Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Abstract

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3+, CD4+ and γδ T-cells; whereas CD8+ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.

Figure 1

Examples of different scores of CD3+ cell infiltration in placentomes from N. caninum-inoculated animals. (a) Score 0: no infiltration of CD3⁺ cells in a placentome of a negative control cow. Counterstained with haematoxylin. Original magnification 100×. (b) Score 1: minimal infiltration of CD3⁺ cells in the maternal caruncles surrounding some necrotic foetal villi in the placentome of a N. caninum-inoculated cow culled 14 dpi. Counterstained with haematoxylin. Original magnification 100×. (c) Score 2: mild infiltration of CD3⁺ cells surrounding area of detachment of fetal trophoblast and mesenchyme of the maternal caruncle of the placentome of an inoculated cow culled 14 dpi. Counterstained with haematoxylin. Original magnification 100×. (d) Score 3: focal aggregation of CD3⁺ cells surrounding focal necrotic area in the maternal caruncle of the placentome of an inoculated cow culled 14 dpi. Counterstained with haematoxylin. Original magnification 100 × .

Figure 2

Examples of different immune cell infiltration in placentomes from N. caninum-inoculated animals. (a) Severe infiltration of macrophages in the base of maternal caruncle of a N. caninum inoculated cow culled 14 dpi. Counterstained with haematoxylin. Original magnification 100×. (b) Mild aggregate of T helper cells (CD4⁺) surrounding a blood vessel in maternal caruncle of a placentome collected from an inoculated cow culled 28 dpi. Counterstained with haematoxylin. Original magnification 200×. (c) Minimal infiltration of cytotoxic T-cells (CD8⁺) in a necrotic focus in maternal caruncle of a N. caninum challenged cow culled 28 dpi. Counterstained with haematoxylin. Original magnification 200×. (d) Mild aggregate of γδ T-cells in the base of maternal caruncle of an inoculated cow culled 14 dpi. Counterstained with haematoxylin. Original magnification 200×. (e) Rare presence of NK-cells in connective tissue in the maternal caruncle surrounding necrotic and mineralized foetal villi, observed in a placenta of an inoculated cow culled 28 dpi. Counterstained with haematoxylin. Original magnification 200×. (f) Rare presence of CD79_αcy⁺ cells aligned with endometrial epithelial cells in the maternal caruncle and in some foetal villi, observed in the placentome of a N. caninum inoculated cow culled 28 dpi. Original magnification 400×. Counterstained with haematoxylin.

Figure 3

Mean of the infiltration scores of the different phenotype of inflammatory cells on placentomes. CD68⁺ (macrophages), CD3⁺ (total T cells), CD4⁺ (T helper), CD8⁺ (cytotoxic T cells), γδTCR⁺ (γδ T cells), NKp46⁺ (NK cells) and CD79_αcy⁺ labelled cells in the placentas. Numbers in the horizontal axis represent days post inoculation (dpi). Error bars indicate standard error of the means (SEM).