INTRODUCTION

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Campylobacter fetus, a microaerophilic gram-negative bacterium, is a recognized veterinary and human pathogen (9, 23). This organism has been divided into two closely related subspecies, C. fetus subsp. venerealis and C. fetus subsp. fetus. C. fetus subsp. venerealis is the main cause of bovine genital campylobacteriosis, a disease characterized by infertility and abortion and of major economic concern to the cattle industry in many countries. C. fetus subsp. fetus causes sporadic abortion in cattle and enzootic abortion in sheep and occasionally causes systemic and intestinal infections in humans, particularly in immunocompromised individuals.

Lipopolysaccharide (LPS) is an essential and characteristic component of the outer membrane of gram-negative bacteria (27). The LPS molecule of C. fetus, like those of many other gram-negative bacteria, is composed of three distinct structural domains: a hydrophobic lipid A portion which forms the outer leaflet of the outer membrane and which contains the endotoxic activity (21), a low-molecular-weight oligosaccharide core complex, and a variable-length O-specific polysaccharide chain with repeating oligosaccharide units (O-antigen). The LPS O-antigens are the basis of the C. fetus heat-stable serotyping scheme (25), and two main heat-stable serotypes, designated A and B, are recognized. All C. fetus subsp. venerealis isolates are serotype A, whereas C. fetus subsp. fetus isolates are serotype A or B (4). The biochemical composition of C. fetus LPS (20) and the structures of serotype A (30) and serotype B (29) O-antigens have been analyzed and provide a chemical basis for the heat-stable serotyping scheme.

Bacterial components such as LPS which are used in serotyping schemes must be well characterized in order to provide reliable results and useful information. While LPS is antigenically stable in many organisms, variable expression of O-antigen factors in strains of several bacteria, including Salmonella enterica serovar Typhimurium (17), S. enterica serovar Enteritidis (11), and Helicobacter pylori (3, 10), has been described. Intrastrain instability or phase variation in the lipooligosaccharides (LOS) from mucosal gram-negative bacteria, such as Neisseria gonorrhoeae (2), Neisseria meningitidis (33), Bordetella pertussis (24), and Haemophilus somnus (14), has also been reported. The intrastrain antigenic variation in LPS and LOS has primarily been detected by serological methods, frequently with monoclonal antibodies (MAbs) specific for particular epitopes and by comparison of electrophoretic profiles.

In the present study two serologically distinct O-antigen epitopes were identified in the LPS of C. fetus serotype A strains and characterized using MAbs. Substantial variation in expression of the two O-antigen epitopes was observed, by enzyme-linked immunosorbent assay (ELISA) using MAbs, among weekly isolates of two C. fetus serotype A strains recovered from two experimentally infected heifers. Minimal variation in the expression of these two epitopes was seen among in vitro subcultures of two C. fetus isolates from the infected animals and one C. fetus strain which had previously undergone many passages in the laboratory.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. Forty-three strains of C. fetus subsp. venerealis and C. fetus subsp. fetus were used in this study (Table 1). These included the type strains for C. fetus subsp. venerealis (ATCC 19438, ADRI 554; serotype A) and C. fetus subsp. fetus (ATCC 27374, ADRI 553; serotype B). Identification of the strains was confirmed as described previously (6) using standard cultural and biochemical tests for C. fetus. The strains were grown on Mueller-Hinton agar under microaerophilic conditions as described previously (6). Cells were harvested, resuspended in 0.01 M Tris, pH 7.5, to a concentration of approximately 10¹⁰ CFU/ml (3.5 mg of protein/ml), and stored at 80°C.

MAb production. The methods described previously for production and initial selection of MAbs (7) were used, with minor modification. The whole-cell inoculum for immunization of mice was prepared by growing C. fetus strains 553 and 554 on Mueller-Hinton agar. The cells were harvested, resuspended in saline containing 0.3% formalin to a concentration of approximately 10⁹ cells/ml (McFarland turbidity standard no. 5), and left overnight at room temperature. The cells were washed twice in saline, resuspended in saline to a concentration of approximately 10⁹ cells/ml, and stored at 20°C until used.

Indirect ELISA. Microtiter plates (Nunc; 475094 or 439454) were coated with C. fetus cell suspensions diluted 1 in 1,000 with 0.06 M carbonate buffer, pH 9.6, or whole-cell proteinase K (PK) digests diluted 1 in 140 with 0.06 M carbonate buffer, pH 9.6. PK digests were prepared by adding 0.4 volume of PK (2.5 mg/ml) and 1 volume of PBS to 1 volume of whole-cell suspension (approximately 10¹⁰ CFU/ml) and heating at 60°C for 60 min and then at 100°C for 30 min.

