INFECTION AND IMMUNITY, Sept. 1991, p. 3346-3349 0019-9567/91/093346-04$02.00/0
Vol. 59, No. 9
Comparative Immunochemistry of Lipopolysaccharides from Branhamella catarrhalis Strains JONNA STORM FOMSGAARD,' ANDERS FOMSGAARD,1 NIELS H0IBY,2 BRITA BRUUN,2 AND CHRIS GALANOSl* Max-Planck Institut fur Immunbiologie, Freiburg D-7800, Federal Republic of Germany,' and Department of Clinical Microbiology, Rigshospitalet, Copenhagen DK-2200, Denmark? Received 15 June 1990/Accepted 2 July 1991
Lipopolysaccharides (LPS) were extracted and purified from the type strain and from a clinical isolate of Branhamella catarrhalis. Chemical analysis revealed the presence of glucose, galactose, and glucosamine in different molar proportions in the LPS from these two isolates, whereas there was no difference between the two isolates in the ratios of ketodeoxyoctonate, phosphate, and the fatty acids C12, 3-OH-C12, and 3-OH-Cl1 present. Heptose or 3-OH-C14 was not detectable in either preparation. LPS from both strains appeared semirough according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, presenting a core polysaccharide plus one repeating unit. Immunoblotting, passive hemolysis, and hemolysis inhibition assays using anti-LPS antibodies from immunized rabbits demonstrated cross-reactivity between the LPS preparations; however, antigenic dissimilarities were also found, suggesting that more than one serotype may exist. The lipid A isolated from the two LPS was serologically identical and exhibited cross-reactivity with lipid A of members of the family Enterobacteriaceae. The B. catarrhalis LPS were biologically active, causing lethality in D-galactosamine-sensitized C57/BL6 mice and inducing Limulus amoebocyte lysate gelation.
Branhamella catarrhalis has long been regarded as an occasional nonpathogenic inhabitant of the respiratory tract (22). However, during the last decade its importance as a pathogen in both acute upper and lower respiratory tract infections has become evident (20, 21). Sporadic cases of otitis, meningitis, and septicemia have also been reported (4, 19, 22). B. catarrhalis is a gram-negative diplococcus, placed in the genus Moxarella (1). A serotyping system for B. catarrhalis has not been described, and it is therefore not known whether B. catarrhalis exists as one or more sero-
chronic bronchitis (3). The bacteria were cultured in fluid medium (Trypticase soy broth; BBL Microbiology Systems, Cockeysville, Md.) with shaking at 37°C for 48 h. In addition, B. catarrhalis BBH 56 was cultured overnight on Truche Agar plates at 37°C. After harvesting, the bacteria were washed successively with distilled water, ethanol, acetone, and ether and dried in vacuo. Isolation and purification of LPS. LPS could be extracted from the two strains of B. catarrhalis by the phenol-chloroform-petroleum ether method (9) and also by the hot-phenolwater procedure (23). The extracts were purified by threefold ultracentrifugation, converted to the triethylamine salt form by electrodialysis (8), and lyophilized. Extraction by the phenol-chloroform-petroleum ether method of B. catarrhalis CCUG 353 (8.0 g [dry weight]) and of B. catarrhalis BBH 56 (15.8 g [dry weight]) cultured in fluid medium led to yields of 0.5 and 1.95% (wt/wt), respectively. Extraction by the phenol-water method of B. catarrhalis CCUG 353 (7.6 g) and B. catarrhalis BBH 56 (13 g) both led to lower yields, 0.05 and 0.32% (wt/wt), respectively. Thus, the higher yields obtained by the phenol-chloroform-petroleum ether method, which was especially developed for LPS extraction from rough mutants, suggest the presence of more lipophilic LPS. Free lipid A was isolated after hydrolysis of the LPS with 1% acetic acid at 100°C for 45 min, washed three times in 1% acetic acid, and lyophilized. Analytical methods. Neutral sugars were liberated by hydrolysis in 0.1 M HCl at 100°C for 48 h, converted to their alditol-acetate derivatives, and analyzed by gas-liquid chromatography (with a Varian 2400 chromatograph) (18). Glucosamine was measured colorimetrically (15) and in an amino acid analyzer (model D-500; Durmun) following hydrolysis of LPS in 4 M HCl at 100°C for 10 h. Ketodeoxyoctonate was measured by the thiobarbituric acid method as modified by Karkharis et al. (12) after its release by treatment with 0.25 M sulfuric acid at 100°C for 8 h. Fatty acids were liberated from LPS and from purified lipid A by hydrolysis in 4 M HCI at 100°C for 4 h and transesterified in
types.
