Antibody responses of cattle immunized with the Tf190 adhesin of Tritrichomonas foetus

Abstract

The antibody response patterns of cattle after subcutaneous and intranasal immunizations with adhesin Tf190 of Tritrichomonas foetus were investigated. Reactions of antibody from cattle parenterally immunized with Tf190 revealed antigen specificity and Tf190 sensitization in the majority of the animals, as determined by Western blotting. The results also demonstrated strong preimmune immunoglobulin G2 (IgG2) binding to T. foetus antigens not seen in IgG1 profiles. Subcutaneous injections of Tf190 resulted in significant (P < 0.05) increases in serum IgG1 and IgG2 titers over time, as determined by parasite specific enzyme-linked immunosorbent assay. Immune sera also significantly inhibited parasite adhesion to mammalian cell lines compared to the level of inhibition obtained with preimmune sera (P < 0.05). Intranasal immunization with Tf190 failed to produce measurable parasite-specific antibody in serum; however, this immunization route did result in significant (P < 0.05) increases in parasite-specific IgA titers in cervical mucus secretions from immunized animals that were more resistant to intravaginal challenge with T. foetus than controls. These results suggest that systemic immunization with Tf190 results in serum antibody production and antiparasitic adhesin antibodies. Additionally, the results of challenge experiments with intranasally immunized animals suggests that Tf190 primes protective immune responses that lead to lower rates of infection among these animals.

FIG. 1

Reactions of antibody from Tf190-immunized cattle reveal antigen specificity and Tf190 sensitization. All animals were immunized subcutaneously on days 0, 20, and 125 with Tf190 plus alum. Whole T. foetus was separated by SDS-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and probed with preimmunization serum obtained on day 0 (lanes 1, 3, 5, and 7) or immune serum obtained on day 125 (lanes 2, 4, 6, and 8) (both at dilutions of 1:50), followed by probing with mouse anti-bovine heavy chain-specific IgG1 antibody (VMRD) (1:500) and detection with anti-mouse HRP-labeled secondary antibody (1:1,000). Lane 9, 60- and 140-kDa bands detected by Tf190-specific MAb 32.3B3.5 (1:50); lane 10, no primary antibody control. The data shown are representative of those for the seven cows demonstrating Tf190 sensitization.

FIG. 2

Comparison of bovine antibody reactions reveals strong preimmunization IgG2 binding to T. foetus antigens not seen in IgG1 profiles. Whole extracts of T. foetus subjected to SDS-PAGE followed by Western blotting were probed with Tf190 immune sera (day 147) or preimmunization sera (day 0) from animals in experiment 1, followed by probing with anti-IgG1 or anti-IgG2 antibodies and secondary sheep anti-bovine. Lanes 1 and 5, preimmunization sera; lanes 2 and 6, immune sera probed with anti-IgG1; lanes 3 and 7, preimmunization sera; lanes 4 and 8, immune sera with probed with anti-IgG2. The numbers on the right indicate the major bands of Tf190 established by the reactivity pattern of Tf190-specific MAb 32.3B3.5.

FIG. 3

Subcutaneous immunization with Tf190 influences levels of parasite-specific IgG1 (A) and IgG2 (B) antibodies. ELISA was performed as described in Materials and Methods with preimmunization sera (day 0) and immune sera (day 125) from animals in experiment 1. The data shown are for two representative animals (animals 4 and 9), with statistically significant differences between preimmune (P) and immune (I) sera detected at the indicated dilutions (∗, P < 0.05). OD and Od, optical density.

FIG. 4

Tf190 immune antibodies in the sera of animals inhibit parasite adhesion to mammalian target cell lines. (A) Parasite strain Tf 330.1 and HeLa cells; (B) parasite strain TFC-5-1 and HeLa cells; (C) parasite strain Tf 330.1 and bovine macrophage cell line M617. Parasites were treated with immune sera (day 125; black bars) or preimmunization sera (day 0; shaded bars) of animals from experiment 1. Numbers above the solid bars indicate percent inhibition, and statistically significant differences were detected as indicated (∗, P < 0.05).

FIG. 5

Parasite-specific, mucosal IgA antibody responses increase in animals intranasally immunized with Tf190. Animals in experiment 2 were given Tf190 (black bars) or control immunizations (alum, CT-B) (shaded bars) and were then challenged intravaginally with T. foetus. Four of six Tf190-immunized animals included in this study (mean of Tf190) cleared the infection, while four of six control animals (mean of control) maintained the infection. Day 0 indicates the day of intravaginal challenge; day 30 indicates 30 days after challenge. Statistically significant differences were detected between the indicated experimental groups (∗, P < 0.05) compared to the results obtained for the control group at day 30 (black bars). OD, optical density