TYPING OF GROUP A STREPTOCOCCI BY IMMUNOFLUORESCENCE

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1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 6, pp June, 1964 Copyright 1964 by the Anmerican Society for Microbiology Printed in U.S.A. TYPING OF GROUP A STREPTOCOCCI BY IMMUNOFLUORESCENCE I. PREPARATION AND PROPERTIES OF TYPE 1 FLUORESCEIN-LABELED ANTIBODY WALTER W. KARAKAWA, EARLE K. BORMAN, AND CLARENCE R. McFARLAND Laboratory Division, Connecticut State Department of Health, Hartford, Connecticut Received for publication 17 January 1964 ABSTRACT KARAKAWA, WALTER W. (Connecticut State Department of Health, Hartford), EARLE K. BORMAN, AND CLARENCE R. McFARLAND. Typing of group A streptococci by immunofluorescence. I. Preparation and properties of type 1 fluorescein-labeled antibody. J. Bacteriol. 87: Unadsorbed, fluorescein-labeled globulins derived from rabbits immunized with acid-extracted M-protein of type 1 streptococci (plus adjuvant) were found to have high fluorescentantibody (FA) staining titers and to be considerably more type-specific than were similar preparations derived from whole-cell immunization. Appropriate adsorption rendered the anti-m reagent entirely type-specific without appreciable loss of titer; whole-cell reagent was appreciably weakened in FA titer by comparable adsorption. Type-specificity was confirmed by parallel bactericidal, long-chain, and precipitin studies. Removal of reactivity by adsorption with homologous M protein was complete, confirming that the FA reaction was truly a manifestation of an M anti-m protein system. The data indicate that the development of FA reagents specific for the streptococcal types is feasible. The M proteins of group A streptococci, upon which serological type-specificity is dependent, were reported to be weak immunizing antigens (Barkulis and Jones, 1957). Recently, others (Kantor and Cole, 1960; Hayashi and Walsh, 1961) reported reasonably high concentrations of anti-m antibodies in rabbit antisera produced by immunization with purified M protein and tested by methods more sensitive than conventional precipitin tests. However, the application of fluorescent-antibody (FA) methods to this problem has not been reported. FA methods for detecting streptococci of Lancefield group A in clinical material have been adopted widely since their applicability was first demonstrated (Moody, Ellis, and Updyke, 1958). However, critical epidemiological and clinical 1377 studies of streptococcal infections and their sequelae could be made more readily if a simple, sensitive, and rapid method for typing group A streptococci were available. It is the purpose of this report to present experimental data demonstrating that a method fulfilling those criteria can be developed with the use of type-specific, fluorescein-labeled conjugates. MATERIALS AND METHODS Streptococcal strains and culture medium. Typable cultures of 39 of the 47 serotypes of group A streptococci were secured from M. D. Moody, Communicable Disease Center, U.S. Public Health Service. Types not available were 7, 10, 16, 20, 21, 34, 44, and 45. Nine different type 1 strains were included in the series. Cultures for antigen production and testing were grown in Todd- Hewitt broth. Immunizing antigens. A type 1 strain (SS 70), rendered highly virulent by 12 serial mouse passages, was used for all immunizations. Whole-cell vaccines were prepared by the conventional method with formalinized cells (Lancefield and Perlman, 1952). For M-protein vaccine preparation, cells were grown in a rotary shaker (New Brunswick, model 25C) in 6-liter lots at 37 C for 18 hr with constant agitation at 200 rev/min. Cells were sedimented in a Sharples centrifuge. Packed cells were resuspended in 100 ml of buffered saline (ph 7.4), and were sedimented in a conventional centrifuge at 1,860 X g. After repeating the washing procedure twice, packed cells were resuspended in 20 ml of buffered saline, and were sonically disrupted for 2 hr in a Raytheon 9-kc sonic oscillator. The sonic extract was sedimented at 2,000 X g for 30 min, washed once in buffered saline, and resuspended to the original volume with buffered saline. After adjustment to ph 2, M protein was extracted by the method of Lancefield and Perlman (1952). Analyses of the M-protein prepara-

