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Toxoplasma gondii RH Ankara: Production of evolving tachyzoites using a novel cell culture method
Experimental Parasitology 128 (2011) 1–8
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Toxoplasma gondii RH Ankara: Production of evolving tachyzoites using a novel cell culture method Aysu Deg˘irmenci a, Mert Dösßkaya a,⇑, Aysße Caner a, Candan Çiçek b, Metin Korkmaz a, Yüksel Gürüz a, Ahmet Üner a a b
Department of Parasitology, Ege University Medical School, Bornova/Izmir 35100, Turkey Department of Medical Microbiology, Ege University Medical School, Bornova/Izmir 35100, Turkey
a r t i c l e
i n f o
Article history: Received 2 August 2010 Received in revised form 26 January 2011 Accepted 27 January 2011 Available online 4 February 2011 Keywords: Evolving Toxoplasma gondii strains Cell culture HeLa Antigen
a b s t r a c t Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem. The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain tachyzoites to harvest abundant tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12–0.5 106/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3 106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12 106/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55 106/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2–99.4% and 0.31– 0.37 106/ml, respectively. ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays’ antigen from local evolving strains. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Toxoplasma gondii is one of the most successful protozoan parasites infecting all warm-blooded animals, including humans and causes serious clinical presentations including congenital toxoplasmosis, toxoplasmic retinochoroiditis and toxoplasmic encephalitis in immunologically impaired individuals. T. gondii tachyzoites can be grown in vivo and in vitro (Buddhirongawatr et al., 2006; Hughes et al., 1986). Currently, cell culture systems are replacing animal models because of ethical issues, infrastructural deficiencies such as the lack of experienced person-
⇑ Corresponding author. Fax: +90 232 388 1347. E-mail address: [email protected] (M. Dösßkaya). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.01.019
nel and/or standardized vivariums in most research centers. In addition, startup and maintenance costs of a cell culture system are lower than animal models (Ashburn et al., 2003; Chatterton et al., 2002; Evans et al., 1999). Various cell types and culture systems are in use as a tachyzoite source to develop diagnostic assays, vaccine strategies and drugs as well as research into the genomics, proteomics, immunology, pathology and biochemical pathways of the parasite (Ashburn et al., 2001; Domzig et al., 1993; Hughes et al., 1986; Sibley and Boothroyd, 1992; Rodgers et al., 2005). Many centers are using T. gondii tachyzoites, derived from cell cultures, as antigen source in serological diagnostic assays (Ashburn et al., 2003; Buddhirongawatr et al., 2006; Chatterton et al., 2002; Hughes et al., 1986). Initially, the aim of these studies was to produce high levels of viable tachyzoites free from host cells. However, host cell contamination
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emerged as a problem in continuous passaging. Although several improved methods have been developed that aim to remove host cells from the cell culture supernatant, the ultimate target, perfect antigen quality, has not been reached (Chatterton et al., 2002; Evans et al., 1999; Grimwood et al., 1979; Hassl and Aspöck, 1990). As the host cells die, the cell culture milieu deteriorates causing a decrease in tachyzoite viability and host cell invasion capacity. In addition, gene expression alterations may occur during the adaptation of tachyzoites to this deteriorating cell culture environment (Dösßkaya et al., 2006; Guo et al., 1997; Howe and Sibley, 1994; Klein et al., 1998; Mavin et al., 2004). Further, the evaluation of serological assays that use antigen derived from cell culture, can be hampered due to host cell contamination (Chatterton et al., 2002; Dösßkaya et al., 2006; Mavin et al., 2004). Thus, current research has focused on decreasing host cell contamination while producing abundant viable T. gondii in cell culture (Hughes et al., 1986; Evans et al., 1999; Chatterton et al., 2002; Domzig et al., 1993). The present study aimed to develop a novel HeLa cell culture strategy for T. gondii RH Ankara strain tachyzoites in order to harvest abundant viable tachyzoites with minimal host cell contamination and antigenic variation at predetermined dates for use as an antigen source for serological assays and to facilitate reduction in animal use. The quality of cell culture derived antigen was assessed by serological assays in comparison with animal derived antigen.
of 25-cm2 flasks (each group contained two flasks) and incubated with 5 ml growth medium at 37 °C for 8 hours with 5% CO2. As the monolayer formed in each flask, growth medium was changed to maintenance medium containing 2% FBS and incubated for an additional 16 hours. Then 5 105 viable T. gondii RH Ankara strain tachyzoites, harvested from previously infected BALB/c mice peritoneums (in 0.9% physiological serum), were inoculated into each flask and incubated for 3 days at 37 °C with 5% CO2. The number of viable tachyzoites and amount of host cell contamination was determined with a haemocytometer by using 0.4% trypan blue dye. 2.2.2. Optimization of tachyzoite inoculum Based on the previous experiment, 2 sets of four 25-cm2 flasks (each group comprising two flasks) containing 5 105 HeLa cells were inoculated with 2.5 105; 5 105; 10 105 and 40 105 tachyzoites in maintenance medium. The two sets of flasks were incubated at 37 °C with 5% CO2 for 2 and 3 days, respectively. 2.2.3. Subcultures of tachyzoites The group with highest viable tachyzoite amount and lowest host cell contamination was subcultured 4 times continuously with the specified amount of T. gondii tachyzoites in maintenance medium and used as an antigen source in serological assays. Each subculture was performed with eight individual flasks. 2.3. Serological assays
2. Materials and methods 2.1. T. gondii RH Ankara strain and HeLa cell culture T. gondii RH Ankara strain used in the present study was isolated from a newborn with congenital toxoplasmosis in Turkey and has been continuously passaged in BALB/c mice for over 30 years (Ekmen et al., 1974). It is a virulent isolate that causes death in BALB/c mice approximately in 3–4 days, similar to RH strain (Dösßkaya et al., 2006). T. gondii RH Ankara strain was found to be Africa 1 genotype according to microsatellite genotyping using multiplex PCR assay designed for multilocus strain typing based on length polymorphism of microsatellite markers (Ajzenberg et al., 2005; Dösßkaya et al., 2009). HeLa (human cervix carcinoma) cells, obtained from German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), were grown in Basal Medium Eagle (BME with Earle’s Balanced Salt and 2.2 g/l NaHCO3) containing 10 mM Hepes solution, 2 mM Lglutamine, 10% fetal bovine serum (FBS) Penicillin (10 U/ml), Streptomycin (10 lg/ml), Gentamicin (2 lg/ml) and Amphotericin B (1 lg/ml) in 25 and 75 cm2 flasks (Greiner, Germany) at 37 °C in a 5% CO2 atmosphere. As the monolayer formed in 10% FBS containing growth medium, flasks were washed once with Hank’s balanced salt solution (HBSS), incubated with 0.25% trypsin for 5 min at 37 °C and tapped sharply to release the cells from the bottom of the flasks. Subsequently BME was added to the trypsinized cells. The cells were counted with a haemocytometer and viability was determined with 0.4% trypan blue dye (Applichem, Germany). Appropriate amount 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. Unless noted otherwise, the entire cell culture media were obtained from Biochrom, Germany. 2.2. Optimization of HeLa cell culture for in vitro T. gondii production 2.2.1. Optimization of HeLa cell inoculum During HeLa cell culture optimization, 1.25 105; 2.5 105; 5 105; 10 105 and 20 105 HeLa cells were added to 5 groups
The quality of the antigens obtained from HeLa cell culture were compared with antigen derived from previously infected BALB/c mice by using Immunofluorescence (IFA), Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. To evaluate the quality of antigens prepared from subsequent subcultures, a cohort of thirty seven serum samples (30 seropositive and 7 seronegative) were used in serological assays. These serum samples had been previously tested for anti-Toxoplasma antibodies using IFA and ELISA assays in Department of Parasitology, Ege University Medical School. IFA and ELISA were performed as described with some modifications (Engvell and Perlman, 1971; Francis et al., 1988; Fulton and Voller, 1964; Gürüz et al., 1996; Payne et al., 1987; van Loon et al., 1983; Voller, 1964; Voller et al., 1976). In addition, Western blot strips containing BALB/c mouse and HeLa culture derived antigens were probed with the sera from seven seropositive newborns and their mothers. Among these pairs, five were composed of seropositive mothers and newborns. During serological follow-up, seropositive newborns became seronegative due to clearance of passively acquired maternal IgG antibodies. In the remaining mother/newborns pairs, mothers were seronegative and newborns were seropositive. During serological follow up, newborns became seronegative. The medical history of remaining two newborns revealed post partum blood transfusion indicative of passive transmission of antibodies from donor to newborns. 2.3.1. Immunofluorescence T. gondii RH Ankara strain tachyzoites obtained from HeLa cell culture and BALB/c mice were passed through a 27 gauge needle, centrifuged at 500g for 5 minutes. The supernatant was centrifuged for 10 minutes at 3000g and washed 3 times with PBS (pH: 7.4). The tachyzoites were counted with a haemocytometer and coated on slides. Immunofluorescence antibody (IFA) testing as performed as described with some modifications (Fulton and Voller, 1964; Gürüz et al., 1996; van Loon et al., 1983; Voller, 1964). Briefly, the slides coated with HeLa cell culture and BALB/c derived tachyzoites were probed with anti-Toxoplasma IgG positive patient serum samples at dilutions of 1:16, 1:64, 1:128, 1:256, 1:512 and 1:1024 for 30 minutes at 37 °C, washed 3 times with PBS. Then,
