Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay

Abstract

The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

FIG 1

Kinetics of the humoral responses to C. abortus antigens of experimentally infected sheep with and without abortion. Antibody responses to surface (A), virulence-associated (B), and hypothetical (C) protein antigens were determined at different times (0, 2, 5, 12, 21, 29, and 47 weeks) following inoculation. Different lines and symbols indicate different infectious doses. The abortion group included 5 animals inoculated with 5 × 10³ IFU and 5 animals inoculated with 5 × 10⁵ IFU, and the lambing group included 10 animals inoculated with 5 × 10⁷ IFU of C. abortus EBs. Mating took place 8 weeks after inoculation, and abortion or lambing occurred 29 weeks after inoculation, respectively. The ordinate shows the median cutoff-adjusted OD with the first (lower margin) and third (upper margin) quartiles of the data calculated from OD measurements of the reactivity of serum samples to the different antigens and the cutoff OD of the corresponding line assay. *, P ≤ 0.02.

FIG 2

Representative line immunoassays of animal serum samples. Serum samples from experimentally infected sheep that had (A) or had not (B) aborted and the corresponding negative-control group consisting of healthy, uninfected sheep (C) were investigated. The values on the left are the numbers of weeks after inoculation. Abortion (A) or lambing (B, C) took place at 29 weeks after inoculation. The lowercase letters on the right denote individual sheep inoculated with 5 × 10³ (a), 5 × 10⁵ (b), or 5 × 10⁷ (c and c′) IFU of infectious C. abortus EBs, respectively. The lowercase letters d and d′ denote healthy, uninfected sheep. (D) Serum samples from sheep that aborted because of natural C. abortus infection. (E) Serum samples from asymptomatic, C. abortus PCR-negative sheep with healthy offspring. (F) Specificity control serum samples from sheep infected with C. pecorum or C. psittaci. Each line assay represents an individual animal. RK, reaction control.

FIG 3

Representative line immunoassays of human serum samples. (A) Samples from two women who had aborted because of severe septic C. abortus infection were examined. (B) Specificity control serum samples from males and females infected with C. trachomatis (C. tr.), C. pneumoniae (C. pn.), or C. psittaci (C. ps.). (C) Serum samples from shepherds and veterinarians exposed to C. abortus in high-prevalence flocks. One of them (out of 88) showed reactivity to the surface antigens MOMP, MIP, and pmp13G, as well as to virulence-associated protein SINC. Each line assay represents one individual. RK, reaction control.