INFECTION AND IMMUNrry, Dec. 1977, p. 666-672Copyright i 1977 American Society for Microbiology

Vol. 18, No. 3Printed in U.S.A.

Group B Streptococcal Long-Chain ReactionPRUDENCE STEWARDSON-KRIEGER, KEITH ALBRANDT, ROBERTO R. KRETSCHMER, AND

SAMUEL P. GOTOFF*

Department ofPediatrics, Michael Reese Hospital and Medical Center, Pritzker School ofMedicine,University of Chicago, Chicago, Illinois 60616

Received for publication 18 July 1977

The streptococcal long-chain reaction was adapted for the measurement oftype-specific antibodies to group B beta-hemolytic streptococci (GBBHS). Rabbitantisera incubated with homologous but not heterologous GBBHS producedchains that were 18 to 33 times longer than chains produced by normal rabbitsera. The long chains were easily apparent, in most instances, by scanning theslides. Human sera with mouse protective and opsonic activity against GBBHSserotype Ia produced chains that were always significantly longer than thoseproduced by incubation in nonimmune human sera. Absorption of rabbit orhuman sera with homologous but not heterologous organisms inhibited thecapacity 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 rapididentification of anti-GBBHS antibodies.

The group B beta-hemolytic streptococci(GBBHS) are currently a major cause of seriousperinatal sepsis (2, 13). These organisms aresubdivided into five serotypes, Ia, Ib, Ic, 11, andIII, based on polysaccharide and protein deter-minants. AlR have been associated with humaninfections, although types III and Ia predomi-nate (2, 13). Studies in animal models haveshown that immunity to GBBHS is type specific(11). However, investigation ofhuman immunityto GBBHS has been hindered by the lack ofreadily available assays for detecting type-spe-cific anti-GBBHS antibodies. The indirect op-sonophagocytic assay (1, 5, 9, 10, 12) requiresthe use of fresh, nonimmune donor blood as asource of leukocytes and complement, and itscomplexity makes it unsuitable for a routinelaboratory. Radioactive antigen-binding meth-ods have been described for the detection ofanti-GBBHS antibodies (4, 17), but they requirethe use of purified, radiolabeled polysaccharideantigen extracts and are technically cumber-some. Recently, a mouse protection test hasbeen used to detect human immunity to GBBHStype Ia, (13a), but the need for animals limitsits clinical applicability, and, furthernore, miceare apparently resistant to infection withGBBHS type III (2).To provide a simple, inexpensive method for

investigating humoral immunity to GBBHS, wehave adapted the streptococcal long-chain re-action (14) for the measurement of antibodiesto GBBHS in rabbit and human sera.

MATERIALS AND METHODSStreptococcal strains. GBBHS strains IaSS615

(Lancefield number 090), IISS619 (Lancefield number18RS21), and HISS620 (Lancefield number D136C)were obtained from Hazel Wilkinson (Center for Dis-ease Control, Atlanta, Ga.). Strains IaSS615 andIIISS620 were passed serially in mice 28 and 50 times,respectively (11), and the corresponding mouse-passedvariants were designated IaSS615/28, which is highlyvirulent for mice, and IIISS620/50, which is not viru-lent for mice. Mouse-passed GBBHS strains IaO90/14,IbH36B/60/2, and II18RS21/67/1 were donated byRebecca Lancefield (Rockefeller University, NewYork). Studies also were performed with strainIaGAR, isolated from the cerebrospinal fluid of a 5-day-old infant with meningitis. Organisms were grownto mid-log phase (optical density at 550 nm = 0.3) inTodd-Hewitt-0.5% dextrose broth and stored at-70°C until used. Over 95% of the organisms remainedviable after freezing, and mouse-virulent strains havebeen maintained for up to 2 years without dissociationto a nonvirulent phase.

