A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

Abstract

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

The in vitro development of Neospora caninum bradyzoites

“…Tylosin (Anti PPLO agent, GIBCO-BRL) was added to some cultures at a concentration of 60 μg ml -1 . ME49 and H7 (a BAG1 knockout [19]) strains of T. gondii were maintained by twice weekly passage in human fibroblasts as previously described [13]. In vivo cysts of N. caninum NC-Liv were purified from infected corticosteroid-treated mice as previously described [20].…”

“…Except where noted, equal amounts of protein and/or organisms (by counting of extracellular organisms in a haemocytometer) were loaded onto 10% SDS-PAGE gels, electrophoresed and transferred to nitrocellulose as previously described [13]. The amount of bradyzoite-specific antigen was ascertained by immunoblot analysis as previously described using mAb 74.1.8 [13]. Detection of bound antibody was performed using the Western Light Kit (Tropix), employing chemiluminescence with CSPD and an alkaline phosphatase-labelled secondary anti-mouse IgG antibody (1:10 000 dilution).…”

“…Organisms purified from human fibroblasts by rupture with a 27-gauge needle followed by filtration through a 3.0 μM Nucleopore filter were split into equal samples, which were then assayed for protein concentration (BioRad) and dissolved in gel sample buffer as previously described [13]. Except where noted, equal amounts of protein and/or organisms (by counting of extracellular organisms in a haemocytometer) were loaded onto 10% SDS-PAGE gels, electrophoresed and transferred to nitrocellulose as previously described [13].…”

“…Several studies have demonstrated that T. gondii bradyzoites can develop in vitro and that the development of cyst-like structures in vitro can be demonstrated by TEM [6][7][8][9][10][11] as well as by bradyzoite-specific mAbs, such as mAb 74.1.8 [12][13][14]. The T. gondii bradyzoitespecific antigen BAG1/hsp30 (also known as BAG5) was cloned using mAb 74.1.8 [15].…”

Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus

“…there are more bradyzoites under these conditions than would be expected from a simple inhibition of tachyzoite replication (Bohne et al, 1999;Weiss and Kim, 2000;Lyons et al, 2002). Temperature stress (43 °C (Soete et al, 1994)), pH stress (pH 6.8 or 8.2 (Soete et al, 1994;Weiss et al, 1995)) or chemical stress (sodium arsenite, nitric oxide, sodium nitroprusside (Bohne et al, 1993;Soete et al, 1994;Weiss et al, 1995;Soete and Dubremetz, 1996)) result in an increase in bradyzoite antigen expression and the development of cyst-like structures of T. gondii in vitro. In bone marrow derived murine macrophage lines, interferon gamma (IFN-γ) increased bradyzoite antigen expression and this appeared to be related to nitric oxide (NO) production (Bohne et al, 1993).…”