The protozoan parasite Neospora caninum causes fetal death after experimental infection of pregnant cattle in early gestation, but the fetus survives a similar infection in late gestation. An increase in Th1-type cytokines in the placenta in response to the presence of the parasite has been implicated as a contributory factor to fetal death due to immune-mediated pathological alterations. We measured, using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, the levels of cytokines in the placentas of cattle experimentally infected with N. caninum in early and late gestation. After infection in early gestation, fetal death occurred, and the levels of mRNA of both Th1 and Th2 cytokines, including interleukin-2 (IL-2), gamma interferon (IFN-␥), IL-12p40, tumor necrosis factor alpha (TNF-␣), IL-18, IL-10, and IL-4, were significantly (P < 0.01) increased by up to 1,000-fold. There was extensive placental necrosis and a corresponding infiltration of CD4 ؉ T cells and macrophages. IFN-␥ protein expression was also highly increased, and a modest increase in transforming growth factor ␤ was detected. A much smaller increase in the same cytokines and IFN-␥ protein expression, with minimal placental necrosis and inflammatory infiltration, occurred after N. caninum infection in late gestation when the fetuses survived. Comparison of cytokine mRNA levels in separated maternal and fetal placental tissue that showed maternal tissue was the major source of all cytokine mRNA except for IL-10 and TNF-␣, which were similar in both maternal and fetal tissues. These results suggest that the magnitude of the cytokine response correlates with but is not necessarily the cause of fetal death and demonstrate that a polarized Th1 response was not evident in the placentas of N. caninum-infected cattle.

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Experimental infection of cattle with Neospora caninum in early gestation causes foetal death, but the foetus survives infection in late gestation. An immunological mechanism of abortion has been suggested; therefore changes in the maternal immune response during pregnancy could account for these differences. We have investigated the peripheral immune responses of pregnant cattle following an intravenous inoculation with 10(7) N. caninum tachyzoites in early and late gestation. Percentages of CD2+ and CD4+ T-cells in peripheral blood mononuclear cells (PBMC) increased 1-2 weeks after infection in both early (day 70) and late (day 210) gestation, and percentages of CD8+ T-cells increased 1-2 weeks after infection at day 70. Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression in PBMC increased 1-2 weeks after infection at day 210 and IL-4 increased 1-2 weeks after infection at day 70. Immunomagnetic isolation of CD4+ cells from PBMC showed that they were a major source of IL-4 and IFN-gamma, and expression of both cytokines increased in CD4+ cells after infection in early and late gestation. These results suggest that CD4+ cells proliferate and express IL-4 and IFN-gamma in response to N. caninum irrespective of the stage of gestation when infection occurs.

Neospora caninum is an intracellular protozoan parasite which is a major cause of abortion in cattle worldwide. It forms persistent infections which recrudesce during pregnancy leading to foetal infection and in a proportion of cases, abortion. The mechanisms underlying abortion are not understood. In this study, recrudescence of a persistent infection in eight naturally infected cows occurred between 20 and 33 weeks of gestation. Animals were killed at the time of recrudescence and parasites were detected in the placentae and foetuses. An active maternal immune response consisting of an infiltration of CD4+ and CD8+ T cells and a 46–49 fold increase in interferon-γ and interleukin-4 mRNA was detected. Other cytokines, notably interleukin-12 p40, interleukin-10 and tumour necrosis factor-α were also significantly increased and Major Histocompatibility Class II antigen was expressed on maternal and foetal epithelial and stromal fibroblastoid cells. Significantly, despite the presence of an active maternal immune response in the placenta, all the foetuses were alive at the time of maternal euthanasia. There was evidence of parasites within foetal tissues; their distribution was restricted to the central nervous system and skeletal muscle and their presence was associated with tissue necrosis and a non-suppurative inflammatory response involving lymphocytes and macrophages, irrespective of the gestational age of the foetus. Whilst an active maternal immune response to a pathogen in the placenta is generally considered to be damaging to the foetal trophoblast, our findings suggest that the presence of a parasite-induced maternal immune response in the placenta is not detrimental to foetal survival but may contribute to the control of placental parasitosis.