Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture.

Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.

Cytologic detection of Toxoplasma gondii in the cerebrospinal fluid of a dog and in vitro isolation of a unique mouse-virulent recombinant strain.

Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.

In contrast to Toxoplasma gondii, Neospora caninum tachyzoites did not sustain multiplication in vitro at increased incubation temperatures.

Neospora caninum and Toxoplasma gondii are coccidian parasites that infect a wide range of mammalian and avian species. While viable T. gondii has been in vitro isolated in natural infections from wild and domestic birds, attempts to isolate N. caninum from naturally-infected birds were unsuccessful. We speculate that body temperatures of birds, which are usually higher than those of mammals, may impair the multiplication of N. caninum. In contrast to N. caninum, T. gondii can grow in vitro at temperatures higher than 37°C. To test the hypothesis that N. caninum tachyzoites are impaired to grow in vitro at high temperatures, three strains of N. caninum (NC-1, NC-Liverpool, and NC-Bahia) and three of T. gondii (RH, ME-49 and NED) were cultivated at gradually increasing temperatures starting at 37°C up to 41.5°C. A permanent chicken cell line was chosen for the study. Parasites were observed microscopically and their presence in culture was evaluated by species-specific conventional PCRs. In a second experiment, growth rates of T. gondii (RH strain) and N. caninum (NC-1 strain) were evaluated after direct passage of tachyzoites from 37°C to 41.5°C, and quantified by real-time PCR. In addition to comparisons between N. caninum and T. gondii, growth rates of three T. gondii strains were compared at high temperatures. Neospora caninum tachyzoites could not sustain multiplication at temperatures between 39°C and 41.5°C. Toxoplasma gondii tachyzoites continued to multiply at the same experimental conditions. Direct passage of N. caninum tachyzoites from 37°C to 41.5°C caused a significant decrease in the number of parasites during 96h of observation, while T. gondii had a significant increase in the number of stages after the same period of time. T. gondii RH strain (clonal type I) presented a different growth rate at 41.5°C when compared with type II and type III strains. In conclusion, multiplication of N. caninum tachyzoites in vitro was inhibited at temperatures similar to those of chickens, what may be one of the reasons that isolation of the parasite is difficult in avian species. In contrast to N. caninum, T. gondii continued to grow at 41.5°C.