meningitidis passages from the original source material by storing the cultures at -65 C (18) in Trypticase Soy Broth

Transcription

1 JOURNAL OP BACTERIOLOGY, Apr. 1968, p Vol. 95, No American Society for Microbiology Printed in U.S.A. A New Serological Group (E) of Neisseria meningitidis NEYLAN A. VEDROS, JAMES NG, AND GARTH CULVER U.S. Naval Medical Research Unit One, Naval Biological Laboratory, University ofcalifornia, Berkeley, California Received for publication 19 January 1968 A new serological group of encapsulated Neisseria meningitidis, tentatively classified as group E, produced halo precipitates with homologous antiserum. Groupspecific complement-fixing antibodies were produced in rabbits by inoculation with lysed cells. The group E isolates are immunologically related to group C, as shown by precipitation and quantitative agglutination. Antisera to group E strains did not protect mice challenged with groups A, B, or C organisms. Until now, a comparison of group E strains with the X, Y, and Z strains of Slaterus has not been conducted. In 1950, the Nomenclature Committee of the International Association of Microbiologists classified Neisseria meningitidis into four serological groups (2). Group A corresponded to the old types I and III of Gordon and Murray (8); group B corresponded to type II of Gordon and Murray. Group C was defined as an independent serological group by agglutination, presence of a capsule (typical group B strains do not exhibit capsules), and inability of group C antiserum to protect mice challenged with group B strains (5). Group D corresponded, to some extent, with the type IV of Gordon and Murray, but it occurred so rarely that its relationship with other serogroups is vague. Since 1963, the incidence of meningococcal infections in the United States has increased (7). The majority of isolates, from patients with meningococcal disease and healthy carriers of these bacteria, were typed as group B. Occasionally group C isolates were found, and very rarely isolates were of group A. In the past 2 years, however, there has been an increasing number of isolates that could not be typed by groups A, B, C, or D antisera obtained from commercial sources or prepared in our laboratory. At present these nontypable isolates constitute a new serogroup of N. meningitidis, and we suggest that they be called group E. MATERIALS AND METHODS Microorganisms. The strains of N. meningitidis chosen for use in our study were only strains that were virulent for mice so that satisfactory mouse protective antibody studies could be carried out (Table 1). The isolates were maintained within two to five 1300 passages from the original source material by storing the cultures at -65 C (18) in Trypticase Soy Broth (TSB; BBL). Initial serological grouping was based on macroscopic agglutination with rabbit antisera to the following prototype strains: group A (strain 1027), group B (strain 2091), group C (strain 1628), group D (strain M168), and group E (strain SD4C). The last strain was isolated from a patient in Group D was dropped from the study because no recent isolates were available, and the two strains obtained from the collection of the late Sarah Branham (strains M168 and M 169) were avirulent for mice (LDS > 106 bacteria). Halo precipitation was determined by incorporation of 0.8 ml of antiserum per 10 ml of Mueller-Hinton (MH) agar (Difco) in plates (13), and wet, India ink preparations were made as described by Duguid (6). Virulence assay. Virulence for mice was established by the technique of Miller (12) with 4% gastric mucin (Nutritional Biochemicals Corp., Cleveland, Ohio; lot 9801). Groups of six mice per dilution of bacteria were observed for 96 hr; all the dead animals were examined for intercurrent infection by smear of peritoneal exudate. Sera. Rabbits were inoculated with lysed cell preparations of the isolates listed in Table 1. The isolates were cultured on MH slants overnight (37 C, 8% C02, 100% humidity), and the growth was harvested with TSB. The inoculum was adjusted to give a final concentration of 107 cells per ml in 100 ml of TSB. After 4-hr incubation at 37 C with shaking, the cells were harvested in the log phase; they were washed twice with saline and were resuspended in saline to 109 cells per ml. Complete autolysis occurred after 7- day storage at 4 C. Each week, rabbits received a 1- ml dose of this mixture intraperitoneally followed 24 hr later by 1-ml doses intravenously daily for 2 days. This schedule was repeated for 3 weeks followed by 1 week of rest; then it was repeated for 1 week. Sera

