Serological Comparison Between Twenty-Five Bovine Ureaplasma (T-Mycoplasma) Strains by Immunofluorescence

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1 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Apr. 197, p. 119 Copyright International Association of Microbiological Societies Vol. 2. No. 4 Printed in U.S.A. Serological Comparison Between TwentyFive Bovine Ureaplasma (TMycoplasma) Strains by Immunofluorescence C. J. HOWARD, R. N. GOURLAY, AND JACQUELINE COLLINS Agricultural Research Council, Institute for Research on Animal Diseases, Cornpton, Near New bury, Berkshire, England A serological examination, by the indirect immunofluorescence.test, was made of twentyfive bovine Ureaplasma (Tmycoplasma) strains. Two of the 2 strains were apparently identical and did not crossreact with any of the others. None of the other remaining 23 strains were identical but each crossreacted with at least one other strain, sometimes by oneway reactions. The percentage of similarity of strains was calculated and a similarity matrix was constructed. By this method, the 2 strains were found to fall into three groups. One group contained 11 strains, the second contained 12 strains, whereas the third group contained the two strains that were apparently identical but unrelated to any of the other 23. Certain strains within the two larger groups had a low percentage of similarity to some strains in the other group. Based on these results, a serological method of typing bovine Ureaplasma strahs is proposed, using antisera to eight strains with which all 2 strains react. None of these eight antisera reacted with any of the eight proposed serotypes of Ureaplasma urealyticum. Serology is one of the most important Strains. All strains, unless otherwise stated, were methods of defining species of typical, large isolated at Compton. Ureaplasma strains A417, F1, colonyforming mycoplasmas and of identifying Gra383, D48, Vic9, 013, Bu2, and Mmb143 have been isolates. Eight serotypes of Ureaplasma urealy described previously (6). Strains T234, T2, T288, ticurn, the name given to the human urea T9, Mmb167, D, F161, and D32 were isolated from calf lungs or trachea. Strains B1, T31, T311, U12, plasmas (Tmycoplasmas), have been proposed T73374, T488, T44, and T49 were isolated from the (12). However, since the guanine and cytosine urogenital tracts of cattle. Strain T49 was isolated content of bovine ureaplasmas (7) has been from the milk of a cow with spontaneous mastitis. found to be different from that of the human Strains T234, T2, and T73374 were provided by L. ureaplasmas (2), it was suggested that the bo Ruhnke, Guelph, Canada. Strains T288, T9, and vine ureaplasmas may be a different species of T488 were provided by C. Livingston, San Angelo, the genus Ureaplasrna or a different subspecies Tex. All of the strains were purified by filtering of U. urealyticum (7). suspensions and picking single colonies of microorganisms on three successive occasions as described Based on the results of a comparison of eight previously (6) and as recommended by the Subcombovine ureaplasma strains by three serological mittee on the Taxonomy of Mycoplasmatales (14). tests, it was suggested that although these Serology. Antisera were prepared as previously microorganisms are serologically heterogeneous, described (6); all were incubated at room temperature groups of similar but not identical strains might with an equal volume of medium for 1 h before use. exist (6). It also appeared from these results The indirect immunofluorescence method of Rosendal that immunofluorescence would be the most and Black (11) using unfixed colonies on agar was useful of the three techniques used for grouping followed. Titres are given as the reciprocal of the strains. highest dilution of antisera which caused fluores Since no suitable scheme exists at present for cence. Each of the 2 antisera was tested against the 2 Ureaplasma strains and, to determine the similarserotyping bovine ureaplasmas, we have comity between strains, use was made of the formula pared a number of bovine Ureaplasma isolates proposed by Sneath (13) for determining the percentby immunofluorescence to determine whether a age of similarity of strains for the construction of taxa. system for the serological identification of these However, serological reactions were not scored simply strains could be evolved. as positive or negative; a titre of < was scored as 0, titres of and were scored as +, titers of and MATERIALS AND METHODS as ++, and titres of 3 as +++. To calculate the percentage of similarity between two strains, the Media. The media used were as described previ reactions of all 2 antisera with the two strains were ously (). listed in the semiquantitative manner outlined 1 IP: On: Sat, 19 Jan 19 16:3:4

