Toxoplasma gondii is an apicomplexan protozoan parasite that can infect numerous warm-blooded animals including humans, other mammals, and birds (Dubey, 2010). Although it can infect a wide variety of host species, only members of the family Felidae can shed oocysts following sexual replication. The parasite is one of the leading causes of foodborne illness in the United States, and T. gondii infection is considered one of the five neglected parasitic infections by the Centers for Disease Control and Prevention (CDC, USA) due to insufficient research funding being available (https://www.cdc.gov/parasites/npi/npi.html). Infection can occur through a multitude of routes including ingestion of sporulated oocysts in water or food that is contaminated by feces of infected and shedding cats, or undercooked meat containing bradyzoites, the slow-replicating forms of the parasite (Benenson et al., 1982).

Toxoplasma gondii can readily grow in cell cultures (Chang and Gabrielson, 1984). This is a major advantage, enabling e.g. production of antigens for diagnostics, study of the biology of these organisms using cutting-edge methodologies, and cryopreservation of strains. For epidemiology, genetics, and population studies, it is often useful to isolate T. gondii from infected hosts. Bioassay, using cats and mice, has been the standard for isolation of T. gondii from infected hosts for the past several decades (Garcia et al., 2006, Dubey, 2010), and has been an essential tool in studying the biology of T. gondii in general. Both cats and mice are natural hosts and susceptible to T. gondii infection, and can clear bacterial and fungal contamination in source animal tissues. Due to high sensitivity to bradyzoite infection and the ability to ingest a large number of animal tissues, bioassay using cats has been the gold standard for isolation of T. gondii strains. However, due to the high cost, strict biosafety requirements to handle oocysts shed in the feces of infected cats, and ethical considerations and limitations of using cats as laboratory animals, isolation of T. gondii by bioassay in cats has been used in a limited number of research laboratories. For these reasons, mice have been the standard choice for bioassay. The key advantages of mouse bioassay are lower cost and lower biosafety requirements since the parasites are not shed by infected mice. Most T. gondii isolates reported in the currently available literature have been isolated using bioassay in mice. However, successful application of cell culture-based methods to isolate T. gondii from biological samples has been reported in the literature, but not widely applied. In this article, we discuss isolation of T. gondii in cell culture, describe a set of experiments we carried out, and provide future perspectives.