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the slides were probed with anti-Human IgG antibody conjugated with fluorescein (Biomerieux, France) at a dilution of 1:1250 for 30 minutes at 37 °C. Thereafter, slides were washed and examined under an immunofluorescence microscope (Olympus, USA). 2.3.2. Enzyme linked immunosorbent assay ELISA was performed as described with some modifications (Engvell and Perlman, 1971; Francis et al., 1988; Gürüz et al., 1996; Payne et al., 1987; Voller et al., 1976). Equal numbers of T. gondii RH Ankara strain tachyzoites obtained from HeLa cell culture and BALB/c mice, prepared and counted as described in Section 2.3.1., were centrifuged at 14,000g for 15 minutes. After centrifugation, supernatant was discarded and the pellet was resuspended in distilled water followed by several cycles of freezing and thawing. Then, the suspensions were centrifuged again at 14,000g for 15 minutes. Supernatants were passed through 0.22 lm filter (Macherey-Nagel, Germany) and used as antigen in ELISA and Western blotting. Briefly, each well of the flat bottom microtitre plates (Linbro, MP Biomedicals, USA) was coated with 100 ll antigen suspension containing 5.7 104 tachyzoites. Plates were incubated for 1.5 hours at room temperature. Next, serum samples at dilution of 1:256 in 0.5% casein buffer (diluted in 1 PBS, pH: 7.5) were added to each well, incubated for one hour at room temperature and washed with 1 PBS. Then, the wells were probed with anti-Human IgG antibody conjugated with peroxidase (Sigma, Germany) at a dilution of 1:10,000 for 30 minutes at room temperature. Thereafter, bound antibodies were visualized after adding 3, 30 , 5, 50 tetramethylbenzidine (TMB) substrate. Reaction was stopped by adding 75 ll of 2 N sulfuric acid and the results were evaluated in a micro titer plate reader (Bio-Tek ELx808, U.S.A.) at 450 nm. Samples were considered positive if the absorbance value (AV) of the serum sample exceeded the mean AV + 5S.D. of the negative control serum samples. 2.3.3. Sodium dodecyl sulfate-polyacrylamide gels and Western blotting To compare the antigen quality of HeLa cell culture and BALB/c mice derived T. gondii RH Ankara strain tachyzoites, antigen solutions with equal amount of tachyzoites (prepared as described in Section 2.3.2.) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The separated protein was transferred to a polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, U.S.A.) and blocked by 6.25% non fat dry milk containing 1 TBS-T buffer (Tris buffered saline containing Tween 20; 20 mM Tris-Cl pH: 7.8, 0.5 M NaCl, 0.5% Tween 20) for 30 minutes at room temperature. The membranes containing equal number of HeLa cell culture and BALB/c mice derived T. gondii RH Ankara strain tachyzoite lysates were probed with a 1:100 dilution of positive and negative serum sample pools in 1 TBS-T. In addition, PVDF strips were probed with sera obtained from the seven pairs of newborns and their mothers to compare the band patterns of HeLa cell culture and BALB/c mice derived antigens. Next, the membranes were washed three times with 1 TBS-T and probed with a 1:2500 dilution of alkaline phosphatase-conjugated goat anti-Human IgG antibody (Sigma, Germany) in 1 TBS-T. Thereafter, the membranes were washed three times with 1 TBS-T and 1 TBS before the blot was developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro-BT (Applichem, Germany) diluted in diethanolamine buffer% 10 Dietanolamin, 0.5 mM MgCl26H2O,% 70 (v/v) pH: 9.8.
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correlation between HeLa cell culture and BALB/c mice derived antigen absorbance values were evaluated by Pearson correlation and linear regression analyses. A two-tailed unpaired t test with 95% confidence interval was used to determine the significance between the results of assays. 3. Results 3.1. Optimization of HeLa cell culture for in vitro T. gondii production 3.1.1. Optimization of HeLa cell inoculum The optimization of HeLa cell culture for in vitro T. gondii RH Ankara strain tachyzoite production was performed in three stages. The initial experiment showed that the supernatant with highest tachyzoite viability rate accompanied with least host cell contamination was obtained from the 5 105 HeLa cell flask inoculated with 5 105 tachyzoites (Table 1). 3.1.2. Optimization of Tachyzoite inoculum Over three days of incubation in the flasks with HeLa-tachyzoite ratio 1/1 and 2/1, HeLa contamination was >1 106/ml, tachyzoite yields and viability rates were 9.4 106/ml, 78.7% and 6 106/ml, 83.3% respectively. In the flask with HeLa-tachyzoite ratio 1/2, although HeLa contamination was 1 106/ml, tachyzoite yield and viability rate were 5 106/ml and 40%, respectively. In the flask with HeLa-tachyzoite ratio 1/8, although tachyzoite yield increased to 6 106/ml, HeLa contamination was >1 106/ml, and viability rate decreased to 16.6% (Table 2). The amount of tachyzoites obtained from the flask with 1/1 HeLa-tachyzoite ratio incubated for 3 days was 9.4 106/ml, viability rate and host cell contamination was 78.7 and 1.18 106/ml, respectively (Tables 2 and 3). In all flasks incubated for two days, viability rate was 100% and HeLa cell contamination decreased to levels between 0.12-0.5 106/ml. In the flasks with HeLa–tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3 106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12 106/ml) in HeLa contamination in the supernatant (Table 3). 3.1.3. Subcultures of Tachyzoites Highest tachyzoite yield was 3.55 106/ml, obtained from the first passage. Until the end of third subculture, viability rates and HeLa cell contaminations ranged between 98.2–99.4% and 0.31– 0.37 106/ml, respectively. Continuous production ceased at the end of the fourth subculture due to a decrease in tachyzoite yield and viability rate and sudden increase in HeLa cell contamination to 0.5 106/ml, indicative of a decrease in the antigen quality (Table 4). 3.2. Serological assays
2.4. Statistical analysis
3.2.1. Immunofluorescence and ELISA The IFA sensitivity of the first cell culture derived antigen was 90% (27/30) and in the following three subcultures the sensitivity decreased considerably to 73.3% (22/30), 40% (12/30), and 30% (9/30), respectively. The ELISA results were similar to IFA and sensitivity rates were 100% (30/30), 76.6% (23/30), 40% (12/30), and 10% (3/30), respectively. The specificity of both assays was 100%. The highest correlation coefficient giving positive linear relation: r = 0.9281 (P < 0.0001) with the following linear regression equation: y = 0.3309 (+)34.01 were obtained from the first HeLa subculture derived antigen (Fig. 1).
Data obtained during the study were processed using Prism 3.03 (GraphPad, San Diego, CA). IFA and ELISA sensitivity and specificity using HeLa cell culture derived antigen was calculated. The
3.2.2. Western blotting The quality of the antigens derived from four subsequent HeLa subcultures were compared with antigen harvested from BALB/c
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Table 1 The amount of viable tachyzoites and host cell contamination obtained from flask supernatants during the interaction of 5 105 T. gondii RH Ankara strain tachyzoites with variable amount of HeLa cells. Amount of HeLa cells added to 25-cm2 flasks 1.25 105 2.5 105 5 105 10 105 20 105 a
HeLa/tachyzoite ratio
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
1/4 1/2 1/1 2/1 4/1
0.5 1 7.4 1.5 3
4 0.5 2 6.5 2
4.5 1.5 9.4 8 5
11.1 66.6 78.7 18.75 60
1 1 1.25 2 3.5
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa cells and tachyzoites are average values obtained from two individual flasks.
Table 2 The amount of viable tachyzoites and host cell contamination obtained from flask supernatants incubated with 5 105 number of HeLa cells with variable amount of T. gondii RH Ankara strain tachyzoites, 3 days post infection. Amount of tachyzoites added to 25-cm2 flasks 2.5 105 5 105 10 105 40 105 a
HeLa/tachyzoite ratio
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
2/1 1/1 1/2 1/8
5 7.4 2 1
1 2 3 5
6 9.4 5 6
83.3 78.7 40 16.6
1.16 1.18 1.00 1.33
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa and tachyzoites are average values obtained from two flasks.
Table 3 The amount of viable tachyzoites and host cell contamination obtained from flask supernatants incubated with 5 105 number of HeLa cells with variable amount of T. gondii RH Ankara strain tachyzoites, 2 days post infection. Amount of tachyzoites added to 25-cm2 flasks 2.5 105 5 105 10 105 40 105 a
HeLa/ tachyzoite ratio
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
2/1 1/1 1/2 1/8
1.4 1.6 2.1 3.0
0 0 0 0
1.4 1.6 2.1 3.0
100 100 100 100
0.5 0.37 0.37 0.12
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa and tachyzoites are average values obtained from two flasks.