2 1378 tions by a modified biuret test (Daughaday et al., 1952) gave readings of 2.0 to 3.5 mg/ml of protein. Incomplete Freund adjuvant was then added, as recommended by Hayashi and Walsh (1961). Immunization procedures. Young New Zealand rabbits weighing approximately 3 kg were used for antiserum production. Conventional injection schedules were used to immunize rabbits with the whole-cell vaccines. However, with the M-protein preparations, the procedure of Hayashi and Walsh (1961) was utilized as follows. Animals were injected intramuscularly every 4 days for a period of 4 weeks with 0.5 ml of M-protein vaccine, each dose containing approximately 1.40 mg of protein. Blood samples were obtained at intervals by intravenous puncture; care was taken to maintain sterility so that the serum could be assayed for specific antibody by the long-chain test. The animals were bled 5 weeks after the initial injection, and the sera were separated from the blood cells and stored at 4 C. Adsorption reagents. A nontypable group A streptococcus, designated A-60 (SS 132; furnished by E. L. Updyke, Communicable Disease Center, U.S. Public Health Service) was used as the principal adsorbing agent. This strain was selected because it lacked detectable M protein. Antibody determinations. Precipitin tests were performed by the capillary tube method (Swift, Wilson, and Lancefield, 1943). The direct bactericidal method of Kantor and Cole (1960) was used to demonstrate the presence of protective antibodies. For convenience, the bactericidal index was used to express results, the index being the ratio of the inhibition of growth in the presence of immune blood to the inhibition of growth in normal blood. Indices were considered to have the following signifi- TABLE 1. of fluorescein-antibody conjugates (anti-type 1) KARAKAWA, BORMAN, AND McFARLAND Comparative fluorescent-antibody titers Immunizing vaccine Rabbit FA titers Whole cells ,280 M protein ,560 M protein ,280 M protein Titer expressed as reciprocal of highest dilution giving 4+ staining intensity. J. BACTERIOL. cance: less than 25, questionable; 25 to 50, weakly positive; 50 to 200, positive. The long-chain method introduced by Stollerman and Ekstedt (1957) also was used to demonstrate the presence of type-specific antibodies. A long-chain index (ratio of mean chain length in immune serum to that in normal serum) greater than 3 was considered significant. FA studies were performed on smears prepared from appropriate 6-hr broth cultures, with the use of reagents and methods described below. Preparation of globulin and FA reagents. Typespecific antisera were obtained by adsorption with A-60 cells. With an equal volume of twicewashed and packed A-60 cells were mixed 20 ml of antiserum. After mixing, the suspension was incubated for 2 hr at 37 C, after which sorption was allowed to continue for 18 hr at 4 C. Cells were then sedimented at 1,860 X g for 30 min, and the supernatant fluid was transferred to a rubber-capped bottle to be stored at 4 C. Globulin fractions were prepared by precipitation at 4 C with 50% saturated (NH4)2SO4. After dialysis against normal saline, globulins were labeled with fluorescein isothiocyanate (BBL, lot no ), according to the method of Marshall, Eveland, and Smith, (1958). FA staining. Stained preparations were examined under a Reichert microscope equipped with a mercury vapor lamp (HBO 200), a pass filter (BG 12), and a barrier filter (OG 1). Cell-wall staining intensities were recorded as follows: 4+, intensely bright staining; 3+, bright staining; 2 +, moderate staining; and 1 +, dull staining. RESULTS Immunization studies. The value of using M protein rather than whole cells for the production of a type-specific FA reagent is clearly shown in Tables 1 and 2. Comparative immune responses to M protein and whole-cell vaccines are shown in Table 1. Anti-M protein sera from rabbits immunized with a total dose of only 9.8 mg of protein yielded FA conjugates with titers comparable to that obtained from an antiserum derived by wholecell immunization. The superiority of conjugates prepared from antisera resulting from M-protein immunization over those resulting from whole-cell immunization is clearly shown in Table 2. Unadsorbed FA

3 VOL. 87, 1964 TYPING OF GROUP A STREPTOCOCCI 1379 TABLE 2. Fluorescent-antibody staining of homologous and heterologous types of streptococci with unadsorbed and adsorbed type 1 conjugates Reciprocal Staining intensity for streptococcal types Conjugate FA titer of dilution used Others Anti-whole cell, un- 1,280t All 4+ adsorbed. Anti-whole cell, adsorbedt All 0 Anti-M, unadsorbed.. 1, types, 0 5 types, 41 8 types, 1+ Anti-M, adsorbed... 1, All 0 Types 1 through 47 with exception of 7, 10, 16, 20, 21, 34, 44, and 45 were tested. t Group as well as type antibodies were present; precipitin test with C carbohydrate was positive. t Two adsorptions with strain A-60. reagent prepared from whole-cell antiserum showed complete lack of type-specificity, and stained all heterologous types of group A streptococci in 1:128 dilution. Conversely, unadsorbed anti-m protein FA reagent reacted strongly with only 7 of the 38 heterologous types at the same dilution. These results suggested that the homologous titer of the whole-cell FA reagent was due in large part to group (anti-c) antibodies present in the serum. This was verified by precipitin tests with purified C carbohydrate extracted from A-60 by the