SDS-PAGE and immunoblotting. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting procedures described by Brooks et al. (6) were used. For some experiments, cells of selected C. fetus strains were suspended in 4 volumes of 0.06 M carbonate buffer, pH 9.6, and 1 volume of 0.01 M Tris buffer, pH 7.5, and not heated or heated at 100°C for 15 min. PK digests of the bacterial cell suspensions were prepared by the procedure of Hitchcock and Brown (13). Discontinuous SDS-PAGE was performed using a 6% stacking gel and a 12% separating gel. C. fetus components separated by SDS-PAGE were electrophoretically transferred from gels to nitrocellulose membranes. For immunological detection, the membranes were blocked with 3% fraction V bovine serum albumin (Sigma) in 0.02 M Tris-buffered saline (TBS; 0.02 M Tris, 0.14 M NaCl, 2.7 mM KCl; the pH was adjusted to 7.4 with HCl), incubated for 16 h with MAbs in tissue culture fluid diluted 1 in 10 with TBS containing 0.05% Tween 20 (TBST), washed, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Zymed Laboratories) diluted 1 in 1,000 with TBST. After being washed, the membranes were incubated with 5-bromo-4-chloro-3-indolylphosphate-p-nitroblue tetrazolium chloride (Kirkegaard and Perry) substrate.

Phenol-water extraction of C. fetus LPS. Briefly, LPS was obtained by hot phenol-water extraction of C. fetus 553 and C. fetus 554 cells essentially in accordance with the procedure of Westphal and Jann as described by Sprott et al. (32). The aqueous extract was dialyzed, using a 3,500-molecular-weight cutoff, against distilled water for 3 days. The phenol extract was further processed with methanol and trichloroacetic acid (22) and then dialyzed, using a 3,500-molecular-weight cutoff, against distilled water for 3 days. The dialyzed aqueous and phenol extracts were lyophilized.

Two-antibody sandwich ELISA. Microtiter plates (Nunc; 439454) were coated with 100 µl of an anti-C. fetus polyclonal rabbit antiserum/well diluted 1 in 5,000 with 0.06 M carbonate buffer, pH 9.6. The polyclonal serum had been prepared using a mixture of cells of C. fetus serotype A and B strains. The plates were incubated overnight at RT and then washed with PBST. Lyophilized aqueous and phenol LPS extracts from C. fetus strains 553 and 554 were suspended in deionized water (aqueous phase extract, 1.0 mg/ml; phenol phase extract, 4.2 mg/ml) and then further diluted with 0.01 M PBS, pH 7.2, or 0.1 M acetate buffer, pH 4.5, or 0.06 M carbonate buffer, pH 9.6. Aliquots of the diluted LPS preparations were left unheated or were heated at 100°C for 15 min and then cooled to RT; 100 µl was added to the antibody-coated wells, and the plates were incubated for 1 h at RT. The plates were washed with PBST, MAbs (hybridoma tissue culture fluid diluted 1 in 100 with PBST) were added (100 µl/well), and the plates were incubated for 2 h at RT. The plates were washed with PBST, horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1 in 10,000 with PBST was added (100 µl/well), and the plates were incubated for 1 h at RT. The plates were washed again, 100 µl of 3,3',5,5'-tetramethylbenzidine-hydrogen peroxide substrate/well was added, and the plates were shaken continuously for 10 min. The OD₆₅₅ was determined. Within- and between-assay variations for triplicate samples were determined by calculating the percent coefficient of variation.

	RESULTS

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Immunoblot characterization of MAbs. After initial selection, cloning, and isotype determination, 27 IgG MAbs and a polyclonal antiserum prepared against a pool of C. fetus serotype A and B strains were compared on immunoblots of C. fetus PK digests. Clusters of high-molecular-mass (HMM) LPS components in a ladderlike arrangement characteristic of O-chains with increasing numbers of oligosaccharide repeat units and low-molecular-mass core LPS components were seen in the immunoblots of C. fetus strains 554 (serotype A) and 553 (serotype B) with the polyclonal antiserum (Fig. 1, lanes 1 and 2, respectively). The relative mobility of the clusters of O-chain bands was slower for strain 554 than for strain 553. Eight MAbs which reacted on immunoblots of C. fetus 554 were identified, and two immunoblot patterns were observed. In both patterns a reaction was evident at the level of the O-chain but not the core region. The number of bands seen in each cluster of HMM bands, most evident in the middle region of the nitrocellulose strip, distinguished the two patterns and was used to divide the eight MAbs into two groups. Six MAbs had a pattern with 6 to 8 bands per cluster (group 1), and two MAbs had a pattern with 2 to 4 bands per cluster (group 2). MAb M1177 was selected as representative of group 1 (Fig. 1, lane 3), and M1194 was selected as representative of group 2 (Fig. 1, lane 4). Both M1177 and M1194 are of the IgG1 isotype. No reaction was observed with M1177 (Fig. 1, lane 6), M1194 (Fig. 1, lane 7), and the other six MAbs on immunoblots of C. fetus 553. 

