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Seriological groups of diphtheria bacilli Kilpatrick, Myrtle Esther 1923

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THE SEROLOGICAL GROUPS OF DIPHTHERIA BACILLI. by M y r t l e E. K i l p a t r i c k . 1 9 2 3 . SEROLOGICAL GROUPS OF DIPHTHERIA BACILLI. by Myrtle Esther Kilpatrick. A Theaia submitted for the Degree of MASTER OF ARTS in the Department of BACTERIOLOGY. THE UNIVERSITY OF BRITISH COLUMBIA MAY 1923. SEROLOGICAL GROUPS OF DIPHTHERIA BACILLI. Charrin and Rogers in 1889 were the first to find the phenomenon of agglutination. Gruber and Durham in 1896 noted that the serum of those suffering from certain infections, particularly typhoid fever, contained a specific substance which caused its homologous micro-organisms, uniformly suspended in fluid, to lose their motility and clump. This substance is known as agglutinin. Later it was found that certain bacteria exactly similar in cultural and pathogenetic characters might differ in their agglutination reactions. Gordon, (1) was one of the first to study the question deeply. He found that the majority of bacteria from cases of cerebro-spinal meningitis could be divided into four main groups which he designates as type I, II, III and IV respectively. Into a fifth group he put cases which were not agglutinated by an immune serum from any of the strains I, II, III and IV and yet were not found in sufficient numbers to justify putting them in a group of their own. Cole (2) and Dochez (^ ) have done a good deal, of work on the grouping of pneumococcus according to its agglutination reaction. They believe there are four groups. Group I and II contain typical, pneumococci. Cont'd... -2-SEROLOC-ICAL GROUPS OF DIPHTHERIA BACILLI Group III pneumococcal mucosus. Group IV contains heterogenous strains, like Gordon's group V of meningo-cocci. More lately attempts have been made to group diphtheria bacilli. J. Nicolas, Lesieur and L. Martin established the possibility of grouping them but Durand (4) was the first to get results of any importance. He was able to classify his 103 strains of diphtheria bacilli into four groups. Group A contained six types:- agglutinated by serum A at least to 1/32,000. This group also gave arough agglutination with sera B, C and D when they had not been saturated with Bacillus A. Group B contains 23 types:- agglutinated by serum B to at least 1/3200. The sera A, C and D are without action, at least to the titre of 1/400 or 1/100. Group C contains seventeen tyoes:- agglutinated by serum C to a titre of 1/3200. The sera A, B and D show no reaction. Group D contains 24 types:- agglutinated by serum D to the titre of 1/3200. In this group the serum agglutinates 22 types varying from a titre of 1/400 to 1/1,600. The sera A, B and C show no reaction with this group. There is also a fifth heterogenous group which contains 26 diphtheria bacilJi. They gave a stable emulsion but were not agglutinated by any sera of the -3-Cont'd.... SEROLOGICAL GROUPS OF PIPHTiTCRIA BACILLI, former groups. In a later paper, Durand has more definitely completed hie classification. He now claims that there are six groups. The first five contain only homogenous strains - for example in group A all the strains are agglutinated to a high titre by serum A alone. The sixth group contains isolated types or groups containing a very small number of strains which are not numerous enough to warrant a group of their own. Havens (5) has also attempted to classify diphtheria bacilli accord-ing to their agglutinating powers. From among his 206 strains, he took a granular form corresponding to "C" of Weetrook's classification and produced an immune serum with a titre of 1/4680; 169 strains were agglutinated by this serum and 37 showed no reaction. An immune serum was produced with one of the 37 strain* and all of the 37 were agglutinated by it. Havens thinks that diphtheria bacilli can be divided into two groups according to their agglutinating reactions. In the paper published by Park and Williams, (6) in 1922, further observations were made. They ottained immune sera with Durand'8 Type strains from two epidemics of diphtheria, - one near New York, the other near Toronto. With these sera they were able to corroborate the work of Durand. Cont'd.... -4-SERQLQGICAL GROUPS OF DIPHTHERIA BACILLI. Park and Mlliams point out that Havens drev: his material from a comparatively small city and that in all probability there only were two types of diphtheria bacilli present, whereas "Durand's and their own material was drawn from a much wider source and consequently there was a much greater chance for more types to be present. The grouping of pathogenic micro-organisms according to their agglutinating powers has proved to be of great v%lue in the serum therapy of diseases caused by certain micro-organisms, most particularly meningococci, pneumococci and gonococci. By "typing" the causative organism it is possible to select and give an homologous serum, For many ye=>ra the antitoxin prepared from the toxin of Park "Ho. fa" Has been thought quite adequate to protect against all strains of diphtheria bacilli. Havens is of a different opinion. He clains that standard antitoxin did not protect pigs inoculated with strains''from his group II, but he showed that there wes a certain amount of group antitoxin present which protected against both his groups if large enough Quantities of antitoxing were given. He also clains that group antitoxins differed in different batches. However, Park and Williams, (5) showed quite conclusively Cont 'd.... -5* SEROLOGICAL GROUPS OF DIPHTHERIA BACILLI. that antitoxin prepared from the strain Park MlTo. 8" were abl e to protect against any group. Durand's Technique: Durand met with a certain number of practical difficulties in the course of his work which causes him to give his technique in some detail. In preparing his immune sera, he used rabbits, horses and goats. Horses were found to be the most satisfactory giving the highest titre serum. Goats were satisfactory and rabbits only fairly so. Bacterial T-ftnulsion : He made a saline emulsion from a 24 hour growth on Loefflers blood serum. The emulsion was kept overnight on ice. He found that the great majority of strains had a more or less abundant sediment with a homogenous emulsion on top. This emulsion was pipetted off and diluted till it contained about one mg. of bacteria per cc. Certain strains, however, were entirely settled, - due to spontaneous agglutination and nothing could be found to prevent it. In the present work, similar practical d difficulties were encountered; 125 strains were isolated, - the majority from clinical cases showing symptoms of the disease, a few from carriers. I am indebted to Park and Williams for immune sera of the types "Benjamin, • *Jo1et,w and "Park IIo. 8.M Also for cultures of the strains, "Benjamin," "Bodet," Cont'd... . -6-SEROLOGICAL GROUPS OF DIPHTHERIA BACILLI. "Park No« 8," " Durand," and "Sirbeaux.• Immune sera of the groups Durand and Sirbeaux are in the course of preparation using rabbits. Agglutination reactions were carried out in accordance with the method of Durand against the immune sera of Hodet, Benjamin, and Park "No.8." The results could not be noted because of the occurrence of spontaneous agglutination. Attempts were then made to overcome this difficulty. Cultures of diphteria strains were made in (a) salt free broth, (b) salt broth, (c) peptone water, (d) Kp HPO4 broth, with no better results. Cultures were then sown on agar and Loefflers blood serum, - these cultures were transferred for 10 days in succession. A suspension was then made in saline, with the same results. A saline suspension from solid media was made and shaken for one-half hour in a mechanical shaker; it was noted that the diphtheria bacilli were more tightly clumped than before. This confirms a phenomenon referred to recently by Eliding Bergstrand (7). Our difficulty was finally overcome by taking a loopful of diphtheria bacilli, which had been grown on Loefflers blood serum for 24 hours and carefully emulsifying it in 1/1000 solution of saline. In this way a good even emulsion was prepared. The agglutination test was again attempted with the above mentioned immune sera; this -7-Cont'd.... SEROLOGICAL GROUPS OF DIPHTH3RIA BACILLI. time no agglutination occurred, the strain failing to be agglutinated by its known homogenous serum. Apparently some error or variation in technique is interfering with the reaction, or it is possible that the immune sera on hand have deteriorated. The cause of the discrepancy between these results and those of other observers is still under investigation and will be reported later. t I I ! t I 1 -8-BIBLIOGRAPHY. (1) Gordon (2) (3) (4) Cole & Dochez. Durand, Jour.Roy.Army Med.Corps 1915 XXV also Jour.Hyg. 1918 XVII Arch. Int. Med. Jour. 3xp. Med. 1914 XIV 56 1912 XVI 663 (5) Havens "Biologic Studies of B.Diphtheria" Jour. Inf. Die. 3 920 (6) Park & Williams "Pathogenic Micro-organisms" 1920, (7) Hilding Bergstrand "On the Variations of Bacterium Coli" Jour, of Baet. Vol.VIII No. 2.March 1923. ! I I t t t I 

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