Preparation of rabbit antisera. Adult male NewZealand white rabbits (Lesser's Rabbit Tree, UnionGrove, Wis.) were injected intravenously with For-malin-killed vaccines of GBBHS strains IaSS615,IISS619, and IIISS620 by the method of Wilkinsonand Moody (18) and were test bled after each seriesof three injections. When strong capillary precipitinreactions with HCl extracts of the immunizing sero-type were obtained, their sera were collected andstored at -70°C. Rabbit antisera were always diluted1:10 with normal rabbit serum for the long-chain re-actions, since undiluted antisera produced streptococ-cal chains too long to quantitate.

Collection and preparation of human sera.666

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GROUP B STREPTOCOCCAL LONG-CHAIN REACTION 667

Blood samples were obtained from 24 normal adultwomen between 20 and 40 years of age, and the serawere frozen immediately at -70°C. For some studies,sera were heat inactivated for 30 min at 56°C. Theindirect bactericidal and mouse protection assays per-formed with these same sera are described elsewhere(13a).

Absorption of sera with GBBHS. Sera were ab-sorbed by a modification of the method of Lancefieldet al. (11), using overnight cultures of GBBHS sero-types Ia, Ib, II, or III in modified Todd-Hewitt broth(3). The organisms were heat killed at 560C for 30min and packed by centrifugation. Rabbit sera diluted1:10 were mixed in a 10:1 ratio with bacteria, incubatedat 00C for 30 min, centrifuged at 4°C, and sterilizedby passage through 0.45-gLm membrane filters (Mili-pore Corp., Bedford, Mass.). Absorbed sera werestored at -70°C until used.Long-chain reaction. Fresh, mid-log-phase cul-

tures of GBBHS in Todd-Hewitt-0.5% dextrose brothwere diluted to approximately 3.5 x 10' colony-formingunits per ml with phosphate-buffered saline, and 0.05ml of bacteria in phosphate-buffered saline was addedto each of a series of tubes containing 0.2 ml of testserum and 0.1 ml of modified Todd-Hewitt broth.Control tubes containing homologous rabbit antiserumdiluted 1:10 and undiluted normal rabbit serum wereincluded in each assay. Forsemiquantitation of results,sera were diluted in fresh frozen nonimmune humanserum (PS), so the amount of serum remained con-stant in all tubes. The tubes were incubated withoutagitation in a 37°C water bath. After 3 h of incubation,a 0.02-mi sample was extracted from each tube andgently spread onto a 0.22-,um membrane filter (Milli-pore Corp.). The filters were dried, stained with meth-ylene blue, and mounted on glass slides. The meannumber of cocci per 50 to 100 streptococcal chainswas counted at a magnification of x480. In addition,the mean chain length of the mid-log-phase bacterialinoculum was determined for each assay. In some

experiments, a class divisor method was used for scor-

ing the long-chain reaction (7). In the long-chain re-

action with GBBHS strain TaSS615/28, the mean

chain length plus 3 standard deviations of organismsgrown in the 17 human sera that gave negative long-chain reactions, namely, 20 cocci per chain, was se-lected as the class divisor. Each of 100 chains in thetubes 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'st test for two means.

RESULTSLong-chain reactions with GBBHS and

rabbit antisera. Mouse-passed GBBHS sero-

types Ia, II, and III incubated in media contain-ing homologous rabbit antiserum grew in chainsthat were significantly longer (P< 0.01) than thechains formed by organisms in normal rabbitserum (Table 1). This effect could be absorbedwith homologous, but not heterologous, orga-

nisms (Table 2). Bacteria incubated with heter-ologous antisera grew in chains that were notsignificantly longer than the chains of organismsincubated with normal rabbit sera. Differencesin chain lengths of organisms incubated withhomologous antisera as compared with controlswere easily apparent in all instances by scanningthe slides (Fig. 1A and B). Experiments per-formed with the same serum and organism ondifferent days yielded mean chain lengths thatwere frequently different from, but always longerthan, the concurrent controls.Long-chain reaction with GBBHS