formamide method (Fuller, 1938). The adsorption results further suggest the superiority of M-protein vaccine in the preparation of type-specific FA reagents, although the original titer obtained with whole-cell vaccine was of the same order. After two adsorptions with A-60, the anti-m FA reagent was markedly type-specific, without having suffered an ap- TABLE 3. Bactericidal test indices with anti-type 1 blood against homologous and heterologous types of streptococci Immune blood Bactericidal indices it A-60 Anti-whole cell Anti-M protein Bactericidal index = growth in normal blood/growth in immune blood (type 1). t Indicates type number of streptococci. TABLE 4. Long-chain indices of eight types of streptococci grown in the presence of type 1 antisera Antisera Long-chain indices It A-60 Type 1 anti-m Type 6 whole cell Type 24 whole cell Long chain index = mean chain length in antiserum/mean chain length in normal serum; a ratio > 3 was considered significant. t Indicates type number of streptococci. preciable loss of titer. By contrast, comparable adsorption of the whole-cell FA reagent resulted in a marked drop in homologous titer. Immunological tests. Bactericidal activities of whole-cell and M-protein antisera are shown in Table 3. The results demonstrate the presence of type-specific antibodies in blood from animals immunized with either type of vaccine. The homologous bactericidal activity of type 1 anti-m blood was approximately 56 times that of normal blood, but activity against heterologous types was not significant. Even though not significantly different from anti-whole cell blood in this respect, the anti-m blood showed marked typespecificity by this test. Long-chain studies are shown in Table 4. Type

4 1380 KARAKAWA, BORMAN, AND McFARLAND J. BACTERIOL. 1 streptococci grown in homologous anti-m serum had a mean chain length which was four times that of the same strain grown in normal serum; heterologous types gave chain-length indices of less than 2. Because a long-chain index greater than 3 is considered significant, the type-specificity of the anti-m serum again is self-evident. Various strains of type 1 streptococci were tested against the anti-m serum by both the long-chain and the FA methods. Comparative results are shown in Table 5. All type 1 strains tested by the long-chain method gave indices comparable to that of the strain from which the M protein used for immunization was derived. Significantly similar results were obtained with the FA method. Strains of streptococci with indices greater than 4 were significantly stained by higher dilutions of the conjugate than were those with lower indices. The one strain with an index below 3 was not as reactive as the others. Ml anti-al involvement in FA staining. More direct evidence of the involvement of a true M anti-m system in the FA reaction is presented in Tables 6 and 7. The M-protein preparation used in all the experiments was found to be serologi- %cally active and specific (Table 6). A potent type 1 precipitin serum (obtained from M. D. Moody) gave a strong precipitin reaction with this M-protein antigen. Furthermore, when adsorbed with M protein, the serum lost all its activity in the precipitin test. Similar adsorption with heterologous M proteins (only results with type 5 are TABLE 5. Comparison of long-chain indices and fluorescent-antibody titers of type 1 anti-m antibody against nine strains of type 1 streptococci Strain Long-chain index FA-staining titert SS ,560 DS ,280 DS ,280 DS ,280 DS DS ,280 SS , ,560 T ,560 Immunizing strain. t Reciprocal of highest intensity of 3+ or greater. dilution giving an TABLE 6. Precipitin reactions with antisera unadsorbed and adsorbed with purified M protein Type 1 antisera Antigen concn (purified type 1 M protein in mg/ml) o -o No No o0 CDC, unadsorbed i CDC, adsorbed with type 1M protein CDC, adsorbed with type 5 M protein Anti-M unadsorbed Anti-M, adsorbed with type 1M protein Anti-M, adsorbed with type 5Mprotein Type 1 precipitin serum obtained from MI. D. Moody. TABLE 7. Inhibition of homologous fluorescentantibody reactions by specific antiserum FA reagent Anti-M globulin, type 1 (1:100) Inhibiting reagent Normal serum Type 1 anti-m Type 1 typing serum (CDC) FA staining intensities Dilutions of inhibiting reagent eq ~o shown) failed to remove the precipitin activity. Comparable adsorption studies with one of o'ur antisera prepared by using M-protein injections gave similar results, although the initial precipitin titer was noticeably lower. The one-step FA inhibition test results shown in Table 7 clearly show the correlation between the two antisera in terms of type-specific antibodies. The capacity of both sera to block the FA-staining reaction was found to be similar, indicating that both sera contained an appreciable concentration of type-specific antibodies to inhibit the AM anti-m reaction.

