Vol. 18, No. 3 Printed in U.S.A.
INFECTION AND IMMUNrry, Dec. 1977, p. 666-672 Copyright i 1977 American Society for Microbiology
Group B Streptococcal Long-Chain Reaction PRUDENCE STEWARDSON-KRIEGER, KEITH ALBRANDT, ROBERTO R. KRETSCHMER, AND SAMUEL P. GOTOFF* Department ofPediatrics, Michael Reese Hospital and Medical Center, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60616
Received for publication 18 July 1977
The streptococcal long-chain reaction was adapted for the measurement of type-specific antibodies to group B beta-hemolytic streptococci (GBBHS). Rabbit antisera incubated with homologous but not heterologous GBBHS produced chains that were 18 to 33 times longer than chains produced by normal rabbit sera. The long chains were easily apparent, in most instances, by scanning the slides. Human sera with mouse protective and opsonic activity against GBBHS serotype Ia produced chains that were always significantly longer than those produced by incubation in nonimmune human sera. Absorption of rabbit or human sera with homologous but not heterologous organisms inhibited the capacity to induce the formation of long chains. The long-chain assay is a simple, rapid, and reproducible test that could constitute a valuable tool for the rapid identification of anti-GBBHS antibodies. The group B beta-hemolytic streptococci (GBBHS) are currently a major cause of serious perinatal sepsis (2, 13). These organisms are subdivided into five serotypes, Ia, Ib, Ic, 11, and III, based on polysaccharide and protein determinants. AlR have been associated with human infections, although types III and Ia predominate (2, 13). Studies in animal models have shown that immunity to GBBHS is type specific (11). However, investigation of human immunity to GBBHS has been hindered by the lack of readily available assays for detecting type-specific anti-GBBHS antibodies. The indirect opsonophagocytic assay (1, 5, 9, 10, 12) requires the use of fresh, nonimmune donor blood as a source of leukocytes and complement, and its complexity makes it unsuitable for a routine laboratory. Radioactive antigen-binding methods have been described for the detection of anti-GBBHS antibodies (4, 17), but they require the use of purified, radiolabeled polysaccharide antigen extracts and are technically cumbersome. Recently, a mouse protection test has been used to detect human immunity to GBBHS type Ia, (13a), but the need for animals limits its clinical applicability, and, furthernore, mice are apparently resistant to infection with GBBHS type III (2). To provide a simple, inexpensive method for investigating humoral immunity to GBBHS, we have adapted the streptococcal long-chain reaction (14) for the measurement of antibodies to GBBHS in rabbit and human sera. 666
MATERIALS AND METHODS Streptococcal strains. GBBHS strains IaSS615 (Lancefield number 090), IISS619 (Lancefield number 18RS21), and HISS620 (Lancefield number D136C) were obtained from Hazel Wilkinson (Center for Disease Control, Atlanta, Ga.). Strains IaSS615 and IIISS620 were passed serially in mice 28 and 50 times, respectively (11), and the corresponding mouse-passed variants were designated IaSS615/28, which is highly virulent for mice, and IIISS620/50, which is not virulent for mice. Mouse-passed GBBHS strains IaO90/14, IbH36B/60/2, and II18RS21/67/1 were donated by Rebecca Lancefield (Rockefeller University, New York). Studies also were performed with strain IaGAR, isolated from the cerebrospinal fluid of a 5day-old infant with meningitis. Organisms were grown to mid-log phase (optical density at 550 nm = 0.3) in Todd-Hewitt-0.5% dextrose broth and stored at -70°C until used. Over 95% of the organisms remained viable after freezing, and mouse-virulent strains have been maintained for up to 2 years without dissociation to a nonvirulent phase. Preparation of rabbit antisera. Adult male New Zealand white rabbits (Lesser's Rabbit Tree, Union Grove, Wis.) were injected intravenously with Formalin-killed vaccines of GBBHS strains IaSS615, IISS619, and IIISS620 by the method of Wilkinson and Moody (18) and were test bled after each series of three injections. When strong capillary precipitin reactions with HCl extracts of the immunizing serotype were obtained, their sera were collected and stored at -70°C. Rabbit antisera were always diluted 1:10 with normal rabbit serum for the long-chain reactions, since undiluted antisera produced streptococcal chains too long to quantitate. Collection and preparation of human sera.
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GROUP B STREPTOCOCCAL LONG-CHAIN REACTION
Blood samples were obtained from 24 normal adult women between 20 and 40 years of age, and the sera were frozen immediately at -70°C. For some studies, sera were heat inactivated for 30 min at 56°C. The indirect bactericidal and mouse protection assays performed with these same sera are described elsewhere (13a). Absorption of sera with GBBHS. Sera were absorbed by a modification of the method of Lancefield et al. (11), using overnight cultures of GBBHS serotypes Ia, Ib, II, or III in modified Todd-Hewitt broth (3). The organisms were heat killed at 560C for 30 min and packed by centrifugation. Rabbit sera diluted 1:10 were mixed in a 10:1 ratio with bacteria, incubated at 00C for 30 min, centrifuged at 4°C, and sterilized by passage through 0.45-gLm membrane filters (Milipore Corp., Bedford, Mass.). Absorbed sera were stored at -70°C until used. Long-chain reaction. Fresh, mid-log-phase cultures of GBBHS in Todd-Hewitt-0.5% dextrose broth were diluted to approximately 3.5 x 10' colony-forming units per ml with phosphate-buffered saline, and 0.05 ml of bacteria in phosphate-buffered saline was added to each of a series of tubes containing 0.2 ml of test serum and 0.1 ml of modified Todd-Hewitt broth. Control tubes containing homologous rabbit antiserum diluted 1:10 and undiluted normal rabbit serum were included in each assay. Forsemiquantitation of results, sera were diluted in fresh frozen nonimmune human serum (PS), so the amount of serum remained constant in all tubes. The tubes were incubated without agitation in a 37°C water bath. After 3 h of incubation, a 0.02-mi sample was extracted from each tube and gently spread onto a 0.22-,um membrane filter (Millipore Corp.). The filters were dried, stained with methylene blue, and mounted on glass slides. The mean number of cocci per 50 to 100 streptococcal chains was counted at a magnification of x480. In addition, the mean chain length of the mid-log-phase bacterial inoculum was determined for each assay. In some
667
experiments, a class divisor method was used for scoring the long-chain reaction (7). In the long-chain reaction with GBBHS strain TaSS615/28, the mean chain length plus 3 standard deviations of organisms grown in the 17 human sera that gave negative longchain reactions, namely, 20 cocci per chain, was selected as the class divisor. Each of 100 chains in the tubes containing test serum was scored as more or less than 20 cocci, and the result was expressed as the percentage of chains longer than 20 cocci per chain. Statistical analysis was performed by Student's t test for two means.
