Long-term investigations on Toxoplasma gondii-infected primary chicken macrophages

Abstract

Toxoplasma (T.) gondii is known to infect various cell types including macrophages. In the present study, we generated monocyte-derived macrophage cultures from chicken blood. By flow cytometrical analysis, 84.5% of the cultivated cells showed typical macrophage properties. Macrophage cultures were cultivated at either 37 °C or 40 °C, respectively, and were infected 72 to 96 h post isolationem with tachyzoites of the T. gondii type II strain ME49 at a rate of 7.5 tachyzoites per host cell. Light microscopical investigations revealed incorporation of tachyzoites into the macrophages and gradual destruction of the infected macrophage culture. Parasite multiplication was observed by a quantitative real time PCR (qPCR) based on the 529-bp fragment specific for T. gondii. Samples were drawn 1 h post infectionem (p.i.), as well as 12, 24, 36, 48, and 72 h p.i. The parasite replication curve showed a transient decrease of parasite stages 12 h p.i. followed by a tachyzoite multiplication. The comparison of different culture conditions showed a significantly higher replication rate of T. gondii at 37 °C (median value 48 h p.i., 289.2% of the initial tachyzoite number) compared to cultures incubated at 40 °C (median value 48 h p.i., 73.1% of the initial tachyzoite number) throughout the observation period (P < 0.05). In general, replication rates were significantly lower than in a standard VERO cell cultures at 37 °C (P < 0.05). The observed differences were attributed to the physiological chicken macrophage reaction at 40 °C probably approximating the situation in vivo.