Endotoxins (lipopolysaccharides [LPS]) are biologically active components in gram-negative bacteria, being responsible for many of the symptoms and pathophysiological characteristics of infections by gram-negative bacteria (16). LPS is also the most important antigen of gram-negative bacteria and determines the serological 0 group of the bacteria. In the family Enterobacteriaceae, LPS is composed of the 0 chain built up of repeating oligosaccharide units, the core oligosaccharide, and the biologically active lipid A (16). In contrast to LPS from S-form bacteria, LPS from rough mutant bacteria lacks the 0 chain, thus containing only the core, or fragments of it, plus lipid A. Semirough LPS contains in addition to the core one oligosaccharide unit. So far, LPS of B. catarrhalis has not been immunochemically characterized, and it is not known whether such LPS would be structurally and immunologically similar among different strains. This study was therefore undertaken to examine two strains of B. catarrhalis for the presence of LPS and to investigate their chemical composition, immunological characteristics, and biological properties. Bacterial strains. The type strain of B. catarrhalis (CCUG 353, NCTC 11020) was obtained from the Culture Collection of the University of Goteborg. B. catarrhalis BBH 56 was a clinical isolate from a patient with acute exacerbation of *
Corresponding author. 3346
VOL. 59, 1991
NOTES
3347
TABLE 1. Chemical composition of B. catarrhalis LPS nmol of (per mg of LPS):
B. catarrhalis t .rai strain
CCUG 353 BBH 56 a
Sugar Glucose
Galactose
KDOa
Glucosamine
Phosphate
C12
1,135 2,365
391 887
256 258
638 409
754 714
32 47
Fatty acid 3-OH-C11
27 36
3-OH-C12
463 496
KDO, ketodeoxyoctonate.
2 M HCl-methanol at 85°C for 10 h. The methyl esters thus obtained were measured with a gas-liquid chromatograph (model 3700) equipped with a capillary column (SE 54; Weeke, Muilheim, Federal Republic of Germany). Total phosphorus was quantitated as described by Lowry and Tinsley (14). Chemical analysis of the LPS material extracted revealed the presence of chemical components that are typical of LPS (Table 1). There were considerable differences in the amounts of neutral sugars present in the LPS of B. catarrhalis BBH 56 and CCUG 353 (58 and 28%, respectively). Only two neutral sugars were present in the LPS, their molar concentrations in the B. catarrhalis BBH 56 LPS being twice as high as in the preparation of CCUG 353 LPS. Heptose, which has been found in many LPS, was not present in either preparation. Glucosamine was present in both LPS, amounting to 11% (by weight) in strain CCUG 353 and 7% in strain BBH 56. Both LPS contained 6.0% ketodeoxyoctonate and 6.7% phosphate. Thus, the molar ratio of glucosamine relative to ketodeoxyoctonate was 2.5 for B. catarrhalis CCUG 353 and 1.6 for strain BBH 56. Three different fatty acids were present in lipid A in different molar concentrations. The relative ratios of the three fatty acids were, however, similar in the two LPS. 3-Hydroxydodecanoic acid was the main constituent. This is in agreement with the results of Johnson et al. (11). Recently, an atypical strain of B. catarrhalis has been described which contained oleic acid (C18) and palmitoleic acid (C16) as the main cellular fatty acids (24); however, this strain was atypical in showing negative nitrate and nitrite reactions and positive -y-glutamyl aminopeptidase reactions (24). The composition of lipid A of Branhamella strains CCUG 353 and BBH 56 has strong similarities to that of an Acinetobacter strain which also lacks heptose in the core (2). These similarities are interesting, since rRNA-DNA hybridization studies have also shown a 70% homology between Branhamella and Acinetobacter species (1). 