possessed the H formula d - e, n.. which is known to exist in the Amersfoort as Salmonella salinatis, was identified by Dr. Welch as a member of the

A DESCRIPTION OF AN UNUSUAL SALMONELLA TYPE WITH SPECIAL REFERENCE TO THE EVOLUTION OF SALMONELLA SPECIES1 P. R. EDWARDS AND D. W. BRUNER Department of Animal Pathology, Kentucky Agricultural Experiment Station, Lexington, Kentucky Received for publication December 21, 1941 The bacillus described here was isolated by Dr. Henry Welch from rat feces collected in the vicinity of Salinas, California. The organism, which is designated as Salmonella salinatis, was identified by Dr. Welch as a member of the genus Salmonella and sent to the writers for further study. On examination it proved to be a motile rod which possessed the morphological, tinctorial, and biochemical characters of Salmonella. The bacillus produced hydrogen sulfide, did not form indole, and failed to liquefy gelatin. Glucose, arabinose, maltose, rhamnose, trehalose, xylose, sorbitol and dulcitol were fermented promptly with the production of acid and gas. Lactose, sucrose, inositol, adonitol, and salicin were not attacked. Dextro-tartrate, levo-tartrate, mucate and citrate were utilized by the bacillus but utilization of meso-tartrate could not be demonstrated. Serological examination of the organism revealed that it was a member of group B of the Kauffmann-White classification and possessed the somatic antigens IV, XII... An agglutinating serum was prepared for S. salinatis and it was found that all 0 agglutinins were removed from the serum by absorption with the Sandiego type (IV, XII...: e, h - e, n, Z15). The determination of the flagellar antigens of the bacillus presented greater difficulties. Broth cultures were sluggishly motile. When diluted with formalized saline and tested with serums representing the various H factors of the Kauffmann-White schema, the broth cultures were flocculated slowly by serum derived from Eberthella typhosa (d) and still more slowly by serum derived from the beta phase of the Mikawashima type (e, n, z15). The organism was passed through semi-solid agar until a very motile culture was obtained. This culture was flocculated rapidly and to the titre of E. typhosa serum as well as all other serums containing agglutinins for the antigen d. It was also actively flocculated by all serums containing agglutinins for antigens e, n... and to a lesser extent by e, h serums. This suggested that the organism was diphasic and possessed the H formula d - e, n.. which is known to exist in the Amersfoort type. Such a culture on plating should give rise to colonies some of which contain antigen d while others contain antigen e, n... This was not true. The culture was plated on a number of occasions and several hundred individual colonies examined. Each colony examined contained antigen d as the major H 1 The investigation reported in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is published by permission of the Director. 289

290 P. R. EDWARDS AND D. W. BRUNER constituent. Some of the colonies also were agglutinated actively by e, n... serums and to a lesser degree by e, h serums. When tested with single factor h and n serums, colonies of this sort were flocculated by n serum but not by h serum. Other colonies when tested on a slide, or when planted in broth and used as antigens in tube tests, reacted only with d serum. If the latter colonies were picked to semi-solid agar and then transferred to broth to insure very active motility, it was found that the organisms were flocculated not only by d serum but that they also reacted in low dilutions of e, h and e, n... serums. When tested with h and n serums, these colonies flocculated slowly and slightly in h serum but were unaffected by n serum. The agglutinative reactions of the isolated colonies and of the cultures used for comparison are given in table 1. TABLE 1 Agglutination reactions of phases of S. salinatis SERUMS ANTIGENS S. S. E S. Mikasalinatis, salinalis, E.* anatum, washima, h n phase 1 phase 2 sa phase i phase 2 S. salinatis, phase1.20,000 20,000 20,000 2,000 1,000 + - S. salinatis, phase2.20,000 20,000 20,000 1,000 10,000 - + E. typhosa (d).20,0 0 0... 