TaSS615/28 and human sera. When incu-bated with 17 human sera (Table 3, numbers 8

TABLE 1. Long-chain reaction with homologous and heterologous GBBHS strains and rabbit seraMean no. of cocci per chain ± SDa

SerumIaSS615/28 I118RS21/67/1 IIISS620/50

2.2 ± 1.5b 1.8 ± 1.0 1.6 ± 0.8Normal rabbit 8.8 ± 7.0 3.1 ± 2.0 2.9 ± 1.4anti-GBBHS Ia 182.6 ± 131.8 3.9 ± 2.8 3.3 ± 1.8anti-GBBHS II 6.6 ± 5.1 103.6 ± 103.0 3.0 ± 1.7anti-GBBHS HI 7.7 ± 3.8 4.5 ± 3.2 51.3 + 35.9

aSD, Standard deviation.b Mid-log-phase inocula.

TABLE 2. Long-chain reaction with homologous GBBHS strains and absorbed rabbit antiseraMean no. of cocci per chain ± SDa

Absorbing strainAnti-Ia + IaSS615/28 Anti-Il + II18RS21/67/1 Anti-Ill + IIISS620/50

No absorption 153.1 ± 95.5 218.4 ± 194.0 66.1 ± 72.4IaSS615/28 9.0 ± 5.2 247.2 ± 136.1 57.4 ± 41.5IbH36B/60/2 172.7 ± 85.4 234.7 ± 170.5 68.3 ± 39.6II18RS21/67/1 145.1 ± 95.8 2.9 ± 1.7 57.9 ± 44.9IIISS620/50 169.0 ± 155.1 262.6 ± 153.3 3.0 ± 1.9

°SD, Standard deviation.

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668 STEWARDSON-KRIEGER ET AL.

B

-I-.

...-- /

.* /7>

*.~~~~~~~I

C.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 ofhomologous rabbit antiserum; (C) clumping of bacteria observed after 3 h of incubation with a 1:32 dilutionof human serum JM; (D) disruption of chains and clumping of bacteria observed after 4 h of incubationwith human serum MN.

to 24) lacking opsonic or mouse protective activ-ity for GBBHS IaSS615/28, IaSS615/28 orga-nisms grew in chains that were not significantlylonger than chains produced in normal rabbitserum. In contrast, organisms incubated withsix human sera (numbers 1 to 6) containingopsonic and mouse protective activity forGBBHS IaSS615/28 grew in significantly longerchains (P < 0.01). These sera produced signifi-cantly longer chains (P < 0.05) than those pro-duced by nonimmune controls in dilutions rang-ing from 1:2 (JS) to 1:32 (JM). In high dilutions,a variable degree of chain fragmentation andgeneralized agglutination was frequently appar-ent (Fig. 10). Heat inactivation of sera at 560Cfor 30 min had no effect on the long-chain reac-

tion. Organisms incubated with serum BZ (num-ber 7) gave intermediate results.

Effect of absorption of human sera onthe long-chain reaction. Six undiluted humansera that gave positive long-chain reactions withGBBHS IaSS615/28 were absorbed with variousstrains of GBBHS (Table 4). The long-chainreaction was completely inhibited by absorptionwith type Ia organisms in all six sera. Absorptionwith a heterologous strain, GBBHSIbH36B/60/2, resulted in the formation of sig-nificantly shorter chains than those formed withunabsorbed sera (P < 0.05) in three instances(BJ, LL, MN), although the chains were stilllonger than those formed when organisms wereincubated with normal rabbit serum (P < 0.01).