3-Hydroxytetradecanoic acid, which is normally present in lipid A of members of the family Enterobacteriaceae and "true" Neisseria spp. (16), was not found in the two Branhamella strains. Since the pattern of lipid A fatty acids for a given bacterial genus or family exhibits constant characteristics (17), these findings would support the classification of B. catarrhalis as a member of the genus Moraxella. SDS-PAGE analysis. LPS samples were treated with 0.05 Tris-HCl (pH 6.8)-2% sodium dodecyl sulfate (SDS)-10% sucrose-0.01% bromphenol blue at 100°C for 5 min and fractionated on SDS gels containing 5% acrylamide in the stacking gel and 12.5% acrylamide in the separating gel (13). Polyacrylamide gel electrophoresis (PAGE) was carried out at 5 mA in the stacking gel and 20 mA in the separating gel. After electrophoresis, LPS were detected by silver staining (6). As shown in Fig. 1, both LPS exhibited bands only in the low-molecular-weight region. Their migration was comparable to that of semirough LPS. The LPS appeared semirough
irrespective of the cultivating method (plates versus fluid medium) and the extraction method. Interestingly, a similar LPS structure has been observed for LPS of noncapsulate Haemophilus influenzae, Bordetella pertussis, and strains of Neisseria meningitidis, which also cause respiratory infections.
Immunoreactivity of B. catarrhalis LPS. The immunoreactivity of the B. catarrhalis LPS was investigated by immunoblotting. Specific anti-LPS antibodies were raised in rabbits by immunizing them with heat-killed bacteria as described earlier (5). LPS of B. catarrhalis BBH 56 and CCUG 353 as well as LPS from Klebsiella pneumoniae, Shigella sonnei, Escherichia coli 0111:B4, E. coli E-100 Ra, Salmonella abortus equi, Pseudomonas aeruginosa 0:3, and Acinetobacter calcoaceticus were resolved by SDS-PAGE and transferred to nitrocellulose by electroblotting at 36 V for 1.5 h (5). After blocking of nonspecific binding sites in incubation buffer (0.05 M Tris-HCl, 0.1 M NaCl, 1% [wt/vol] gelatin, pH 7.4) overnight at 22°C, the nitrocellulose was
1
2
FIG. 1. Silver-stained SDS-PAGE pattern of LPS from the type strain, CCUG 353 (NCTC 11020) (lane 1), and a clinical strain, BBH 56 (lane 2), of B. catarrhalis. LPS (2.5 jig) was applied to each well. Both LPS appear rough, with no high-molecular-weight S-form LPS components.
3348
NOTES
A t i -56 n
INFECT. IMMUN.
Anti -353
TABLE 2. Passive hemolysis: reactivity of B. catarrhalis BBH 56 and CCUG 353 LPS with homologous and heterologous rabbit antiserum before and after absorption with homologous and heterologous LPS Rabbit antiserum
56 353
56
353
FIG. 2. LPS immunoblotting with specific rabbit antisera against B. catarrhalis strains. Antibodies in anti-BBH 56 (rabbit no. II) and anti-CCUG 353 (rabbit no. V) reacted with their homologous LPS (LPS 56 and LPS 353, respectively) but only weakly with the heterologous LPS.