Table 4 The amount of viable tachyzoites and host cell contamination obtained from four subcultures of T. gondii RH Ankara tachyzoites incubated with 4 106 HeLa cells performed at 2 days intervals.
a
Subculture
Amount of HeLa cells added to 25-cm2 flasks (106)
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
1 2 3 4
4 4 4 4
3.5 2.2 1.9 0.84
0.05 0.04 0.01 0.36
3.55 2.24 1.91 1.2
98.5 98.2 99.4 70
0.37 0.37 0.31 0.5
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa cells and tachyzoites are average values obtained from eight individual flasks.
mice by Western blotting, using equal amount of tachyzoite containing PVDF strips probed with seropositive serum sample pools. The antigen obtained from first HeLa subculture showed comparable band quality with BALB/c mice derived antigen. In subsequent subcultures, the band signals decreased and some bands faded simultaneously (Fig. 2). Then, antigen obtained from first HeLa subculture and BALB/c mice were probed with seropositive serum samples obtained from seven newborns and their mothers. The Western blot band patterns of the first five newborns and their mothers were similar in strips prepared from HeLa cell culture and BALB/c mice. In the remaining groups, the band patterns of newborns were different than the seronegative mothers. In addition, the band signals were weaker in HeLa subculture derived strips compared to BALB/c derived strips (Fig. 3).
4. Discussion Serological assays have major value in the diagnosis of human toxoplasmosis. Traditionally, T. gondii tachyzoites produced in animal models are being used as the antigen source in serological diagnostic assays in many laboratories. T. gondii tachyzoites produced in cell culture systems are favored because of easy management, low maintenance cost and ethical values compared to animal models (Ashburn et al., 2003; Buddhirongawatr et al., 2006; Chatterton et al., 2002; Evans et al., 1999). Cell culture systems aimed to produce abundant viable tachyzoites with low host cell contamination (Evans et al., 1999; Chatterton et al., 2002; Domzig et al., 1993; Grimwood et al., 1979; Hassl and Aspöck, 1990; Valkoun, 1983). Currently, the Scottish Toxoplasma Reference Laboratory has published several data sets and methods of
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Fig. 1. Pearson correlation and linear regression analyses of ELISA IgG absorbance values (AV) of antigens derived from four subsequent HeLa subcultures at two day intervals and BALB/c mice. Cut-off value: the mean AV + 5S.D. of the negative control serum samples. Cut-off values of ELISAs using BALB/c derived antigen and four subsequent cell culture derived antigen were 73 + 5(16), 55 + 5(13), 56 + 5(10), 55 + 5(10), 62 + 5(9), respectively.
production of T. gondii tachyzoites in different cell cultures and have used cell culture derived tachyzoites in routine Sabin Feldman Dye tests and ELISA (Ashburn et al., 2003; Ashburn et al., 2001; Chatterton et al., 2002; Evans et al., 1999; Ho-Yen, 2010; Evans et al., 1999) produced T. gondii RH strain tachyzoites in several cell lines. The highest amount of tachyzoites (P1 106/ml tachyzoite in culture supernatant) were obtained from HeLa and LLC flasks with 1:1 cell:tachyzoite ratio after 72 hours inoculation while the viability was 96% and 90%, respectively. During production under these conditions in HeLa cells, harvesting time was between 72–216 hours in initial passages and the amount of tachyzoites and viability rate ranged between 0.2–3.7 106/ml and 71–99%, respectively. In subsequent passages, harvesting time was shortened to 72–144 hours, the amount of tachyzoites decreased to 0.2–2.7 106/ml, and viability rate stabilized between 88–98%. The decrease in harvesting times, amount of tachyzoites and viability rates observed in subsequent passages were associated with the adaptation process of T. gondii to HeLa cell culture environment (Evans et al., 1999). In the later study of the Scottish Toxoplasma Reference Laboratory, harvesting time of the tachyzoites from HeLa cell culture varied between 48-144 hours which created difficulties in planning routine assays due to the lack of tachyzoites when required. To resolve this issue, eight flasks incubated at 37 °C for 54 hours until maximally infected, were moved to 25 °C and maintained for between 96-312 hours. In three flasks that were incubated for 312 hours, HeLa contamination and the amount of tachyzoites were between 0.52–0.97 106/ml and 2–4.1 106/ml, respectively whereas the viability rates were <10%. In the remaining flasks, maintained at 25 °C between 96–192 hours, the amount of tachyzoites increased to 3.1–5.9 106/ml, HeLa contamination
Fig. 2. Western blot of antigens derived from four subsequent HeLa subcultures compared with antigen obtained from BALB/c mice. Lane 1. Antigen obtained from mice; Lanes 2, 3, 4, 5. Antigens obtained from four subcultures, respectively.
decreased to 0.28–0.85 106/ml and viability rate increased to 92–98% (Chatterton et al., 2002). Although, several methods have been developed previously to remove abundant host cells from cell culture supernatant, currently Chatterton et al. (2002) introduced the above described cell culture method to decrease host cell contamination during the production of abundant viable T. gondii
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Fig. 3. The band patterns obtained from (A) BALB/c mice and (B) first HeLa subculture antigens probed with anti-Toxoplasma IgG antibody positive serum samples obtained from seven groups of newborns and their mothers. M: mother; B: newborn; N: negative control; P: positive control. Arrowhead shows protein bands with approximately 22 kDa molecular weight.
tachyzoites (Chatterton et al., 2002; Domzig et al., 1993; Grimwood et al., 1979; Hassl and Aspöck, 1990). Host cell contamination created by destruction of monolayers or remnants of invaded host cells disturb the cell culture media and cause rapid tachyzoite death. The half life of a tachyzoite is 10 hours without invading a host cell (Chatterton et al., 2002). During this process, protein expression and antigenic structure changes may occur due to stress. Altogether, unfiltered host cell remnants and protein expression changes occurring in dying tachyzoites may disturb cell culture media and affect the quality of antigen used in serological assays (Dösßkaya et al., 2006; Klein et al., 1998; Mavin et al., 2004). T. gondii RH strain protein expression has changed since 1939 and genetic variability of various RH strain stocks from different laboratories have been demonstrated by Restriction Fragment Length Polymorphism and Random Amplified polymorphic DNA analyses (Guo et al., 1997; Howe and Sibley, 1994; Klein et al., 1998; Mavin et al., 2004). Behavior of T. gondii RH Ankara strain tachyzoites was investigated in several cell lines. During continuous production,the highest yield and reduction in size was achieved in myeloma cell culture. The least decrease in tachyzoite size was observed in HeLa cell cultures. The results of the virulence assay in BALB/c mice suggested that, as the size of the tachyzoites decreased, the virulence in mice was reduced. The authors highlighted the importance of cell culture derived T. gondii RH Ankara strain tachyzoites protein expression and reliability of serologic assays using cellculture derived tachyzoites (Dösßkaya et al., 2006). Overall, current research has focused on increasing the amount and quality of the T. gondii antigen while decreasing host cell contamination additional to the minimum antigenic variation arising from protein expression modifications of the tachyzoite in disturbed cell culture media (Dösßkaya et al., 2006; Mavin et al., 2004). In the present study, a novel HeLa cell culture strategy was developed to reduce animal use and produce abundant viable T. gondii RH Ankara strain tachyzoites, with least host cell contamination and minimum antigenic variation at prescheduled test dates,
suitable for serological assays. Initially, 5 105 T. gondii RH Ankara strain tachyzoites were initially inoculated with different amount of HeLa cells and optimum conditions were achieved from the flasks with HeLa:tachyzoite ratio 1:1 (Table 1). These results were lower than the viability (>90% in culture supernatant) and production (P1 106/ml in culture supernatant) threshold levels of previously published data (Evans et al., 1999). Then, two sets of 5 105 HeLa cells were inoculated with variable amount tachyzoites and incubated for two and three days, respectively. During 2 days incubation, viability rate increased to 100% and HeLa cell contamination decreased to levels between 0.12–0.5 106/ml in all flasks. Among these, the tachyzoite yield and viability ratio in the flasks with 1/8 HeLa-tachyzoite ratio were 3 106/ml and 100% compared to 6 106/ml yield and 16.6% viability in the flask incubated for 3 days. In addition, HeLa cell contamination decreased approximately 10 fold to 0.12 106/ml in the flask incubated for 2 days (Tables 2 and 3). These results were compatible with required criteria of P1 106 tachyzoites/ml and P90% viability in cell culture supernatant (Evans et al., 1999). According to these data, continuous production of T. gondii RH Ankara strain tachyzoite production was performed at two days intervals in eight flasks with HeLa-tachyzoite ratio 1/8. Highest tachyzoite yield was obtained from the first passage (3.55 106/ml) and until the end of third subculture, viability rates and HeLa cell contaminations ranged between 98.2–99.4% and 0.31–0.37 106/ml, respectively. Continuous production ceased at the end of the fourth subculture due to decrease in tachyzoite yield, viability rate and increase in HeLa cell contamination (0.5 106/ml), indicative of a decrease in the antigen quality (Table 4). To determine the antigenic discrepancy, antigens obtained from subcultures were compared to BALB/c mice derived antigen using serological assays. The ELISA and IFA sensitivities achieved by the first subculture derived antigen were 100% and 90%, respectively and decreased consecutively in subsequent subcultures. The lower sensitivity observed in the IFA compared to ELISA using first subculture derived antigen may be due to the down regulation of
