INTRODUCTION
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis. T. gondii is one of the most successful protozoan parasites because it has a very broad host range, infecting all warm-blooded animals, including humans. The most important clinical presentations of toxoplasmosis are congenital toxoplasmosis, which leads to birth defects in unborn children, as well as reactivation, which causes fatal toxoplasmic encephalitis in immunologically impaired individuals (Montoya and Liesenfeld, 2004).
Currently, various cell cultures and T. gondii strains are used as a source of fresh tachyzoites in all aspects of research, from the development of diagnostic tests to the evaluation of new therapeutics. Initially, research has focused on the continuous production of abundant pure tachyzoites in cell culture. Buckley has achieved continuous production in a Vero cell line (Buckley, 1973), Braveny, Winter and Disko (1978) have accomplished abundant production in a Hep-2 cell line, and Grimwood et al. (1979) have purified cell culture-derived tachyzoites. Hughes, Hudson and Fleck (1986) have produced tachyzoites in African Green Monkey Kidney cells for use in the Dye test. Mavin et al. (2003) maximized the efficient retrieval of T. gondii tachyzoites from frozen stocks using chilled reagents and fresh parasites, a procedure that improved the retrieval rate of tachyzoites from 36% to 100% and which was available for diagnostic and experimental usage. Hermentin and Aspock (1987) developed a method for abundant production and purification of tachyzoites. In 1993, a new purification method, which has only 0·17% host cell debris, was developed (Domzig, Seguela and Binz, 1993). Roos et al. (1994) established a cell culture method for continuous production T. gondii tachyzoites in human foreskin fibroblast (HFF) cells and Chatteron et al. (2002) optimized the cell culture conditions for continuous production of HeLa cells. In another study, serum-free cell culture medium was considered a good alternative to grow T. gondii tachyzoites (De Meerschman et al. 2002). In recent years, the research emphasis has shifted to optimization of diagnostic assays of the virulent T. gondii RH strain tachyzoites grown in cell culture, Obwaller et al. (1995) used serum-free cell culture-derived tachyzoites in enzyme-linked immunosorbent assay, Hassl and Aspock (1990) used cell culture-derived tachyzoites in an indirect immunofluorescent antibody test, and a research group from Scotland concluded that HeLa cell culture is most convenient for the continuous production of fresh tachyzoites for the Dye test (Evans et al. 1999; Ashburn et al. 2000).
The Toxoplasma Diagnosis Laboratory at Ege University Medical Faculty in Turkey uses animal-derived T. gondii RH Ankara strain tachyzoites as a source of fresh tachyzoites for diagnostic assays. The present study examines the properties of T. gondii RH Ankara strain tachyzoites during continuous production in myeloma X63.Ag8.653, HeLa, Hep-2, and Vero cell lines. In addition, a preliminary study of the virulence of cell-cultured tachyzoites in outbred BALB/c mice has been performed to determine whether size affects any properties of tachyzoites. The ultimate objective is to determine whether the cell culture-derived tachyzoites are of sufficient quality to allow cessation of tachyzoite production in animals as a source for diagnostic assays.
MATERIALS AND METHODS
Stock cell cultures
Myeloma X.63 Ag8658 cells were kindly obtained from the Naval Medical Research Institute, Bethesda, Maryland by Mucide AK. HeLa, Hep-2 and Vero cells were obtained from the Departments of Parasitology and Microbiology, Ege University. The stock cell cultures were prepared using 1× Minimum Essential Medium Eagle with Earle's Balanced Salt with 25 mM Hepes solution (EMEM/Hepes), and foetal calf serum (FCS) (Biological Industries) in 25 cm2 flasks (Miles Laboratories) at 37 °C in a 5% CO2 atmosphere. The cells were initially grown in growth medium (EMEM/Hepes containing 10% FCS) and then transferred to maintenance medium (EMEM/Hepes containing 2% FCS) when a confluent monolayer was obtained. Myeloma cells were removed from the flask by gentle tapping and subcultured. Confluent HeLa, Hep-2 and Vero cells were washed with 1× Hank's balanced salt solution (HBSS) (Cellgro) and then trypsinized with 0·5 ml 25% trypsin (Cellgro) for 5 min at 37 °C to release the cells from the bottom of the flasks. Subsequently, 4·5 ml of 1× EMEM/Hepes were added to the trypsinized cells in 25 cm2 flasks. Appropriate numbers of cells were subcultured every 3–4 days into new flasks with growth medium and then incubated at 37 °C in a 5% CO2 atmosphere to form the confluent monolayer.