2 VOL. 95, 1968 GROUP E NFISSERIA MENINGITIDIS 1301 employed in this study were obtained 10 days after the last injection. The mouse-protective capacity of sera was determined by the technique of Branham (4). At 0.5 to 2 hr before intraperitoneal injection of the test bacteria suspended in mucin, 1 ml of various dilutions of heatinactivated serum in saline was injected intraperitoneally. Mice were challenged with 100 to 10,000 minimal lethal doses (MLD). The number of mouseprotective units was defined as the reciprocal of the highest serum dilution which protected 50% of the challenged mice. Mice in separate control groups were inoculated with 0.15 M saline or a 1:10 dilution of normal rabbit serum before receiving the challenge dosage of bacteria. Serology. Macroscopic agglutination was performed by mixing 0.05 ml of cell suspension (0.15 M phosphate buffer with 0.5% phenol, ph 6.8) with 0.05 ml of undiluted serum. We read the results after 5 min at room temperature. Normal rabbit serum and saline were used as controls. We employed the same complement-fixation (CF) technique used by Terzin et al. (16). The CF titers of all sera were determined with 4 units of antigen. The precipitin content of sera was established by the capillary technique (15). The cell debris in the lysed cell preparation was removed by centrifugation, and equal volumes of the supernatant liquid and heatinactivated sera were drawn into the capillary. The amount of precipitate was recorded after incubation for 4 hr at 37 C, after it had been refrigerated overnight. Total agglutinin nitrogen and group-specific agglutinin nitrogen were determined by quantitative agglutination, as described by Kabat and Mayer (9), by use of a modified Folin-phenol technique (11). Preliminary experiments indicated that the total agglutinin nitrogen of group E antisera was extracted with group E cells after four absorptions. Heat-inactivated (56 C,30 min) group E antisera were treated ina similar manner with groups A, B, and C cells. Equal amounts of bacteria (approximately 500,ug of bacterial nitrogen) were used for each absorption; the amount of bacterial nitrogen recovered from control tubes containing normal rabbit serum was employed to correct for total agglutinin nitrogen. All determinations were run in duplicate. RESULTS Growth characteristics and morphology. During the past 2 years, 625 isolates from healthy carriers of meningococci and 72 isolates from pateints with meningococcal disease were received by this laboratory. Of these isolates, 237 (38%) from carriers and 8 (11 %) from patients could not be classified serologically with antisera to groups A, B, or C. Initial colony growth of these nontypable isolates (tentatively classified as group E) on MH agar or on a selective medium (17) was similar to that of groups A, B, and C. These colonies were glistening and lenticular, and their diameter measured 1 to 2 mm. As shown in Table 1, the groups A, C, and E strains produced halo precipitates when spotinoculated on agar containing their respective antisera. These precipitates were well defined, and none of the strains tested yielded precipitates with antisera other than their own group. Precipitates did not develop with any of the group B isolates. Capsules were noted in wet, India ink prepara- TABLE 1. Source and characteristics of Neisseria meningitidis cultures Sulfadijazine Group Strain Source precipitate Halo Capsule sensitivity (MIC)a LD=J6 A 791 Morocco + + <0.05 1,200 CL 4 North Carolina + + < K-36c Korea + + < B L-1 Louisiana _ - >10.0 <10 NOR 25 Norfolk <0.( SDIC San Diego < C PTS-5 Portsmouth + + <0.05 2,000 LAIC Los Angeles ,000 SD3C San Diego <10 E SD4C San Diego + + < Bo-6c Washington, D.C. + + < NOR 28 Norfolk + + <0.05 <10 a MIC is the minimal inhibitory concentration in milligrams per 100 ml. b The LD50 is the number of microorganisms that caused death in mice within 96 hr. c Strains K-36 and Bo-6 were isolated from healthy carriers. The other strains were isolated from the cerebrospinal fluid of patients with meningococcal disease.