20,000 20,000 20,000 - - _ S. anatum, phase1 (e, h).500 1,000-40,000 500 + - Mkikawashima, phase 2 (e, n, z15).500 5,000-500 20,000 - + Figures indicate highest dilution at which agglutination occurred. - indicates absence of agglutination at 1-100. + indicates agglutination in h or n serum at 1-500. It is evident that two antigenically different phases are present in the culture and that these can be separated by colony selection. In both phases d is the dominant antigen and this dominance tends to mask their differential characters. This is particularly true of phase 1, in which the minor components are present only in very slight amount. The results indicate that the antigens d, e, h and n... are present and that phase 1 carries d, e, h while phase 2 contains d, e, n... This supposition is confirmed by the absorption tests given in table 2. Serum derived from phase 1 of S. salinatis, after absorption with Salmonella anatum, phase 1 (e, h), still agglutinates phases 1 and 2 of S. salinatis and E. typhosa strongly because of the high d titre of the serum. However it no longer agglutinates e, h or e, n... forms. If the serum is absorbed with phase 2 of the Mikawashima type (e, n, zis) it still retains its d content and agglutinates S. salinatis and E. typhosa. In addition there is a slight residue of agglutinins left for e, h phases but none for e, n... phases. This demonstrates that in addition to the dominant antigen d, phase 1 contains very small amounts of e, h and that the phase may be expressed as d, e, h. The e, h component of this phase is so poorly developed that when serum derived from phase 1 of S. anatum (e, h) is absorbed with phase 1 of S. salinatis a pronounced residue of e, h agglutinins

DESCRIPTION OF UNUSUAL SALMONELLA TYPE remains even though very large absorbing doses are used. This might be interpreted as indicating that the e, h antigens of phase 1 were not typical and that failure to absorb was due to a qualitative rather than a quantitative difference. It is believed that this interpretation is not correct since the results given in the second part of the paper demonstrate that a perfectly typical e, h phase can be obtained from phase 1 of S. salinatis. The lack of absorbing power apparently is due only to the slight amount of e, h antigens in phase 1. If serum derived from phase 2 of S. salinatis is absorbed by phase 1 of S. anatum the dominant d agglutinin for S. salinatis and E. typhosa remains. All agglutinins for the e, h phases are removed but a well-defined residue of agglutinins for the e, n... phases remains. If the serum is absorbed with Salmonella TA1BLE 2 Agglutinin absorption tests with phases of S. salinatis SERlUMS S. salinatis, pbase 1 S. salinatis, phase 2 absorbed by absorbed by 291 ANTIGENS - lb 0.~~~~~~~o. S. salinatis, phase 1 (d, e, h)... 20,000 20,000 10,000 10,000 10,000 - - S. salinatis, phase 2 (d, e, n, zii)... 10,000 20,000 20,000 10,000 10,000-100 E. typhosa (d)... 10,000 10,000 10,000 10,000 - - S. anatum, phase I (e, h)...- 200 _ - - - 5000 Newport, phase 1 (e, h)...- 200 _ - - - 5000 S. abortus-equi, phase 2 (e, n, x)... - - 2,000 - - - 200 Mikawashima, phase 2 (e, n, Z16)...- - 2,000 200 - - 200 Figures indicate highest dilution at which agglutination occurred. - indicates absence of agglutination at 1-100. abortus-equi (e, n, x), agglutinins for the e, h phases and for the absorbing strain are removed but a small amount of agglutinin acting on phase 2 of Mikawashima (e, n, zi5) remains. This indicates that phase 2 of S. salinatis contains the antigens e, n, z15. This view is strengthened by the results of the absorption of the serum with phase 2 of Mikawashima, which leaves only the agglutinins for antigen d in the serum. Finally, absorption of serum derived from phase 2 of Mikawashima by phase 2 of W. salinatis removes all agglutinins for the homologous strain. It may be concluded that phase 2 of S. salinais contains the antigens d, e, n, Z15. All the phases of the genus Salmonella which contain antigen d cross agglutinate in very high dilution, usually to the titres of the serums. In spite of this close relationship of antigen d contained in different types, minimal differences

292 P. R. EDWARDS AND D. W. BRUNER in these d phases can be demonstrated by absorption. These differences have been commented upon by Kauffman (1937), by Hormaeche and Peluffo (1939) and by Edwards and Bruner (1941). In order to determine more exactly the relationships of the d component of S. salinatis to that of other types cross absorption tests were done. The results of these absorptions are given in table 3. The S. salinatis serum used in the tests was derived from phase 1 and was subjected to a previous absorption with the Sandiego type to remove the small amount of e, h agglutinins which it contained. Absorption of S. salinatis serum with the Shangani, Gaminara, or Oregon types removed all H agglutinins for the homologous strain. Absorption with E. typhosa, Muenchen or Stanley left residues of agglutinins in the serum. S. salinatis did not completely remove agglutinins from E. typhosa, Muenchen, or Oregon serums. The results indicate TABLE 3 Serological relationships of antigen d of S. salinatis SERUMS S. salinatis absorbed by 2.., ANTIGENS a03 -e0a ; - E. typhosa.