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tachyzoite surface antigens. According to annual reports of the Scottish Toxoplasma Reference Laboratory published between 2006 and 2009, more effective removal of HeLa cells contamination was achieved and antigens for the in house IgG ELISA and IgG avidity tests have been successfully produced from cell culture and are in use routinely (Ho-Yen, 2010). Comparison of Western blotting of antigens derived from HeLa subcultures with antigen harvested from BALB/c mice showed that antigen obtained from first HeLa subculture had comparable band quality with BALB/c mice derived antigen (Fig. 2). Antigens derived from first HeLa subculture and BALB/c mice were probed with serum samples obtained from seven groups of newborns and their mothers. Probed strips revealed bands with 20, 30 and 37 kDa molecular weight typical for T. gondii (Contini et al., 1998; Franck et al., 2008; Gallego-Marín et al., 2006; Remington et al., 2004; Robert-Gangneux et al., 2000). Among the mother and newborn pairs, five of them displayed similar bands patterns possibly due to passively acquired maternal IgG antibodies. In the remaining two pairs, 20, 30 and 37 kDa molecular weight bands appeared in seropositive newborns whereas these bands did not appear in seronegative mothers, indicative of a source of IgG antibody in newborns reactive with T. gondii other than the mothers (Fig. 3). In the third mother/newborn pair, a strong extra band with approximately 22 kDa molecular weight appeared in strips prepared by BALB/c mice derived antigen but was absent from strips prepared by HeLa subculture derived antigen. This might be due to down-regulation of protein expression of the 22 kDa protein in HeLa culture (Fig. 3, arrowhead) or cross reactivity of the IgG antibody with T. gondii antigens. Cross reactivity of IgG or IgM antibodies with T. gondii antigens may occur (Araujo et al., 1971; Naot et al., 1981; Payne et al., 1987). Overall, the signals of the displayed bands in HeLa subculture derived strips were weaker than BALB/c derived strips and the results of the serological assays show that the quality of the antigen, derived from the first HeLa subculture was suitable for serological assays. Gene expression changes in various laboratory stocks of T. gondii RH strain tachyzoites were demonstrated due continuous culturing since 1939 (Howe and Sibley, 1994). Mavin et al. (2004) demonstrated that among three lineages of RH strain tachyzoites that were derived from continuous HeLa culturing; two of them had stable gene expression and tachyzoite yield. In contrast, the gene expression of the remaining lineage was unstable and tachyzoite yield was unpredictable after multiple passaging, indicative of an evolving strain. These results suggested that lineages still evolving in continuous T. gondii cell cultures should be avoided for serological assays (Mavin et al., 2004). Some laboratories use local evolving T. gondii strains as an antigen source for in house serological assays. The cell culture strategy to be used for the production of local evolving strains and reduction in animal use appears as an issue that needs to be resolved. The present study showed that T. gondii RH Ankara strain tachyzoites are still evolving in cell culture and not suitable to fully replace animal sources. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of antigen from local evolving strains. Laboratories may inoculate evolving T. gondii strains from an animal model or cryopreserved sample to a convenient cell culture, incubate for two days at ambient conditions, and produce abundant tachyzoites and use in routine serological assays. Acknowledgments The research was supported by the grant given by Scientific and Technical Research Council of Turkey (TUBITAK) (Project no: SBAG 105S022) and partially by the grant given by Ege University Science and Technology Centre (EBILTEM) (Project no: 2006 BIL 012).
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References Ajzenberg, D., Dumètre, A., Dardé, M.L., 2005. Multiplex PCR for typing strains of Toxoplasma gondii. Journal of Clinical Microbiology 43, 1940–1943. Araujo, F.G., Barnett, E.V., Gentry, L.O., Remington, J.S., 1971. False-positive antiToxoplasma fluorescent-antibody tests in patients with antinuclear antibodies. Applied Microbiology 22, 270–275. Ashburn, D., Evans, R., Chatterton, J.M., Joss, A.W., Ho-Yen, D.O., 2003. Toxoplasma dye test using cell culture derived tachyzoites. Journal of Clinical Pathology 53, 630–633. Ashburn, D., Chatterton, J.M., Evans, R., Joss, A.W., Ho-Yen, D.O., 2001. Success in the Toxoplasma dye test. The Journal of Infection 42, 16–19. Buddhirongawatr, R., Tungsudjai, S., Chaichoune, K., Sangloung, C., Tantawiwattananon, N., Phonaknguen, R., Sukthana, Y., 2006. Detection of Toxoplasma gondii in captive wild felids. The Southeast Asian Journal of Tropical Medicine and Public Health 3, 15–17. Chatterton, J.M., Evans, R., Ashburn, D., Joss, A.W., Ho-Yen, D.O., 2002. Toxoplasma gondii in vitro culture for experimentation. Journal of Microbiological Methods 51, 331–335. Contini, C., Fainardi, E., Cultrera, R., Canipari, R., Peyron, F., Delia, S., Paolino, E., Granieri, E., 1998. Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting. Journal of Neuroimmunology 92, 29–37. Domzig, W., Séguéla, J.P., Binz, H., 1993. A method to obtain large quantities of Toxoplasma gondii tachyzoites with extreme purity. The Journal of Parasitology 79, 613–615. Dösßkaya, M., Caner, A., Ajzenberg, D., Deg˘irmenci, A., Dardé, M.L., Can, H., Erdog˘an, D.D., Korkmaz, M., Üner, A., Güngör, Ç., Altıntasß, K., Gürüz. Y., 2009. Türkiye’de _ saptanan Toxoplasma gondii Isolatlarının Genotiplendirilmesi (Genotyping Toxoplasma gondii isolates detected in Turkey). Abstract book of 16th National Parasitology Congress held in Adana, Turkey, p. 211. Dösßkaya, M., Deg˘irmenci, A., Çiçek, C., Ak, M., Korkmaz, M., Gürüz, Y., Üner, A., 2006. Behaviour of Toxoplasma gondii RH Ankara strain tachyzoites during continuous production in various cell lines. Parasitology 132, 315–319. Ekmen, H., Altay, G., Altıntasß, K., 1974. Konjenital toxoplasmosizli bir bebekten Toxoplasma gondii izolmanı (Isolation of Toxoplasma gondii from a newborn with congenital toxoplasmosis). Abstract book of 16th Turkish Microbiology Congress held in Izmir, Turkey, pp. 291–294. Engvell, E., Perlman, P., 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874. Evans, R., Chatterton, J.M., Ashburn, D., Joss, A.W., Ho-Yen, D.O., 1999. Cell-culture system for continuous production of Toxoplasma gondii tachyzoites. European Journal of Clinical Microbiology & Infectious Diseases 18, 879–884. Francis, J.M., Payne, R.A., Joynson, D.H.M., 1988. Rapid indirect enzyme-linked immunosorbent assay (ELISA) for detecting antitoxoplasma IgG: comparison with the dye test. Journal of Clinical Pathology 41, 802–805. Franck, J., Garin, Y.J., Dumon, H., 2008. LDBio-Toxo II immunoglobulin G Western blot confirmatory test for anti-toxoplasma antibody detection. Journal of Clinical Microbiology 46, 2334–2338. Fulton, J.D., Voller, A., 1964. Evaluation of immunofluorescent and direct agglutination methods for detection of specific Toxoplasma antibodies. British Medical Journal 2, 1173–1175. Gallego-Marín, C., Henao, A.C., Gómez-Marín, J.E., 2006. Clinical validation of a western blot assay for congenital toxoplasmosis and newborn screening in a hospital in Armenia (Quindio) Colombia. Journal of Tropical Pediatrics 52, 107– 112. Grimwood, B.G., Hechemy, K., Stevens, R.W., 1979. Toxoplasma gondii: purification of trophozoites propagated in cell culture. Experimental Parasitology 48, 282– 286. Guo, Z.G., Gross, U., Johnson, A.M., 1997. Toxoplasma gondii virulence markers identified by random amplified polymorphic DNA polymerase chain reaction. Parasitology Research 83, 458–463. Gürüz, A.Y., Ok, U.Z., Korkmaz, M., 1996. Assessment of latex indirect agglutination test (Toxolatex Fumouze) for the detection of Toxoplasma specific antibodies in human sera in Turkey. Journal of the Egyptian Society of Parasitology 26, 367– 374. Hassl, A., Aspöck, H., 1990. A rapid and simple method of purification of Toxoplasma gondii trophozoites originating from tissue culture for use in the indirect immunofluorescent antibody test. Zentralblatt für Bakteriologie 272, 509– 513. Howe, D.K., Sibley, L.D., 1994. Toxoplasma gondii: analysis of different laboratory stocks of the RH strain reveals genetic heterogeneity. Experimental Parasitology 78, 242–245. Ho-Yen, D.O., Annual Reports, 2005–2009, Scottish Toxoplasma Reference Laboratory. http://www.hps.scot.nhs.uk/reflab/RefLabDetail.aspx?id=21 (Active, 2 August 2010). Hughes, H.P., Hudson, L., Fleck, D.G., 1986. In vitro culture of Toxoplasma gondii in primary and established cell lines. International Journal for Parasitology 16, 317–322. Klein, H., Anduleit, M., Bornhak, M., Fischer, M., Gross, U., Löschner, B., Nicol, S., Reiter-Orwona, I.I., Zyto, N., Montag-Lessing, T., 1998. Characteristics of Toxoplasma gondii tachyzoites from different culture systems. ALTEX 15, 37–39. Mavin, S., Joss, A.W., Ball, J., Ho-Yen, D.O., 2004. Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage? Journal of Clinical Pathology 57, 609–611.