incubated for 3.5 h at 22°C in rabbit antiserum diluted (1:50) in the incubation buffer. The sheets were then washed three times and incubated for 1.5 h at 22°C with peroxidaseconjugated swine anti-rabbit immunoglobulins (Dakopatts, Glostrup, Denmark) diluted (1:50) in the incubation buffer. After three washings, the color developed in Tris-saline0.003% (wt/vol) 4-chloro-1-naphtol-0.05% (vol/vol) H202. Anti-B. catarrhalis BBH 56 LPS antibodies reacted strongly with the homologous LPS (two bands) but only weakly with the heterologous B. catarrhalis CCUG 353 LPS and vice versa (Fig. 2). Antisera of some rabbits immunized with B. catarrhalis did bind to both Branhamella LPS but also to LPS of other gram-negative bacteria (A. calcoaceticus, E. coli 0111:B4, and S. sonnei). However, after absorption of these antisera with the homologous LPS, the reaction to both B. catarrhalis strains disappeared, whereas the reaction to the other bacterial LPS remained. This indicates that the anti-B. catarrhalis LPS antibodies did not cross-react with LPS from the other species and that the presence of antibodies to these unrelated LPS was coincidental. Thus, immunoblotting demonstrated that the LPS of one B. catarrhalis strain possesses both individual and common antigenic determinants which differed from those of the other LPS preparation tested. The demonstration of cross-reactivity between the two Branhamella LPS is in agreement with the similar chemical composition differing only in the molar ratios of the sugar components. To investigate the degree of cross-reactivity between B. catarrhalis CCUG 353 LPS and BBH 56 LPS, passive hemolysis and hemolysis inhibition analyses were performed as described previously (10). Fresh guinea pig serum served as a source of complement and was preabsorbed with sheep erythrocytes (SRBC) coated with one of the LPS. Likewise, test sera were preabsorbed with SRBC. For the specific absorption of antibodies, each antiserum was allowed to interact with SRBC coated with the corresponding LPS for 1 h at 4°C and centrifuged and the supernatant was used for cross-reactivity tests. LPS of S. abortusequi was used as a negative control. Antiserum to B. catarrhalis BBH 56 hemolysed SRBC coated with B. catarrhalis BBH 56 LPS and CCUG 353 LPS (Table 2), the titers to the homologous LPS being higher. Absorption of anti-B. catarrhalis BBH 56 antiserum with B. catarrhalis CCUG 353 LPS reduced the
LPS antigen
Anti-LPS titera Absorbed Absorbed Unabsorbed with BBH with CCUG 353 LPS 56 LPS
Anti-BBH 56
BBH 56 CCUG 353
1,280 640
-2 -2
20 s2
Anti-CCUG 353
BBH 56 CCUG 353
320 1,280
Vol. 59, No. 9
Comparative Immunochemistry of Lipopolysaccharides from Branhamella catarrhalis Strains JONNA STORM FOMSGAARD,' ANDERS FOMSGAARD,1 NIELS H0IBY,2 BRITA BRUUN,2 AND CHRIS GALANOSl* Max-Planck Institut fur Immunbiologie, Freiburg D-7800, Federal Republic of Germany,' and Department of Clinical Microbiology, Rigshospitalet, Copenhagen DK-2200, Denmark? Received 15 June 1990/Accepted 2 July 1991
Lipopolysaccharides (LPS) were extracted and purified from the type strain and from a clinical isolate of Branhamella catarrhalis. Chemical analysis revealed the presence of glucose, galactose, and glucosamine in different molar proportions in the LPS from these two isolates, whereas there was no difference between the two isolates in the ratios of ketodeoxyoctonate, phosphate, and the fatty acids C12, 3-OH-C12, and 3-OH-Cl1 present. Heptose or 3-OH-C14 was not detectable in either preparation. LPS from both strains appeared semirough according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, presenting a core polysaccharide plus one repeating unit. Immunoblotting, passive hemolysis, and hemolysis inhibition assays using anti-LPS antibodies from immunized rabbits demonstrated cross-reactivity between the LPS preparations; however, antigenic dissimilarities were also found, suggesting that more than one serotype may exist. The lipid A isolated from the two LPS was serologically identical and exhibited cross-reactivity with lipid A of members of the family Enterobacteriaceae. The B. catarrhalis LPS were biologically active, causing lethality in D-galactosamine-sensitized C57/BL6 mice and inducing Limulus amoebocyte lysate gelation.