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VOL. 18, 1977 GROUP B STREPTOCOCCAL LONG-CHAIN REACTION 669

TABLE 3. Comparison ofmouse protection test, bactericidal assay, and long-chain reaction with GBBHSIaSS615/28 and human sera

Mouse protec- Indirect bacteri- Long-chain reactionsSerum tion test (Mice cidal assay (Bac- (Mean no. of cocci per chain ± SD,)

protected [%]) teria inhibited[%Jl) Lowest value Highest value

2.2 ± 1.5b 5.8 ± 4.6h

Rabbit anti-Ia 100 ND" 153.1 ± 95.5 182.6 ± 131.8

Normal rabbit serum 0 ND 5.1 ± 3.2 8.8 ± 7.0

Human1 BJ 100 >99 93.1 ± 54.6 126.0 ± 70.82 LL 92 >99 70.3 ± 59.1 148.9 ± 103.53 JM 100 >99 127.0 ± 86.6 202.2 ± 133.84 MN 100 >99 48.9 ± 35.3 104.9 ± 81.85 CP 94 >99 20.7 ± 18.4 40.4 ± 77.96 JS 92 >99 16.6 ± 9.4 54.0 ± 31.77 BZ 36 96 8.1 ± 7.4 12.0 ± 11.88 CM 25 82 4.0± 2.99 GK 25 63 5.4 ± 4.410 GP 25 39 4.2± 2.711 DP 25 8 6.1±4.312 AP 13 30 4.2 ± 4.0 6.6 ± 5.613 DA 0 18 6.4 ± 5.714 AB 0 62 4.8± 2.915 ND 0 77 4.2 ± 2.816 DE 0 35 5.8 ± 5.317 LF 0 36 4.5±3.318 EL 0 52 5.9 ± 4.1 7.8 ± 7.019 BM 0 74 4.1 ± 3.020 PM 0 26 5.1±3.221 KS 0 8 5.6±4.022 PS 0 12 4.0±4.0 8.0±4.523 JU 0 24 3.9±3.7 5.6±4.724 JV 0 34 4.9± 3.9

a SD, Standard deviation.b Mid-log-phase inocula.c ND, Not done.

TABLE 4. Effect of absorption ofhuman sera on the long-chain reaction with GBBHS IaSS615/28Mean no. of cocci per chain ± SD" Concurrent

Serum Strain of GBBHS used for absorption nrabbitUnaborbdmsrabmiUnabsorbed Ia Ia Ia lb II III serum

SS615 SS615/28 GAR He6B/60/2 18RS21/67/1 SS620/50 control

BJ 121.3 ± 89.0 5.6 ± 3.6 53.1 ± 39.0 126.8 ± 82.1 124.1 ± 92.4 8.5 ± 5.6126.0 ± 70.8 5.6 ± 5.3 6.2 ± 5.0 7.0 ± 4.4

LL 112.1 ± 89.6 5.0 ± 3.5 74.0 ± 77.0 101.8 ± 79.6 112.9 ± 85.5 8.5 ± 5.6104.7 ± 53.6 4.5 ± 2.6 4.7 ± 2.8 7.0 ± 4.4

JM 127.0 ± 86.6 7.5 ± 5.3 131.4 ± 94.2 127.1 ± 103.5 8.5 ± 5.6202.2 ± 133.8 199.3 ± 109.7 6.3 ± 3.2153.8 ± 87.6 5.2 ± 2.8 5.3 ± 4.0 7.0 ± 4.4

MN 83.0 ± 98.7 5.3 ± 3.1 17.0 ± 10.5 95.8 ± 77.8 12.*9 ± 7.8 8.5 ± 5.666.2 ± 54.6 5.6 ± 3.5 4.3 ± 2.8 7.0 ± 4.4

CP 32.4 ± 30.4 7.6 ± 5.8 36.7 ± 45.1 41.6 ± 28.5 8.5 ± 5.635.1 ± 28.4 6.8 ± 3.5 6.3 ± 3.220.7 ± 18.4 5.2 ± 4.6 5.2 ± 3.2 7.0 ± 4.4

JS 38.4 ± 35.5 6.2 ± 3.2 41.5 ± 27.6 38.4 ± 24.7 45.3 ± 36.5 8.5 ± 5.629.7 ± 25.1 5.5 ± 3.0 5.8 ± 4.2 7.0 ± 4.4

aSD, Standard deviation.