RESULTS reactions with GBBHS and Long-chain rabbit antisera. Mouse-passed GBBHS serotypes Ia, II, and III incubated in media containing homologous rabbit antiserum grew in chains that were significantly longer (P < 0.01) than the chains formed by organisms in normal rabbit serum (Table 1). This effect could be absorbed with homologous, but not heterologous, organisms (Table 2). Bacteria incubated with heterologous antisera grew in chains that were not significantly longer than the chains of organisms incubated with normal rabbit sera. Differences in chain lengths of organisms incubated with homologous antisera as compared with controls were easily apparent in all instances by scanning the slides (Fig. 1A and B). Experiments performed with the same serum and organism on different days yielded mean chain lengths that were frequently different from, but always longer than, the concurrent controls. Long-chain reaction with GBBHS TaSS615/28 and human sera. When incubated with 17 human sera (Table 3, numbers 8
TABLE 1. Long-chain reaction with homologous and heterologous GBBHS strains and rabbit sera Mean no. of cocci per chain ± SDa
Serum
Normal rabbit anti-GBBHS Ia anti-GBBHS II anti-GBBHS HI
IaSS615/28 2.2 ± 1.5b 8.8 ± 7.0 182.6 ± 131.8 6.6 ± 5.1 7.7 ± 3.8
I118RS21/67/1 1.8 ± 1.0 3.1 ± 3.9 ± 103.6 ± 4.5 ±
2.0 2.8 103.0 3.2
IIISS620/50 1.6 ± 0.8 2.9 ± 1.4 3.3 ± 1.8 3.0 ± 1.7 51.3 + 35.9
aSD, Standard deviation. b
Mid-log-phase inocula.
TABLE 2. Long-chain reaction with homologous GBBHS strains and absorbed rabbit antisera Mean no. of cocci per chain ± SDa
Absorbing strain
Anti-Ia IaSS615/28 153.1 ± 95.5 9.0 ± 5.2 172.7 ± 85.4 145.1 ± 95.8 169.0 ± 155.1 +
No absorption IaSS615/28
IbH36B/60/2 II18RS21/67/1 IIISS620/50 °SD, Standard deviation.
Anti-Il + II18RS21/67/1 218.4 ± 247.2 ± 234.7 ± 2.9 ± 262.6 ±
194.0 136.1 170.5 1.7 153.3
Anti-Ill + IIISS620/50 66.1 ± 72.4 57.4 ± 41.5 68.3 ± 39.6 57.9 ± 44.9 3.0 ± 1.9
668
INFECT. IMMUN.
STEWARDSON-KRIEGER ET AL.
B
-I-. ...--
/
.* /7>
'I
o.
*.~~~~~~~I
C.
D
FIG. 1. Long-chain reactions with GBBHS strain IaSS615/28 (x336). (A) Short chains formed after 3 h of incubation with normal rabbit serum; (B) long chain produced after 3 h of incubation with a 1:10 dilution of homologous rabbit antiserum; (C) clumping of bacteria observed after 3 h of incubation with a 1:32 dilution of human serum JM; (D) disruption of chains and clumping of bacteria observed after 4 h of incubation with human serum MN.
to 24) lacking opsonic or mouse protective activity for GBBHS IaSS615/28, IaSS615/28 organisms grew in chains that were not significantly longer than chains produced in normal rabbit serum. In contrast, organisms incubated with six human sera (numbers 1 to 6) containing opsonic and mouse protective activity for GBBHS IaSS615/28 grew in significantly longer chains (P < 0.01). These sera produced significantly longer chains (P < 0.05) than those produced by nonimmune controls in dilutions ranging from 1:2 (JS) to 1:32 (JM). In high dilutions, a variable degree of chain fragmentation and
generalized agglutination was frequently apparent (Fig. 10). Heat inactivation of sera at 560C for 30 min had no effect on the long-chain reac-
tion. Organisms incubated with serum BZ (number 7) gave intermediate results. Effect of absorption of human sera on the long-chain reaction. Six undiluted human sera that gave positive long-chain reactions with GBBHS IaSS615/28 were absorbed with various strains of GBBHS (Table 4). The long-chain reaction was completely inhibited by absorption with type Ia organisms in all six sera. Absorption with a heterologous strain, GBBHS IbH36B/60/2, resulted in the formation of significantly shorter chains than those formed with unabsorbed sera (P < 0.05) in three instances (BJ, LL, MN), although the chains were still longer than those formed when organisms were incubated with normal rabbit serum (P < 0.01).
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GROUP B STREPTOCOCCAL LONG-CHAIN REACTION
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TABLE 3. Comparison of mouse protection test, bactericidal assay, and long-chain reaction with GBBHS IaSS615/28 and human sera Mouse protection test (Mice
Serum
protected [%])
Rabbit anti-Ia
2
3 4 5
6 7 8 9
teria inhibited
[%Jl)
100
Normal rabbit serum Human 1
Indirect bacteri-
cidal assay (Bac-
BJ LL JM MN CP JS
BZ CM
GK GP DP 12 AP 13 DA 14 AB 15 ND 16 DE 17 LF 18 EL 19 BM 20 PM 21 KS 22 PS 23 JU 24 JV a SD, Standard deviation. b Mid-log-phase inocula. ND, Not done.
10 11
Long-chain reactions Lowest value
SD,) Highest value
2.2 ± 1.5b
5.8 ± 4.6h
(Mean no. of cocci per chain ±
ND"
153.1 ± 95.5
182.6 ± 131.8
0
ND
5.1 ± 3.2
8.8 ± 7.0
100
>99
93.1 ± 54.6
92
>99
100 100
>99 >99
94 92 36 25
>99 >99 96 82
25 25 25 13 0 0 0 0 0 0 0 0 0 0 0 0
63 39 8 30
18 62 77 35 36
70.3 ± 127.0 ± 48.9 ± 20.7 ± 16.6 ± 8.1 ±
126.0 ± 70.8
59.1
148.9 ± 103.5
86.6 35.3
202.2 ± 133.8 104.9 ± 81.8
18.4
40.4 ± 77.9 54.0 ± 31.7
9.4 7.4 4.0± 2.9 5.4 ± 4.4
12.0 ± 11.8
4.2± 2.7 6.1±4.3 4.2 ± 4.0 6.4 ± 5.7
6.6 ± 5.6
4.8± 2.9 4.2 ± 2.8 5.8 ± 5.3 4.5±3.3
52
5.9 ± 4.1
74 26 8 12 24 34
4.1 ± 3.0 5.1±3.2 5.6±4.0 4.0±4.0 3.9±3.7 4.9± 3.9
7.8 ± 7.0
8.0±4.5 5.6±4.7
c
TABLE 4. Effect of absorption of human sera on the long-chain reaction with GBBHS IaSS615/28 Mean no. of cocci per chain ± SD" Concurrent Serum
Strain of GBBHS used for absorption nrabbit Unaborbdmsrabmi Unabsorbed serum Ia Ia Ia II lb III
SS615
BJ
LL JM MN
CP JS
121.3 ± 89.0 126.0 ± 70.8 112.1 ± 89.6 104.7 ± 53.6 127.0 ± 86.6 202.2 ± 133.8 153.8 ± 87.6 83.0 ± 98.7 66.2 ± 54.6 32.4 ± 30.4 35.1 ± 28.4 20.7 ± 18.4 38.4 ± 35.5 29.7 ± 25.1
aSD, Standard deviation.
