THE ANTIGENIC RELATIONSHIP OF THE HMMOPHILIC BACILLI 801 



of them. Strains that had been trained to grow on agar, however, failed to agglutinate 

 with the sera prepared against the recently isolated strains, and sera prepared against 

 the agar strains failed to agglutinate the strains grown on the Bordet-Gengou medium. 

 These observations were confirmed and extended by Bordet (1912). The change in 

 antigenic structure was ascribed to the medium, but it was noted that a similar change 

 might occur on a blood- containing medium after repeated subculture. Most observers 

 have confirmed Bordet' s findings that all recently isolated strains belong to a single sero- 

 logical tjrpe (see Kjistensen 1922, 1927). A few have recorded the existence of two different 

 tjrpes among strains maintained permanently on a blood-containing medium (Krumwiede 

 et al. 1923) ; but there is little doubt that such findings have been due to the slow occurrence 

 of an antigenic variation that takes place more rapidly when smooth strains are grown 

 on an unsuitable medium. Leshe and Gardner (1931) made a careful study of 32 strains 

 of H. pertussis, none of which had been regarded as rough variants. They found that 

 these strains fell into four different antigenic groups, to which they refer as Phases I, II, 

 III and IV. Of 20 recently isolated strains 18 fell into Phase I, and 2 were intermediate 

 between Phase I and Phase II. Of 7 laboratory strains that had been maintained on an 

 egg medium, 3 were in Phase III and 4 in Phase IV. Of 5 other laboratory strains, one was 

 intermediate between Phases II and III, 3 were in Phase III and 1 in Phase IV. Studies 

 by later workers (Shibley and Hoelscher 1934, Toomey et al. 1935) have been in general 

 agreement with LesUe and Gardner's findings, though they regarded the various phases 

 as arbitrary stages in the course of an S -> R variation, depending on the amount of 

 Phase I antigen on the bacillary surface (see also Toomey, Takacs and Ranta 1936, Toomey 

 and Takacs 1937). Flosdorf, Dozois and KimbaU (1941) on the other hand, find, like 

 LesHe and Gardner, that the phases differ quahtatively, and suggest the following antigenic 

 structure for three of the four phases they studied : 



Phase. Major antigens. Minor antigens. 



I ........ . a b, c 



III ......... b c or d 



IV c, d 



They also described a new Phase X, related to III and IV. Taking these studies as a 

 whole it seems safe to conclude that H. pertussis, in the form in which it exists in the 

 tissues or in recent cultures on Bordet-Gengou medium, belongs to a single, homogeneous 

 antigenic type ; but that an S — > R variation occurs somewhat readily, even in cultures 

 kept on a blood- containing medium, and very readily on less favourable media. This 

 variation is apparently step-like, so that intermediate stages exist between the normal 

 smooth form, which corresponds to Leslie and Gardner's Phase I, and the fully developed 

 rough form which corresponds to their Phase IV. It was noted by Leshe and Gardner 

 that there is no very obvious and striking colonial difference between the rough and the 

 smooth forms, nor any constant and measurable difference in salt sensitiveness, though 

 strains in their Phase III and Phase IV are, on the average, rougher in colonial appear- 

 ance and less stable in saline suspensions than strains in Phase I or II. The S form is 

 capsulated (Lawson 1933). The capsular substance, which determines the agglutination 

 of the S form by smooth antisera, is readily removed by washing (Miller 1937). Flosdorf, 

 Kimball and Chambers (1939) and Flosdorf and Kimball (1940a, b) have studied this 

 soluble agglutinogen extensively. It removes agglutinating antibodies from smooth 

 antisera. It may be Uberated by sonic vibrations. It is non-toxic and induces agglutinins 

 in the rabbit and stimulates pertussis immunity. By tryptic digestion of H. pertussis, 

 Cruickshank and Freeman (1937) obtained a carbohydrate-containing water-soluble fraction 

 capable of inducing active immimity to experimental infection in mice. It is probable 

 that this fraction is the same as the " agglutinogen " of later workers, mixed perhaps with 

 some toxin. Smolens and Mudd (1943) obtained a large yield of agglutinogen by acid 

 extraction of the bacilli. 



Eldering and Kendrick (1937, 1938) and Bradford and Slavin (1937) isolated an organism 

 from cases of whooping cough which differed from H. pertussis in producing definite 



P.B. D D 