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670 STEWARDSON-KRIEGER ET AL.

Absorption of these six sera with GBBHSI118RS21/67/1 had no effect on the long-chainreaction. However, absorption with GBBHSstrain IIISS620/50 completely inhibited thelong-chain reaction in one serum (CP) and sig-nificantly reduced it (P< 0.01) in another (MN).

Effect of duration of incubation on thelong-chain reaction. Eight human sera incu-bated with GBBHS IaSS615/28 were sampledat 30-min intervals between 1 and 5 h (Fig. 2).In six sera that had produced positive long-chainreactions, maximum chain lengths occurred be-tween 2.5 and 3 h of incubation. The chainlengths of organisms incubated in one serum(JM) continued to increase beyond 3 h, butbecame too long to quantitate. No increase inchain length was observed at any time intervalin the control serum (PS). Streptococcal chainssampled between 1 and 3 h of incubation withsix of seven sera with positive long-chain reac-tions exhibited only slight fragmentation and nogeneralized agglutination, whereas chains sam-pled between 3.5 and 4.5 h of incubation withthese same sera were often fragmented and ag-glutinated (Fig. 1D). Generalized agglutinationwas no longer observed at 5 h of incubation.Class divisor method for scoring long-

chain reactions. The mean class divisor scoresof the 17 human sera that gave negative long-chain reactions with GBBHS IaSS615/28 werenot greater than 3%, whereas the mean classdivisor scores of the seven sera that gave positivelong-chain reactions ranged from 12 (BZ) to 93%(BJ) (Table 5). The class divisor scores de-creased progressively with increasing dilutionsof sera.Long-chain reactions with different

O JMO LLA JS

200 *MNZ* BJ

100AC

MEANNUMBER

OFCOCCI/CHAIN

I0

HOURS OF INCUBATION

FIG. 2. Effect of incubation time on the formationof long chains. For identification of sera, see Table 3.

strains of GBBHS type Ta. To determinewhether GBBHS Ia strains other than SS615/28were capable of forming long chains in antisera,the assay was performed with the mouse-viru-lent strain IaO90/14 obtained from Dr. Lance-field's laboratory, with the fresh isolate IaGARand with IaSS615 organisms that had not beenpassed in mice (Table 6). As expected, the long-chain reaction with IaO90/14 gave results thatwere similar to those obtained with IaSS615/28.When incubated with homologous rabbit an-

tiserum or immune human serum, GBBHSIaGAR formed chains that were significantlylonger than the result obtained with normalrabbit serum or nonimmune human serum (P <0.01). However, the magnitude of the long-chainreaction with IaGAR was considerably less thanwith either of the two mouse-passed variantsand was not readily apparent by scanning theslides.The IaSS615 strain that had not been passed

in mice also grew in longer chains when incu-bated with sera containing antibody to serotypeIa, but spontaneously formed very long chainsin all sera with which it was incubated. At the

TABLE 5. GBBHS IaSS615/28 long-chain reactionassessed by the class divisor method

Serum

Normal rabbit

Rabbit anti-Ia

HumanBJLLJMMNCPJSBZCMGKGPDPAPDAABNDDELFELBMPMKSPSJUJv

Mean classdivisorscorea ±SDb

4.5 ± 3.1

95.6 ± 3.3

93.4 ± 4.988.8 ± 8.785.6 ± 11.084.0 ± 6.843.8 ± 13.062.8 ± 19.512.2 ± 57

1200

1.5 ± 0.730010

1.5 ± 2.1013

2.7 ± 2.40.5 + 0.7

1

No. oftimes se-rum sam-ple wasstudied

8

10

558586411

1212111112111621

Range ofclass div-

isorscores

0-8

91-100

86-9875-9770-10076-9225-6937-867-19

1-2

0-3

0-70-1

aClass divisor score = percentage of streptococcal chainswith more than 20 cocci.