SS615/28
GAR
5.6 ± 3.6 5.6 ± 5.3
He6B/60/2
18RS21/67/1
SS620/50
control
53.1 ± 39.0
126.8 ± 82.1
124.1 ± 92.4
74.0 ± 77.0
101.8 ± 79.6
112.9 ± 85.5
131.4 ± 94.2
127.1 ± 103.5
8.5 ± 5.6 7.0 ± 4.4 8.5 ± 5.6 7.0 ± 4.4 8.5 ± 5.6 6.3 ± 3.2 7.0 ± 4.4 8.5 ± 5.6 7.0 ± 4.4 8.5 ± 5.6 6.3 ± 3.2 7.0 ± 4.4 8.5 ± 5.6 7.0 ± 4.4
6.2 ± 5.0
5.0 ± 3.5 4.5 ± 2.6
4.7 ± 2.8 7.5 ± 5.3
199.3 ± 109.7
5.2 ± 2.8
5.3 ± 4.0 5.3 ± 3.1
17.0 ± 10.5
95.8 ± 77.8
36.7 ± 45.1
41.6 ± 28.5
41.5 ± 27.6
38.4
4.3 ± 2.8
5.6 ± 3.5 7.6 ± 5.8
12.*9 ± 7.8 6.8 ± 3.5
5.2 ± 3.2
5.2 ± 4.6 6.2 ± 3.2 5.5 ± 3.0
5.8 ± 4.2
± 24.7
45.3 ± 36.5
670
INFECT. IMMUJN.
STEWARDSON-KRIEGER ET AL.
Absorption of these six sera with GBBHS I118RS21/67/1 had no effect on the long-chain reaction. However, absorption with GBBHS strain IIISS620/50 completely inhibited the long-chain reaction in one serum (CP) and significantly reduced it (P < 0.01) in another (MN). Effect of duration of incubation on the long-chain reaction. Eight human sera incubated with GBBHS IaSS615/28 were sampled at 30-min intervals between 1 and 5 h (Fig. 2). In six sera that had produced positive long-chain reactions, maximum chain lengths occurred between 2.5 and 3 h of incubation. The chain lengths of organisms incubated in one serum (JM) continued to increase beyond 3 h, but became too long to quantitate. No increase in chain length was observed at any time interval in the control serum (PS). Streptococcal chains sampled between 1 and 3 h of incubation with six of seven sera with positive long-chain reactions exhibited only slight fragmentation and no generalized agglutination, whereas chains sampled between 3.5 and 4.5 h of incubation with these same sera were often fragmented and agglutinated (Fig. 1D). Generalized agglutination was no longer observed at 5 h of incubation. Class divisor method for scoring longchain reactions. The mean class divisor scores of the 17 human sera that gave negative longchain reactions with GBBHS IaSS615/28 were not greater than 3%, whereas the mean class divisor scores of the seven sera that gave positive long-chain reactions ranged from 12 (BZ) to 93% (BJ) (Table 5). The class divisor scores decreased progressively with increasing dilutions of sera. Long-chain reactions with different
O JM O LL A JS 200
*MNZ * BJ
100AC MEAN NUMBER OF COCCI/CHAIN
I0
strains of GBBHS type Ta. To determine whether GBBHS Ia strains other than SS615/28 were capable of forming long chains in antisera, the assay was performed with the mouse-virulent strain IaO90/14 obtained from Dr. Lancefield's laboratory, with the fresh isolate IaGAR and with IaSS615 organisms that had not been passed in mice (Table 6). As expected, the longchain reaction with IaO90/14 gave results that were similar to those obtained with IaSS615/28. When incubated with homologous rabbit antiserum or immune human serum, GBBHS IaGAR formed chains that were significantly longer than the result obtained with normal rabbit serum or nonimmune human serum (P < 0.01). However, the magnitude of the long-chain reaction with IaGAR was considerably less than with either of the two mouse-passed variants and was not readily apparent by scanning the slides. The IaSS615 strain that had not been passed in mice also grew in longer chains when incubated with sera containing antibody to serotype Ia, but spontaneously formed very long chains in all sera with which it was incubated. At the TABLE 5. GBBHS IaSS615/28 long-chain reaction assessed by the class divisor method
SDb
ple was studied
Normal rabbit
4.5 ± 3.1
8
0-8
Rabbit anti-Ia
95.6 ± 3.3
10
91-100
93.4 ± 4.9 88.8 ± 8.7 85.6 ± 11.0 84.0 ± 6.8 43.8 ± 13.0 62.8 ± 19.5 12.2 ± 57 1 2 0 0 1.5 ± 0.7 3 0 0 1
5 5 8 5 8 6 4 1 1
86-98 75-97 70-100 76-92 25-69 37-86 7-19
Serum
Human BJ LL JM MN CP JS BZ CM GK GP DP AP DA AB ND DE LF EL BM PM KS PS
JU
Jv
HOURS OF INCUBATION
FIG. 2. Effect of incubation time on the formation of long chains. For identification of sera, see Table 3.
No. of times serum sam-
Range of class divisor scores
Mean class divisor scorea ±
0 1.5 ± 2.1 0 1 3 2.7 ± 2.4 0.5 + 0.7 1
1 221 1 1 1 1 1 2 1 1 1 6 2 1
1-2
0-3
0-7 0-1
aClass divisor score = percentage of streptococcal chains with more than 20 cocci. 'SD, Standard deviation.
A
GROUP B STREPTOCOCCAL LONG-CHAIN REACTION
VOL. 18, 1977
671
TABLE 6. Long-chain reaction with different strains of GBBHS serotype Ia Serum
IaSS615/28 Normal rabbit Rabbit anti-Ia Human PS JU BZ CP JM a SD, Standard deviation. b
Mean no. of cocci per chain ± SDa IaO90/14 IaGAR 4.8 ± 2.8b 4.1 ± 1.6b 9.1 _ 5.8
1.6 ± 0.8
IaSS615 12.8 ± 6.2b 100.2 ± 81.9
182.6 ± 131.8
256.3 ± 174.3
10.3 ± 5.8
TLTC'
8.0 ± 4.5 5.6 ± 4.7 12.0 ± 9.9 40.4 ± 77.9 202.2 ± 133.8
6.7 6.2 11.5 55.7 334.5
1.8 ± 1.2
47.3 ± 41.2 ND 79.7 ± 58.8 137.4 ± 116.3 TLTC
5.8 ± 4.6b 8.8 ± 7.0
± 8.3 ± 3.2 ± 8.1 ± 77.6 ± 201.5
NDd 3.0 ± 2.5 4.9 ± 3.5 10.8 ± 7.1
Mid-log-phase inocula.
e TLTC, Too long to count. d
ND, Not done.