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Naot, Y., Barnett, E.V., Remington, J.S., 1981. Method for avoiding false-positive results occurring in immunoglobulin M enzyme-linked immunosorbent assays due to presence of both rheumatoid factor and antinuclear antibodies. Journal of Clinical Microbiology 14, 73–78. Payne, R.A., Joynson, D.H., Balfour, A.H., Harford, J.P., Fleck, D.G., Mythen, M., Saunders, R.J., 1987. Public health laboratory service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody. Journal of Clinical Pathology 40, 276–281. Remington, J.S., Thulliez, P., Montoya, J.G., 2004. Recent developments for diagnosis of toxoplasmosis. Journal of Clinical Microbiology 42, 941–945. Robert-Gangneux, F., Amrein, C., Lavarde, V., Botterel, F., Dupouy-Camet, J., 2000. Neosynthesized IgG detected by western blotting in Toxoplasma-seropositive heart or lung transplant recipients. Transplant International 13, 448–452. Rodgers, L., Wang, X., Wen, X., Dunford, B., Miller, R., Suzuki, Y., 2005. Strains of Toxoplasma gondii used for tachyzoite antigens to stimulate spleen cells of
infected mice in vitro affect cytokine responses of the cells in the culture. Parasitology Research 97, 332–335. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82–85. Valkoun, A., 1983. Continual cultivation of Toxoplasma gondii on HeLa cells. Folia Parasitologica 30, 289–294. van Loon, A.M., van der Logt, J.T., Heessen, F.W., van der Veen, J., 1983. Enzymelinked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: comparison with indirect immunofluorescence and double-sandwich enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 17, 997–1004. Voller, A., 1964. Fluorescent antibody methods and their use in malaria research. Bulletin of the World Health Organization 30, 343–354. Voller, A., Bidwell, D.E., Bartlett, A., Fleck, D.G., Perkins, M., Oladehin, B., 1976. A microplate enzyme-immunoassay for toxoplasma antibody. Journal of Clinical Pathology 29, 150–153.
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Toxoplasma gondii RH Ankara: Production of evolving tachyzoites using a novel cell culture method Aysu Deg˘irmenci a, Mert Dösßkaya a,⇑, Aysße Caner a, Candan Çiçek b, Metin Korkmaz a, Yüksel Gürüz a, Ahmet Üner a a b
Department of Parasitology, Ege University Medical School, Bornova/Izmir 35100, Turkey Department of Medical Microbiology, Ege University Medical School, Bornova/Izmir 35100, Turkey
a r t i c l e
i n f o
Article history: Received 2 August 2010 Received in revised form 26 January 2011 Accepted 27 January 2011 Available online 4 February 2011 Keywords: Evolving Toxoplasma gondii strains Cell culture HeLa Antigen
a b s t r a c t Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem. The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain tachyzoites to harvest abundant tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12–0.5 106/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3 106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12 106/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55 106/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2–99.4% and 0.31– 0.37 106/ml, respectively. ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays’ antigen from local evolving strains. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Toxoplasma gondii is one of the most successful protozoan parasites infecting all warm-blooded animals, including humans and causes serious clinical presentations including congenital toxoplasmosis, toxoplasmic retinochoroiditis and toxoplasmic encephalitis in immunologically impaired individuals. T. gondii tachyzoites can be grown in vivo and in vitro (Buddhirongawatr et al., 2006; Hughes et al., 1986). Currently, cell culture systems are replacing animal models because of ethical issues, infrastructural deficiencies such as the lack of experienced person-
⇑ Corresponding author. Fax: +90 232 388 1347. E-mail address: [email protected] (M. Dösßkaya). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.01.019
nel and/or standardized vivariums in most research centers. In addition, startup and maintenance costs of a cell culture system are lower than animal models (Ashburn et al., 2003; Chatterton et al., 2002; Evans et al., 1999). Various cell types and culture systems are in use as a tachyzoite source to develop diagnostic assays, vaccine strategies and drugs as well as research into the genomics, proteomics, immunology, pathology and biochemical pathways of the parasite (Ashburn et al., 2001; Domzig et al., 1993; Hughes et al., 1986; Sibley and Boothroyd, 1992; Rodgers et al., 2005). Many centers are using T. gondii tachyzoites, derived from cell cultures, as antigen source in serological diagnostic assays (Ashburn et al., 2003; Buddhirongawatr et al., 2006; Chatterton et al., 2002; Hughes et al., 1986). Initially, the aim of these studies was to produce high levels of viable tachyzoites free from host cells. However, host cell contamination
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emerged as a problem in continuous passaging. Although several improved methods have been developed that aim to remove host cells from the cell culture supernatant, the ultimate target, perfect antigen quality, has not been reached (Chatterton et al., 2002; Evans et al., 1999; Grimwood et al., 1979; Hassl and Aspöck, 1990). As the host cells die, the cell culture milieu deteriorates causing a decrease in tachyzoite viability and host cell invasion capacity. In addition, gene expression alterations may occur during the adaptation of tachyzoites to this deteriorating cell culture environment (Dösßkaya et al., 2006; Guo et al., 1997; Howe and Sibley, 1994; Klein et al., 1998; Mavin et al., 2004). Further, the evaluation of serological assays that use antigen derived from cell culture, can be hampered due to host cell contamination (Chatterton et al., 2002; Dösßkaya et al., 2006; Mavin et al., 2004). Thus, current research has focused on decreasing host cell contamination while producing abundant viable T. gondii in cell culture (Hughes et al., 1986; Evans et al., 1999; Chatterton et al., 2002; Domzig et al., 1993). The present study aimed to develop a novel HeLa cell culture strategy for T. gondii RH Ankara strain tachyzoites in order to harvest abundant viable tachyzoites with minimal host cell contamination and antigenic variation at predetermined dates for use as an antigen source for serological assays and to facilitate reduction in animal use. The quality of cell culture derived antigen was assessed by serological assays in comparison with animal derived antigen.