Continuous Toxoplasma gondii culture
Confluent myeloma X63.Ag8.653, HeLa, Hep-2 and Vero cells were inoculated with fresh T. gondii RH Ankara strain tachyzoites, which were obtained from the peritoneal exudates of previously infected BALB/c mice. T. gondii RH Ankara strain tachyzoites were diluted to 1×106/ml with maintenance medium.
After the confluent monolayer for each cell culture type had formed, the medium was aspirated and confluent cells were washed with 1× HBSS. Subsequently, 25 cm2 flasks were inoculated with 1×106 ml fresh tachyzoites in 1 ml of maintenance medium and incubated for 30 min at 37 °C in a 5% CO2 atmosphere. Then, 4 ml of maintenance medium were added to the 25 cm2 flasks and incubated at 37 °C in a 5% CO2 atmosphere for 120–156 h for myeloma cells and 72–96 h for the HeLa, Hep-2 and Vero cells. When the cultures reached an estimated concentration of 1×106/ml tachyzoites or greater, the flasks were sharply tapped from the sides to release the cells. The amount and viability of tachyzoites harvested from each cell culture type in the 5 ml of decanted supernatant were determined with a haemocytometer under light microscopy (×40 magnification) after staining with 0·1% Eosin. Harvests containing less than 1×106/ml viable tachyzoites were concentrated by centrifugation at 500 g for several min to eliminate the cell culture debris and then again at 3000 g for 10 min to concentrate the tachyzoites. Subsequently, tachyzoites harvested from 4 different cell culture supernatants were diluted to 1×106/ml with maintenance medium. Then, freshly grown monolayers of myeloma X.63 Ag8658, HeLa, HEp-2 and Vero cells in 25 cm2 flasks were inoculated with 1×106 tachyzoites in 1 ml of maintenance medium. Continuous production of T. gondii RH Ankara strain in 4 different cell culture types was performed for approximately 2 months. During 2 months of continuous production of T. gondii RH Ankara strain tachyzoites 2 separate 25 cm2 flasks were inoculated for passaging of each cell culture. In addition, during this time 16–18 passages were achieved for HeLa, HEp-2 and Vero cell cultures and 10 passages were performed for myeloma cell cultures.
Virulence assay
Outbred BALB/c male mice, 6–10 weeks of age, were used in the virulence assay. Four groups (3 mice in each group) of mice were inoculated once per week intraperitoneally with lethal doses of fresh T. gondii RH Ankara strain tachyzoites obtained from 4 different cell cultures and a control group was inoculated intraperitonealy with lethal doses of fresh T. gondii RH Ankara strain tachyzoites obtained from continuous passaging in mice. The mice were observed for 30 days.
Statistical calculations
Where appropriate, T. gondii yield data were analysed by the Chi-square test with Yates' correction. The differences in width and height size of T. gondii Ankara strain tachyzoites during continuous production were analysed by Kruskal Wallis test (KW).
RESULTS
Tachyzoite yield from continuous cell culture
T. gondii RH Ankara strain tachyzoites were continuously produced in myeloma, HeLa, Hep-2 and Vero cells cultures for 2 months. During the 2 months, the amount of tachyzoites, observed in each harvest slightly decreased compared to the plentiful yields harvested at the onset of continuous production, with the exception of tachyzoites derived from the myeloma cell culture. Initially, and in every subculture, all cell cultures in 25 cm2 flasks were inoculated with 1×106 viable tachyzoites diluted in 1 ml of maintenance medium. During the continuous production, average viable tachyzoite yields harvested from myeloma, Vero, Hep-2, and HeLa cell cultures were 5·1×106/ml, 0·75×106/ml, 1·16×106/ml, and 1·33×106/ml, respectively. Although there was a significant difference between the average tachyzoite yields harvested from each cell culture supernatant (P<0·1), there were no significant differences between the average tachyzoite yields harvested from Vero, Hep-2, and HeLa cell cultures. However, the average tachyzoite yield harvested from myeloma cell culture was significantly greater than the tachyzoites harvested from Vero, Hep-2, and HeLa cell cultures (P<0·1).