end of the 3-h incubation period, the chains of IaSS615 organisms incubated with rabbit antiserum or serum JM were too long to be quantitated accurately. Serum CP produced chains of IaSS615 organisms that were gnificantly longer than chains incubated in normal rabbit serum (P < 0.05). The chain length of IaSS615 organisms incubated with serum BZ was not significantly different from the normal rabbit serum control, whereas the chains produced by serum PS were significantly shorter than those formed with normal rabbit serum and serum BZ (P < 0.05). DISCUSSION The long-chain reaction was shown to be as sensitive as the indirect bactericidal assay for the detection of type-specific immunity to both group A streptococci and Streptococcus pneumoniae (6,14,15). Hahn and Cole demonstrated that the reaction depends on the presence of bivalent antibody to streptococcal antigens, and they proposed an end-to-end agglutination model with antibody bridges to explain the phenomenon (7, 8). However, the lack of side-toside agglutination in their long-chain reaction could not be explained. In the original description by Stolierman et al., the long-chain assay required the counting of a large number of streptococcal chains (15). Subsequently, Hahn and Cole introduced the more convenient class divisor method for evaluating test results (7). We found this simplified method equally applicable to the quantitation of GBBHS long-chain reactions. Our investigation demonstrates that the longchain reaction is a sensitive method for detecting type-specific GBBHS antibody in both hyperimmune rabbit antisera and human sera. The 3-h incubation period was optimal for the longchain studies with GBBHS IaSS615/28, as sam-
ples taken at this time interval produced a density of chains on the filters that permitted rapid counting. There were no discrepancies between the long-chain reaction with GBBHS serotype Ia and either the mouse protection or indirect bactericidal assays with the same organism. Although the long-chainreactionwith GBBHS serotype Ia can be detected by using a streptococcal strain that is not mouse passed and a strain isolated from a clinical source, better results are obtained by using organisms that have been passed in mice. Quality and quantity of both antigen and antibody, as well as other nonimmunological factors, may influence the long-chain reaction. Hence, the long-chain reaction should be performed with a standardized strain. Similar results were reported previously for the long-chain reaction with group A streptococci (14). Storage of GBBHS in portions at -70°C provides a convenient and reproducible source of bacteria. It appears that, at low ratios of antigen to antibody, streptococcal chains are not prone to fragmentation, and therefore side-to-side agglutination occurs infrequently. At antigen excess ratios, chains tend to fragment and agglutinate, presumably because fewer bivalent antibody bridges are present to bind them end-to-end at the time of cell division. Two of six human sera with antibody to GBBHS Ia contained antibody that cross-reacted with GBBHS serotype III. Antibody that cross-reacts with GBBHS Ia and GBBHS III has been described, using unabosrbed rabbit antiserum to serotype Ia (5), but has not been reported with human sera. Three of the six human sera that we studied contained antibody cross-reactive with GBBHS Ib, which is known to share minor carbohydrate determinants with
GBBHS Ia (16).
672
STEWARDSON-KRIEGER ET AL.
The long-chain assay for GBBHS requires small amounts of serum and is simple, reproducible, and rapid to perform, using minimal laboratory equipment. It could constitute a valuable addition to the presently available methods for investigating humoral immunity to GBBHS. ACKNOWLEDGMENTS This work was supported by grants from the Chicago Community Trust and The Jennie Singer League for Pediatric Research. We thank Diane April for her excellent secretarial assistance.
LITERATURE CITED 1. Anthony, B. F. 1976. Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Tb, and Ic. J. Exp. Med. 143:1186-1198. 2. Baker, C. J. 1977. Summary of the workshop on perinatal infections due to Group B streptococcus. J. Infect. Dis. 136:137-152. 3. Baker, C. J., and D. L. Kasper. 1976. Microcapsule of type III strains of group B streptococcus: production and morphology. Infect. Immun. 13:189-194. 4. Baker, C. J., D. L. Kasper, I. B. Tager, A. Paredes, S. Alpert, W. M. McCormack, and D. Goroff. 1977. Quantitative determination of antibody to capsular polysaccharide in infection with type III strains of group B streptococcus. J. Clin. Invest. 59:810-818. 5. Baltimore, R. S., D. L. Kasper, C. J. Baker, and D. K. Goroff. 1977. Antigenic specificity of opsonophagocytic antibodies in rabbit antisera to group B streptococci. J. Immunol. 118:673-678. 6. Ekstedt, R. D., and G. H. Stollerman. 1960. Factors affecting the chain length of group A streptococci. I. Demonstration of a metabolically active chain-splitting system. J. Exp. Med. 112:671-686. 7. Hahn, J. J., and R. M. Cole. 1962. Time and concentration relationships in the long-chain reaction of group A streptococci in homologous antiserum and an improved method for evaluation of test results. J. Bacte-
INFECT. IMMUN. riol. 83:85-96. 8. Hahn, J. J., and R. M. Cole. 1963. Studies on the mechanism of the long chain phenomenon of group A streptococci. J. Exp. Med. 117:583-594. 9. Heming, V. G., R. T. Hall, P. G. Rhodes, A. 0. Shigeoka, and H. R. Hill. 1976. Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence. J. Clin. Invest. 58:1379-1387. 10. Klesius, P. H., R. A. Zimmerman, J. H. Mathews, and D. H. Krushak. 1973. Cellular and humoral immune response to group B streptococci. J. Pediatr. 83:926-932. 11. Lancefield, R. C., M. McCarty, and W. N. Everly. 1975. Multiple mouse-protective antibodies directed against group B streptococci. J. Exp. Med. 142:165-179. 12. Mathews, J. H., P. H. Klesius, and R. A. Zimmerman. 1974. Opsonin system of the group B streptococcus. Infect. Immun. 10:1315-1320. 13. Patterson, M. J., and A. E. B. Hafeez. 1976. Group B streptococci in human disease. Bacteriol. Rev. 40:774-792. 13a.Stewardson-Krieger, P. B., K. Albrant, T. Nevin, R. R. Kretschmer, and S. P. Gotoff. 1977. Perinatal immunity to group B beta hemolytic streptococcus type 1A. J. Infect. Dis. 136:649-654. 14. Stolierman, G. H., and R. Ekstedt. 1957. Long chain formation by strains of group A streptococci in the presence of homologous antiserum: a type-specific reaction. J. Exp. Med. 106:345-355. 15. Stolierman, G. H., A. C. Siegel, and E. E. Johnson. 1959. Evaluation of the "long chain reaction" as a means for detecting type-specific antibody to group A streptococci in human sera. J. Exp. Med. 110:887-897. 16. Wilkinson, H. W. 1975. Immunochemistry of purified polysaccharide type antigens of group B streptococcal types Ia, Ib, and Ic. Infect. Immun. 11:845-852. 17. Wilkinson, H. W., and W. L. Jones. 1976. Radioimmunoassay for measuring antibodies specific for group B streptococcal types Ia, Ib, Ic, II, and III. J. Clin. Microbiol. 3:480-485. 18. Wilkinson, H. W., and M. D. Moody. 1969. Serological relationships of type I antigens of group B streptococci. J. Bacteriol. 97:629-634.