Branhamella catarrhalis has long been regarded as an occasional nonpathogenic inhabitant of the respiratory tract (22). However, during the last decade its importance as a pathogen in both acute upper and lower respiratory tract infections has become evident (20, 21). Sporadic cases of otitis, meningitis, and septicemia have also been reported (4, 19, 22). B. catarrhalis is a gram-negative diplococcus, placed in the genus Moxarella (1). A serotyping system for B. catarrhalis has not been described, and it is therefore not known whether B. catarrhalis exists as one or more sero-
chronic bronchitis (3). The bacteria were cultured in fluid medium (Trypticase soy broth; BBL Microbiology Systems, Cockeysville, Md.) with shaking at 37°C for 48 h. In addition, B. catarrhalis BBH 56 was cultured overnight on Truche Agar plates at 37°C. After harvesting, the bacteria were washed successively with distilled water, ethanol, acetone, and ether and dried in vacuo. Isolation and purification of LPS. LPS could be extracted from the two strains of B. catarrhalis by the phenol-chloroform-petroleum ether method (9) and also by the hot-phenolwater procedure (23). The extracts were purified by threefold ultracentrifugation, converted to the triethylamine salt form by electrodialysis (8), and lyophilized. Extraction by the phenol-chloroform-petroleum ether method of B. catarrhalis CCUG 353 (8.0 g [dry weight]) and of B. catarrhalis BBH 56 (15.8 g [dry weight]) cultured in fluid medium led to yields of 0.5 and 1.95% (wt/wt), respectively. Extraction by the phenol-water method of B. catarrhalis CCUG 353 (7.6 g) and B. catarrhalis BBH 56 (13 g) both led to lower yields, 0.05 and 0.32% (wt/wt), respectively. Thus, the higher yields obtained by the phenol-chloroform-petroleum ether method, which was especially developed for LPS extraction from rough mutants, suggest the presence of more lipophilic LPS. Free lipid A was isolated after hydrolysis of the LPS with 1% acetic acid at 100°C for 45 min, washed three times in 1% acetic acid, and lyophilized. Analytical methods. Neutral sugars were liberated by hydrolysis in 0.1 M HCl at 100°C for 48 h, converted to their alditol-acetate derivatives, and analyzed by gas-liquid chromatography (with a Varian 2400 chromatograph) (18). Glucosamine was measured colorimetrically (15) and in an amino acid analyzer (model D-500; Durmun) following hydrolysis of LPS in 4 M HCl at 100°C for 10 h. Ketodeoxyoctonate was measured by the thiobarbituric acid method as modified by Karkharis et al. (12) after its release by treatment with 0.25 M sulfuric acid at 100°C for 8 h. Fatty acids were liberated from LPS and from purified lipid A by hydrolysis in 4 M HCI at 100°C for 4 h and transesterified in
types.
Endotoxins (lipopolysaccharides [LPS]) are biologically active components in gram-negative bacteria, being responsible for many of the symptoms and pathophysiological characteristics of infections by gram-negative bacteria (16). LPS is also the most important antigen of gram-negative bacteria and determines the serological 0 group of the bacteria. In the family Enterobacteriaceae, LPS is composed of the 0 chain built up of repeating oligosaccharide units, the core oligosaccharide, and the biologically active lipid A (16). In contrast to LPS from S-form bacteria, LPS from rough mutant bacteria lacks the 0 chain, thus containing only the core, or fragments of it, plus lipid A. Semirough LPS contains in addition to the core one oligosaccharide unit. So far, LPS of B. catarrhalis has not been immunochemically characterized, and it is not known whether such LPS would be structurally and immunologically similar among different strains. This study was therefore undertaken to examine two strains of B. catarrhalis for the presence of LPS and to investigate their chemical composition, immunological characteristics, and biological properties. Bacterial strains. The type strain of B. catarrhalis (CCUG 353, NCTC 11020) was obtained from the Culture Collection of the University of Goteborg. B. catarrhalis BBH 56 was a clinical isolate from a patient with acute exacerbation of *
Corresponding author. 3346
VOL. 59, 1991
NOTES
3347
TABLE 1. Chemical composition of B. catarrhalis LPS nmol of (per mg of LPS):
B. catarrhalis t .rai strain
CCUG 353 BBH 56 a
Sugar Glucose
Galactose
KDOa
Glucosamine
Phosphate
C12
1,135 2,365
391 887
256 258
638 409
754 714
32 47
Fatty acid 3-OH-C11
27 36
3-OH-C12
463 496
KDO, ketodeoxyoctonate.