3 1302 VEDROS, NG, AND CULVER J. BAcrmRoL. tions of groups A, C, and E strains but not in group B. The capsules appeared to be similar in size for all three groups (approximately 0.5 A). In several field studies of a 3-month duration, we frequently observed, on weekly culturing of meningococci from the nasopharynx of a chronic carrier, that the serotypes isolated from a given individual changed. For example, for several weeks isolates would type as group B, but suddenly, subsequent isolates agglutinated only with group E antisera. The reverse (group E to B) was not observed nor was this phenomenon apparent with carriers of group C meningococci. Several strains of groups B and E isolates were passed daily on chocolate-blood-agar (CA) for 25 days and by blind passage (intraperitoneally) in mice for 10 days to determine whether frequent passage in vitro and in vivo affected the agglutinating specificity. After passage in vitro and in vivo, the test strains retained their group specificity, as demonstrated by macroscopic agglutination. Colonial morphology did not change even after mouse or CA passage. Serology. Group-specific complement-fixing antibodies were produced in rabbits inoculated with lysed cell preparations (Table 2). Although some cross-reactions occurred among the four serogroups, the homologous titers were 16- to 512-fold higher than the heterologous titers. These sera produced by inoculation with lysed cells were more group specific than were the antisera to whole cells (18). Precipitin reactions are summarized in Table 3. Some cross-reaction occurred between group C antiserum and group E antigen. There were also some group C precipitins in group E antisera. Essentially, no cross-reaction was observed among groups A, B, or E. The serological identity of the group E isolates and their relationship with the other serogroups were further demonstrated by cross-absorption experiments using the quantitative agglutination technique. The results (Table 4) show that group C cells removed only approximately 20% as much TABLE 2. Complement-fixing titers of rabbit antisera to lysed Neisseria meningitidis cells Antigen A B C E A 4,096a B 64 2, C , E ,048 a Reciprocal of highest dilution of serum reacting with four units of antigen. TABLE 3. Precipitin reaction of rabbit antisera to lysed Neisseria meningitidis cells" Antigen.- A B C E A ++b B C E a Cell debris removed from lysed cell preparation and equal volume of supernatant fluid mixed with equal volume of heat-inactivated sera. & The symbols 0 to ++++ indicate increasing amounts of precipitate after 4 hr at 37 C and overnight at 4 C. TABLE 4. Absorption of agglutinins to group E (strain SD4C) of Neisseria meningitidisa Cells (serogroup) Nitrogen removedb Agglutinin nitrogen (mg/mi of serum) absorbed'(% A B C E a All rabbit sera inactivated at 56 C for 30 min and absorbed four times at 4 C for 4 days for each absorption. Each 1 ml of sera absorbed with 500 jug of bacterial nitrogen. b Average of two determinations. agglutinin nitrogen from group E antisera as from the homologous group E cells. Very little crossreaction occurred among group E antisera and groups A or B cells. Mouse-protection studies. Rabbit antisera to the four serogroups protected mice, as shown in Table 5. The mouse-protective units in the homologous antisera ranged from 28 to 160. Group E antisera did not protect any of the mice challenged with groups A, B, or C. Mice challenged with strain SD4C were better protected by the homologous antiserum than they were by group E, strain Bo-6 antiserum. Mice challenged with group E strain NOR-28 were not protected by 1 :10 dilution of antisera to groups A, B, or C (Table 6). However, the mouseprotective units numbered 70 in the homologous antiserum. DIscussIoN Our finding of nontypable meningococcal isolates is somewhat analogous to what was noted in the late 1930's. At that time, an increasing number of isolates could not be typed with standard anti-