5 l'ol. 87, 1964 TYPING OF GROUP A STREPTOCOCCI 1381 DISCUSSION The procedures for the production of type 1 conjugate detailed herein were applied to the production of conjugates for types 6 and 18. Results paralleled those reported for type 1. The data demonstrate the feasibility of using the FA method for typing group A streptococci. WVith this method, acid-extracted type 1 M protein combined with an adjuvant was consistently found to elicit good immune responses when injected into rabbits intramuscularly. Enhancement by mouse passage of the virulence of the immunizing strain is an essential prerequisite for the production of suitable M-protein extracts. The similarity of anti-m titers of sera and conjugates derived from rabbits immunized with M antigen as compared with those derived from whole-cell immunization brings into focus certain significant points. The anti-m serum, although weakly positive by the precipitin test, yielded a conjugate giving strong FA staining in high dilution. On the other hand, the anti-whole cell serum gave a stronger precipitin test, but the FAstaining titer of its conjugate was not significantly different from that of anti-m serum. Logically, one might expect the anti-whole cell serum to give a stronger FA reaction than does the anti-m serum because of its greater precipitin reactivity. Because this is not the case, it is probable that the precipitin test measures something in addition to the presence of type-specific M protein. Furthermore, it appears that precipitin test results are not truly indicative of the titer of type-specific antibodies in an antiserum, and may even lead to a false conclusion that they are absent when actually present in high titer, as is demonstrable if one prepares and tests a suitable FA conjugate. FA methods are so sensitive that they should be more fully explored for the detection of either type-specific substance or its antibody. Other conventional methods for the demonstration of type-specific antibodies are principally dependent upon the presence of anti-m antibodies. The bactericidal test (Hirsh, 1960) depends upon the phagocytosis and destruction of streptococci in their presence. Another method, the long-chain test (Stollerman and Ekstedt, 1957), depends upon the chaining out of streptococci grown in the presence of the specific antibodies. Although considered to be the most reliable tests available to demonstrate the M anti-m reaction, these methods as well as the precipitin test are laborious and sometimes difficult to interpret. Data presented show clearly that M anti-m reactions shown by FA studies closely parallel those demonstrable by conventional methods; our studies suggest strongly that they have the same serological basis. The type-specificity of the FA reagents developed in our study is most promising. By analogy with other methods, one might have anticipated a greater degree of nonspecificity with such a highly sensitive serological test. However, this was not the case with the FA method. Fluorescein-labeled, type 1 anti-m antibodies gave good type-specificity when tested against 38 heterologous types of group A streptococci. Type-specificity of this magnitude with a procedure as sensitive as the FA method clearly substantiates the concept that type-specificity resides in the M-protein fraction of group A streptococci. The development of high-titered, type-specific, FA-labeled antibodies could have much practical importance. Kaplan (1962) already demonstrated localization of M protein in tissue with FA reagents. Availability of better typing conjugates would facilitate this and other avenues of investigation. Before type-specific fluorescein reagents can be fully developed and widely applied, certain problems must be solved, the most critical being the isolation and purification of M protein relatively free of other cellular components. Our data confirm the findings of Hayashi and Walsh (1961) that high-titered antisera can be obtained by immunizing