C. S. Guy

Neospora caninum -associated abortion in cattle: the time of experimentally-induced parasitaemia during gestation determines foetal survival

The parasite, Neospora caninum is an important cause of abortion in cattle. It is transmitted vertically or horizontally and infection may result in abortion or the birth of a live, healthy but infected calf at full-term. Only a proportion of infected cattle abort and the pathogenesis of abortion is not understood. Groups of cattle were infected with 10(7) N. caninum tachyzoites intravenously at different times relative to gestation. Intravenous inoculation was chosen to reproduce the putative haematogenous spread of N. caninum following either recrudescence of endogenous infection or de novo infection. In all cattle, infection was accompanied by high gamma-interferon and lymphoproliferative responses, and a biased IgG2 response indicating that N. caninum infection is accompanied by a profound Th1 helper T cell-like response. Infection at 10 weeks gestation resulted in foetopathy and resorption of foetal tissues 3 weeks after infection in 5 out of 6 cows. Infection at 30 weeks gestation resulted in the birth of asymptomatic, congenitally-infected calves at full term in all 6 cows, whereas the 6 cows infected before artificial insemination gave birth to live, uninfected calves. These results suggest that the reason some cows abort is related to the time during gestation when they become infected or an existing infection recrudesces.

Neospora caninum in persistently infected, pregnant cows: spontaneous transplacental infection is associated with an acute increase in maternal antibody

Nine cows which were naturally and persistently infected with Neospora caninum were housed and observed intensively throughout pregnancy. No recrudescence of a latent infection was detected by PCR tests on maternal blood but fetal infection, implying a recrudescence of maternal parasitosis, was associated with a marked increase in maternal antibody. The increase occurred in the second half of pregnancy in five cows which infected their calves, and before mid-pregnancy in one cow which aborted. There was no change in the avidity of the antibody, which remained high and characteristic of long-term infection. In three infected cows that gave birth to uninfected calves there was no marked increase in maternal antibody. Antigen-specific interferon gamma responses of peripheral blood mononuclear cells were observed in all the infected cattle but they did not vary significantly either during pregnancy, or whether the cows did or did not infect their calves, although the responses were consistently higher in the latter. There was no change in the plasma concentrations of cortisol or acute phase proteins associated with the recrudescence of the parasite. Three uninfected cows housed with the infected cows remained uninfected throughout the experiment. No immunosuppressive event was detected which might have provoked parasite recrudescence but the acute antibody rise associated with transplacental infection provides a valuable, non-invasive marker for further studies to investigate the cause and consequences of parasite recrudescence in N caninum infection in cattle.

Immunization of Cattle with Live Tachyzoites of Neospora caninum Confers Protection against Fetal Death

ABSTRACT Neospora caninum is an obligate intracellular protozoan parasite that causes abortion in cattle. It is normally found as a latent infection controlled by a T-helper-cell type 1 response involving CD4+ cytotoxic T cells and gamma interferon. Cattle may be infected by two different routes: transplacentally as a result of activation of the latent infection in the mother causing congenital infection or abortion and by ingestion of oocysts, which, if it occurs during gestation, can also result in abortion. Here, for the first time, we establish proof that live vaccination protects against fetal death, whereas immunization using whole-tachyzoite lysate in different adjuvants fails to protect against fetal death. Strong antibody responses were induced in all the vaccinated groups, and the quality and magnitude of these responses were similar in the live- and the lysate-vaccinated groups. In contrast, only the group immunized with live tachyzoites had strong cellular and gamma interferon responses prior to challenge, and these responses correlated with protection against fetopathy. These results suggest that a cellular immune response may be important in the mechanisms involved in protection against N. caninum-associated abortions.

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection.

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.

Neospora caninum: oocyst challenge of pregnant cows.

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.

Upregulation of Cytokines Is Detected in the Placentas of Cattle Infected with Neospora caninum and Is More Marked Early in Gestation When Fetal Death Is Observed

ABSTRACT 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-γ), IL-12p40, tumor necrosis factor alpha (TNF-α), 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-γ protein expression was also highly increased, and a modest increase in transforming growth factor β was detected. A much smaller increase in the same cytokines and IFN-γ 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-α, 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.

Peripheral immune responses in pregnant cattle following Neospora caninum infection

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 107 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‐γ (IFN‐γ) 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‐γ, 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‐γ in response to N. caninum irrespective of the stage of gestation when infection occurs.