Briefly, 3 time-points were chosen to reveal the difference of viable tachyzoite yields harvested from 5 ml of supernatants during the continuous production. At the second week of continuous production, there were no significant differences between the tachyzoite yields obtained from myeloma, Vero, Hep-2, and HeLa cell cultures which were 2·5×106/ml, 1·25×106/ml, 2×106/ml, and 2×106/ml, respectively. After 4 weeks of continuous production, there were no significant differences between the tachyzoite yields harvested from Vero, Hep-2, and HeLa cell cultures which were 0·5×106/ml, 1×106/ml, 1×106/ml, respectively. However, the tachyzoite yield harvested from myeloma cell culture was 5×106/ml and significantly higher than others (P<0·1). At the end of the second month, the tachyzoite yield harvested from myeloma cell culture was 8×106/ml and significantly greater than the tachyzoite yields harvested from Vero, Hep-2, and HeLa cell cultures which were 0·5×106/ml, 0·5×106/ml, and 1×106/ml, respectively (P<0·001).
Size change of tachyzoites during continuous production in cell culture
To determine the change in shape and size, T. gondii RH Ankara strain tachyzoites were harvested from the 4 different cell cultures and a minimum of 10 tachyzoites from each cell culture were examined with immersion microscopy after staining with Giemsa dye. The average size of tachyzoites obtained from the peritoneal space of mice, which was used for the initial inoculation of each cell culture, was determined to be 3×5·7 μm (SDwidth: 0·22, SDheight: 0·34). During the continuous production, the size of tachyzoites harvested from each cell culture gradually decreased (Table 1). The average size of the tachyzoites derived from the second myeloma subculture after the second week was 2·8×5·7 μm (SDwidth: 0·26, SDheight: 0·27). The size of the tachyzoites obtained from the fifth myeloma cell subculture was 1×2·3 μm (SDwidth: 0·18, SDheight: 0·25) at the end of first month. Thereafter, the average size of tachyzoites slightly decreased to 1×2·1 μm (SDwidth: 0·16, SDheight: 0·25) that were obtained from the ninth myeloma cell subculture at the end of 2 months. During the continuous production, the decrease in the size of tachyzoites obtained from the myeloma cell culture was significant (KWwidth: 29·8, P<0·001; KWheight: 29·7, P<0·001, D.F.: 3).
The average size of tachyzoites obtained from third and seventh Vero cell subcultures decreased to 2×4·8 μm (SDwidth: 0·32, SDheight: 0·41) and 1·9×3·8 μm (SDwidth: 0·25, SDheight: 0·4) at the end of 2 weeks and 1 month respectively. At the end of the second month, the average size of tachyzoites obtained from the fourteenth Vero cell subculture slightly decreased to 1·8×3·5 μm (SDwidth: 0·29, SDheight: 0·23). As a result, the size of continuously produced tachyzoites harvested from the Vero cell culture significantly decreased (KWwidth: 23·3, P<0·001; KWheight: 32·04, P<0·001, D.F.: 3). A similar decrease in size was observed in tachyzoites harvested from Hep-2 cell culture. However, the average size of tachyzoites obtained from Hep-2 cell culture decreased faster than the other cell culture-derived tachyzoites. The average size of tachyzoites obtained from the fourth subculture was 1·6×2·6 μm (SDwidth: 0·2, SDheight: 0·26) at the end of the first 2 weeks. The average tachyzoite size obtained from the seventh subculture was 1·3×1·9 μm (SDwidth: 0·29, SDheight: 0·25) at the end of first month and tachyzoite size obtained from the seventeenth subculture was 1·2×1·7 μm (SDwidth: 0·26, SDheight: 0·26) at the end of two months. Overall, the decrease in size of the continuously produced tachyzoites obtained from Hep-2 cell culture was significant (KWwidth: 26·8, P<0·001; KWheight: 32·7, P<0·001, D.F.: 3).
Although the tachyzoite yield obtained from the HeLa cell culture was also low compared to myeloma cell culture tachyzoite yields, the average size of the tachyzoites was larger than the tachyzoite size observed in the other cell culture types. The average size of the tachyzoites obtained from the third HeLa cell subculture decreased rapidly to 2×3·9 μm (SDwidth: 0·24, SDheight: 0·32) at the end of the first 2 weeks, the average tachyzoite size obtained from HeLa subcultures did not change before the end of continuous production which was 2×3·8 μm (SDwidth: 0·14, SDheight: 0·2). However, the size of the tachyzoites was also significantly less than the initial size of the tachyzoites obtained from mouse peritoneum (KWwidth: 21·8, P<0·001; KWheight: 22·4, P<0·001, D.F.: 3).