2 16 HOWARD, GOURLAY, AND COLLINS INT. J. SYST. BACTERIOL. above. Properties were scored additively and negative matches were not taken into account in the calculation. The formula used was percentage of similarity = [number of characters positive for both strains + (number of characters positive for both strains + number of characters positive for only one strain)] x 0 (13). RESULTS The titres of the antisera against the 2 strains examined in reciprocal crossimmunofluorescence tests are given in Table 1. With the exception of antisera to strain T234, all homologous titres were >SO. A considerable amount of crossreaction was observed between strains, titres against heterologous strains ranging from < to. Two of the strains, T44 and T49, appeared identical and distinct from the other 23 strains. All these 23 strains showed crossreactions, sometimes one way, with at least one other strain. However, none of these 23 strains appeared identical to any of the others. The percentage of similarity of the strains to each other is shown in Fig. 1. The 2 strains form three distinguishable groups. These groups are comprised of: the eleven strains a417 to Bu2 (Fig. 1); the twelve strains Mmb143 to T73374; and the two apparently identical strains T44 and 1'49 which are unrelated to any of the other 23. Each of the strains within the larger two of the three groups has a similarity of % to at least one other strain within that group. However, each of these two groups are heterogeneous, and within them smaller groups of strains with a higher percentage of similarity exist, e.g., strains A417, U12, B1, and Gra383 form a group of strains with a percentage of similarity to each other of >\60%. Certain of the strains in the two major groups crossreact with strains in the other group (Table 1);thus a low percentage of similarity ( <%) was obtained between strains in these two different groups. Antisera to eight of the 2 strains (listed in Table 2) were found to react with all 2 strains at titres of 9 (Table 1). It seemed that these eight strains might be useful as serotypes for the bovine ureaplasmas (see discussion). These eight antisera were therefore tested against the eight serotypes proposed for U. urealyticum (1, 12). All eight antisera had titres of < to all the U. urealyticum serotypes I to VIII. DISCUSSION The results indicate that these 2 bovine Ureaplasma strains, isolated from cattle in Britain, Canada, and the USA, comprise at least two distinguishable but slightly related heterogeneous groups and a third distinct group com prising strains T44 and T49. Certain of the strains were also compared by the metabolism inhibition test (unpublished observations). This test appeared more specific than immunofluorescence, as was found before (6), and was not as useful as immunofluorescence for the serological grouping of strains. Similar conclusions were reached in a study of Mycoplasrna hyorhinis (8). By using the formula derived from Sneath (13), a strain's reactions with all 2 antisera are taken into account when the percentage of similarity is calculated. This method incorporates more of the available information than do calculations involving homologous and heterologous reactions only (3, 9). Several alternative methods of typing new isolates can be proposed as a result of these findings. First, all new isolates could be tested with all 2 antisera. Although this method would yield the most information about the serology of a strain, it seems impractical because of the amount of labour involved. Second, pools of antisera could be made which give positive reactions with organisms comprising the three groups; further serogroups could be formed as new strains are isolated which do not react with these pools of antisera. This method would be useful only for identifying pure cultures. Since serologically distinguishable strains fall into the same group, information on the distribution of strains within a population of animals would not become evident. Third, a few strains whose antisera react with all 2 strains could be chosen, These few strains could then be regarded as serotypes with which all new isolates could be compared. The eight strains listed in Table 2 are suggested as being suitable for this purpose. Thus, if standard dilutions of antisera are used, all 2 strains tested react with at least one of these eight antisera. Although this method of typing strains does not provide as much information about new strains as would the use of all 2 antisera, it does provide a practical means of serotyping bovine ureaplasmas. This method could also be used to differentiate strains in mixed cultures since fluorescing and nonfluorescing colonies would be seen. New serotypes could be added to these eight as strains are found which do not react with the antisera, and moreover the system could be modified in the future if it became necessary. U. urealyticum (human ureaplasmas) has been shown to be serologically heterogeneous with many crossreactions between strains (9, ). However, human strains which are distinct have been found and eight serotypes of U. urealyticum have been proposed (1, 4, 12). The IP: On: Sat, 19 Jan 19 16:3:4

3 Antiserum against strain: A417 u12 BlOl Gra383 F 1 T488 T314 T9 T288 Mmb167 Bu2 Mmb Vic9 D48 D32 T2 T311 D F161 T234 T31 T73374 T44 T49 TABLE 1. Titres by immunofluorescence of antisera to 2 bovine ureaplasma strains tested against the same 2 strains A41: BlOl Gra383 F1 16C F 8C 16C 1c ia r48 Strain used as antigen Bui 01: Vies D4f Mmbl6; klmb14: D32 17 ' D2( F16: r23 T73374 T44 T49 t. IP: On: Sat, 19 Jan 19 16:3:4