-... _ + + + Gaminara,phasel1.-. _.... -.. +.. -. + - + + Stanley,phasel1.-. _.. _.. _.... -. + + + + Muenchen,phasel1.-. _.. _.. _.. _...- + + + Shangani,phasel1.-. _.... -.. + _... + + + + Wichita.-... -_ + _ - +. -_+ _ - + + + Amersfoort,phase... + + + + Oregon,phasel1.-... + agglutination at1-100. - no agglutination at 1-100. that the antigen d of S. salinatis is very closely related to that of Oregon, GaminaS, and Shangani but that it lacks some of the minor components of those types. Twelve transplants of sa.i.atis, S. each of which was isolated from a single colony, were maintained on agar slants and transferred at weekly intervals. When isolated, six of these subcultures were phase 1, the other six were phase 2. After three transfers, each of the subcultures was, plated and the antigens of individual colonies were determined. It was found that the six subcultures originally isolated from phase 1 colonies gave rise to colonies, the majority of which were phase 1. However, some phase 2 colonies were found in each of the suboultures. The number of phase 2 colonies in the different subcultures varied from 5 per cent to 30 per cent of the colonies examined. The subcultures isolated from phase 2 colonies all gave rise to some phase 1 colonies when they were

DESCRIPTION OF UNUSUAL SALMONELLA TYPE 293 plated. As in the case of the phase 1 subcultures, the percentage of colonies -which reverted to the other phase was variable. These results demonstrate that the culture is diphasic and exhibits natural phase variation. It may be concluded that S. salinatis is a diphasic Salmonella and that it is represented by the antigenic formula IV, XII...: d, e, h - d, e, n, z15. INDUCED VARIATION IN SALMONELLA SALINATIS It should be emphasized that the foregoing results were obtained with naturally occurring phases of the bacillus and that in no instance were the organisms cultivated in immune serums in order to induce variation. The writers (Bruner and Edwards, 1939, 1941; Edwards and Bruner, 1939, 1940) have demonstrated that suppressed phases of the monophasic nonspecific types, of Salmonella paratyphi A and of S. abortus-equi could be made apparent by the cultivation of the bacilli in the presence of serums containing agglutinins for their dominant phases. The results obtained in these studies supported the view of White (1926) that current Salmonella types were loss variants of antigenically complex parent strains. Since the antigenic composition of S. salinatis was more complex than that of any previously described Salmonella type it seemed an excellent strain with which to attempt the production of loss variation. In attempting to produce variation a modification of the method of Gard (1937) was used. This procedure was described by Bruner and Edwards (1939) and is based on the immobilizing effect of agglutinating serum upon motile organisms. Semi-solid agar containing agglutinating serum for the organism under study is inoculated by stabbing. The resulting growth is confined to the line of stab unless changes occur in the flagellar antigens, in which case the bacilli migrate through the medium. The organism was first cultivated in semisolid agar containing e, h; e, n; h; n; e, h + e, n; and h + n serums, respectively. The serums were used in a dilution of one fifth of the agglutinin titre, i.e. a serum having a titre of 1 to 20,000 was used in a dilution of 1-4000. When these serums were used singly or in combination the organisms migrated through the medium. The results obtained with phases 1 and 2 in semi-solid agar containing e, h; e, n, Z15 and e, h + e, n, z,5 serums are given in table 4. The results obtained with absorbed single factor h and n serums were the same. When phase 1 (d, e, h) was cultivated in semi-solid agar with an e, h serum, both phase 1 and phase 2 were recovered from the spreading growth. When phase 1 was cultivated in e, n, z,5 serum only phase 1 migrated through the medium. If both e, h and e, n... serums were added to the medium, phase 1 still spread through the agar. The amounts of serum were increased tenfold and phase 1 still spread through the medium. The S. anatum serum, which had a titre of 1-40,000 was then added to the medium in amount of 1 ml. to 3 ml. of semi-solid agar while the e, n, z15 serum was added in the amount previously used. Even in this high concentration of serum, phase 1 continued to spread. The small amount of the e, h components in phase 1 was commented upon above. Apparently these antigens are present in such slight amount that the phase is not immobilized by e, h serums regardless of the concentration in which they are used.