2 M HCl-methanol at 85°C for 10 h. The methyl esters thus obtained were measured with a gas-liquid chromatograph (model 3700) equipped with a capillary column (SE 54; Weeke, Muilheim, Federal Republic of Germany). Total phosphorus was quantitated as described by Lowry and Tinsley (14). Chemical analysis of the LPS material extracted revealed the presence of chemical components that are typical of LPS (Table 1). There were considerable differences in the amounts of neutral sugars present in the LPS of B. catarrhalis BBH 56 and CCUG 353 (58 and 28%, respectively). Only two neutral sugars were present in the LPS, their molar concentrations in the B. catarrhalis BBH 56 LPS being twice as high as in the preparation of CCUG 353 LPS. Heptose, which has been found in many LPS, was not present in either preparation. Glucosamine was present in both LPS, amounting to 11% (by weight) in strain CCUG 353 and 7% in strain BBH 56. Both LPS contained 6.0% ketodeoxyoctonate and 6.7% phosphate. Thus, the molar ratio of glucosamine relative to ketodeoxyoctonate was 2.5 for B. catarrhalis CCUG 353 and 1.6 for strain BBH 56. Three different fatty acids were present in lipid A in different molar concentrations. The relative ratios of the three fatty acids were, however, similar in the two LPS. 3-Hydroxydodecanoic acid was the main constituent. This is in agreement with the results of Johnson et al. (11). Recently, an atypical strain of B. catarrhalis has been described which contained oleic acid (C18) and palmitoleic acid (C16) as the main cellular fatty acids (24); however, this strain was atypical in showing negative nitrate and nitrite reactions and positive -y-glutamyl aminopeptidase reactions (24). The composition of lipid A of Branhamella strains CCUG 353 and BBH 56 has strong similarities to that of an Acinetobacter strain which also lacks heptose in the core (2). These similarities are interesting, since rRNA-DNA hybridization studies have also shown a 70% homology between Branhamella and Acinetobacter species (1). 3-Hydroxytetradecanoic acid, which is normally present in lipid A of members of the family Enterobacteriaceae and "true" Neisseria spp. (16), was not found in the two Branhamella strains. Since the pattern of lipid A fatty acids for a given bacterial genus or family exhibits constant characteristics (17), these findings would support the classification of B. catarrhalis as a member of the genus Moraxella. SDS-PAGE analysis. LPS samples were treated with 0.05 Tris-HCl (pH 6.8)-2% sodium dodecyl sulfate (SDS)-10% sucrose-0.01% bromphenol blue at 100°C for 5 min and fractionated on SDS gels containing 5% acrylamide in the stacking gel and 12.5% acrylamide in the separating gel (13). Polyacrylamide gel electrophoresis (PAGE) was carried out at 5 mA in the stacking gel and 20 mA in the separating gel. After electrophoresis, LPS were detected by silver staining (6). As shown in Fig. 1, both LPS exhibited bands only in the low-molecular-weight region. Their migration was comparable to that of semirough LPS. The LPS appeared semirough
irrespective of the cultivating method (plates versus fluid medium) and the extraction method. Interestingly, a similar LPS structure has been observed for LPS of noncapsulate Haemophilus influenzae, Bordetella pertussis, and strains of Neisseria meningitidis, which also cause respiratory infections.