'SD, Standard deviation.A

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GROUP B STREPTOCOCCAL LONG-CHAIN REACTION

TABLE 6. Long-chain reaction with different strains of GBBHS serotype IaMean no. of cocci per chain ± SDa

SerumIaSS615/28 IaO90/14 IaGAR IaSS615

5.8 ± 4.6b 4.1 ± 1.6b 4.8 ± 2.8b 12.8 ± 6.2bNormal rabbit 8.8 ± 7.0 9.1 _ 5.8 1.6 ± 0.8 100.2 ± 81.9

Rabbit anti-Ia 182.6 ± 131.8 256.3 ± 174.3 10.3 ± 5.8 TLTC'

HumanPS 8.0 ± 4.5 6.7 ± 8.3 1.8 ± 1.2 47.3 ± 41.2JU 5.6 ± 4.7 6.2 ± 3.2 NDd NDBZ 12.0 ± 9.9 11.5 ± 8.1 3.0 ± 2.5 79.7 ± 58.8CP 40.4 ± 77.9 55.7 ± 77.6 4.9 ± 3.5 137.4 ± 116.3JM 202.2 ± 133.8 334.5 ± 201.5 10.8 ± 7.1 TLTC

a SD, Standard deviation.b Mid-log-phase inocula.e TLTC, Too long to count.d ND, Not done.

end of the 3-h incubation period, the chains ofIaSS615 organisms incubated with rabbit anti-serum or serum JM were too long to be quanti-tated accurately. Serum CP produced chains ofIaSS615 organisms that were gnificantly longerthan chains incubated in normal rabbit serum (P< 0.05). The chain length of IaSS615 organismsincubated with serum BZ was not significantlydifferent from the normal rabbit serum control,whereas the chains produced by serum PS weresignificantly shorter than those formed with nor-mal rabbit serum and serum BZ (P < 0.05).

DISCUSSIONThe long-chain reaction was shown to be as

sensitive as the indirect bactericidal assay forthe detection of type-specific immunity to bothgroup A streptococci and Streptococcus pneu-moniae (6,14,15). Hahn and Cole demonstratedthat the reaction depends on the presence ofbivalent antibody to streptococcal antigens, andthey proposed an end-to-end agglutinationmodel with antibody bridges to explain the phe-nomenon (7, 8). However, the lack of side-to-side agglutination in their long-chain reactioncould not be explained. In the original descrip-tion by Stolierman et al., the long-chain assayrequired the counting of a large number of strep-tococcal chains (15). Subsequently, Hahn andCole introduced the more convenient class divi-sor method for evaluating test results (7). Wefound this simplified method equally applicableto the quantitation of GBBHS long-chain reac-tions.Our investigation demonstrates that the long-

chain reaction is a sensitive method for detectingtype-specific GBBHS antibody in both hyper-immune rabbit antisera and human sera. The3-h incubation period was optimal for the long-chain studies with GBBHS IaSS615/28, as sam-

ples taken at this time interval produced a den-sity of chains on the filters that permitted rapidcounting. There were no discrepancies betweenthe long-chain reaction with GBBHS serotypeIa and either the mouse protection or indirectbactericidal assays with the same organism.Although the long-chainreactionwithGBBHS

serotype Ia can be detected by using a strepto-coccal strain that is not mouse passed and astrain isolated from a clinical source, better re-sults are obtained by using organisms that havebeen passed in mice. Quality and quantity ofboth antigen and antibody, as well as othernonimmunological factors, may influence thelong-chain reaction. Hence, the long-chain re-action should be performed with a standardizedstrain. Similar results were reported previouslyfor the long-chain reaction with group A strep-tococci (14). Storage of GBBHS in portions at-70°C provides a convenient and reproduciblesource of bacteria.