294 P. R. EDWARDS AND D. W. BRUNER The results obtained by the cultivation of phase 2 in e, h; e, n, Z15 and e, h + e, n, z1u serums were similar to those obtained with phase 1. Phase 2 spread unchanged through semi-solid agar containing e, h serum. If agar containing e, n, zis serum, or e, h + e, n, z15 serum was inoculated with phase 2 the organisms of that phase were immobilized and phase 1 was recovered from the spreading growth. The addition of e, h and e, n, zi5 serums to the medium did nothing more than cause the organism to exhibit normal phase variation. The organisms which had been exposed to these serums, and which migrated through the medium in their presence were apparently unchanged. After their isolation from the tubes containing large amounts of serum they continued to display phase TABLE 4 Agglutination of S. salinatis after growth in presence of agglutinating serum SERUMS ANTIGENS SERUMS ADDED TO MEDIUM Mika- E. typhosa S. anatum, washima, h n hae1phase 2 S. anatum, phase 1 (e, h)... 20,000 1,000 5,000 + + Mikawashima, phase 2 (e,n,zn)... 20,000 1,000 500 + - S. salinatis, S. anatum, phase 1 + Mikawashima, phase 1 culti- phase 2... 10,000 1,000 200 + - vated in E. typhosa..20,000 500 + - E. typhosa + h.. 1,000 20,000 - + E. typhosa + n 20,000 500 + - S. anatum, phase 1 (e, h). 10,000 500 5,000 - + Mikawashima, phase 2 (e, n, Z15)... 20,000 1,000 500 + - S. salinatis, S. anatum, phase 1 + Mikawashima, phase 2 culti- phase 2... 10,000 1,000 500 + - vated in E. typhosa... _ 1,000 20,000 - + E. typhosa + h... _ 1,000 20,000 - + E. typhosa + n... _ 20,000 500 + - Figures indicate highest dilution at which agglutination occurred. - indicates absence of agglutination at 1-100. + indicates agglutination in h or n serum at 1-500. variation in the absence of serum. The failure to induce variation must be attributed to the failure of e, h serum to immobilize the d, e, h phase of the bacillus. Entirely different results were obtained when the organisms were grown m semi-solid agar containing E. typhosa serum or any other serum that contained agglutinins for antigen d. The results of these experiments are summarized in table 4. If phase 1 of S. salinatis (d, e, h) is placed in semi-solid agar containing E. typhosa serum the organisms spread rapidly through the medium. Cultures isolated from the spreading growth have undergone profound changes in their antigenic characters. They no longer are agglutinated by E. typhosa serum but they are agglutinated to the titre of S. anatum (e, h) serum and to a lesser extent

DESCRIPTION OF UNUSUAL SALMONELLA TYPE 295 by Mikawashima (e, n, z15) serum. When tested with single factor h and n serums, they are agglutinated by h serum but not by n serum. They behave as a typical e, h phase, which occurs naturally in phase 1 of many Salmonella types. If, in addition to E. typhosa serum, single factor n serum is added to the medium phase 1 behaves in the same way. If, in addition to E. typhosa serum, single factor h serum is added to the medium the organisms spread through the agar but the spreading growth possesses different antigenic characters. It is not agglutinated by E. typhosa serum and is agglutinated only in low dilution with S. anatum serum. It reacts to the titre of Mikawashima serum and when tested with single factors, reacts with n serum but not with h serum. It resembles the naturally occurring e, n... antigens of phase 2 of many Salmonella types. When the experiments described above were repeated with phase 2 of S. salinatis (d, e, n, z15) comparable results were obtained. When cultivated in E. typhosa serum, or E. typhosa + h serum, an e, n... phase was isolated from the spreading growth. If E. typhosa serum + n serum were added to the medium an e, h phase was isolated. If either phase were planted in semi-solid agar containing h and n serums in addition to E. typhosa, serum, no spreading occurred. Further, either e, h serum or e, n... serum, when used in conjunction with E. typhosa serum retarded spreading of the organisms. Apparently the agglutinin in the serums for the antigen e which is common to both of the phases was sufficiently active to cause this retardation of migration. It was demonstrated previously that when phase 1 (d, e, h) was placed in e, h serum it was not immobilized and that phase 1 as well as phase 2 could be isolated from the