4 18 HOWARD, GOURLAY, AND COLLINS INT. J. SYST. BACTERIOL. FIG. 1. Percentage of similarity of 2 bovine Ureaplasma strains by immunofluorescence. The hatchings indicate similarities of 0, to 99, 60 to 79, to 9, to 39, and 1 to 19%. TABLE 2. Crossreactions, by immunofluorescence, between eight proposed serotypes of bovine ureaplasmas An tiserum Titre against strain: against strain: A417 T9 T288 Mmb167 Bu2 D48 T31 T44 A417 T9 T288 Mmb167 Bu2 D48 T31 T44 system proposed for the bovine ureaplasmas would be analogous to that proposed for U. urealyticum, in which crossreactions between certain of the proposed serotypes have been observed (1). As in the case of the human ureaplasmas, it seems unreasonable to propose that the different serotypes of bovine ureaplasmas be regarded as separate species (12). The usefulness of this proposed system, using the eight bovine strains in Table 2 as serotypes, will depend on the percentage of new isolates that can be typed by these antisera and also on the number of serotypes eventually found. Should the number of serotypes necessary to type all bovine ureaplasmas become too large, then serological methods of identifying bovine ureaplasmas may have to be declared impractical. LITERATURE CITED 1. Black, F. T Serological methods for classification of human Tmycoplasmas. Fifth Int. Congr. Infect. Dis. Vienna 1: Black, F. T., C. Christiansen, and G. Askaa Genome size and base composition of deoxyribonucleic acid from eight human Tmycoplasmas. Int. J. Syst. Bacteriol. 22: Clyde, W. A Mycoplasma species identification IP: On: Sat, 19 Jan 19 16:3:4

5 VOL. 2, 197 COMPARISON OF BOVINE UREAPLASMA STRAINS 19 based upon growth inhibition by specific antisera. J. Immunol. 92: Ford, D. K Relationships between mycoplasma and the etiology of nongonococcal urethritis and Reiter s syndrome. Ann. N.Y. Acad. Sci Gourlay, R. N., J. Brownlie, and C. J. Howard Isolation of Tmycoplasmas from goats, and the production of subclinical mastitis in goats by the intramammary inoculation of human Tmycoplasmas. J. Gen. Microbiol. 76: Howard, C. J., and R. N. Gourlay Serological comparison of bovine Tmycoplasmas. J. Gen. Microbiol. 79: Howard, C. J., R. N. Gourlay, D. J. Garwes, D. H. Pocock, and J. Collins Base composition of deoxyribonucleic acid from bovine Tmycoplasmas. Int. J. Syst. Bacteriol. 24: Jansson, R Evaluation of some serological techniques for the identification of Mycoplasma hyorhinis and Mycoplasma suipneumoniae. Acta Vet. Scand. 1:274m2. 9. Lin, JS. L., M. I. Kendrick. and E. H. Kass Serological typing of human genital Tmycoplasmas by a complementdependent mycoplasmacidal test. J. Infect. Dis. 126: Purcell, R. H., R. M. Chanock, and D. TaylorRobinson Serology of the mycoplasmas of Man, p In L. Hayflick (ed.), The mycoplasmatales and the L phase of bacteria. AppletonCenturyCrofts, New York. 11. Rosendal, S., and F. T. Black Direct and indirect immunofluorescence of unfixed and fixed mycoplasma colonies. Acta Pathol. Microbiol. Sect. B : Shepard, M. C., C. D. Lunceford, D. K. Ford, R. H. Purcell, D. TaylorRobinson, S. Razin, and F. T. Black Ureaplasma urealyticum gen. nov., sp. nov.: proposed nomenclature for the human T (Tstrain) mycoplasmas. Int. J. Syst. Bacteriol. 24: Sneath, P. H. A The construction of taxonomic groups. SOC. Gen. Microbiol. Symp. 12: Subcommittee on the Taxonomy of Mycoplasmatales Proposal for minimal standards for descriptions of new species of the order Mycoplasmatales. Int. J. Syst. Bacteriol. 24: IP: On: Sat, 19 Jan 19 16:3:4