of 25-cm2 flasks (each group contained two flasks) and incubated with 5 ml growth medium at 37 °C for 8 hours with 5% CO2. As the monolayer formed in each flask, growth medium was changed to maintenance medium containing 2% FBS and incubated for an additional 16 hours. Then 5 105 viable T. gondii RH Ankara strain tachyzoites, harvested from previously infected BALB/c mice peritoneums (in 0.9% physiological serum), were inoculated into each flask and incubated for 3 days at 37 °C with 5% CO2. The number of viable tachyzoites and amount of host cell contamination was determined with a haemocytometer by using 0.4% trypan blue dye. 2.2.2. Optimization of tachyzoite inoculum Based on the previous experiment, 2 sets of four 25-cm2 flasks (each group comprising two flasks) containing 5 105 HeLa cells were inoculated with 2.5 105; 5 105; 10 105 and 40 105 tachyzoites in maintenance medium. The two sets of flasks were incubated at 37 °C with 5% CO2 for 2 and 3 days, respectively. 2.2.3. Subcultures of tachyzoites The group with highest viable tachyzoite amount and lowest host cell contamination was subcultured 4 times continuously with the specified amount of T. gondii tachyzoites in maintenance medium and used as an antigen source in serological assays. Each subculture was performed with eight individual flasks. 2.3. Serological assays
2. Materials and methods 2.1. T. gondii RH Ankara strain and HeLa cell culture T. gondii RH Ankara strain used in the present study was isolated from a newborn with congenital toxoplasmosis in Turkey and has been continuously passaged in BALB/c mice for over 30 years (Ekmen et al., 1974). It is a virulent isolate that causes death in BALB/c mice approximately in 3–4 days, similar to RH strain (Dösßkaya et al., 2006). T. gondii RH Ankara strain was found to be Africa 1 genotype according to microsatellite genotyping using multiplex PCR assay designed for multilocus strain typing based on length polymorphism of microsatellite markers (Ajzenberg et al., 2005; Dösßkaya et al., 2009). HeLa (human cervix carcinoma) cells, obtained from German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), were grown in Basal Medium Eagle (BME with Earle’s Balanced Salt and 2.2 g/l NaHCO3) containing 10 mM Hepes solution, 2 mM Lglutamine, 10% fetal bovine serum (FBS) Penicillin (10 U/ml), Streptomycin (10 lg/ml), Gentamicin (2 lg/ml) and Amphotericin B (1 lg/ml) in 25 and 75 cm2 flasks (Greiner, Germany) at 37 °C in a 5% CO2 atmosphere. As the monolayer formed in 10% FBS containing growth medium, flasks were washed once with Hank’s balanced salt solution (HBSS), incubated with 0.25% trypsin for 5 min at 37 °C and tapped sharply to release the cells from the bottom of the flasks. Subsequently BME was added to the trypsinized cells. The cells were counted with a haemocytometer and viability was determined with 0.4% trypan blue dye (Applichem, Germany). Appropriate amount 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. Unless noted otherwise, the entire cell culture media were obtained from Biochrom, Germany. 2.2. Optimization of HeLa cell culture for in vitro T. gondii production 2.2.1. Optimization of HeLa cell inoculum During HeLa cell culture optimization, 1.25 105; 2.5 105; 5 105; 10 105 and 20 105 HeLa cells were added to 5 groups
The quality of the antigens obtained from HeLa cell culture were compared with antigen derived from previously infected BALB/c mice by using Immunofluorescence (IFA), Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. To evaluate the quality of antigens prepared from subsequent subcultures, a cohort of thirty seven serum samples (30 seropositive and 7 seronegative) were used in serological assays. These serum samples had been previously tested for anti-Toxoplasma antibodies using IFA and ELISA assays in Department of Parasitology, Ege University Medical School. IFA and ELISA were performed as described with some modifications (Engvell and Perlman, 1971; Francis et al., 1988; Fulton and Voller, 1964; Gürüz et al., 1996; Payne et al., 1987; van Loon et al., 1983; Voller, 1964; Voller et al., 1976). In addition, Western blot strips containing BALB/c mouse and HeLa culture derived antigens were probed with the sera from seven seropositive newborns and their mothers. Among these pairs, five were composed of seropositive mothers and newborns. During serological follow-up, seropositive newborns became seronegative due to clearance of passively acquired maternal IgG antibodies. In the remaining mother/newborns pairs, mothers were seronegative and newborns were seropositive. During serological follow up, newborns became seronegative. The medical history of remaining two newborns revealed post partum blood transfusion indicative of passive transmission of antibodies from donor to newborns. 2.3.1. Immunofluorescence T. gondii RH Ankara strain tachyzoites obtained from HeLa cell culture and BALB/c mice were passed through a 27 gauge needle, centrifuged at 500g for 5 minutes. The supernatant was centrifuged for 10 minutes at 3000g and washed 3 times with PBS (pH: 7.4). The tachyzoites were counted with a haemocytometer and coated on slides. Immunofluorescence antibody (IFA) testing as performed as described with some modifications (Fulton and Voller, 1964; Gürüz et al., 1996; van Loon et al., 1983; Voller, 1964). Briefly, the slides coated with HeLa cell culture and BALB/c derived tachyzoites were probed with anti-Toxoplasma IgG positive patient serum samples at dilutions of 1:16, 1:64, 1:128, 1:256, 1:512 and 1:1024 for 30 minutes at 37 °C, washed 3 times with PBS. Then,
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the slides were probed with anti-Human IgG antibody conjugated with fluorescein (Biomerieux, France) at a dilution of 1:1250 for 30 minutes at 37 °C. Thereafter, slides were washed and examined under an immunofluorescence microscope (Olympus, USA). 2.3.2. Enzyme linked immunosorbent assay ELISA was performed as described with some modifications (Engvell and Perlman, 1971; Francis et al., 1988; Gürüz et al., 1996; Payne et al., 1987; Voller et al., 1976). Equal numbers of T. gondii RH Ankara strain tachyzoites obtained from HeLa cell culture and BALB/c mice, prepared and counted as described in Section 2.3.1., were centrifuged at 14,000g for 15 minutes. After centrifugation, supernatant was discarded and the pellet was resuspended in distilled water followed by several cycles of freezing and thawing. Then, the suspensions were centrifuged again at 14,000g for 15 minutes. Supernatants were passed through 0.22 lm filter (Macherey-Nagel, Germany) and used as antigen in ELISA and Western blotting. Briefly, each well of the flat bottom microtitre plates (Linbro, MP Biomedicals, USA) was coated with 100 ll antigen suspension containing 5.7 104 tachyzoites. Plates were incubated for 1.5 hours at room temperature. Next, serum samples at dilution of 1:256 in 0.5% casein buffer (diluted in 1 PBS, pH: 7.5) were added to each well, incubated for one hour at room temperature and washed with 1 PBS. Then, the wells were probed with anti-Human IgG antibody conjugated with peroxidase (Sigma, Germany) at a dilution of 1:10,000 for 30 minutes at room temperature. Thereafter, bound antibodies were visualized after adding 3, 30 , 5, 50 tetramethylbenzidine (TMB) substrate. Reaction was stopped by adding 75 ll of 2 N sulfuric acid and the results were evaluated in a micro titer plate reader (Bio-Tek ELx808, U.S.A.) at 450 nm. Samples were considered positive if the absorbance value (AV) of the serum sample exceeded the mean AV + 5S.D. of the negative control serum samples. 2.3.3. Sodium dodecyl sulfate-polyacrylamide gels and Western blotting To compare the antigen quality of HeLa cell culture and BALB/c mice derived T. gondii RH Ankara strain tachyzoites, antigen solutions with equal amount of tachyzoites (prepared as described in Section 2.3.2.) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The separated protein was transferred to a polyvinylidene difluoride (PVDF) transfer membrane (Immobilon-P, Millipore, U.S.A.) and blocked by 6.25% non fat dry milk containing 1 TBS-T buffer (Tris buffered saline containing Tween 20; 20 mM Tris-Cl pH: 7.8, 0.5 M NaCl, 0.5% Tween 20) for 30 minutes at room temperature. The membranes containing equal number of HeLa cell culture and BALB/c mice derived T. gondii RH Ankara strain tachyzoite lysates were probed with a 1:100 dilution of positive and negative serum sample pools in 1 TBS-T. In addition, PVDF strips were probed with sera obtained from the seven pairs of newborns and their mothers to compare the band patterns of HeLa cell culture and BALB/c mice derived antigens. Next, the membranes were washed three times with 1 TBS-T and probed with a 1:2500 dilution of alkaline phosphatase-conjugated goat anti-Human IgG antibody (Sigma, Germany) in 1 TBS-T. Thereafter, the membranes were washed three times with 1 TBS-T and 1 TBS before the blot was developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro-BT (Applichem, Germany) diluted in diethanolamine buffer% 10 Dietanolamin, 0.5 mM MgCl26H2O,% 70 (v/v) pH: 9.8.
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correlation between HeLa cell culture and BALB/c mice derived antigen absorbance values were evaluated by Pearson correlation and linear regression analyses. A two-tailed unpaired t test with 95% confidence interval was used to determine the significance between the results of assays. 3. Results 3.1. Optimization of HeLa cell culture for in vitro T. gondii production 3.1.1. Optimization of HeLa cell inoculum The optimization of HeLa cell culture for in vitro T. gondii RH Ankara strain tachyzoite production was performed in three stages. The initial experiment showed that the supernatant with highest tachyzoite viability rate accompanied with least host cell contamination was obtained from the 5 105 HeLa cell flask inoculated with 5 105 tachyzoites (Table 1). 3.1.2. Optimization of Tachyzoite inoculum Over three days of incubation in the flasks with HeLa-tachyzoite ratio 1/1 and 2/1, HeLa contamination was >1 106/ml, tachyzoite yields and viability rates were 9.4 106/ml, 78.7% and 6 106/ml, 83.3% respectively. In the flask with HeLa-tachyzoite ratio 1/2, although HeLa contamination was 1 106/ml, tachyzoite yield and viability rate were 5 106/ml and 40%, respectively. In the flask with HeLa-tachyzoite ratio 1/8, although tachyzoite yield increased to 6 106/ml, HeLa contamination was >1 106/ml, and viability rate decreased to 16.6% (Table 2). The amount of tachyzoites obtained from the flask with 1/1 HeLa-tachyzoite ratio incubated for 3 days was 9.4 106/ml, viability rate and host cell contamination was 78.7 and 1.18 106/ml, respectively (Tables 2 and 3). In all flasks incubated for two days, viability rate was 100% and HeLa cell contamination decreased to levels between 0.12-0.5 106/ml. In the flasks with HeLa–tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3 106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12 106/ml) in HeLa contamination in the supernatant (Table 3). 3.1.3. Subcultures of Tachyzoites Highest tachyzoite yield was 3.55 106/ml, obtained from the first passage. Until the end of third subculture, viability rates and HeLa cell contaminations ranged between 98.2–99.4% and 0.31– 0.37 106/ml, respectively. Continuous production ceased at the end of the fourth subculture due to a decrease in tachyzoite yield and viability rate and sudden increase in HeLa cell contamination to 0.5 106/ml, indicative of a decrease in the antigen quality (Table 4). 3.2. Serological assays
2.4. Statistical analysis
3.2.1. Immunofluorescence and ELISA The IFA sensitivity of the first cell culture derived antigen was 90% (27/30) and in the following three subcultures the sensitivity decreased considerably to 73.3% (22/30), 40% (12/30), and 30% (9/30), respectively. The ELISA results were similar to IFA and sensitivity rates were 100% (30/30), 76.6% (23/30), 40% (12/30), and 10% (3/30), respectively. The specificity of both assays was 100%. The highest correlation coefficient giving positive linear relation: r = 0.9281 (P < 0.0001) with the following linear regression equation: y = 0.3309 (+)34.01 were obtained from the first HeLa subculture derived antigen (Fig. 1).