spreading growth. In contrast, when E. typhosa serum, as well as h serum, is added to the medium the e, h components no longer spread, as evidenced by the isolation of pure e, n... phases under these conditions. This difference in action apparently is due to the change in the e, h antigens from a very minor 4bomponent of the natural phase 1 to a major component under the influence of E. typhosa serum. After the e, h and e, n... phases were isolated they were plated and subcultures were made from isolated colonies. Six colonies of each phase were selected and transferred to agar slants. The subcultures were transferred at weekly intervals and after three weeks they were plated and the colonies were examined. In each of the subcultures which originated from single e, h colonies, there appeared some e, n... colonies. The proportion of these e, n... colonies in the different subcultures varied from 10 per cent to 40 per cent. In each of the subcultures which were isolated from e, n... colonies some e, h colonies appeared when they were plated. As in the case of the subcultures which originated from e, h colonies, the percentage of colonies of the opposite phase varied in different cultures. None of the colonies from e, h or e, n... subcultures was agglutinated by E. typhosa serum. It would appear that S. salinatis, which in its original state exhibited d, e, h - d, e, n, z16 phase variation, after passage through E. typhosa (d) serum had lost antigen d and now displayed e, h - e, n... phase variation. The induced e, n... phase was examined more closely to determine its minor antigens. It was not agglutinated by x serum, but flocculated actively with zu serum. It

296 P. R. EDWARDS AND D. W. BRUNER left a pronounced residue of agglutinins in S. abortus-equi (e, n, x) serum but completely removed agglutinins from phase 2 Mikawashima (e, n, z15) serum when used as antigen in absorption tests. The induced phase, therefore, may be expressed as e, n, z15. Repeated attempts were made to recover the d, e, h - d, e, n, z15 form of the original culture from the e, h - e, n, z16 form of the variant. These attempts included allowing cultures to age and then plating them and the maintenance of cultures with regular periods of transfer and plating at various intervals. In addition, numerous cultures were planted in semi-solid agar containing e, h and e, n, z15 serums or h and n serums. These cultures were transferred many times in semi-solid agar containing these serums. Serial transfer in such mediums is the most effective method known for the isolation of suppressed antigens. These induced e, h - e, n, z15 phases have been maintained in the laboratory for a TABLE 5 Serological characteristics of induced phases of S. salinatis SERUMS ANTIGENS e., S. salinatis e 1 Sandiego, Sandiego, e, h phase 'h' phase 1 phase 2 E. typhosa... - - - - S. salinatis, phase 1... 2,000 2,000 1,000 1,000 S. salinatis, phase 2... 5,000 5,000 2,000 5,000 S. salinatis; e, h phase... 20,000 10,000 5,000 2,000 S. salinatis; e, n, Z16 phase... 10,000 40,000 2,000 5,000 Sandiego, phase 1... 20,000 10,000 5,000 2,000 Sandiego, phase 2... 5,000 20,000 2,000 5,000 Figures indicate highest dilution at which agglutination occurred. - indicates absence of agglutination at 1-100. year and during that time attempts to revert them to the original phase or to recover antigen d from them have been carried on almost constantly. In no instance has any variation from the induced e, h - e, n, z15 pattern been observed. Therefore the transformation of the d, e, h - d, e, n, z15 phases to the e, h -e, n, zli phases must be considered as an irreversible change. It should be emphasized that no change occurred in the somatic antigens of the bacillus during the experiments. The antigenic formula of the original culture was IV, XII...: d, e, h - d, e, n, z15 while that of the variant was IV, XII...: e, h-e, n,z15. Agglutinating serums were prepared from the induced e, h and e, n, z15 phases and from phase 1 and phase 2 of the Sandiego type (IV, XII...: e, h - e, n, z15) of Kauffmann (1940). The reactions obtained with these serums are given in table 5. No differences are discernible in the action of the serums derived from the induced phases of S. salinatis and those derived from the Sandiego type. It is worthy of note that serums derived from the induced phases contained no flocculating agglutinins for E. typhosa. This is further evidence of the complete

