Phages. The Tbilisi phage (Vershilova and

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1 DIFFERENTIATION OF BRUCELLAE BY THE AID OF PHAGES J6ZEF PARNAS Department of Microbiology, Academy of Medicine, Lublin, Poland ABSTRACT PARNAS, J6ZEF (Academy of Medicine, Lublin, Poland). Differentiation of brucellae by the aid of phages. J. Bacteriol. 82: There appears to be no absolute specificity of phages for the three species of the genus Brucella. The phage test is only supplementary to biochemical and serological methods for species differentiation. Pickett and Nelson (1950) were the first to examine brucellaphage. Drozewkina, Uralewa, and Czerczenkowa (1955) made fundamental examinations of brucellaphages that they isolated. Others (Mamacaszwili, 1958; Vershilova and Ostrowskaja, 1956; Dubrovskaia, Ostrovskaia, and Glubdzina, 1958) also made important observations on brucellaphage. In 1956 I obtained from the Institute of Gamaleja the "Tbilisi" phage which, according to Vershilova and Ostrowskaja (1956), lysed cultures of Brucella abortus but not Brucella suis or Brucella melitensis. This was an important observation and use of the standard World Health Organization strains (B. melitensis strain 16M, B. suis strain 1330, and B. abortus strain 544) confirmed the action of "Tbilisi" phage (Parnas, 1957). Accordingly, at the request of the World Health Organization, the Tbilisi phage was sent to Stableforth at the Central Veterinary Laboratory (Weybridge, England), van Drimmelen at the University of Pretoria, Stinebring and Braun at Rutgers University, and Jones at the University of Wisconsin. Data from these laboratories (Brinley-Morgan, Kay, and Bradley, 1960; Stinebring and Braun, 1959; van Drimmelen, 1959; Jones, 1960) are in general accord with the observations on Tbilisi phage; that is, this brucellaphage selectively lysed B. abortus and is useful in differentiating B. abortus from B. melitensis and B. suis. However, Brinley-Morgan et al. (1960) isolated from cattle in England 40 strains of Brucella which were B. melitensis Received for publication December 2, when tested by the methods of Huddleson (1943) and Wilson and Miles (1932), yet they were lysed by Tbilisi phage. This observation was also made by Jones (1960). The purpose of the present paper is to present data on examinations made from 1957 to 1960 of 44 brucellaphages on over 329 cultures of brucellae from my own collection. After examination of such a large number of brucellae, the initial optimism concerning the possibility of the use of phages to differentiate the species of the genus Brucella appears not to be entirely justified. Brucellaphage is useful in differentiating the brucellae, but only as a supplement for the methods of Huddleson (1943) and Wilson and Miles (1932), not as the sole determinative test. MATERIALS AND METHODS Phages. The Tbilisi phage (Vershilova and Ostrowskaja, 1956), phages P, Mp1p, and 2XIt (Drozewkina et al., 1955), and 34 other phages were isolated in my laboratory. (I omit data in this communication on other phages isolated by me.) Brucella cultures. Nearly 329 cultures from my collection and from foreign culture collections were used. The cultures were in the smooth phase when tested against the phages. All 329 strains were subjected to virulent phages. Not all strains were used in the tests on the activity of individual phages, and the differeniees are explained by the numbers enclosed in the text and in the tables. Isolation of brucellaphages. Phages were isolated from stock cultures 6 months old or older and, in five cases, from manure gathered from farms on which there were Brucella-infected animals. To these materials was added liver broth at ph 7.0 to 7.2 in the proportion of 1:10. This was kept in an incubator at 37 C for 10 days. The ph was adjusted to 7.0 to 7.2 and the broth then filtered, first through a Berkefeld (57 mm) filter and then through a Chamberland (160 mm)

