Lymphocyte depletion in ileal Peyer's patch follicles in lambs infected with Eimeria ovinoidalis

Abstract

A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer's patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5'- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer's patch follicles.

FIG. 1.

Dome (d) of IPP of a lamb (no. 14) infected with E. ovinoidalis. The FAE covering the dome shows fusions with adjacent absorptive epithelium (arrowheads). Hematoxylin-eosin. Bar, 50 μm.

FIG. 2.

Additional aspects of the dome (d) of IPP of a lamb (no. 14) infected with E. ovinoidalis. Numerous macrogametes and oocysts are present in the FAE and in the dome region (arrowheads). Hematoxylin-eosin. Bar, 50 μm.

FIG. 3.

IPP of lamb no. 16 infected with E. ovinoidalis. The lymphoid follicles (f) are reduced in size and depleted of lymphocytes. Hematoxylin-eosin. Bar, 205 μm.

FIG. 4.

Mean and standard error of the mean area of the follicles of the IPP of age-matched control lambs and infected lambs, as determined using computer-assisted morphometric analysis. (a) Infected lambs (no. 1 to 8) and control lambs (no. 9 to 13) killed 2 weeks after infection. (b) Infected lambs (no. 14 to 19) and control lambs (no. 20 to 23) killed 3 weeks after infection.

FIG. 5.

5′-Nucleotidase staining of IPP. (a) Reticular staining in the ileal lymphoid follicles of a control lamb (no. 23). Bar, 440 μm. (b) Lamb no. 18 infected with E. ovinoidalis. Moderate follicle atrophy is present. The lymphoid follicles show reticular staining for 5′-nucleotidase in the peripheral zone, whereas the central zone in many follicles stains more intensely. Bar, 440 μm. (c) Coccidium-infected lamb (no. 16) with severe follicle atrophy. Even, intense staining due to condensation of stromal cells and lymphocyte depletion is present in the lymphoid follicles. Bar, 460 μm.

FIG. 6.

Carbonic anhydrase staining of the IPP. (a) Lymphoid follicles of a control lamb (no. 22). The FAE (arrows) shows reactivity along the apical and lateral plasma cell membranes. Note the granular staining of the follicle centers (F). Bar, 205 μm. (b) Coccidium-infected lamb (no. 16) with severe atrophy. The FAE (arrows) shows focal reactivity at the luminal cell membrane, whereas staining along the lateral cell membrane is absent or weak. The follicle centers (F) show diffuse, weak staining. Bar, 205 μm.

FIG. 7.

Staining with the TUNEL method for apoptotic cells. (a) IPP of a control lamb (no. 22). Numerous apoptotic cells are present in the lymphoid follicles (F). Bar, 205 μm. (b) IPP of lamb no. 16 infected with E. ovinoidalis. Few apoptotic cells are present in the follicles (F). Bar, 102 μm.

FIG. 8.

IHC analysis for leukocyte common antigen (CD45). Avidin-biotin-peroxidase complex method. Hematoxylin counterstain. (a) IPP of a control lamb (no. 23). The majority of cells in the follicles (F) are CD45 positive. Bar, 480 μm. (b) IPP of lamb no. 16 infected with E. ovinoidalis. The atrophic ileal follicles (F) contain scattered CD45-positive cells. Bar, 490 μm.

FIG. 9.

IHC analysis for B-cell marker CD21. Avidin-biotin-peroxidase complex method. Hematoxylin counterstain. (a) IPP of a control lamb (no. 20). Numerous positive cells are present in the follicles. Bar, 175 μm. (b) IPP of an infected lamb (no. 16). B cells are scarce in the lymphoid follicles. Bar, 175 μm.

FIG. 10.

IHC analysis for proliferation marker Ki-67. Avidin-biotin-peroxidase complex method. Hematoxylin counterstain. (a) IPP of a control lamb (no. 20). Intense staining of follicle lymphocytes is present. Bar, 480 μm. (b) IPP of an infected lamb (no. 16). Few cells in the follicles (F) are positive. Prominent follicle atrophy is present. Bar, 480 μm.