DESCRIPTION OF UNUSUAL SALMONELLA TYPE absence of antigen d in the induced phases. When reciprocal absorption tests were performed it was found that the induced phases of S. salinatis removed all agglutinins from serums derived from the corresponding phases of the Sandiego type. Likewise the phases of Sandiego removed all agglutinins from the serums derived from the corresponding induced phases of S. salinatis. This removal of agglutinins applies to the 0 antigens as well as the H antigens. Thus the serological identity of the two organisms is proven and the antigenic formula of the induced variant of S. salinatis established as IV, XII...: e, h - e, n, Z16. DISCUSSION 297 The results obtained in the study of S. salinatis naturally raise the question of the purity of the culture. It should be remembered that the culture was plated many times during the course of the work and, of the hundreds of colonies examined serologically, not one was found which did not possess the antigens attributed to the naturally occurring form of S. salinatis. Further, practically all of the work on induced variation was done with cultures isolated from single colonies. More than 100 single colonies were cultivated in semi-solid agar containing E. typhosa serum. In all these colonies antigen d was the major component. From every one the e, h - e, n, z1l form could be obtained. It is inconceivable that in a mixture of two Salmonella types, each of the numerous colonies examined should be mixed. The failure to find e, h - e, n, z15 forms in the original culture and the isolation of such forms from every colony cultivated in E. typhosa serum is proof that a mixture of types was not present in the original culture. The biochemical characteristics of the original culture and the induced forms were identical. The two phases of S. salinatis (d, e, h and d, e, n, zm5) are unusually complex. No other Salmonella has been described in which simiilarly complex phases occur. The antigen d is found in many Salmonella types and in them this antigen alone constitutes a complete phase. It may occur alone in monophasic organisms, as in E. typhosa and the Wichita type (I, XIII, XXIII: d: -) of Schiff and Strauss (1939), or it may constitute one phase of a diphasic type, as in Muenchen (VI, VIII: d - 1, 2) or Amersfoort (VI, VII: d - e, n, x). The e, h and e, n, z15 antigens each constitutes a complete phase of a number of Salmonella types, as in the Sandiego type (IV, XII...: e, h - e, n, z16), S. anatum (III, X, XXVI: e, h - 1, 6) and the Mikawashima type (VI, VII: y - e, n, z16). The fact that d is the dominant antigen in both phases of S. salinatis makes the recognition of the phases more difficult than in other Salmonella types. This difficulty is further complicated by the presence of the common antigen e in the minor components of the two phases. The e, h components of the d, e, h phase are present in such small amount, or are so poorly developed, that their recognition is sometimes most difficult. For some time after the separation of the phases the writers were of the opinion that certain colonies contained only antigen d and that the bacillus existed in three phases which could be expressed as d - d, e, h - d, e, n, z15. Further studies demonstrated that the supposed d phase was actually d, e, h. The failure to recognize the e, h components in these

298 P. R. EDWARDS AND D. W. BRUNER instances was due to lack of maximum development of the H antigens. Certain lots of infusion broth which were being used did not yield cultures which were sufficiently rich in these substances to permit the detection of traces of e, h antigens. It was noted above that when phase 1 of S. salinatis was placed in E. typhosa serum, e, h phases were isolated and that phase 2 under the same conditions yielded e, n, z15 phases. Large numbers of colonies of both phases were examined and only very rarely did a d, e, h phase give rise to an admixture of e, h and e, n, z15 phases. It was found that the supposed d phase, when cultivated in a medium containing E. typhosa serum gave rise to an e, h phase. This occurred in the case of every "d" colony so examined. Serum prepared from such a colony contained traces of e, h agglutinins but not e, n, Z15 agglutinins. When the organisms were