Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis

Abstract

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.

FIG. 1.

Response kinetics of serum antibody specific for LAM-enriched antigen. Sera from M. bovis-infected reindeer (▪; n = 11) were analyzed for reactivity to M. bovis-derived LAM by ELISA. Data are presented as mean (± standard errors of the mean) changes in optical density (ΔOD). Arrows on the x axis indicate time points when purified protein derivatives were injected for skin testing (CCT). Asterisks (*) indicate responses that exceed (P < 0.05) preinfection responses (i.e., week −1).

FIG. 2.

Preparative immunoblots of M. bovis strain 95-1315 WCS antigen probed with sera from a reindeer experimentally infected with M. bovis. Molecular mass markers are indicated in the left margin (in kDa) and weeks postinfection in the lower margin. Arrows in the lower margin indicate time points when purified protein derivatives were injected for skin testing (CCT). Symbols in the lower left margin refer to sera from known noninfected (−) and infected (+) white-tailed deer used as controls for the assay. Numbers in the lower margin refer to weeks relative to infection. Immunoblots were performed on samples from each reindeer at all time points indicated. Responses presented in this figure are representative of 10/11 of the infected reindeer. One infected reindeer (animal 125) had only minimal responses detectable by immunoblot analysis.

FIG. 3.

Antibody responses to recombinant antigens detected by MAPIA in reindeer experimentally infected with M. bovis. Arrows in the upper margin indicate time points when purified protein derivatives were injected for skin testing (CCT). Antigens printed are shown in the right margin. Strips represent different time points during infection when serum samples were collected (shown in weeks in the lower margin). Representative responses by two different M. bovis-infected animals, animal 120 (A) and animal 126 (B), are provided to demonstrate the variability in antigen recognition patterns.

FIG. 4.

Effects of injection of PPDs for skin testing on antibody responses (as measured by MAPIA) by noninfected (A) and infected (B) reindeer to M. bovis antigens. Reindeer were injected with PPDs for skin testing 3 months after challenge with M. bovis and sera collected at indicated time points for analysis of antibody. Numbers in the upper margins indicate animal identification numbers. Numbers in lower margins indicate weeks relative to skin test. Antigens are indicated in the right margin.