Kinetics of interferon gamma production in vivo during infection with the S48 vaccine strain of Toxoplasma gondii.

The kinetics of interferon gamma (IFN gamma) production in vivo was examined in sheep during a primary and secondary infection with the sheep vaccine strain (S48) of Toxoplasma gondii. Efferent lymph plasma from a node draining the site of inoculation was tested for anti-viral activity which could be neutralized with monoclonal antibodies against IFN gamma. Within 2 to 5 days of primary infection IFN gamma was detected in each of five sheep and persisted for 6 to 9 days. Accelerated production of IFN gamma occurred after secondary infection, the cytokine being detected in the first 24 h, and persisting in lymph for a further 4-5 days. From day 6 onwards after primary infection, efferent lymph cells produced IFN gamma when stimulated in vitro with a crude T. gondii antigen. These results show that IFN gamma is induced in sheep after infection with the S48 strain of T. gondii.

Interferon gamma inhibits the intracellular multiplication of Neospora caninum, as shown by incorporation of 3H uracil.

An assay was developed to quantify the growth of two different isolates of the protozoon Neospora caninum within ovine fibroblast cells in vitro by differential uptake of 3H uracil. The NC-1 isolate of N. caninum multiplied more quickly in culture than the NC Liverpool isolate, as reflected by increased incorporation of isotope by the former over a shorter period of time. After the parasites had left the ruptured host cells, there was very little incorporation of isotope. This suggested that multiplication occurred within and not outside the cells. Treatment of the cells with ovine recombinant interferon gamma for 24 h before infection significantly inhibited intracellular multiplication of the parasite.

Induction of CD4+ and CD8+ T cell responses in efferent lymph responding to Toxoplasma gondii infection: analysis of phenotype and function.

The kinetics of induction of T cell responses were examined in efferent lymph from a node draining the site of a primary inoculation of Toxoplasma gondii. The numbers of T cells increased after infection, due initially to an expansion of the CD4+ T cell population followed by an increase in the number of CD8+ T cells which coincided with the peak lymphoblast response. Proliferative responses of CD4+ T cells to T. gondii antigen were detectable from day six after infection and immune efferent lymph cells inhibited the intracellular multiplication of T. gondii in vitro. Optimum inhibition was achieved using CD8+ T cells restimulated in vitro, and the effector function appeared to be directed preferentially against the autologous rather than the allogeneic infected target cell. The results provide unique information on the induction of immune responses to T. gondii in vivo and provide evidence that both CD4+ and CD8+ T cells are necessary for the development of protective immunity induced by the S48 strain of T. gondii which is used as a live vaccine in sheep.

Kinetics of the local and systemic antibody response to primary and secondary infection with S48 Toxoplasma gondii in sheep.

Vaccination of sheep with live tachyzoites of Toxoplasma gondii, strain S48, affords protection against subsequent challenge with the parasite, but the mechanisms of immunity have not been fully determined. To understand better the nature of the antibody response the kinetics of both local and systemic antibody production were monitored in vaccinated sheep by means of an enzyme-linked immunosorbent assay and Western blotting. Local specific IgG production was analysed in efferent lymph obtained from the cannulated pre-femoral lymph node draining the site of infection. Antibody in efferent lymph plasma and peripheral blood serum from animals vaccinated with S48 tachyzoites was monitored and compared with IgG production in vaccinated sheep given a secondary tachyzoite challenge. Secondary challenge resulted in a clear immunological memory response, antibody being detected in the lymph 3 to 4 days after infection as compared with 7 to 8 days after a primary infection. IgG production was dominated by antibody recognizing a protein with an apparent molecular weight of 30 kDa, but other antigens (32, 24 and 11 kDa) were also readily detected.

Primary and secondary responses of the ovine lymph node to Toxoplasma gondii: cell output in efferent lymph and parasite detection.

Efferent lymphatic cannulation was used to study the dissemination of strain S48 of Toxoplasma gondii and the cell output from the prefemoral lymph node, after infection of both "naive" and vaccinated sheep. In the former the mean cell output decreased for 3 days before reaching a peak at 11 and 12 days, but in vaccinated ewes a similar drop in cell output and subsequent peak occurred significantly earlier, at 24 h and 5 days, respectively. The cellular response in both types of sheep was largely due to a marked increase in blast cells. The detection of live toxoplasms and parasite DNA by mouse inoculation and the polymerase chain reaction, respectively, gave similar results; the parasite was demonstrated in lymph from days 3 to 12 during a primary infection but with a sharp cut-off after day 9 coinciding with the peak blast cell response. Very little evidence of T. gondii was found in lymph of vaccinated sheep after challenge. Immunity, which is thought to be largely T-cell mediated and is sustained without continuous antigenic stimulation, suppresses dissemination of the parasite in the lymph and therefore to other sites, which might include the gravid uterus.

The inhibitory effect of ovine recombinant interferon-gamma on intracellular replication of Toxoplasma gondii.

A model for the in vitro infection of ovine cells with Toxoplasma gondii tachyzoites has been developed and used to investigate the effect of treatment with ovine recombinant interferon-gamma (ov.rIFN gamma) on parasite replication. Treatment of both alveolar macrophages and fibroblast cells either 24 h pre-infection or 2 h post-infection with ov.rIFN gamma inhibited replication of T. gondii and was quantified by suppression of 3H uracil uptake by the parasite. Replication of T. gondii in the fibroblast cells was significantly inhibited by treatment with 200-300 U/ml ov.rIFN gamma, whereas concentrations as low as 1 U/ml suppressed parasite replication in the alveolar macrophages.