cultivated in semi-solid agar containing both e, h and e, n, z15 serums the d, e, h phase was invariably isolated from the spreading growth. This also is evidence against the existence of a pure d phase in the culture. The induction of a fully developed, typical e, h phase from such a poorly developed partial antigen is a curious phenomenon. The e, n, z15 antigens of the d, e, n, zir phase are much more fully developed. One would expect, a priori, that the minor components of the two phases would exhibit approximately equal development, particularly since the e, h and e, n, Z15 antigens of the induced phases are equally well developed and typical. The e, h - e, n, z15 phases recovered from S. salinatis by cultivation in a medium containing d serum were surprisingly resistant to further change. Normal phase variation was apparent and the e, h phase was readily isolated from the e, n, z1 phase by cultivation in a medium containing n serum. The reverse change from e, h to e, n, z15 was also easily accomplished. This variation represented the extent of change in the induced forms. When cultivated in e, h + e, n... serums or h + n serums the organisms could not be forced to develop a phase which contained antigen d, as did the original culture. Further, no "artificial" phases, i.e. phases having little or no relationship to the naturally occurring H antigens of the genus, were obtained when the organisms were cultivated serially in a medium containing such serums. The production of artificial phases from E. typhosa was described by Kauffmann (1936) and from the Schleissheim type by Kauffmann and Tesdal (1937). While the attempts to induce variation in the e, h - e, n, z15 forms of S. salinatis were in progress, two cultures of the Sandiego type were used in parallel studies. Both of these Sandiego cultures spread through semi-solid agar containing h + n serum after three transfers in the medium. The induced phases thus obtained had no relationships to the natural H antigens of the genus but were related to the artificial phase of the Schleissheim type (z5). The stability of the e, h - e, n, z15 forms isolated from S. salinatis and the complete absence of d agglutinins in serums prepared from them are evidence of the profundity and permanence of the change that has occurred in the culture. The permanence of this change is even more significant when one considers the results obtained in the study of S. abortus-equi (Edwards and Bruner, 1939) and S. paratyphi A (Bruner and Edwards, 1941). In both of these monophasic types it was possible to induce a

DESCRIPTION OF UNUSUAL SALMONELLA TYPE 299 second phase which resembled the naturally occurring antigens of other types. While the induced phases of these species did not revert spontaneously to the naturally occurring phases, they were reverted by cultivation in mediums containing appropriate serums. This is in direct contrast to the irreversibility of the induced phases of S. salinatis. The change in the latter species apparently is not a simple variation but is a mutation. Since the transformation of S. salinatis from d, e, h - d, e, n, z15 to e, h -e, n, z15 is accomplished so easily in the test tube simply by cultivating it in the presence of d serum, it is not inconceivable that the same change should take place in the body of an animal whose serum contained agglutinins for antigen d. Six guinea pigs which had been injected with killed cultures of phase 1 of the Muenchen type until their d agglutinin titre rose to 1 to 1000 were injected with living cultures of S. salinatis. The animals withstood large amounts of the culture without apparent ill effect and this question was not pursued further. In his classical work on the Salmonella group, White (1926) discussed the evolution of types at some length. He concluded that in view of the numerous antigenic relationships in the genus different types must have had a common ancestry and that the organisms as they exist today have evolved by loss variation. Through this process of loss certain antigens became dominant in some types, other antigens became dominant in other types. The writers have felt that White was correct in his deductions regarding Salmonella phylogeny and have been able to bring forward some evidence to support his theories. Edwards and Bruner (1939) isolated antigen a from the naturally monophasic