It appears that, at low ratios of antigen toantibody, streptococcal chains are not prone tofragmentation, and therefore side-to-side agglu-tination occurs infrequently. At antigen excessratios, chains tend to fragment and agglutinate,presumably because fewer bivalent antibodybridges are present to bind them end-to-end atthe time of cell division.Two of six human sera with antibody to

GBBHS Ia contained antibody that cross-re-acted with GBBHS serotype III. Antibody thatcross-reacts with GBBHS Ia and GBBHS IIIhas been described, using unabosrbed rabbit an-tiserum to serotype Ia (5), but has not beenreported with human sera. Three of the sixhuman sera that we studied contained antibodycross-reactive with GBBHS Ib, which is knownto share minor carbohydrate determinants withGBBHS Ia (16).

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672 STEWARDSON-KRIEGER ET AL.

The long-chain assay for GBBHS requiressmall amounts of serum and is simple, reproduc-ible, and rapid to perform, using minimal labo-ratory equipment. It could constitute a valuableaddition to the presently available methods forinvestigating humoral immunity to GBBHS.

ACKNOWLEDGMENTSThis work was supported by grants from the Chicago

Community Trust and The Jennie Singer League for PediatricResearch.We thank Diane April for her excellent secretarial assist-

ance.

LITERATURE CITED1. Anthony, B. F. 1976. Immunity to the group B strepto-

cocci: interaction of serum and macrophages with typesIa, Tb, and Ic. J. Exp. Med. 143:1186-1198.

2. Baker, C. J. 1977. Summary of the workshop on perinatalinfections due to Group B streptococcus. J. Infect. Dis.136:137-152.

3. Baker, C. J., and D. L. Kasper. 1976. Microcapsule oftype III strains of group B streptococcus: productionand 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 capsularpolysaccharide in infection with type III strains of groupB 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 opsonophago-cytic antibodies in rabbit antisera to group B strepto-cocci. J. Immunol. 118:673-678.

6. Ekstedt, R. D., and G. H. Stollerman. 1960. Factorsaffecting the chain length of group A streptococci. I.Demonstration of a metabolically active chain-splittingsystem. J. Exp. Med. 112:671-686.

7. Hahn, J. J., and R. M. Cole. 1962. Time and concentra-tion relationships in the long-chain reaction of groupA streptococci in homologous antiserum and an im-proved method for evaluation of test results. J. Bacte-

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riol. 83:85-96.8. Hahn, J. J., and R. M. Cole. 1963. Studies on the

mechanism of the long chain phenomenon of group Astreptococci. J. Exp. Med. 117:583-594.

9. Heming, V. G., R. T. Hall, P. G. Rhodes, A. 0. Shi-geoka, and H. R. Hill. 1976. Assessment of group Bstreptococcal opsonins in human and rabbit serum byneutrophil 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 im-mune 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 directedagainst 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 Bstreptococci 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. Perinatalimmunity to group B beta hemolytic streptococcus type1A. J. Infect. Dis. 136:649-654.

14. Stolierman, G. H., and R. Ekstedt. 1957. Long chainformation by strains of group A streptococci in thepresence of homologous antiserum: a type-specific re-action. 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 meansfor detecting type-specific antibody to group A strep-tococci in human sera. J. Exp. Med. 110:887-897.

16. Wilkinson, H. W. 1975. Immunochemistry of purifiedpolysaccharide type antigens of group B streptococcaltypes Ia, Ib, and Ic. Infect. Immun. 11:845-852.

17. Wilkinson, H. W., and W. L. Jones. 1976. Radioim-munoassay for measuring antibodies specific for groupB streptococcal types Ia, Ib, Ic, II, and III. J. Clin.Microbiol. 3:480-485.

18. Wilkinson, H. W., and M. D. Moody. 1969. Serologicalrelationships of type I antigens of group B streptococci.J. Bacteriol. 97:629-634.

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