INFECTION AND IMMUNrry, Dec. 1977, p. 666-672 Copyright i 1977 American Society for Microbiology
Group B Streptococcal Long-Chain Reaction PRUDENCE STEWARDSON-KRIEGER, KEITH ALBRANDT, ROBERTO R. KRETSCHMER, AND SAMUEL P. GOTOFF* Department ofPediatrics, Michael Reese Hospital and Medical Center, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60616
Received for publication 18 July 1977
The streptococcal long-chain reaction was adapted for the measurement of type-specific antibodies to group B beta-hemolytic streptococci (GBBHS). Rabbit antisera incubated with homologous but not heterologous GBBHS produced chains that were 18 to 33 times longer than chains produced by normal rabbit sera. The long chains were easily apparent, in most instances, by scanning the slides. Human sera with mouse protective and opsonic activity against GBBHS serotype Ia produced chains that were always significantly longer than those produced by incubation in nonimmune human sera. Absorption of rabbit or human sera with homologous but not heterologous organisms inhibited the capacity to induce the formation of long chains. The long-chain assay is a simple, rapid, and reproducible test that could constitute a valuable tool for the rapid identification of anti-GBBHS antibodies. The group B beta-hemolytic streptococci (GBBHS) are currently a major cause of serious perinatal sepsis (2, 13). These organisms are subdivided into five serotypes, Ia, Ib, Ic, 11, and III, based on polysaccharide and protein determinants. AlR have been associated with human infections, although types III and Ia predominate (2, 13). Studies in animal models have shown that immunity to GBBHS is type specific (11). However, investigation of human immunity to GBBHS has been hindered by the lack of readily available assays for detecting type-specific anti-GBBHS antibodies. The indirect opsonophagocytic assay (1, 5, 9, 10, 12) requires the use of fresh, nonimmune donor blood as a source of leukocytes and complement, and its complexity makes it unsuitable for a routine laboratory. Radioactive antigen-binding methods have been described for the detection of anti-GBBHS antibodies (4, 17), but they require the use of purified, radiolabeled polysaccharide antigen extracts and are technically cumbersome. Recently, a mouse protection test has been used to detect human immunity to GBBHS type Ia, (13a), but the need for animals limits its clinical applicability, and, furthernore, mice are apparently resistant to infection with GBBHS type III (2). To provide a simple, inexpensive method for investigating humoral immunity to GBBHS, we have adapted the streptococcal long-chain reaction (14) for the measurement of antibodies to GBBHS in rabbit and human sera. 666
MATERIALS AND METHODS Streptococcal strains. GBBHS strains IaSS615 (Lancefield number 090), IISS619 (Lancefield number 18RS21), and HISS620 (Lancefield number D136C) were obtained from Hazel Wilkinson (Center for Disease Control, Atlanta, Ga.). Strains IaSS615 and IIISS620 were passed serially in mice 28 and 50 times, respectively (11), and the corresponding mouse-passed variants were designated IaSS615/28, which is highly virulent for mice, and IIISS620/50, which is not virulent for mice. Mouse-passed GBBHS strains IaO90/14, IbH36B/60/2, and II18RS21/67/1 were donated by Rebecca Lancefield (Rockefeller University, New York). Studies also were performed with strain IaGAR, isolated from the cerebrospinal fluid of a 5day-old infant with meningitis. Organisms were grown to mid-log phase (optical density at 550 nm = 0.3) in Todd-Hewitt-0.5% dextrose broth and stored at -70°C until used. Over 95% of the organisms remained viable after freezing, and mouse-virulent strains have been maintained for up to 2 years without dissociation to a nonvirulent phase. Preparation of rabbit antisera. Adult male New Zealand white rabbits (Lesser's Rabbit Tree, Union Grove, Wis.) were injected intravenously with Formalin-killed vaccines of GBBHS strains IaSS615, IISS619, and IIISS620 by the method of Wilkinson and Moody (18) and were test bled after each series of three injections. When strong capillary precipitin reactions with HCl extracts of the immunizing serotype were obtained, their sera were collected and stored at -70°C. Rabbit antisera were always diluted 1:10 with normal rabbit serum for the long-chain reactions, since undiluted antisera produced streptococcal chains too long to quantitate. Collection and preparation of human sera.
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Blood samples were obtained from 24 normal adult women between 20 and 40 years of age, and the sera were frozen immediately at -70°C. For some studies, sera were heat inactivated for 30 min at 56°C. The indirect bactericidal and mouse protection assays performed with these same sera are described elsewhere (13a). Absorption of sera with GBBHS. Sera were absorbed by a modification of the method of Lancefield et al. (11), using overnight cultures of GBBHS serotypes Ia, Ib, II, or III in modified Todd-Hewitt broth (3). The organisms were heat killed at 560C for 30 min and packed by centrifugation. Rabbit sera diluted 1:10 were mixed in a 10:1 ratio with bacteria, incubated at 00C for 30 min, centrifuged at 4°C, and sterilized by passage through 0.45-gLm membrane filters (Milipore Corp., Bedford, Mass.). Absorbed sera were stored at -70°C until used. Long-chain reaction. Fresh, mid-log-phase cultures of GBBHS in Todd-Hewitt-0.5% dextrose broth were diluted to approximately 3.5 x 10' colony-forming units per ml with phosphate-buffered saline, and 0.05 ml of bacteria in phosphate-buffered saline was added to each of a series of tubes containing 0.2 ml of test serum and 0.1 ml of modified Todd-Hewitt broth. Control tubes containing homologous rabbit antiserum diluted 1:10 and undiluted normal rabbit serum were included in each assay. Forsemiquantitation of results, sera were diluted in fresh frozen nonimmune human serum (PS), so the amount of serum remained constant in all tubes. The tubes were incubated without agitation in a 37°C water bath. After 3 h of incubation, a 0.02-mi sample was extracted from each tube and gently spread onto a 0.22-,um membrane filter (Millipore Corp.). The filters were dried, stained with methylene blue, and mounted on glass slides. The mean number of cocci per 50 to 100 streptococcal chains was counted at a magnification of x480. In addition, the mean chain length of the mid-log-phase bacterial inoculum was determined for each assay. In some
667
experiments, a class divisor method was used for scoring the long-chain reaction (7). In the long-chain reaction with GBBHS strain TaSS615/28, the mean chain length plus 3 standard deviations of organisms grown in the 17 human sera that gave negative longchain reactions, namely, 20 cocci per chain, was selected as the class divisor. Each of 100 chains in the tubes containing test serum was scored as more or less than 20 cocci, and the result was expressed as the percentage of chains longer than 20 cocci per chain. Statistical analysis was performed by Student's t test for two means.