Data obtained during the study were processed using Prism 3.03 (GraphPad, San Diego, CA). IFA and ELISA sensitivity and specificity using HeLa cell culture derived antigen was calculated. The
3.2.2. Western blotting The quality of the antigens derived from four subsequent HeLa subcultures were compared with antigen harvested from BALB/c
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Table 1 The amount of viable tachyzoites and host cell contamination obtained from flask supernatants during the interaction of 5 105 T. gondii RH Ankara strain tachyzoites with variable amount of HeLa cells. Amount of HeLa cells added to 25-cm2 flasks 1.25 105 2.5 105 5 105 10 105 20 105 a
HeLa/tachyzoite ratio
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
1/4 1/2 1/1 2/1 4/1
0.5 1 7.4 1.5 3
4 0.5 2 6.5 2
4.5 1.5 9.4 8 5
11.1 66.6 78.7 18.75 60
1 1 1.25 2 3.5
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa cells and tachyzoites are average values obtained from two individual flasks.
Table 2 The amount of viable tachyzoites and host cell contamination obtained from flask supernatants incubated with 5 105 number of HeLa cells with variable amount of T. gondii RH Ankara strain tachyzoites, 3 days post infection. Amount of tachyzoites added to 25-cm2 flasks 2.5 105 5 105 10 105 40 105 a
HeLa/tachyzoite ratio
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
2/1 1/1 1/2 1/8
5 7.4 2 1
1 2 3 5
6 9.4 5 6
83.3 78.7 40 16.6
1.16 1.18 1.00 1.33
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa and tachyzoites are average values obtained from two flasks.
Table 3 The amount of viable tachyzoites and host cell contamination obtained from flask supernatants incubated with 5 105 number of HeLa cells with variable amount of T. gondii RH Ankara strain tachyzoites, 2 days post infection. Amount of tachyzoites added to 25-cm2 flasks 2.5 105 5 105 10 105 40 105 a
HeLa/ tachyzoite ratio
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
2/1 1/1 1/2 1/8
1.4 1.6 2.1 3.0
0 0 0 0
1.4 1.6 2.1 3.0
100 100 100 100
0.5 0.37 0.37 0.12
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa and tachyzoites are average values obtained from two flasks.
Table 4 The amount of viable tachyzoites and host cell contamination obtained from four subcultures of T. gondii RH Ankara tachyzoites incubated with 4 106 HeLa cells performed at 2 days intervals.
a
Subculture
Amount of HeLa cells added to 25-cm2 flasks (106)
Amount of viable tachyzoites (106/ml)a
Amount of dead tachyzoites (106/ml)a
Amount of total tachyzoites (106/ml)a
Viability rate (%)
HeLa cell contamination (106/ml)a
1 2 3 4
4 4 4 4
3.5 2.2 1.9 0.84
0.05 0.04 0.01 0.36
3.55 2.24 1.91 1.2
98.5 98.2 99.4 70
0.37 0.37 0.31 0.5
5 ml Growth medium was used in 25-cm2 flasks. The amount of HeLa cells and tachyzoites are average values obtained from eight individual flasks.
mice by Western blotting, using equal amount of tachyzoite containing PVDF strips probed with seropositive serum sample pools. The antigen obtained from first HeLa subculture showed comparable band quality with BALB/c mice derived antigen. In subsequent subcultures, the band signals decreased and some bands faded simultaneously (Fig. 2). Then, antigen obtained from first HeLa subculture and BALB/c mice were probed with seropositive serum samples obtained from seven newborns and their mothers. The Western blot band patterns of the first five newborns and their mothers were similar in strips prepared from HeLa cell culture and BALB/c mice. In the remaining groups, the band patterns of newborns were different than the seronegative mothers. In addition, the band signals were weaker in HeLa subculture derived strips compared to BALB/c derived strips (Fig. 3).
4. Discussion Serological assays have major value in the diagnosis of human toxoplasmosis. Traditionally, T. gondii tachyzoites produced in animal models are being used as the antigen source in serological diagnostic assays in many laboratories. T. gondii tachyzoites produced in cell culture systems are favored because of easy management, low maintenance cost and ethical values compared to animal models (Ashburn et al., 2003; Buddhirongawatr et al., 2006; Chatterton et al., 2002; Evans et al., 1999). Cell culture systems aimed to produce abundant viable tachyzoites with low host cell contamination (Evans et al., 1999; Chatterton et al., 2002; Domzig et al., 1993; Grimwood et al., 1979; Hassl and Aspöck, 1990; Valkoun, 1983). Currently, the Scottish Toxoplasma Reference Laboratory has published several data sets and methods of
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Fig. 1. Pearson correlation and linear regression analyses of ELISA IgG absorbance values (AV) of antigens derived from four subsequent HeLa subcultures at two day intervals and BALB/c mice. Cut-off value: the mean AV + 5S.D. of the negative control serum samples. Cut-off values of ELISAs using BALB/c derived antigen and four subsequent cell culture derived antigen were 73 + 5(16), 55 + 5(13), 56 + 5(10), 55 + 5(10), 62 + 5(9), respectively.
production of T. gondii tachyzoites in different cell cultures and have used cell culture derived tachyzoites in routine Sabin Feldman Dye tests and ELISA (Ashburn et al., 2003; Ashburn et al., 2001; Chatterton et al., 2002; Evans et al., 1999; Ho-Yen, 2010; Evans et al., 1999) produced T. gondii RH strain tachyzoites in several cell lines. The highest amount of tachyzoites (P1 106/ml tachyzoite in culture supernatant) were obtained from HeLa and LLC flasks with 1:1 cell:tachyzoite ratio after 72 hours inoculation while the viability was 96% and 90%, respectively. During production under these conditions in HeLa cells, harvesting time was between 72–216 hours in initial passages and the amount of tachyzoites and viability rate ranged between 0.2–3.7 106/ml and 71–99%, respectively. In subsequent passages, harvesting time was shortened to 72–144 hours, the amount of tachyzoites decreased to 0.2–2.7 106/ml, and viability rate stabilized between 88–98%. The decrease in harvesting times, amount of tachyzoites and viability rates observed in subsequent passages were associated with the adaptation process of T. gondii to HeLa cell culture environment (Evans et al., 1999). In the later study of the Scottish Toxoplasma Reference Laboratory, harvesting time of the tachyzoites from HeLa cell culture varied between 48-144 hours which created difficulties in planning routine assays due to the lack of tachyzoites when required. To resolve this issue, eight flasks incubated at 37 °C for 54 hours until maximally infected, were moved to 25 °C and maintained for between 96-312 hours. In three flasks that were incubated for 312 hours, HeLa contamination and the amount of tachyzoites were between 0.52–0.97 106/ml and 2–4.1 106/ml, respectively whereas the viability rates were <10%. In the remaining flasks, maintained at 25 °C between 96–192 hours, the amount of tachyzoites increased to 3.1–5.9 106/ml, HeLa contamination
Fig. 2. Western blot of antigens derived from four subsequent HeLa subcultures compared with antigen obtained from BALB/c mice. Lane 1. Antigen obtained from mice; Lanes 2, 3, 4, 5. Antigens obtained from four subcultures, respectively.
decreased to 0.28–0.85 106/ml and viability rate increased to 92–98% (Chatterton et al., 2002). Although, several methods have been developed previously to remove abundant host cells from cell culture supernatant, currently Chatterton et al. (2002) introduced the above described cell culture method to decrease host cell contamination during the production of abundant viable T. gondii
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Fig. 3. The band patterns obtained from (A) BALB/c mice and (B) first HeLa subculture antigens probed with anti-Toxoplasma IgG antibody positive serum samples obtained from seven groups of newborns and their mothers. M: mother; B: newborn; N: negative control; P: positive control. Arrowhead shows protein bands with approximately 22 kDa molecular weight.