2 320 PARNAS [VOL. 82 filter. The filtrate was examined for the presence of brucellaphage by placing some filtrate on the surface of medium containing 1.4% agar after the surface had been seeded with a culture of Brucella. World Health Organization standard strains used were: melitensis 16M, suis 1330, bovis 544, and other indicated strains, with slight sensitivity to phage activity. The phages were also titrated in this manner. In this way one phage from manure and 33 phages from old cultures were isolated. The phages were propagated on the indicated Brucella cultures in liver broth at ph 7.2. The phages were preserved by freezing at -20 C, and diluted 10-3 to 10-9 for determination of activity on various cultures of brucellae. RESULTS The Tbilisi phage lysed the standard B. abortus strain 544, but not B. melitensis strain 16M and B. suis strain 1330 (Table 1). The use of Tbilisi phage for the examination of 236 cultures of brucellae showed that the specificity of the phage for B. abortus was not absolute because only 101 TABLE 1. Action of Russian phages on Brucella cultures Lysis of standard strains No. of cultures lysed/no. examined Phage Phage source B. mel- B. abortus B. melitensis B. suir B.a- B. el- B. bortus den- ss M 1330 Typical AtYi- Typi- AtYja- Typical AtYyi- Tbilisi Vershilova, and Ostrow /164 9/23 6/13 4/4 14/22 3/10 skaja, 1956 P 89/130 14/27 2/14 2/9 22/46 4/18 Mt Drozewkina et al., /130 12/27 2/14 2/9 24/46 0/18 Mp 5/130 3/30 0/14 0/9 0/43 0/18 TABLE 2. Examination of Brucella strains with 14 phages isolated in Poland, No. of cultures lysed/no. examined Phage no. Phage source B. abortus B. melitensis B. suis Typical Atypical Typical Atypical Typical Atypical I1* Bovis typical 103/121 8/9 3/3 4/4 13/20 9/11 II Bovis typical 28/121 7/9 2/3 4/4 13/20 8/11 IX Mixture of strains 84/121 8/9 3/3 1/4 11/20 8/11 VIIa Mixture of strains 102/121 8/9 3/3 2/4 16/20 8/11 VI/10 Mixture of strains 102/121 7/9 1/3 2/4 16/20 6/11 XIVa Mixture of strains 81/121 7/9 0/3 2/4 11/20 5/11 VIlla Mixture of strains 94/121 6/9 0/3 3/5 12/20 7/11 XVI Mixture of strains 97/121 7/9 0/3 2/3 13/20 7/11 VII Mixture of strains 90/121 7/9 3/3 1/4 13/20 8/11 VIII Mixture of strains 96/121 8/9 3/3 3/4 16/20 7/11 XIa Bovis typical 101/121 8/9 0/3 3/4 16/20 9/11 IV/10 Mixture of strains 95/121 8/9 0/3 3/4 16/20 8/11 XI Mixture of strains 98/121 8/9 1/3 2/4 16/20 9/11 XIV Mixture of strains 109/121 8/9 0/3 3/4 18/20 11/11 * Each Roman numeral corresponds to a group of ten muzeal strains-determined as "mixture strains." Each means that in a group of ten strains there were different variants: bovis, melitensis, and suis typical and atypical.

3 1961] PHAGE DIFFERENTIATION OF BRUCELLAE 321 of 164 cultures of B. abortus were lysed. Thirtytwo strains of B. suis and 17 of B. melitensis were not lysed. Parnas (1958) reported that atypical Brucella strains behaved differently with Tbilisi phage. The data from the examination of phages P, Mt, and Mp on 250 strains of brucellae are given (Table 1). Table 2 shows the results of the examination of Brucella strains collected from 1957 to 1958 with 14 phages isolated from typical and atypical strains of Brucella bovis and B. suis. In 1959, I isolated 17 phages from old Brucella cultures (strains bovis and suis), and these were tested on standard strains of brucellae (Table 3). In addition, ten phages were tested on 205 brucellae strains (Table 4). Table 5 gives data on three phages isolated. When these were tested on the standard cultures, the phages acted like the Tbilisi phage, and, when tested on a great number of other brucellae, they were found to lyse some strains of species other than B. abortus. But the greatest percentage of cultures lysed with B. abortus. The photographs presented here illustrate some problems. In Fig. 1 the action of three phages (10/I, 24/II, and 212/XV) is visible on typical strains of B. bovis. Figure 2 illustrates the action of phages 212/XV and 24/II on typical strains of B. bovis. Phage 24/II in the titer 10-3 is especially strong. In the last sector (E) on the right in Fig. 2 there is complete stoppage of growth. Figures 3 and 4 show the action of phages 24/II and 212/XV on the standard strains, B. melitensis 16M, B. bovis 544, and B. suis Only the bovis strain shows sensitivity in the presence of the phages. On this basis, we can confirm that both phages are able to differentiate Brucella types. But this is an illusion only. When a greater number of strains Examination of Brucella strains with 17 phages isolated in Poland TABLE 3. Phage no. S 4/I 7/I 18/Il 21/II 92/VII 99/VIII 100/VIII 106/VIII 112/IX 116/IX 187/XIV 202/XV 205/XV 219/XVI 220/XVI 222/VIII Phage source manure * s.a. m.t. s.t. s.t. b.a. b.a. R. abortus 544 Lysis of standard strains B. melitensi.s 16M B. suis 1330 B. abortus 19 * = bovis typical, b.a. = bovis atypical, s.t. = suis typical, s.a. = suis atypical, m.t. = melitensis typical. TABLE 4. Examination of ten of my own phages on 205 Brucella strains Phage no. Source of No. of cultures lysed/no. examined phage Bovis Melitensis Suis Bovis Melitensis Stuis typical typical typical atypical atypical atypical 7/I * 97/205 4/205 10/205 10/205 1/205 3/205 18/II 77/205 4/205 7/205 8/205 1/205 5/205 21/II 109/205 2/205 13/205 8/205 1/205 1/ /VIII 62/205 2/205 2/205 5/205 1/205 2/ /VIII 7/205 0/205 0/205 0/205 0/205 0/ /XIV 76/205 1/205 5/205 7/205 1/205 1/ /XV s.t. 20/205 3/205 3/205 3/205 0/205 0/ /XVI b.a. 70/205 1/205 5/205 8/205 0/205 5/ /XVI b.a. 89/205 1/205 24/205 11/205 1/205 5/205 S manure 58/205 2/205 1/205 4/205 1/205 4/205 * = bovis typical, s.t. = suis typical, b.a. = bovis atypical.

