Identification, characterization, and variation in expression of two serologically distinct O-antigen epitopes in lipopolysaccharides of Campylobacter fetus serotype A strains

Abstract

Monoclonal antibodies (MAbs) to the lipopolysaccharide (LPS) O-antigens of Campylobacter fetus serotype A and B strains were produced. Eight MAbs specific for serotype A LPS were characterized on immunoblots of C. fetus serotype A LPS. Two immunoblot patterns were observed and were used to divide the eight MAbs into two groups. MAbs M1177 and M1194 were selected as representative of the two groups and were used in an enzyme-linked immunosorbent assay (ELISA) to examine the LPS O-antigen epitopes of 37 serotype A C. fetus subsp. fetus and C. fetus subsp. venerealis strains. Thirty-three strains (89%) reacted with both M1177 and M1194, 2 strains reacted only with M1177, and 2 strains reacted only with M1194. To further characterize the O-antigen epitopes, purified serotype A LPS was treated using various temperature and pH conditions and the effect of the treatments on the reactivity of the LPS with MAbs M1177 and M1194 was evaluated by ELISA. While no difference among several treatments was observed, heating serotype A LPS under alkaline conditions decreased the reaction with M1177 to background levels and increased the reaction with M1194. MAbs M1177 and M1194 were also used with ELISA to investigate in vivo and in vitro expression of the two O-antigen epitopes. There was substantial variation in expression of the two epitopes among weekly isolates of two C. fetus serotype A strains recovered from experimentally infected heifers. There was minimal variation in expression of the two epitopes in successive subcultures of three C. fetus serotype A strains.

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.

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.

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.

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.

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.