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Immunoblots of C. fetus 553 and C. fetus 554 PK digests with MAbs M1177, M1183, and M1194. Lanes 1, 3, 4, and 5, PK digests of C. fetus 554 cells in 0.01 M Tris, pH 7.5; lanes 2, 6, 7, and 8, PK digests of C. fetus 553 cells in 0.01 M Tris, pH 7.5. Lanes 1 and 2, polyclonal rabbit anti-C. fetus serum; lanes 3 and 6, M1177; lanes 4 and 7, M1194; lanes 5 and 8, M1183. Note differences in banding patterns observed with M1177 and M1194 and C. fetus 554 PK (bracketed regions). Images were acquired using a Hewlett-Packard ScanJet 4c/T flatbed scanner and labeled using CorelDraw, version 8.0.

Strain characterization using MAbs and ELISA. Forty-three C. fetus strains were examined on indirect ELISA with M1183 and the eight MAbs that reacted on immunoblots with C. fetus 554. Six strains (553, 809, 811, 830, 832, and 1810) reacted only with M1183 (mean OD ± standard deviation, 1.6 ± 0.8) and were considered to be serotype B. With the other 37 strains, the reactions observed with M1177 and M1194 were representative of the reactions with the MAbs in immunoblot groups 1 and 2, respectively. Thirty-three (89%) of these 37 strains reacted with both M1177 and M1194 (ODs,1.7 ± 0.5 and 2.0 ± 0.5, respectively). Two strains (502 and 523) reacted with M1177 (OD, 1.1 ± 0.7) but not with M1194. Two strains (536 and 1358) reacted with M1194 (OD, 1.6 ± 2.0) but not with M1177. The 37 strains were considered to be serotype A and did not react with M1183.

Effect of temperature and pH on C. fetus serotype A LPS reactivity with MAbs. The effect of various temperature and pH conditions on purified C. fetus serotype A LPS was investigated using a two-antibody sandwich ELISA. M1177 and M1194 reacted with LPS, heated (100°C, 15 min) or unheated (RT, 15 min), in PBS, pH 7.0, or acetate buffer, pH 4.5, and similar titration curves were observed (data not shown). M1177 and M1194 also reacted with unheated LPS in carbonate buffer, pH 9.6 (Fig. 2). In contrast, the reaction of M1177 with LPS heated in carbonate buffer, pH 9.6, was reduced to background levels while that of M1194 increased from an OD of 0.8 to 1.9 at the highest LPS concentration examined (Fig. 2). The results with phenol phase LPS are shown in Fig. 2, and the results with aqueous-phase LPS (not shown) were similar. The within-assay coefficients of variation were less than 5% with LPS dilutions of 1/100,000, and the between-assay coefficients of variation were less than 10%.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Two-antibody sandwich ELISA for measurement of the reactivity of MAbs M1177 and M1194 with C. fetus 554 LPS, either not heated () or heated at 100°C for 15 min (), in 0.06 M carbonate buffer, pH 9.6. Polyclonal rabbit anti-C. fetus serum was used to capture extracted phenol phase LPS (4.2 mg/ml) diluted in carbonate buffer. Carbonate buffer (carb) with no LPS was included as a control. Monoclonal antibodies M1177 and M1194 and a polyclonal goat anti-mouse serum conjugated with horseradish peroxidase were used for detection. Data represent the means of triplicates.