4 322 PARNAS [VOL. 82 TABLE 5. Examination of Brucella strains with three phages isolated in Poland Lysis of standard strains No. of cultures lysed/no. examined Phage no. source B. abor- B. mel- B. B. abortus B. melilensis B. suis tus itensis suis 'M 1330 Typical Atypical Typical Atypical Typical Atypical 10/I _ - 100/162 11/28 5/28 2/10 20/59 5/23 24/II _ 97/162 14/28 5/28 2/10 15/59 2/23 212/XV s.t /162 10/28 4/28 1/10 17/59 6/23 * = bovis typical, s.t. = typical. FIG. 1. Six strains of Brucella bovis treated with brucellaphages 10/1 (sectors A), 24/II (sectors B), and 212/XV (sectors C). is examined, the sensitivity is variable and has no regularity. CONCLUSIONS In old cultures of brucellae, we frequently found phages. Certain phages showed lytic activity for Brucella abortus and these same phages often would not lyse Brucella melitensis or Brucella sutis. The brucellae that are atypical for a species behaved somewhat differently than did typical cultures. The Tbilisi phage of Vershilova lysed most but not all B. abortus strains and no B. suis or B. melitensis. Phages P and AMt of Drozewkina lysed some typical and atypical strains of B. abortus, B. suis, and B. melitensis, whereas phage Mp lysed a few B. abortus and atypical B. abortus. In this

5 .S....i t FIG. 2. Five strains of Brucella bovis infected with brucellaphages 212/IV (sectors A, B, and C) and 24/II (sectors D and E). FIG. 3. Action of brucellaphage 24/lI on Brucella suis strain 1330 (upper three areas of growth), Brucella bovis strain 544 (middle three areas of growth), and Brucella melitensis strain M16 (lower three areas of growth. 323

6 324 PARNAS [VOL..82 FIG. 4. Action of brucellaphabe 212/XV on Brucella suis strain 1330 (upper three areas of growth), Brucella bovis strain 544 (middle three areas of growth), and Brucella melitensis strain M16 (lower three areas of growth). respect phages P and Mt are polyvalent but phage Mp is monovalent. My own phages, 10/I, 24/1I, and 212/XV, appeared to be specific for B. abortus when tested on the standard strains, but were polyvalent when tested on a large number of typical and atypical strains of the three Brucella species. In conclusion, there appears to be no absolute specificity of phage to species. The phage test is useful as a supplementary test to the biochemical and serological methods (Huddleson, 1943; Wilson and Miles, 1932) of species differentiation. Examination by phage of more brucellae strains from all parts of the world is necessary. LITERATURE CITED BRINLEY-MORGAN, W. J., D. KAY, AND D. E. BRADLEY Brucella bacteriophage. Nature 188: DROZEWKINA, M. S., W. S. URALEWA, AND N. A. CZERCZENKOWA Opyt promienienija bruceloznowo bakteriofaga dlja idientifikacji hemokultur, wydielennych ot bolnoj bruciellozomljudiej. Trudy Rost. Gosud. Naucz. Islied. Prot. Inst., IX: DUBROVSKATA, I. I., N. N. OSTROVSKAIA, AND A. I. GLUBOKINA Izmenenie khimicheskogo sostava brutesell pod viliianiem faga. Biokhimiya 23: HUDDLESON, B. F Brucellosis in man and animals, p. 23. Commonwealth Fund, New York. JONES, L. M Comparison of phage typing with standard methods of species differentiation in brucellae. Bull. World Health Organization 23:130. MAMACASZWILI, E. G Brucelloznyj bakteriofag. Med. Zurn. Uzbechistana. 1:28. PARNAS, J La diff6renciation des vari6tes de brucella A l'aide du phage. Bull. World Health Organization/Bruc/177, Lima, Peru. PARNAS, J Nouvelles recherches sur la variabilit6 des Brucella. Arch. Inst. Pasteur Tunis 35:179. PICKETT, M. J., AND E. L. NELSON Brucella bacteriophage. J. Hyg. 48:

7 19611 PHAGE DIFFERENTIATION OF BRUCELLAE 325 STINEBRING, W. R., AND W. BRAUN Brucella phage. J. Bacteriol. 78: VAN DRIMMELEN, G. C Bacteriophage typing applied to strains of Brucella organism. Nature 184 (suppl. 14):1079. VERSHILOVA, P. A., AND N. N. OSTROWSKAJA Personal communication. Moscou. WILSON, G. S., AND A. A. MILES The serological differentiation of smooth strains of the Brucella group. Brit. J. Exptl. Pathol. 13:1.