339 and 88 were not removed by repeated absorption with three Flexner "X" strans and two strains of Flexner "Y." Agglutinins for all other Shigella, with TABLE Reaction of paracolon strains 889 and 88 with Shigella antisera, unabsorbed 8NMA DILUTIONS 0? fisa 1:0 1:80 1:160 1:320 1:60 1:1,280 1:2,360 1:5,120 1:10,20 S. paradysenterias Flexner "V"... 1 Flexner"W". 3 2 _ Flexner "X"... =fi Flexner "Z"... 2 _. Flexner "Y" Boyd 103. Boyd P _ Boyd _ Boyd 170 _... 3 Boyd P _ BoydD I... i: Boyd D t Boyd P _ A Boyd P S. allcalescens... S.sonnei... S.ambigua... S. dysenteriae... Incubation, 52 C, water bath, 18 hours. Paracolon strains were in the "S" phase and were not agglutinated by saline. - complete agglutination. -. the exception of Boyd P 13, were removed. Further absorption of this antiserumwith339 and 88 orgms did not remove specific agglutinins for Boyd P 13, although complete removal of agglutinins for the paracolon strains was effected. In order to rule out the possibility that the reaction of 339 and 88 with both absorbed and unabsorbed Boyd P 13 antisera was due to "natural" agglutinins, a new antiserum was prepared against Boyd P 13 orgams.2 The rabbit to be 1 terminology of Boyd (190) is used here to denote types Flexner V, Z, W, X, Y, Boyd 103, Boyd P 119, and Boyd 88. 'Source of culture: Dr. C. A. Peluffo, Instituto de Higiene, Montevideo.

5 PARACOLON CULTURES AND SHIGELLA PARADYSENTERIAE immunized was tested before inoculation and found to have no agglutinins for either 339 or 88. Following immunization, the new Boyd P 13 antiserurm agglutinated the homologous strain in a dilution of 1:5,120; it agglutinated the paracolon strains in a dilution of 1:60. It is evident that a Boyd P 13 antiserum prepared by inoculation of the strain used by us will, if absorbed with Shigella strains only, contain agglutinins for the paracolon strains 339 and 88. DISCUSSION The foregoing description of the antigenic relationship of paracolon strains 339 and 88 with types of Shigella parady8enteriae is additional evidence of the lack of sharp antigenic demarcation between organisms of the Enterobacteriaceae. How commonly paracolon strains of the type described may be encountered can only be conjectured. It is probable the occurrence is rare. The chief interest to the medical bacteriologist in these organisms lies in the fact that their antigenic relationship with Boyd P 13 led to an error in the rapid slide agglutination test. We do not believe that antigenic relationships such as those described in this paper invalidate the use of absorbed Shigella antisera for rapid identification by slide technique. Our experience with such sera, together with biochemical confirmation tests, leads us to believe that the percentage of error is very low. The lack of reciprocal agglutinins in the paracolon antisera for Boyd P 13 orgasms is not an unusual finding. Berger (195) has described a similar phenomenon in his analysis of a new antigenic type of Shige71a paradysenteriae, differing only in degree from the relationship described here. It appears from our experience in producing an absorbed Boyd P 13 antiserum that the agglutinins present for 339 and 88 strains of paracolon are distinct from the type-specific fraction and are not removed by absorption with other Shigella strains which are agglutinated by this serum in low dilution. SUMMARY Two paracolon strain, isolated from the same patient,.that bear identical antigens and that share minor antigens with the Flexner group of Shigella paradysenteriae are described. In addition, a nonreciprocal relationship of Boyd P 13 with the paracolon strains is described. ACKNOWLEDGMENT Our thanks are due to Mr. Arnold Juenker of the Salmonella Typing Station, Michigan Department of Health, for tests conducted on paracolon strains 339 and 88 with Salmonella "O" antisera. REFERENCES BERGE5R, F. M. 195 A new non-mannite-fermenting dysentery organism of the Flexner group. J. Hyg.,, ill

6 112 W. W. FERGUSON AND WARREN E. WHEELER BORNsTEIN, S., SAPHRA, I., AND DANIELS, J. B. 191 The occurrence of Salmonella antigens in dysentery bacilli. J. Immunol., 2, BOYD, J. S. K New types of dysentery bacilli. 2nd Intern. Congr. Microbiol., London. BOYD, J. S. K The antigenic structure of the mannitol-fermenting group of dysentery bacilli. J. Hyg., 38, BOYD, J. S. K. 190 Laboratory diagnosis of bacillary dysentery. Trans. Roy. Soc. Trop. Med. Hyg., 33, EDWARDS, P. R., CHERRY, W. B., AND BRUNER, D. W. 193 Further studies on coliform bacteria sqrologically related to the genus Salmonella. J. Infectious Diseases, 73, SAPERA, I., AND SILBERBERG, M. 192 Taxonomy of Salmonella-like coliform organisms. J. Immunol.,, SAPHRA, I., AND WASSERMANN, M. 195 Serological relationship amongst Salmonella and other Enterobacteriaceae. J. Immunol., 50, STUART, C. A., RUSTIGIAN, R., ZIMMERMAN, A., AND CORRIGAN, F. V. 193 Pathogenicity, antigenic relationships and evolutionary trends of Shigella alkalescens. J. Immunol., 7, WHEELER, K. M. 19 Antigenic relationships of Shigella paradysenteriae. J. Immunol., 8, WHEELER, K. M., STUART, C. A., RUSTIGIAN, R., AND BORMAN, E. K. 193 Salmonella antigens of coliform bacteria. J. Immunol., 7, Downloaded from on June 19, 2018 by guest