Immunoblot analysis of LPS from selected C. fetus strains. The effect of temperature and alkaline pH on the LPS immunoblot profiles of three selected C. fetus strains, 554, 1352 and 523, was examined using MAbs M1177 and M1194. M1177 reacted with LPS from unheated C. fetus 554 cells in carbonate buffer, pH 9.6, and a ladderlike pattern was observed with 6 to 8 bands per cluster of HMM bands (Fig. 3, lane 1). M1177 did not react with LPS from C. fetus 554 cells heated in carbonate buffer (Fig. 3, lane 2). M1194 reacted with LPS from unheated C. fetus 554 cells or cells heated in carbonate buffer, and a ladderlike pattern with 2 to 4 bands per cluster of HMM bands was seen. The clusters of bands migrated slightly faster with the heated LPS (Fig. 3, lane 4) than with the unheated LPS (Fig. 3, lane 3). M1177 did not react with C. fetus 1352 LPS, unheated or heated, in carbonate buffer (Fig. 3, lanes 5 and 6). M1194 reacted with C. fetus 1352 LPS, unheated or heated, in carbonate buffer (Fig. 3, lanes 7 and 8, respectively), and the patterns were very similar to those observed with C. fetus 554 LPS. M1177 reacted with unheated C. fetus 523 LPS in carbonate buffer, and a ladderlike pattern with three bands per cluster of HMM bands was observed (Fig. 3, lane 9). The mobility of the bands was similar to that of the fastest-migrating bands in the clusters seen with unheated C. fetus 554 LPS. M1177 did not react with C. fetus 523 LPS heated in carbonate buffer (Fig. 3, lane 10), and M1194 did not react with C. fetus 523 LPS, unheated or heated, in carbonate buffer (Fig. 3, lanes 11 and 12). The patterns observed with MAbs M1177 and M1194 and unheated C. fetus 554, 1352, and 523 LPS in carbonate buffer were almost identical with those seen using unheated LPS in Tris, pH 7.5 (C. fetus 554, Fig. 1, lanes 3 and 4; C. fetus 1352 and 523, data not shown).

View larger version (119K): [in this window] [in a new window]   FIG. 3.   Immunoblots of C. fetus 554, C. fetus 1352, and C. fetus 523 PK digests of cells, unheated or heated, in 0.06 M carbonate buffer, pH 9.6, with MAbs M1177 and M1194. Lanes 1 to 4, PK digest of C. fetus 554; lanes 5 to 8, PK digest of C. fetus 1352; lanes 9 to 12, PK digest of C. fetus 523. Lanes 1, 3, 5, 7, 9, and 11, unheated cells; lanes 2, 4, 6, 8, 10 and 12, heated cells. Lanes 1, 2, 5, 6, 9, and 10, M1177; lanes 3, 4, 7, 8, 11, and 12, M1194. Images were acquired using a Hewlett-Packard ScanJet 4c/T flatbed scanner and labeled using CorelDraw, version 8.0.

Variation in expression of O-antigens among C. fetus isolates from infected heifers. PK digests of cell preparations of C. fetus weekly isolates from two experimentally infected heifers were examined by indirect ELISA with M1177 and M1194 to determine whether variation in C. fetus LPS O-antigen expression occurs in vivo. The reproducibility of the assay was examined with multiple replicates assessed with the same test and between tests, using different digests and different dilutions of the same digest for various cell preparations. The within-assay coefficients of variation were less than 5%, and the between-assay coefficients of variation were less than 10%.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Indirect ELISA for measurement of the reactivities of MAbs M1177 and M1194 with PK digests of C. fetus 555 and C. fetus 1352 weekly isolates from experimentally infected heifers. C. fetus was not isolated at weeks 6 and 15 and at weeks 15 and 17 from the animals infected with C. fetus 555 and 1352, respectively. Data represent the means of triplicates.

Examination of LPS from sequential in vitro subcultures of C. fetus strains. To investigate if variation in expression of LPS O-chain determinants occurs in vitro, PK digests of 14 subcultures of two colonies from one high-passage laboratory strain (C. fetus 554) and two low-passage isolates (C. fetus 555 and 1352 week 1 isolates) were examined using indirect ELISA. With four of the colonies, one each from C. fetus 554 and C. fetus 555 (Fig. 5) and two from C. fetus 1352, the reactions observed with M1194 and M1177 were very similar and were consistently strong in cells from all 14 subcultures. The largest difference in reactivity between M1194 and M1177 for any of these four colonies was 0.2 OD unit, and the smallest difference was 0.0 OD units. The highest mean reaction with M1194 for all subcultures was observed with C. fetus 555 (OD, 2.1 ± 0.1), and the lowest was observed with C. fetus 1352 (OD, 1.5 ± 0.1).

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Indirect ELISA for measurement of the reactivities of MAbs M1177 and M1194 with PK digests of sequential in vitro subcultures of one colony each of C. fetus 554 and C. fetus 555. Data represent the means of triplicates.