RESULTS reactions with GBBHS and Long-chain rabbit antisera. Mouse-passed GBBHS serotypes Ia, II, and III incubated in media containing homologous rabbit antiserum grew in chains that were significantly longer (P < 0.01) than the chains formed by organisms in normal rabbit serum (Table 1). This effect could be absorbed with homologous, but not heterologous, organisms (Table 2). Bacteria incubated with heterologous antisera grew in chains that were not significantly longer than the chains of organisms incubated with normal rabbit sera. Differences in chain lengths of organisms incubated with homologous antisera as compared with controls were easily apparent in all instances by scanning the slides (Fig. 1A and B). Experiments performed with the same serum and organism on different days yielded mean chain lengths that were frequently different from, but always longer than, the concurrent controls. Long-chain reaction with GBBHS TaSS615/28 and human sera. When incubated with 17 human sera (Table 3, numbers 8
TABLE 1. Long-chain reaction with homologous and heterologous GBBHS strains and rabbit sera Mean no. of cocci per chain ± SDa
Serum
Normal rabbit anti-GBBHS Ia anti-GBBHS II anti-GBBHS HI
IaSS615/28 2.2 ± 1.5b 8.8 ± 7.0 182.6 ± 131.8 6.6 ± 5.1 7.7 ± 3.8
I118RS21/67/1 1.8 ± 1.0 3.1 ± 3.9 ± 103.6 ± 4.5 ±
2.0 2.8 103.0 3.2
IIISS620/50 1.6 ± 0.8 2.9 ± 1.4 3.3 ± 1.8 3.0 ± 1.7 51.3 + 35.9
aSD, Standard deviation. b
Mid-log-phase inocula.
TABLE 2. Long-chain reaction with homologous GBBHS strains and absorbed rabbit antisera Mean no. of cocci per chain ± SDa
Absorbing strain
Anti-Ia IaSS615/28 153.1 ± 95.5 9.0 ± 5.2 172.7 ± 85.4 145.1 ± 95.8 169.0 ± 155.1 +
No absorption IaSS615/28
IbH36B/60/2 II18RS21/67/1 IIISS620/50 °SD, Standard deviation.
Anti-Il + II18RS21/67/1 218.4 ± 247.2 ± 234.7 ± 2.9 ± 262.6 ±
194.0 136.1 170.5 1.7 153.3
Anti-Ill + IIISS620/50 66.1 ± 72.4 57.4 ± 41.5 68.3 ± 39.6 57.9 ± 44.9 3.0 ± 1.9
668
INFECT. IMMUN.
STEWARDSON-KRIEGER ET AL.
B
-I-. ...--
/
.* /7>
'I
o.
*.~~~~~~~I
C.
D
FIG. 1. Long-chain reactions with GBBHS strain IaSS615/28 (x336). (A) Short chains formed after 3 h of incubation with normal rabbit serum; (B) long chain produced after 3 h of incubation with a 1:10 dilution of homologous rabbit antiserum; (C) clumping of bacteria observed after 3 h of incubation with a 1:32 dilution of human serum JM; (D) disruption of chains and clumping of bacteria observed after 4 h of incubation with human serum MN.
to 24) lacking opsonic or mouse protective activity for GBBHS IaSS615/28, IaSS615/28 organisms grew in chains that were not significantly longer than chains produced in normal rabbit serum. In contrast, organisms incubated with six human sera (numbers 1 to 6) containing opsonic and mouse protective activity for GBBHS IaSS615/28 grew in significantly longer chains (P < 0.01). These sera produced significantly longer chains (P < 0.05) than those produced by nonimmune controls in dilutions ranging from 1:2 (JS) to 1:32 (JM). In high dilutions, a variable degree of chain fragmentation and
generalized agglutination was frequently apparent (Fig. 10). Heat inactivation of sera at 560C for 30 min had no effect on the long-chain reac-
tion. Organisms incubated with serum BZ (number 7) gave intermediate results. Effect of absorption of human sera on the long-chain reaction. Six undiluted human sera that gave positive long-chain reactions with GBBHS IaSS615/28 were absorbed with various strains of GBBHS (Table 4). The long-chain reaction was completely inhibited by absorption with type Ia organisms in all six sera. Absorption with a heterologous strain, GBBHS IbH36B/60/2, resulted in the formation of significantly shorter chains than those formed with unabsorbed sera (P < 0.05) in three instances (BJ, LL, MN), although the chains were still longer than those formed when organisms were incubated with normal rabbit serum (P < 0.01).
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669
TABLE 3. Comparison of mouse protection test, bactericidal assay, and long-chain reaction with GBBHS IaSS615/28 and human sera Mouse protection test (Mice
Serum
protected [%])
Rabbit anti-Ia
2
3 4 5
6 7 8 9
teria inhibited
[%Jl)
100
Normal rabbit serum Human 1
Indirect bacteri-
cidal assay (Bac-
BJ LL JM MN CP JS
BZ CM
GK GP DP 12 AP 13 DA 14 AB 15 ND 16 DE 17 LF 18 EL 19 BM 20 PM 21 KS 22 PS 23 JU 24 JV a SD, Standard deviation. b Mid-log-phase inocula. ND, Not done.
10 11
Long-chain reactions Lowest value
SD,) Highest value
2.2 ± 1.5b
5.8 ± 4.6h
(Mean no. of cocci per chain ±
ND"
153.1 ± 95.5
182.6 ± 131.8
0
ND
5.1 ± 3.2
8.8 ± 7.0
100
>99
93.1 ± 54.6
92
>99
100 100
>99 >99
94 92 36 25
>99 >99 96 82
25 25 25 13 0 0 0 0 0 0 0 0 0 0 0 0
63 39 8 30
18 62 77 35 36
70.3 ± 127.0 ± 48.9 ± 20.7 ± 16.6 ± 8.1 ±
126.0 ± 70.8
59.1
148.9 ± 103.5
86.6 35.3
202.2 ± 133.8 104.9 ± 81.8
18.4
40.4 ± 77.9 54.0 ± 31.7
9.4 7.4 4.0± 2.9 5.4 ± 4.4
12.0 ± 11.8
4.2± 2.7 6.1±4.3 4.2 ± 4.0 6.4 ± 5.7
6.6 ± 5.6
4.8± 2.9 4.2 ± 2.8 5.8 ± 5.3 4.5±3.3
52
5.9 ± 4.1
74 26 8 12 24 34
4.1 ± 3.0 5.1±3.2 5.6±4.0 4.0±4.0 3.9±3.7 4.9± 3.9
7.8 ± 7.0
8.0±4.5 5.6±4.7
c
TABLE 4. Effect of absorption of human sera on the long-chain reaction with GBBHS IaSS615/28 Mean no. of cocci per chain ± SD" Concurrent Serum
Strain of GBBHS used for absorption nrabbit Unaborbdmsrabmi Unabsorbed serum Ia Ia Ia II lb III
SS615
BJ
LL JM MN
CP JS
121.3 ± 89.0 126.0 ± 70.8 112.1 ± 89.6 104.7 ± 53.6 127.0 ± 86.6 202.2 ± 133.8 153.8 ± 87.6 83.0 ± 98.7 66.2 ± 54.6 32.4 ± 30.4 35.1 ± 28.4 20.7 ± 18.4 38.4 ± 35.5 29.7 ± 25.1
aSD, Standard deviation.