4 VOL. 95, 1968 GROUP E NEISSERIA MENINGITIDIS 1303 TABLE 5. Capacity of meningococcal antisera to protect mice challenged with groups A, B, C, anid E Neisseria meningitidis Challenge No. of Homologu Group E Serogroup Strain MLD (MpUmus strain Bo-6 ' (MPU) A 791 1, <10 CL-4 10, <10 B L-1 10, <10 SDIC <10 C LAIC <10 SD3C <10 E SD4C Bc a Mouse-protective units defined as the highest dilution of serum that protects 50% of challenged mice (2). TABLE 6. Capacity of meningococcal antisera to protect mice challenged with group E Neisseria meningitidis Serogroup Strain Group E challengea (MPU)b A CL4 <10 B L-1 <10 C SD3C <10 E NOR a Mice challenged with, 1000 MLD of group E, strain NOR-28 cells. b Mouse-protective units defined as the highest dilution of serum which protects 50% of challenged mice (2). sera to groups A or B, but they appeared to be capsulated; antisera to these strains did not protect mice challenged with group B (3). These strains were thought to be a subgroup of type II (group B) and were classified as type Ila (group C). The majority of current meningococcal infections in the United States have been caused by group B; but, because of the serological variability within this group (1), it was believed that the nontypable isolates were related to group B. Nonetheless, this study illustrates that the group E isolates are serologically distinct and that these isolates constitute a new serogroup of N. meningitidis. The group E strains are more closely related to group C than to either groups A or B, as indicated by precipitin and agglutinin tests. Initial serological grouping of isolates, as they were received, demonstrated no relationship between groups E and D strains. In addition, no crossreaction was noted between group E and group A, B, or C in the mouse-protection tests. This specificity corresponds to group A, which shows no correlation between its agglutinin titer in a serum and the capacity of that serum to protect mice (10). Studies are currently in progress to establish the relationship of group E to groups X, Y, and Z which were identified by Slaterus (14). ACKNOWLEDGMENTS We thank J. Evans for the Bo-6 strain and J. Leedoms for strain LAIC. This investigation was sponsored by the Bureau of of Medicine and Surgery and the Office of Naval Research, U.S. Navy, under a contract with the Regents of the University of California. LITERATURE CITED 1. BRANHAM, S. E Serologic diversity among the meningococci. J. Immunol. 23: BRANHAM, S. E Serological relationships among meningococci. Bacteriol. Rev. 17: BRANHAM, S. E., AND S. A. CARLIN Comments on a new recognized group of the meningococcus. Proc. Soc. Exptl. Biol. Med. 49: BRANHAM, S. E., AND M. PITrMAN A recommended procedure for the mouse protection test in evaluation of antimeningococcal serum. Public Health Rept. (U.S.) 55: BRANHAM, S. E., AND M. F. WORMALD Serologic relationship between the meningococci of the French type C and group II alpha. J. Bacteriol DUGUID, J. P The demonstration of bacterial capsules and slime. J. Pathol. Bacteriol. 63: FELDMAN, H Meningococcal disease. J. Am. Med. Assoc. 196: GORDON, M. H., AND E. G. D. MURRAY J. Roy. Army Med. Corps. 25: KABAT, E. A., AND M. M. MAYER Experimental immunochemistry, 2nd ed., p Charles C Thomas, Publisher, Springfield, Ill. 10. KABAT, E. A., C. P. MILLER, H. KAISER, AND A. Z. FOSTER Chemical studies on bacterial agglutination. VII. A quantitative study of the type-specific and group-specific antibodies in antimeningococcal sera of various species and their relation to mouse protection. J. Exptl. Med. 81: LOWRY, 0. H., N. J. ROSEBROUGH, A. J. FARR, AND R. J. RANDALL Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193: MILLER, C. P Experimental meningococcal infection in mice. Science 78:

5 1304 VEDROS, NG, AND CULVER J. BACTERIOL. 13. PETRE, C. F A specific precipitin reaction associated with growth on agar plates of meningococcus, pneumococcus, and Bacillus dysenteriae (Shiga). Brit. J. Exptl. Pathol. 13: SLATERUS, K. W A serological typing of meningococci by means of micro-precipitation. Antonie Van Leeunwenhoek J. Microbiol. Serol. 27: SWIFT, H. F., A. T. WiLsoN, AND R. C. LANCE- FIELD Typing Group A hemolytic streptococci by M precipitin reactions in capillary pipettes. J. Exptl. Med. 52: TERZIN, A. L., M. R. FORNAZARIC, AND B. V. BIRTASEVIC Demonstration of complement-fixing titers against species-specific trachoma antigen in sera of trachoma patients. J. Hyg. 61: THAYER, J. D., AND J. E. MARTIN Improved medium selective for cultivation of N. gonorrhea and N. meningitidis. Public Health Rept. U.S. 81: VEDROS, N. A., D. H. HUNTER, AND J. H. RUST, JR Studies on immunity in meningococcal meningitis. Military Med. 131: Downloaded from on September 10, 2018 by guest