rabbits with acid-extracted M protein. Reactions were, however, observed with a few heterologous types of streptococci. That these crosses were due to foreign protein extracted in parallel with M protein seems likely, because the reactions could frequently be removed by adsorbing with a heterologous type with only a minimal loss of titer. Observations of this nature fully emphasize the crucial importance of purity of immunizing antigens in producing specific FA reagents. Experience has taught us that the production of antisera relatively free of strong heterologous crosses facilitates the production of highly reactive, fluorescein-labeled globulins of appropriate specificity. A proteinase (Elliott, 1945) poses another

6 1382 KARAKAWA, BORMAN, AND McFARLAND J. BACTERIOL. problem in the development of potent reagents. This substance is an extracellular enzyme produced by many group A streptococci which can destroy M protein while it is being synthesized in broth culture. According to Elliott and Dole (1947), this proteinase is derived from a precursor which is converted into the active enzyme autocatalytically under reduced oxygen tension. Successful inhibition of this reaction appears possible, but our studies directed toward this end are too incomplete for reporting at this time. Nevertheless, the results reported herein and continuing studies in our laoratory encourage us to believe that the typing of group A streptococci by an FA method is feasible, and that success in this development will open up new avenues of epidemiological and clinical investigation. ACKNOWLEDGMENT This work was supported in large part by the Heart Disease Control Program, U.S. Public Health Service (contract no. PH ). LITERATURE CITED BARKULIS, S. S., AND M. F. JONES Studies of streptococcal cell walls. I. Isolation, chemical composition, and preparation of M protein. J. Bacteriol. 74: DAUGHADAY, W. H., 0. H. LOWRY, N. J. ROSE- BROUGH, AND W. S. FIELDS Determination of cerebrospinal fluid protein with the Folin phenol reagent. J. Lab. Clin. Med. 39: ELLIOTT, S. D A proteolytic enzyme produced by group A streptococci with special reference to its effect on the type-specific M antigen. J. Exptl. Med. 81: ELLIOTT, S. D., AND V. P. DOLE An inactive precursor of streptococcal proteinase. J. Exptl. Med. 85: FULLER, A. T The formamide method for the extraction of polysaccharides from hemolytic streptococci. Brit. J. Exptl. Pathol. 19: HAYASHI, J. A., AND G. WALSH Studies of streptococcal cell walls. VI. Effects of adjuvants on the production of type-specific antibodies to cell walls and isolated M protein. J. Bacteriol. 82: HIRSCH, J. H., AND A. B. CHURCH Studies on phagocytosis of group A streptococci by polymorphonuclear leucocytes in vitro. J. Exptl. Med. 111: KANTOR, F. S., AND R. M. COLE Preparation and antigenicity of M protein released from group A, type 1 streptococcal cell walls by phage-associated lysin. J. Exptl. Med. 112: KAPLAN, M. H Localization of streptococcal antigens in tissues. I. Histologic distribution and persistence of M protein, types 1, 5, 12, and 19 in the tissue of the mouse. J. Exptl. Med. 107: LANCEFIELD, R. C., AND G. E. PERLMAN Preparation and properties of type-specific M antigen isolated from group A, type 1 hemolytic streptococcus. J. Exptl. Med. 96 : MARSHALL, J. D., W. C. EVELAND, AND C. W. SMITH Superiority of fluorescein isothiocyanate for fluorescent antibody technique with a modification of its application. Proc. Soc. Exptl. Biol. Med. 98: MOODY, M. D., E. C. ELLIS, AND E. L. UPDYKE Staining bacterial smears with fluorescent antibody. IV. Grouping streptococci with fluorescent antibody. J. Bacteriol. 75: STOLLERMAN, G. H., AND R. EKSTEDT Long chain formation by strains of group A streptococci in the presence of homologous antiserum: a type-specific reaction. J. Exptl. Med. 106: SWIFT, H. F., A. T. WILSON, AND R. C. LANCE- FIELD Typing group A hemolytic streptococci by M precipitin reactions in capillary tubes. J. Exptl. Med. 78: