Notes "Species" of Brucella Characterized by Phage Lysis by Dr G. C. VAN DRIMMELEN, Professor of Bacteriology and Bacterial Diseases, Faculty of Veterinary Science, University of Pretoria, Union of South Africa; Senior State Veterinarian (Research), Onderstepoort, Union of South Africa Susceptibility to phage lysis is usually very specific Methods and related to the surface antigens of taxonomic groups, species, or strains of bacteria.a Serial tenfold dilutions of the phage suspension Especially were spotted on in the determination of Salmonella species b and of plates seeded with laboratory stock the strains of Salmonella strains, to form a " mat " of growing Brucella typhi, phage typing has organisms. been put to great practical use. c Pickett & Nelson d discovered Brucella phages After 24 hours' incubation, when sufficient growth after a thorough search. Anti-brucella phage was was achieved, plaques from the areas attacked by also discovered by Drozewkina & Vershilova, who the virus in limiting dilutions, were picked with isolated it from manure and old cultures according a platinum loop and transplanted on to fresh plates to Parnas, Feltynowski & Bulikowski. e The latter similarly seeded with Brucella bacteria. The resultant obtained from the USSR a phage which did not lyse plaques were scraped to collect the virus and the Br. melitensis or Br. suis. Only strains of Br. abortus scrapings suspended in sterile 0.1 % carboxy-methyl- which did not differ from the standard FAO/WHO cellulose solution. This suspension, which con- strain 544 (Weybridge) were lysed. The fourteen tained some Brucella organisms, was centrifuged at phages isolated by Parnas et al. did not lyse either 3000 r.p.m. for 60 minutes and then filtered through melitensis or suis strains. A Brucella phage strain a collodion membrane of 570 mp pore size. from Russia, which was examined by Braun, was A shake-flask culture of Brucella abortus strain 19, found to lyse smooth Br. abortus, but not rough or which supported the growth of the phages dealt mucoid strains (W. R. Stinebring & W. Braun- with, was inoculated with 1 ml of phage-containing personal communication, 1959). filtrate and incubated on a shaker at 37°C for At a joint CCTA/FAO/WHO course and seminar 48 hours. The culture was harvested, centrifuged on brucellosis, held at Elisabethville, Belgian Congo, and filtered, and the filtrate was heated to 600C for in 1958, a photograph was exhibited showing a phage 60 minutes to ensure freedom from bacterial cells plate in which four strains of phage, as judged by the in every possible way. This was tested by placing plaque size, could be seen. A phage suspension was drops of the filtrate on Brucella media as controls kindly supplied by Dr A. W. Stableforth, Director in every operation. of the Central Veterinary Laboratory, Weybridge, Phage activity was titrated by placing drops of England. phage suspension on agar plates seeded with live The present paper is a report on the application bacteria in suspension. of viruses obtained from this sample to the problem The phage sensitivity of various strains of Brucella of identifying and classifying strains of Brucella was tested by placing drops of filtrate on the surface organisms by phage typing. of agar plates, which had been seeded with Brucella in a Twort, F. W. (1915) Lancet, 2, 1241 suspension and dried the incubator immediately b Schmidt, A. (1931) Zbl. Bakt., 132, 202 before. It was often possible to place different c Crocket, C. G. (1947) J. Hyg. (Lond.), 45, 118 strains of bacteria either in drops or in streaks on d1 Pickett, M. J. & Nelson, E. L. (1950) J. Hyg. (Lond.), the agar surface on one plate, and these were then 48, 500 e Parnas, J., Feltynowski, A. & Bulikowski, W. (1958) exposed to drops of one or more filtrates containing Nature (Lond.), 182, 1610 phage. The tests were repeated several times. 906A - 127 - 128 NOTES A phage plaque in a usually non-susceptible TABLE 2 Brucella culture was isolated and dealt with as above. DIFFERENTIATION OF THE THREE CLASSICAL In this a variant or mutation of the "SPECIES" OF BRUCELLA ORGANISMS BY MEANS OF way original BACTERIOPHAGE STRAINS phage suspension was propagated and kept for WITH SELECTIVE ACTIVITY investigation. Lysis by strains of Brucella bacteriophage Strain of Bruce/Ia The different phage cultures were designated -______ according to the sequence of isolation, preceded by 58-f-I 59-f-2 59-f-3 the letter " f " for filtrate and the year of isolation- for example, 58-f-1, 59-f-2, 59-f-3, etc. Br. melitensis, 16M - Results Br. abortus, 544 ++++ ++++ ++++ Br. Preliminary findings showed that the original suis, 1330 +++ +++ phage suspension consisted of a culture producing Br. abortus, 19 ++++ ++++ ++++ plaques of different sizes. A single plaque isolated from a Brucella abortus strain 19 plate, and propagated on the same bac- terial strain in shake-flasks, yielded a final filtrate 58-f-1 which proved to be lytic on Br. abortus strain TABLE 1 19 and on Br. abortus strain 544 (Weybridge), the SUSCEPTIBILITY OF FIELD STRAINS OF BRUCELLA FAO/WHO reference strain. The phage suspension ORGANISMS TO A HIGH-TITRE SUSPENSION OF had no effect when spotted on Br. melitensis strain BRUCELLA BACTERIOPHAGE FILTRATE 58-f-I 16M (Beltsville) or on Br. suis strain 1330 (Minne- Suscepti- sota). Reference bility to As a result of this finding, the phage suspension lysis numberencenumber ~~~~OriginOrigin10-' dilutionby was immediately applied to a number of field strains of 58-f-1 being examined at the time. The dilution 10-2 was selected for this screening test and the earliest results 0.57. 9.Sw. Sheep foetus; Maltahbhe, South- are listed in Table 1. West Africa A culture derived from the spleen of a guinea-pig, G.57.11.Op. Guinea-pig, Karakul strain; Onder- previously inoculated with semen from an infected stepoort, Union of South Africa bull from which one testicle had been removed, was K.57.17.MI. Milk after abortion, sheep; Marien- ++++ to thal, South-West Africa found be resistant to lysis by the phage suspension at a dilution of 10-2. This result was believed to be 0.58. 6.Gr. Ram semen; Graaff Reinet, Union of +++ South Africa irregular, as the origin of the strain was bovine, but tests 0.58 8.0p. Ram semen; Onderstepoort, Union repeated confirmed it, until a single small of South Africa plaque or " spot " of phage was discovered in a M.58.20.Cf. Br. melitensis, Rev. I strain (Elberg) culture.treated with dilution 10-3. This was picked 0.58.21.Mb. Ram semen; Murraysburg, Union of and propagated on Br. abortus strain 19 to produce South Africa filtrate 59-f-2, and on Br. abortus strain 544 (Wey- 0.58.22.0p. Ram semen; Onderstepoort, Union bridge) to produce filtrate 59-f-3. Both these phage of South Africa suspensions were found to lyse Br. suis strain 1330 C.58.23.Bs. Goat foetus; Brits, Union of South ++++ (Minnesota) and thus a primitive set-up for Africa characterization of the three classical " species " of 0.58.27.Hb. Ram semen; Heidelberg, Union of South Africa Brucella organisms by means of phage lysis was available. This was demonstrated in the results B.58.29.Wk. Bull testicle; Wolwehoek, Union of ++++ South Africa shown in Table 2. The 0.59. 1.Mb. Ram semen; Murraysburg, Union of results were considered to be sufficiently South Africa clear-cut to justify the tentative inclusion of the H.59. 3.Db. Human patient; Durban, Union of phage-susceptibility pattern in the system of strain South Africa characterization of Brucella organisms. A number G.59. 4.0p. Guinea-pig, bovine unilateral cas- of tests were carried out simultaneously with the trate semen; Onderstepoort, Union of South Africa routine serological, cultural and biochemical charac- terization tests. The findings are summarized in SPECIES" OF BRUCELLA CHARACTERIZED BY PHAGE LYSIS 129 TABLE 3 COMPARISON OF PHAGE LYSIS CHARACTERISTICS WITH SEROLOGICAL, CULTURAL AND BIOCHEMICAL CHARACTERISTICS OF LABORATORY AND FIELD STRAINS OF BRUCELLA ORGANISMS Phage lysis Other characteristics Reference _ Urease numberence Monospecific CO2 Dye sensitivity production number f-i f-2 f-3 Magglutination depen- (minutes) A dence FT _ _ _ _ _ _ _ _ _ _-- _M___ __ _ _ _ _ _ _ 0.57.9.Sw. - NC NC - + - + - - G.57.11.Op. _ - _ + _ _ - - 90 K.57.17.MI. +++ +++ +++ - + _ + - 30 0.58.6.Gr. ++++ ++++ ++++ - _ + + - 180 0.58.8.0p. _ _ _ NC N C _ + - M.58.12.Es. _ - _ + _ _ _ _ 90 A.58.13.Es. ++++ ++++ ++++ _ + + + - - S.58.14.Es. - +++ +++ - _ _ _ + 10 M.58.20.Cf. - - _ + _ - _ _ 90 0 .58.21.M b. - _ - _ _ + _ ± _ 0.58.22.0p. - _ _ _ - _ + + _ C.58.23.Bs. ++++ ++++ ++++ _ + + + _ - 0.58.27.Hb. - - - - - + -±- B.58.29.Wk. +++ +++ +++ _ + + + _ 60 0.59.1.Mb. - - - - - +- ±- A.59.3. Db. _ _ _ + _ _ _ _ _ G.59.4.0p. - ++ ++ _ - + 10 M = melitensis A = abortus T = thionine F = fuchsin NC = test not completed Table 3. There was complete agreement with the culture, it is not yet possible to state that we are routine tests. dealing with pure phage cultures. Even the large The findings on Brucella strain G.59.4.0p. are plaques show " rough " edges and numerous particularly interesting. This strain, isolated by " spots " in the vicinity. guinea-pig inoculation of bovine semen, showed " suis " characteristics in most of the tests. It was Discussion resistant to the phage in the first filtrate and formed The finding of a differential susceptibility to the substrate for isolation of the phage in filtrate 2, certain phages in the genus Brucella is of considerable which lyses Br.