	DISCUSSION

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In the present study, MAbs to C. fetus serotype A and serotype B LPS O-chain epitopes were produced. These MAbs were used in ELISA to determine the serotypes of 43 C. fetus strains. The results obtained were in full agreement with serotyping results obtained previously using LPS electrophoretic analysis and immunoblotting with polyclonal antisera (6). These findings indicate that MAbs M1177, M1194, and M1183 are useful to serotype C. fetus strains.

Two serologically distinct O-antigen epitopes were identified in the LPS of C. fetus serotype A strains using MAbs M1177 and M1194. The two epitopes were initially distinguished on the basis of immunoblot patterns observed with M1177 and M1194 and the C. fetus subsp. venerealis type strain LPS as the antigen. The two epitopes were present in 33 of 37 (89%) C. fetus serotype A strains examined. In the other four strains only one of the two O antigen epitopes was detected. The undetected epitope may have been absent or present at a concentration too low to be detected by the ELISA. These results confirm previous observations with polyclonal antisera which suggested the presence of more than one O-antigen epitope in C. fetus serotype A strains (6).

Previous structural studies have shown that the O-specific polysaccharide of C. fetus serotype A LPS is a partially O-acetylated 1, 3-linked -D-mannan (30). Acetylation of the O-antigen is a common form of LPS O-polysaccharide modification found in many bacteria (19, 27). Treatment using hot-alkali conditions, which cleaves all O-acetyl groups (8), has been used to investigate structural and immunological properties of acetylated LPS. In the present study, the observation that heating C. fetus type A LPS under alkaline conditions eliminated the reactivity with M1177 but not M1194 is consistent with an acetyl group being present in the epitope recognized by M1177 and absent in the epitope recognized by M1194. In addition to adding a new epitope, acetylation of an O group has also been shown to mask existing antigenic determinants (15, 31, 37). The increased reactivity of M1194 with serotype A LPS after hot-alkali treatment suggests that some of the epitopes potentially recognized by M1194 are masked by the antigenic determinant recognized by M1177. Acetylation may be a constant part of the LPS structure and responsible for differences among serotypes in a bacterial species (1, 31) or may cause variation within a single bacterial strain (12, 17). The present study suggests that both acetylated and nonacetylated epitopes were present in the LPS O-polysaccharides of the majority (89%) of the 37 serotype A C. fetus strains examined. This is consistent with the finding of Senchenkova et al. (30) that the O-polysaccharide of C. fetus serotype A LPS is partially O-acetylated and extends the observation to other C. fetus subsp. venerealis and C. fetus subsp. fetus serotype A strains. In contrast, the O-specific polysaccharide chain of C. fetus serotype B LPS is a D-rhamnan terminated with 3-O-methyl-D-rhamnose (29), and in the present study only a single O-antigen epitope was identified using M1183 and 18 other MAbs.

In the present study, use of MAbs M1177 and M1194 showed substantial variation in LPS O-antigen expression among isolates of two C. fetus serotype A strains recovered from experimentally infected heifers. Extensive antigenic variation in LOS epitopes of other bacteria such as N. gonorrhoeae (2, 28), Haemophilus influenzae type b (16), and Haemophilus somnus (14) has also been demonstrated using MAbs. Inzana et al. (14) compared levels of LOS epitope expression in disease and commensal preputial isolates of H. somnus. All disease isolates examined were shown to undergo LOS phenotypic phase variation in vivo and in vitro, whereas commensal isolates did not undergo LOS phase variation following in vitro passage. In the present study, in contrast to what was found for the isolates from the experimentally infected animals, there was minimal variation in expression of the two O-antigen epitopes in 14 in vitro subcultures of two C. fetus isolates from the infected animals and one laboratory strain.

Modification of the LPS O polysaccharide by acetylation of specific residues in the O units affects the structure and the immunological properties of the LPS molecule (31) and can be determined by examining phage or chromosomal genes. In Salmonella enterica serovar Typhimurium, for example, O-acetylation of the abequose residue is dependent on chromosomal gene oafA (19) while O-acetylation of the rhamnose residue is determined by genes from lysogenic phages (37). The basis for O-acetylation of the mannose residues in the O units of C. fetus serotype A LPS has yet to be determined.

Phenotypic phase variation in LPS (3, 18) or LOS (14, 26, 34, 36) has been suggested as a virulence mechanism for various bacteria and may play a role in evasion of the host immune defenses, persistence of infection, and invasion. In C. fetus, the HMM S-layer proteins undergo antigenic variation (35) and play a critical role in virulence (5). Further studies are needed to assess the biological relevance of variation in O-antigen expression in C. fetus serotype A strains.