Preliminary virulence assay in outbred BALB/c mice
In the control group, 1×105 T. gondii RH Ankara strain tachyzoites caused death in mice in 4–6 days after inoculation. During the virulence assay, mice in each group were infected with lethal doses of viable fresh tachyzoites. Preliminary virulence assays demonstrated that, tachyzoites obtained from myeloma cell cultures did not cause death in any of the mice after the second week. Virulence of the HeLa cell culture-derived tachyzoites ceased after 4 weeks. Tachyzoites harvested from Vero cell culture did not kill mice after the fifth week. Tachyzoites obtained from the Hep-2 cell culture became virulent after 7 weeks.
DISCUSSION
T. gondii tachyzoites can be continuously produced in vitro or in vivo (Grimwood et al. 1979; Hughes et al. 1986; Sibley and Howe, 1996; Dubey et al. 1999). Currently, serial passaging in the peritoneal cavity of mice is the method of choice for harvesting T. gondii tachyzoites in most laboratories (Evans et al. 1999). However, continuous production of tachyzoites in a cell culture system is attractive because this approach eliminates the need to sacrifice animals for scientific purposes. Recent research has focused on alternative in vitro methods, such as the continuous production of T. gondii tachyzoites in cell culture systems (Evans et al. 1999; Chatterton et al. 2002). Cell culture-derived tachyzoites have been used successfully in the Dye test (Ashburn et al. 2000), the indirect immunofluorescent antibody test (Hassl and Aspock, 1990), and the enzyme linked immunosorbent assay (Obwaller et al. 1995). The results are encouraging but not as good as from mouse-derived tachyzoites (Evans et al. 1999). To investigate possible reasons for the differences in results between cell-culture and animal-derived tachyzoites, the present study was undertaken to observe the behaviour of T. gondii RH Ankara strain tachyzoites obtained from cell culture. Continuous production of myeloma, Vero, Hep-2, and HeLa cell culture systems was used in the study.
The yield of T. gondii tachyzoites was plentiful from each cell culture at the onset of the study. However, the yield decreased slightly during continuous production in each cell culture, except in the myeloma cell line, which actually exhibited an increase in the number of tachyzoites as the production period was extended. However, the average size of the tachyzoites obtained from the myeloma cell culture decreased in size more than the tachyzoites obtained from the other 3 cell cultures compared in this research.
T. gondii RH Ankara strain tachyzoites, which were used in the study, belonged to the Type 1 group since they killed infected mice in 4–6 days (Sibley and Howe, 1996). However, tachyzoites obtained from the cell cultures lost virulence at varying rates depending upon the cell culture type, but at a rate independent of the average tachyzoite size. Virulence was best preserved in tachyzoites obtained from the Hep-2 cell culture system, even though their average size was the second smallest. Tachyzoites from HeLa cell cultures decreased in size the least of all cell culture-derived tachyzoites but had the second fastest rate of virulence loss.
The most important conclusion from the study is that tachyzoites obtained from the 4 cell-culture types are not identical in all properties to the tachyzoites produced in vivo. The average tachyzoite size decreased in all cell types. The preliminary virulence assays also indicate that cell culture-derived tachyzoites are not as virulent as tachyzoites produced in animals. The present results do not suggest a direct correlation between tachyzoite size and virulence, but a larger animal study may definitely rule out such a correlation. Another conclusion from the study is that tachyzoites harvested from HeLa cells produced the best yield with the lowest decrease in size and that tachyzoites from Hep-2 cultured cells retained their virulence longer than the tachyzoites obtained from the other 3 cell cultures.
Howe and Sibley (1994) demonstrated that the pattern of gene expression changes in different laboratory stocks of RH strain tachyzoites after continuous culture in a laboratory environment since 1939. Mavin et al. (2004) investigated 3 lineages of RH strain tachyzoites (B, J, and Q) that were derived from continuous passaging in HeLa cell culture, in terms of their performance in the Dye test and gene expression. They demonstrated that gene expression of B and Q lineages was stable during passaging and suitable for use in the Dye test. In contrast, the gene expression of the J lineage changed with multiple passages and was not suitable for use in the Dye test. As a result, they suggested that T. gondii lineages that are still evolving in cell culture should be avoided in diagnostic assays (Mavin et al. 2004). Thus, the present study raises additional questions to be addressed in future research. Does the size and virulence of the tachyzoites obtained from cell cultures have an effect on protein expression of the parasite and/or interfere with the diagnostic assays? Do other T. gondii strains also exhibit different tachyzoite properties in continuous cell cultures and if so, do the different behaviors affect the results of diagnostic assays?