SS615/28
GAR
5.6 ± 3.6 5.6 ± 5.3
He6B/60/2
18RS21/67/1
SS620/50
control
53.1 ± 39.0
126.8 ± 82.1
124.1 ± 92.4
74.0 ± 77.0
101.8 ± 79.6
112.9 ± 85.5
131.4 ± 94.2
127.1 ± 103.5
8.5 ± 5.6 7.0 ± 4.4 8.5 ± 5.6 7.0 ± 4.4 8.5 ± 5.6 6.3 ± 3.2 7.0 ± 4.4 8.5 ± 5.6 7.0 ± 4.4 8.5 ± 5.6 6.3 ± 3.2 7.0 ± 4.4 8.5 ± 5.6 7.0 ± 4.4
6.2 ± 5.0
5.0 ± 3.5 4.5 ± 2.6
4.7 ± 2.8 7.5 ± 5.3
199.3 ± 109.7
5.2 ± 2.8
5.3 ± 4.0 5.3 ± 3.1
17.0 ± 10.5
95.8 ± 77.8
36.7 ± 45.1
41.6 ± 28.5
41.5 ± 27.6
38.4
4.3 ± 2.8
5.6 ± 3.5 7.6 ± 5.8
12.*9 ± 7.8 6.8 ± 3.5
5.2 ± 3.2
5.2 ± 4.6 6.2 ± 3.2 5.5 ± 3.0
5.8 ± 4.2
± 24.7
45.3 ± 36.5
670
INFECT. IMMUJN.
STEWARDSON-KRIEGER ET AL.
Absorption of these six sera with GBBHS I118RS21/67/1 had no effect on the long-chain reaction. However, absorption with GBBHS strain IIISS620/50 completely inhibited the long-chain reaction in one serum (CP) and significantly reduced it (P < 0.01) in another (MN). Effect of duration of incubation on the long-chain reaction. Eight human sera incubated with GBBHS IaSS615/28 were sampled at 30-min intervals between 1 and 5 h (Fig. 2). In six sera that had produced positive long-chain reactions, maximum chain lengths occurred between 2.5 and 3 h of incubation. The chain lengths of organisms incubated in one serum (JM) continued to increase beyond 3 h, but became too long to quantitate. No increase in chain length was observed at any time interval in the control serum (PS). Streptococcal chains sampled between 1 and 3 h of incubation with six of seven sera with positive long-chain reactions exhibited only slight fragmentation and no generalized agglutination, whereas chains sampled between 3.5 and 4.5 h of incubation with these same sera were often fragmented and agglutinated (Fig. 1D). Generalized agglutination was no longer observed at 5 h of incubation. Class divisor method for scoring longchain reactions. The mean class divisor scores of the 17 human sera that gave negative longchain reactions with GBBHS IaSS615/28 were not greater than 3%, whereas the mean class divisor scores of the seven sera that gave positive long-chain reactions ranged from 12 (BZ) to 93% (BJ) (Table 5). The class divisor scores decreased progressively with increasing dilutions of sera. Long-chain reactions with different
O JM O LL A JS 200
*MNZ * BJ
100AC MEAN NUMBER OF COCCI/CHAIN
I0
strains of GBBHS type Ta. To determine whether GBBHS Ia strains other than SS615/28 were capable of forming long chains in antisera, the assay was performed with the mouse-virulent strain IaO90/14 obtained from Dr. Lancefield's laboratory, with the fresh isolate IaGAR and with IaSS615 organisms that had not been passed in mice (Table 6). As expected, the longchain reaction with IaO90/14 gave results that were similar to those obtained with IaSS615/28. When incubated with homologous rabbit antiserum or immune human serum, GBBHS IaGAR formed chains that were significantly longer than the result obtained with normal rabbit serum or nonimmune human serum (P < 0.01). However, the magnitude of the long-chain reaction with IaGAR was considerably less than with either of the two mouse-passed variants and was not readily apparent by scanning the slides. The IaSS615 strain that had not been passed in mice also grew in longer chains when incubated with sera containing antibody to serotype Ia, but spontaneously formed very long chains in all sera with which it was incubated. At the TABLE 5. GBBHS IaSS615/28 long-chain reaction assessed by the class divisor method
SDb
ple was studied
Normal rabbit
4.5 ± 3.1
8
0-8
Rabbit anti-Ia
95.6 ± 3.3
10
91-100
93.4 ± 4.9 88.8 ± 8.7 85.6 ± 11.0 84.0 ± 6.8 43.8 ± 13.0 62.8 ± 19.5 12.2 ± 57 1 2 0 0 1.5 ± 0.7 3 0 0 1
5 5 8 5 8 6 4 1 1
86-98 75-97 70-100 76-92 25-69 37-86 7-19
Serum
Human BJ LL JM MN CP JS BZ CM GK GP DP AP DA AB ND DE LF EL BM PM KS PS
JU
Jv
HOURS OF INCUBATION
FIG. 2. Effect of incubation time on the formation of long chains. For identification of sera, see Table 3.
No. of times serum sam-
Range of class divisor scores
Mean class divisor scorea ±
0 1.5 ± 2.1 0 1 3 2.7 ± 2.4 0.5 + 0.7 1
1 221 1 1 1 1 1 2 1 1 1 6 2 1
1-2
0-3
0-7 0-1
aClass divisor score = percentage of streptococcal chains with more than 20 cocci. 'SD, Standard deviation.
A
GROUP B STREPTOCOCCAL LONG-CHAIN REACTION
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671
TABLE 6. Long-chain reaction with different strains of GBBHS serotype Ia Serum
IaSS615/28 Normal rabbit Rabbit anti-Ia Human PS JU BZ CP JM a SD, Standard deviation. b
Mean no. of cocci per chain ± SDa IaO90/14 IaGAR 4.8 ± 2.8b 4.1 ± 1.6b 9.1 _ 5.8
1.6 ± 0.8
IaSS615 12.8 ± 6.2b 100.2 ± 81.9
182.6 ± 131.8
256.3 ± 174.3
10.3 ± 5.8
TLTC'
8.0 ± 4.5 5.6 ± 4.7 12.0 ± 9.9 40.4 ± 77.9 202.2 ± 133.8
6.7 6.2 11.5 55.7 334.5
1.8 ± 1.2
47.3 ± 41.2 ND 79.7 ± 58.8 137.4 ± 116.3 TLTC
5.8 ± 4.6b 8.8 ± 7.0
± 8.3 ± 3.2 ± 8.1 ± 77.6 ± 201.5
NDd 3.0 ± 2.5 4.9 ± 3.5 10.8 ± 7.1
Mid-log-phase inocula.
e TLTC, Too long to count. d
ND, Not done.