S. abortusequi (IV, XII...:-: e, n, x) and Bruner and Edwards (1941) recovered 1, 5... phases from S. paratyphi A (I, II, XII... : -). When the suppressed phases of these two bacilli were isolated they did not spontaneously revert to the original phases. These suppressed phases are isolated with difficulty. Long series of transfers in mediums containing serum usually are necessary to cause their appearance. This indicates that the monophasic state is deeply seated in these two species. The lack of phase variation in the suppressed phases after they are brought into dominance supports this view. These facts indicate that once an organism has lost the power of phase variation, that power is not revived even when suppressed phases are brought into dominance. These induced variants of S. paratyphi A and S. abortus-equi are additive variants and, if the theory of White is accepted, are, in a sense, anachronisms. The organisms have not been forced forward in their evolutionary course, but backward. Unlike the changes described in S. abortus-equi and S. paratyphi A, the change induced in S. salinatis results in a loss of antigenic components. This, according It to White's view, represents a step in the natural evolution of the organism. is not surprising, therefore, that the induced phases of S. salinatis should act like those of any other diphasic Salmonella and be subject to natural, spontaneous phase variation. The acceptance of White's theory presupposes the previous existence of an antigenically complex prototype. S. salinatis is, of course, not nearly so complex as this prototype must necessarily have been. However, its phases are

300 P. R. EDWARDS AND D. W. BRUNER more complex than those of any Salmonella yet described. It is undoubtedly a more primitive type than any other now known and its behavior indicates that it is in a highly unstable state. Finally, attention should be called to the fact that in the transformation of S. salinatis from IV, XII...d, e, h - d, e, n, z15 to IV, XII...: e, h - e, n, z15 an organism biochemically and serologically indistinguishable from the Sandiego type has been induced. CONCLUSIONS 1. A new Salmonella type, Salmonella salinatis, is described. The antigenic formula for this type is IV, XII...: d, e, h - d, e, n, Z15. 2. Through cultivation of S. salinatis in semi-solid agar containing agglutinating serum for Eberthella typhosa, an organism having the antigenic formula IV, XII...: e, h - e, n, z,5 was isolated. The latter is biochemically and serologically indistinguishable from the Sandiego type. 3. The bearing of these observations, and of previous experiments on induced variation, on White's theory of Salmonella phylogeny is discussed. REFERENCES BRUNER, D. W., AND EDWARDS, P. R. 1939 A note on the monophasic non-specific Salmonella types. J. Bact., 37, 365-370. BRUNER, D. W., AND EDWARDS, P. R. 1941 The demonstration of nonspecific components in Salmonella paratyphi A by induced variation. J. Bact., 42, 467-478. EDWARDS, P. R., AND BRUNER, D. W. 1939 The demonstration of phase variation in Salmonella abortus-equi. J. Bact., 38, 63-72. EDWARDS, P. R., AND BRUNER, D. W. 1940 The antigenic analysis of Salmonella species derived from domestic animals. Proc. Third Intern. Congr. Microbiol., 629-630. EDWARDS, P. R., AND BRUNER, D. W. 1941 Two new Salmonella types closely related to Salmonella muenchen. Am. J. Hyg., 34, 121-124. GARD, S. 1937 Ein Colistamm mit Salmonella-H-Antigen, zugleich ein Beitrag zur Frage der Definition der Salmonellagruppe. Z. Hyg. Infektionskrankh., 120, 59-65. HORMAECHE, E., AND PELUFFO, C. A. 1939 Estudios sobre Salmonella gaminara. Arch. urug. med., cirugia y especialid, 14, 217-226. KAUFFMANN, F. 1936 tyber die diphasische Natur der Typhusbacillen. Z. Hyg. Infektionskrankh., 119, 104-118. KAUFFMANN, F. 1937 Salmonella Probleme. Z. Hyg. Infektionskrankh., 120, 177-197. KAUFFMANN, F. 1940 tyber den a-3 Phasenwechsel in der Salmonellagruppe. Acta Path. Microbiol. Scand., 17, 430-437. KAUFFMANN, F., AND TESDAL, M. 1937 tvber zwei neue Salmonellatypen mit a-$l Phasenwechsel., Z. Hyg. Infektionskrankh., 120, 168-176. SCHIFF, F., AND STRAUSS, L. 1939 A new Salmonella type (Salmonella wichita) J. Infectious Diseases, 65, 125-126. WHITE, P. B. 1926 Further studies of the Salmonella group. Med. Research Council (Brit.), Special Rept. Series No. 103.