Early dynamics of Toxoplasma gondii infection in sheep inoculated at mid-gestation with archetypal type II oocysts

Abstract

Early abortion is a clinical presentation of ovine toxoplasmosis that occurs in the second week post-infection (pi), which is characterised by placental infarcts, foetal leukomalacia and absence of parasites in the placenta and foetal tissues. The pathogenic mechanism of early abortion is unknown, and descriptions of the early dynamics of T. gondii infection in pregnant sheep are scarce. The aim of this study was to investigate the presence of lesions and parasite DNA in the small intestine, mesenteric lymph nodes and placenta/foetus, that could be key during the first week after oral infection in sheep at mid-pregnancy. In the small intestine, lesions were rare and parasite DNA detection rates were low (3-8%), with the highest parasite DNA detection and burden found on day 6 pi in the Peyer's patches of the medial jejunum. In the mesenteric lymph nodes, adenomegaly and microscopic lesions were mainly observed on day 6 pi. Parasite DNA was detected in 11% and 61.2% of the samples from mesenteric lymph nodes on days 3 and 6 pi, respectively, with higher parasite DNA detection rates and burdens in the medial and distal jejunal lymph nodes on day 6 pi. In the placentomes, on day 6 pi, gross lesions were not observed, although significant histological changes, such as endothelial activation and vascular thrombosis, were found in 18.6% and 8.3% of the placentomes, respectively. These findings lay the groundwork for future research aimed at elucidating the precise mechanisms underlying early abortions following T. gondii infection in pregnant sheep.

Keywords: Toxoplasma gondii; early abortion; early infection dynamics; mesenteric lymph nodes; mid-gestation; placental thrombosis; sheep; small intestine.

Figure 1

Overview of the experimental design. dpi: days post-infection.

Figure 2

Rectal temperatures of T. gondii-infected and uninfected sheep. Each point represents the mean ± S.D. for each group. Rectal temperatures were analysed using two-way repeated-measures ANOVA. For significant differences, (***) indicates P < 0.001 and (****) indicates P < 0.0001.

Figure 3

Anti-T. gondii IgG levels in the dam’s sera. IgG levels are expressed as the relative index percent (RIPC). Each point represents the mean ± S.D. at the different sampling times for each group. The horizontal dashed line in B indicates the cut-off (RIPC ≥ 32.21) for the validated TgSALUVET ELISA 2.0 [35]. Toxoplasma gondii-specific IgG levels in the dams were analysed using two-way repeated-measures ANOVA. For significant differences between the infected and uninfected groups, (****) indicates P < 0.0001.

Figure 4

Macroscopic and microscopic findings in mesenteric lymph nodes. A Adenomegaly of mesenteric lymph nodes (arrow) of sheep 2.1 euthanized on day 6 pi (B) Secondary lymphoid follicles at the cortex of a jejunal mesenteric lymph node in sheep 1.3 (infected/euthanized on day 3 pi). H/E, magnification: 40x. C Expansion of the medullary sinuses due to increased accumulation of macrophages (sinus histiocytosis) in a jejunal mesenteric lymph node in sheep 2.1 (infected/euthanized on day 6 pi). H/E, magnification: 12x. D Granuloma at the cortex of a jejunal mesenteric lymph node (arrowheads) in sheep 3.3 (infected/euthanized on day 28 pi). H/E, magnification: 100x.

Figure 5

Dot-plot graphs of the T. gondii burden in mesenteric lymph nodes from sheep in G2 (infected/euthanized on day 6 pi). Each dot represents an individual value of the estimated parasite burden (number of parasites per milligram of host tissue), and the medians are represented as horizontal lines. Considering that the T. gondii detection limit by real-time PCR is 0.1 parasites, negative samples (0 parasites) were represented on the log scale as < 0.1 (i.e., 10.⁻²). Parasite burdens were analysed via the nonparametric Kruskal‒Wallis test followed by Dunn’s test for comparisons between different lymph nodes, as well as the Mann‒Whitney test for pairwise comparisons. For significant differences between infected groups in each tissue, (*) indicates P < 0.05, (**) indicates P < 0.01, (***) indicates P < 0.001 and (****) indicates P < 0.0001.

Figure 6

Histological lesions in the placentomes. A Hyaline degeneration of a vessel wall in a maternal villus at the placenta denoted by a deposit of homogeneous, eosinophilic material in the wall (arrowheads). Sheep 1.2 (infected/euthanized on day 3 pi). H/E. Magnification: 200x. B Marked extravasation of eosinophilic fibrin, which had undergone polymerisation, forming dense fibrillar deposits within the surrounding stroma (arrowheads). Sheep 2.2 (infected/euthanized on day 6 pi). H/E. Magnification: 200x. C Caruncular vessel within the interdigitated area of the placentome completely occluded by a thrombus (arrowheads). Note the hyaline degeneration of the vascular wall at the affected vessel. Sheep 2.2 (infected/euthanized on day 6 pi). H/E. Magnification: 400x.

Figure 7

Histological lesions in the foetal brains. A Areas of leukomalacia (arrowheads) in the white matter of a foetal brain from an early abortion in G3. B Focus of glial cells (arrowheads) in the brain of a live foetus on day 28 pi in G3.