end of the 3-h incubation period, the chains of IaSS615 organisms incubated with rabbit antiserum or serum JM were too long to be quantitated accurately. Serum CP produced chains of IaSS615 organisms that were gnificantly longer than chains incubated in normal rabbit serum (P < 0.05). The chain length of IaSS615 organisms incubated with serum BZ was not significantly different from the normal rabbit serum control, whereas the chains produced by serum PS were significantly shorter than those formed with normal rabbit serum and serum BZ (P < 0.05). DISCUSSION The long-chain reaction was shown to be as sensitive as the indirect bactericidal assay for the detection of type-specific immunity to both group A streptococci and Streptococcus pneumoniae (6,14,15). Hahn and Cole demonstrated that the reaction depends on the presence of bivalent antibody to streptococcal antigens, and they proposed an end-to-end agglutination model with antibody bridges to explain the phenomenon (7, 8). However, the lack of side-toside agglutination in their long-chain reaction could not be explained. In the original description by Stolierman et al., the long-chain assay required the counting of a large number of streptococcal chains (15). Subsequently, Hahn and Cole introduced the more convenient class divisor method for evaluating test results (7). We found this simplified method equally applicable to the quantitation of GBBHS long-chain reactions. Our investigation demonstrates that the longchain reaction is a sensitive method for detecting type-specific GBBHS antibody in both hyperimmune rabbit antisera and human sera. The 3-h incubation period was optimal for the longchain studies with GBBHS IaSS615/28, as sam-
ples taken at this time interval produced a density of chains on the filters that permitted rapid counting. There were no discrepancies between the long-chain reaction with GBBHS serotype Ia and either the mouse protection or indirect bactericidal assays with the same organism. Although the long-chainreactionwith GBBHS serotype Ia can be detected by using a streptococcal strain that is not mouse passed and a strain isolated from a clinical source, better results are obtained by using organisms that have been passed in mice. Quality and quantity of both antigen and antibody, as well as other nonimmunological factors, may influence the long-chain reaction. Hence, the long-chain reaction should be performed with a standardized strain. Similar results were reported previously for the long-chain reaction with group A streptococci (14). Storage of GBBHS in portions at -70°C provides a convenient and reproducible source of bacteria. It appears that, at low ratios of antigen to antibody, streptococcal chains are not prone to fragmentation, and therefore side-to-side agglutination occurs infrequently. At antigen excess ratios, chains tend to fragment and agglutinate, presumably because fewer bivalent antibody bridges are present to bind them end-to-end at the time of cell division. Two of six human sera with antibody to GBBHS Ia contained antibody that cross-reacted with GBBHS serotype III. Antibody that cross-reacts with GBBHS Ia and GBBHS III has been described, using unabosrbed rabbit antiserum to serotype Ia (5), but has not been reported with human sera. Three of the six human sera that we studied contained antibody cross-reactive with GBBHS Ib, which is known to share minor carbohydrate determinants with
GBBHS Ia (16).
672
STEWARDSON-KRIEGER ET AL.
The long-chain assay for GBBHS requires small amounts of serum and is simple, reproducible, and rapid to perform, using minimal laboratory equipment. It could constitute a valuable addition to the presently available methods for investigating humoral immunity to GBBHS. ACKNOWLEDGMENTS This work was supported by grants from the Chicago Community Trust and The Jennie Singer League for Pediatric Research. We thank Diane April for her excellent secretarial assistance.
LITERATURE CITED 1. Anthony, B. F. 1976. Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Tb, and Ic. J. Exp. Med. 143:1186-1198. 2. Baker, C. J. 1977. Summary of the workshop on perinatal infections due to Group B streptococcus. J. Infect. Dis. 136:137-152. 3. Baker, C. J., and D. L. Kasper. 1976. Microcapsule of type III strains of group B streptococcus: production and morphology. Infect. Immun. 13:189-194. 4. Baker, C. J., D. L. Kasper, I. B. Tager, A. Paredes, S. Alpert, W. M. McCormack, and D. Goroff. 1977. Quantitative determination of antibody to capsular polysaccharide in infection with type III strains of group B streptococcus. J. Clin. Invest. 59:810-818. 5. Baltimore, R. S., D. L. Kasper, C. J. Baker, and D. K. Goroff. 1977. Antigenic specificity of opsonophagocytic antibodies in rabbit antisera to group B streptococci. J. Immunol. 118:673-678. 6. Ekstedt, R. D., and G. H. Stollerman. 1960. Factors affecting the chain length of group A streptococci. I. Demonstration of a metabolically active chain-splitting system. J. Exp. Med. 112:671-686. 7. Hahn, J. J., and R. M. Cole. 1962. Time and concentration relationships in the long-chain reaction of group A streptococci in homologous antiserum and an improved method for evaluation of test results. J. Bacte-
INFECT. IMMUN. riol. 83:85-96. 8. Hahn, J. J., and R. M. Cole. 1963. Studies on the mechanism of the long chain phenomenon of group A streptococci. J. Exp. Med. 117:583-594. 9. Heming, V. G., R. T. Hall, P. G. Rhodes, A. 0. Shigeoka, and H. R. Hill. 1976. Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence. J. Clin. Invest. 58:1379-1387. 10. Klesius, P. H., R. A. Zimmerman, J. H. Mathews, and D. H. Krushak. 1973. Cellular and humoral immune response to group B streptococci. J. Pediatr. 83:926-932. 11. Lancefield, R. C., M. McCarty, and W. N. Everly. 1975. Multiple mouse-protective antibodies directed against group B streptococci. J. Exp. Med. 142:165-179. 12. Mathews, J. H., P. H. Klesius, and R. A. Zimmerman. 1974. Opsonin system of the group B streptococcus. Infect. Immun. 10:1315-1320. 13. Patterson, M. J., and A. E. B. Hafeez. 1976. Group B streptococci in human disease. Bacteriol. Rev. 40:774-792. 13a.Stewardson-Krieger, P. B., K. Albrant, T. Nevin, R. R. Kretschmer, and S. P. Gotoff. 1977. Perinatal immunity to group B beta hemolytic streptococcus type 1A. J. Infect. Dis. 136:649-654. 14. Stolierman, G. H., and R. Ekstedt. 1957. Long chain formation by strains of group A streptococci in the presence of homologous antiserum: a type-specific reaction. J. Exp. Med. 106:345-355. 15. Stolierman, G. H., A. C. Siegel, and E. E. Johnson. 1959. Evaluation of the "long chain reaction" as a means for detecting type-specific antibody to group A streptococci in human sera. J. Exp. Med. 110:887-897. 16. Wilkinson, H. W. 1975. Immunochemistry of purified polysaccharide type antigens of group B streptococcal types Ia, Ib, and Ic. Infect. Immun. 11:845-852. 17. Wilkinson, H. W., and W. L. Jones. 1976. Radioimmunoassay for measuring antibodies specific for group B streptococcal types Ia, Ib, Ic, II, and III. J. Clin. Microbiol. 3:480-485. 18. Wilkinson, H. W., and M. D. Moody. 1969. Serological relationships of type I antigens of group B streptococci. J. Bacteriol. 97:629-634.