Immunoreactivity of B. catarrhalis LPS. The immunoreactivity of the B. catarrhalis LPS was investigated by immunoblotting. Specific anti-LPS antibodies were raised in rabbits by immunizing them with heat-killed bacteria as described earlier (5). LPS of B. catarrhalis BBH 56 and CCUG 353 as well as LPS from Klebsiella pneumoniae, Shigella sonnei, Escherichia coli 0111:B4, E. coli E-100 Ra, Salmonella abortus equi, Pseudomonas aeruginosa 0:3, and Acinetobacter calcoaceticus were resolved by SDS-PAGE and transferred to nitrocellulose by electroblotting at 36 V for 1.5 h (5). After blocking of nonspecific binding sites in incubation buffer (0.05 M Tris-HCl, 0.1 M NaCl, 1% [wt/vol] gelatin, pH 7.4) overnight at 22°C, the nitrocellulose was
1
2
FIG. 1. Silver-stained SDS-PAGE pattern of LPS from the type strain, CCUG 353 (NCTC 11020) (lane 1), and a clinical strain, BBH 56 (lane 2), of B. catarrhalis. LPS (2.5 jig) was applied to each well. Both LPS appear rough, with no high-molecular-weight S-form LPS components.
3348
NOTES
A t i -56 n
INFECT. IMMUN.
Anti -353
TABLE 2. Passive hemolysis: reactivity of B. catarrhalis BBH 56 and CCUG 353 LPS with homologous and heterologous rabbit antiserum before and after absorption with homologous and heterologous LPS Rabbit antiserum
56 353
56
353
FIG. 2. LPS immunoblotting with specific rabbit antisera against B. catarrhalis strains. Antibodies in anti-BBH 56 (rabbit no. II) and anti-CCUG 353 (rabbit no. V) reacted with their homologous LPS (LPS 56 and LPS 353, respectively) but only weakly with the heterologous LPS.
incubated for 3.5 h at 22°C in rabbit antiserum diluted (1:50) in the incubation buffer. The sheets were then washed three times and incubated for 1.5 h at 22°C with peroxidaseconjugated swine anti-rabbit immunoglobulins (Dakopatts, Glostrup, Denmark) diluted (1:50) in the incubation buffer. After three washings, the color developed in Tris-saline0.003% (wt/vol) 4-chloro-1-naphtol-0.05% (vol/vol) H202. Anti-B. catarrhalis BBH 56 LPS antibodies reacted strongly with the homologous LPS (two bands) but only weakly with the heterologous B. catarrhalis CCUG 353 LPS and vice versa (Fig. 2). Antisera of some rabbits immunized with B. catarrhalis did bind to both Branhamella LPS but also to LPS of other gram-negative bacteria (A. calcoaceticus, E. coli 0111:B4, and S. sonnei). However, after absorption of these antisera with the homologous LPS, the reaction to both B. catarrhalis strains disappeared, whereas the reaction to the other bacterial LPS remained. This indicates that the anti-B. catarrhalis LPS antibodies did not cross-react with LPS from the other species and that the presence of antibodies to these unrelated LPS was coincidental. Thus, immunoblotting demonstrated that the LPS of one B. catarrhalis strain possesses both individual and common antigenic determinants which differed from those of the other LPS preparation tested. The demonstration of cross-reactivity between the two Branhamella LPS is in agreement with the similar chemical composition differing only in the molar ratios of the sugar components. To investigate the degree of cross-reactivity between B. catarrhalis CCUG 353 LPS and BBH 56 LPS, passive hemolysis and hemolysis inhibition analyses were performed as described previously (10). Fresh guinea pig serum served as a source of complement and was preabsorbed with sheep erythrocytes (SRBC) coated with one of the LPS. Likewise, test sera were preabsorbed with SRBC. For the specific absorption of antibodies, each antiserum was allowed to interact with SRBC coated with the corresponding LPS for 1 h at 4°C and centrifuged and the supernatant was used for cross-reactivity tests. LPS of S. abortusequi was used as a negative control. Antiserum to B. catarrhalis BBH 56 hemolysed SRBC coated with B. catarrhalis BBH 56 LPS and CCUG 353 LPS (Table 2), the titers to the homologous LPS being higher. Absorption of anti-B. catarrhalis BBH 56 antiserum with B. catarrhalis CCUG 353 LPS reduced the
LPS antigen
Anti-LPS titera Absorbed Absorbed Unabsorbed with BBH with CCUG 353 LPS 56 LPS
Anti-BBH 56
BBH 56 CCUG 353
1,280 640
-2 -2
20 s2
Anti-CCUG 353
BBH 56 CCUG 353
320 1,280
