Early induction of humoral and cellular immune responses during experimental Mycobacterium avium subsp. paratuberculosis infection of calves

Abstract

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-gamma), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4(+) cells from infected calves proliferated, produced IFN-gamma, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of approximately 50 and approximately 60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.

FIG. 1.

Cellular immune response kinetics to M. avium PPDa. Mean (± SEM) DNA synthesis (A), IFN-γ (B), and NO (C) responses by PBMC from M. avium subsp. paratuberculosis-infected calves (closed squares) (n = 3). DNA synthesis was measured by [³H]thymidine uptake, IFN-γ was measured by ELISA, and nitrite (as an indication of NO synthesis) was measured by Griess reaction. *, Differs (P < 0.05) from response at day 0 (i.e., prechallenge response).

FIG. 2.

Cellular immune response kinetics to M. avium subsp. paratuberculosis strain 19698 WCS. Mean (± SEM) DNA synthesis (A), IFN-γ (B), and NO (C) responses by PBMC from M. avium subsp. paratuberculosis-infected calves (closed squares) (n = 3) are depicted. DNA synthesis was measured by [³H]thymidine uptake, IFN-γ was measured by ELISA, and nitrite (as an indication of NO synthesis) was measured by Griess reaction. *, Differs (P < 0.05) from response at day 0 (i.e., prechallenge response.

FIG. 3.

IFN-γ response by CD4⁺ cells to M. avium subsp. paratuberculosis strain 19698 WCS. At 313 days postchallenge, PBMC were cultured with either medium alone (A) or with 10 μg strain 19698 WCS/ml (B) for 7 days (ionomycin, PMA, and brefeldin A were added for the terminal 5 h), harvested, and analyzed by flow cytometry for CD4 expression and intracellular IFN-γ. CD4⁺ and IFN-γ⁺ are in the upper right quadrants. Panel C was stained with the isotype control antibody for IFN-γ.

FIG. 4.

Response kinetics of serum antibody specific for LAM-enriched antigen. Serum from M. avium subsp. paratuberculosis-infected animals was collected at the indicated time points relative to the initial challenge and was analyzed for reactivity to LAM by ELISA. The LAM antigen used for the assay was prepared from strain 19698 of M. avium subsp. paratuberculosis as described previously (26). This procedure involved cell disruption with a French press, protein digestion with proteinase K, and purification by centrifugation and ultrafiltration. The S/P ratios of test samples were calculated from absorbance values by using the formula (sample − negative control)/(positive sample − negative control).

FIG. 5.

Preparative immunoblots of M. avium subsp. paratuberculosis strain K10 WCS antigen probed with sera from experimentally infected cattle. Note that serum samples from the initial three bleed dates prior to challenge show no reactivity. Animal numbers are indicated in the right margin, days relative to initial challenge of sampling are indicated along the top margin, and protein size markers (in kilodaltons) are indicated in the left margin.