Experimental infection model for Johne's disease in sheep

Abstract

Johne's disease in ruminants results in chronic enteritis caused by the pathogenic bacterium Mycobacterium avium subsp. paratuberculosis. This study examined two M. avium subsp. paratuberculosis strains (JD3 and W), using different doses and routes of infection, to establish the optimal time postchallenge when predictable levels of infection, gut lesions, and clinical disease occur in a large proportion of sheep. While a small proportion (25%) of sheep challenged with a low-passage-number laboratory culture of M. avium subsp. paratuberculosis (strain W) became infected, no infection was found in animals exposed to a high-passage-number culture isolate of strain W. In contrast, a primary tissue homogenate of M. avium subsp. paratuberculosis (JD3) resulted in high (90%) infection rates and gut histopathology following oral or intratonsillar challenge. The optimal conditions necessary to produce Johne's disease involve oral inoculation of 3-month-old lambs with four doses of 5 x 10(8) CFU of M. avium subsp. paratuberculosis isolated directly from the gut lymphatic tissues of clinically affected sheep. This resulted in consistent gut histopathology at 9 months and the onset of clinical disease by 11 months postchallenge.

FIG. 1.

Immune response profiles of unchallenged controls and sheep challenged orally with a tissue homogenate of M. avium subsp. paratuberculosis (JD3). (A) Mean lymphocyte transformation response following PPDj stimulation of peripheral blood mononuclear cells (PBMC). (B) IFN-γ production in PPDj-stimulated blood. (C) PPDj-specific antibody levels, as measured by ELISA. Mean data ± standard errors of the means (SEM) are shown (n = 30 challenged and 10 unchallenged animals).

FIG. 2.

Cull rates and immune profiles of sheep challenged orally with M. avium subsp. paratuberculosis (JD3). (A) Elective slaughter rates of challenged sheep due to weight loss starting at 10 months postchallenge. (B) PBMC lymphocyte transformation levels after PPDj stimulation in cells from animals electively slaughtered due to clinical disease at different times after challenge (PC). (C) IFN-γ levels in blood after stimulation with PPDj for animals electively slaughtered due to clinical disease at different times postchallenge (PC). (D) Specific antibody levels to PPDj in electively slaughtered animals. Squares represent animals that developed disease early (at 11 to 12 months postchallenge [PC]). Triangles represent animals that developed disease later (at 14 to 18 months postchallenge [PC]). Mean responses ± SEM are shown for each group (n = 6).

FIG. 3.

Immune profiles for different tissues from clinically diseased or unchallenged animals following necropsy. (A) Lymphocyte proliferation following PPDj stimulation of isolated cells. (B) IFN-γ production following PPDj stimulation of isolated cells. (C) Proportions of CD4⁺ cells from different tissues, as detected by FACS analysis. (D) Proportions of CD8⁺ cells. Measurements are expressed as mean counts or percentages (n = 9 diseased and 3 unchallenged animals) plus the SEM.

FIG. 4.

Immune profiles of animals after experimental challenge via the tonsil with the JD3 tissue homogenate (JD3-IT), represented by closed squares. Animals challenged orally with the tissue homogenate JD3 (JD3-O) are represented by closed triangles. Animals challenged orally with the W strain (W-O) are represented by crosses, and unchallenged controls are represented by open circles. (A) Mean lymphoproliferative responses of PBMC to PPDj. (B) IFN-γ responses in whole blood after stimulation with PPDj. (C) Plasma antibody levels specific to PPDj, as measured by ELISA. Mean responses ± SEM are shown (n = 12).