tachyzoites (Chatterton et al., 2002; Domzig et al., 1993; Grimwood et al., 1979; Hassl and Aspöck, 1990). Host cell contamination created by destruction of monolayers or remnants of invaded host cells disturb the cell culture media and cause rapid tachyzoite death. The half life of a tachyzoite is 10 hours without invading a host cell (Chatterton et al., 2002). During this process, protein expression and antigenic structure changes may occur due to stress. Altogether, unfiltered host cell remnants and protein expression changes occurring in dying tachyzoites may disturb cell culture media and affect the quality of antigen used in serological assays (Dösßkaya et al., 2006; Klein et al., 1998; Mavin et al., 2004). T. gondii RH strain protein expression has changed since 1939 and genetic variability of various RH strain stocks from different laboratories have been demonstrated by Restriction Fragment Length Polymorphism and Random Amplified polymorphic DNA analyses (Guo et al., 1997; Howe and Sibley, 1994; Klein et al., 1998; Mavin et al., 2004). Behavior of T. gondii RH Ankara strain tachyzoites was investigated in several cell lines. During continuous production,the highest yield and reduction in size was achieved in myeloma cell culture. The least decrease in tachyzoite size was observed in HeLa cell cultures. The results of the virulence assay in BALB/c mice suggested that, as the size of the tachyzoites decreased, the virulence in mice was reduced. The authors highlighted the importance of cell culture derived T. gondii RH Ankara strain tachyzoites protein expression and reliability of serologic assays using cellculture derived tachyzoites (Dösßkaya et al., 2006). Overall, current research has focused on increasing the amount and quality of the T. gondii antigen while decreasing host cell contamination additional to the minimum antigenic variation arising from protein expression modifications of the tachyzoite in disturbed cell culture media (Dösßkaya et al., 2006; Mavin et al., 2004). In the present study, a novel HeLa cell culture strategy was developed to reduce animal use and produce abundant viable T. gondii RH Ankara strain tachyzoites, with least host cell contamination and minimum antigenic variation at prescheduled test dates,
suitable for serological assays. Initially, 5 105 T. gondii RH Ankara strain tachyzoites were initially inoculated with different amount of HeLa cells and optimum conditions were achieved from the flasks with HeLa:tachyzoite ratio 1:1 (Table 1). These results were lower than the viability (>90% in culture supernatant) and production (P1 106/ml in culture supernatant) threshold levels of previously published data (Evans et al., 1999). Then, two sets of 5 105 HeLa cells were inoculated with variable amount tachyzoites and incubated for two and three days, respectively. During 2 days incubation, viability rate increased to 100% and HeLa cell contamination decreased to levels between 0.12–0.5 106/ml in all flasks. Among these, the tachyzoite yield and viability ratio in the flasks with 1/8 HeLa-tachyzoite ratio were 3 106/ml and 100% compared to 6 106/ml yield and 16.6% viability in the flask incubated for 3 days. In addition, HeLa cell contamination decreased approximately 10 fold to 0.12 106/ml in the flask incubated for 2 days (Tables 2 and 3). These results were compatible with required criteria of P1 106 tachyzoites/ml and P90% viability in cell culture supernatant (Evans et al., 1999). According to these data, continuous production of T. gondii RH Ankara strain tachyzoite production was performed at two days intervals in eight flasks with HeLa-tachyzoite ratio 1/8. Highest tachyzoite yield was obtained from the first passage (3.55 106/ml) and until the end of third subculture, viability rates and HeLa cell contaminations ranged between 98.2–99.4% and 0.31–0.37 106/ml, respectively. Continuous production ceased at the end of the fourth subculture due to decrease in tachyzoite yield, viability rate and increase in HeLa cell contamination (0.5 106/ml), indicative of a decrease in the antigen quality (Table 4). To determine the antigenic discrepancy, antigens obtained from subcultures were compared to BALB/c mice derived antigen using serological assays. The ELISA and IFA sensitivities achieved by the first subculture derived antigen were 100% and 90%, respectively and decreased consecutively in subsequent subcultures. The lower sensitivity observed in the IFA compared to ELISA using first subculture derived antigen may be due to the down regulation of
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tachyzoite surface antigens. According to annual reports of the Scottish Toxoplasma Reference Laboratory published between 2006 and 2009, more effective removal of HeLa cells contamination was achieved and antigens for the in house IgG ELISA and IgG avidity tests have been successfully produced from cell culture and are in use routinely (Ho-Yen, 2010). Comparison of Western blotting of antigens derived from HeLa subcultures with antigen harvested from BALB/c mice showed that antigen obtained from first HeLa subculture had comparable band quality with BALB/c mice derived antigen (Fig. 2). Antigens derived from first HeLa subculture and BALB/c mice were probed with serum samples obtained from seven groups of newborns and their mothers. Probed strips revealed bands with 20, 30 and 37 kDa molecular weight typical for T. gondii (Contini et al., 1998; Franck et al., 2008; Gallego-Marín et al., 2006; Remington et al., 2004; Robert-Gangneux et al., 2000). Among the mother and newborn pairs, five of them displayed similar bands patterns possibly due to passively acquired maternal IgG antibodies. In the remaining two pairs, 20, 30 and 37 kDa molecular weight bands appeared in seropositive newborns whereas these bands did not appear in seronegative mothers, indicative of a source of IgG antibody in newborns reactive with T. gondii other than the mothers (Fig. 3). In the third mother/newborn pair, a strong extra band with approximately 22 kDa molecular weight appeared in strips prepared by BALB/c mice derived antigen but was absent from strips prepared by HeLa subculture derived antigen. This might be due to down-regulation of protein expression of the 22 kDa protein in HeLa culture (Fig. 3, arrowhead) or cross reactivity of the IgG antibody with T. gondii antigens. Cross reactivity of IgG or IgM antibodies with T. gondii antigens may occur (Araujo et al., 1971; Naot et al., 1981; Payne et al., 1987). Overall, the signals of the displayed bands in HeLa subculture derived strips were weaker than BALB/c derived strips and the results of the serological assays show that the quality of the antigen, derived from the first HeLa subculture was suitable for serological assays. Gene expression changes in various laboratory stocks of T. gondii RH strain tachyzoites were demonstrated due continuous culturing since 1939 (Howe and Sibley, 1994). Mavin et al. (2004) demonstrated that among three lineages of RH strain tachyzoites that were derived from continuous HeLa culturing; two of them had stable gene expression and tachyzoite yield. In contrast, the gene expression of the remaining lineage was unstable and tachyzoite yield was unpredictable after multiple passaging, indicative of an evolving strain. These results suggested that lineages still evolving in continuous T. gondii cell cultures should be avoided for serological assays (Mavin et al., 2004). Some laboratories use local evolving T. gondii strains as an antigen source for in house serological assays. The cell culture strategy to be used for the production of local evolving strains and reduction in animal use appears as an issue that needs to be resolved. The present study showed that T. gondii RH Ankara strain tachyzoites are still evolving in cell culture and not suitable to fully replace animal sources. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of antigen from local evolving strains. Laboratories may inoculate evolving T. gondii strains from an animal model or cryopreserved sample to a convenient cell culture, incubate for two days at ambient conditions, and produce abundant tachyzoites and use in routine serological assays. Acknowledgments The research was supported by the grant given by Scientific and Technical Research Council of Turkey (TUBITAK) (Project no: SBAG 105S022) and partially by the grant given by Ege University Science and Technology Centre (EBILTEM) (Project no: 2006 BIL 012).
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References Ajzenberg, D., Dumètre, A., Dardé, M.L., 2005. Multiplex PCR for typing strains of Toxoplasma gondii. Journal of Clinical Microbiology 43, 1940–1943. Araujo, F.G., Barnett, E.V., Gentry, L.O., Remington, J.S., 1971. False-positive antiToxoplasma fluorescent-antibody tests in patients with antinuclear antibodies. Applied Microbiology 22, 270–275. Ashburn, D., Evans, R., Chatterton, J.M., Joss, A.W., Ho-Yen, D.O., 2003. Toxoplasma dye test using cell culture derived tachyzoites. Journal of Clinical Pathology 53, 630–633. Ashburn, D., Chatterton, J.M., Evans, R., Joss, A.W., Ho-Yen, D.O., 2001. Success in the Toxoplasma dye test. The Journal of Infection 42, 16–19. Buddhirongawatr, R., Tungsudjai, S., Chaichoune, K., Sangloung, C., Tantawiwattananon, N., Phonaknguen, R., Sukthana, Y., 2006. Detection of Toxoplasma gondii in captive wild felids. The Southeast Asian Journal of Tropical Medicine and Public Health 3, 15–17. Chatterton, J.M., Evans, R., Ashburn, D., Joss, A.W., Ho-Yen, D.O., 2002. Toxoplasma gondii in vitro culture for experimentation. Journal of Microbiological Methods 51, 331–335. Contini, C., Fainardi, E., Cultrera, R., Canipari, R., Peyron, F., Delia, S., Paolino, E., Granieri, E., 1998. Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting. Journal of Neuroimmunology 92, 29–37. Domzig, W., Séguéla, J.P., Binz, H., 1993. A method to obtain large quantities of Toxoplasma gondii tachyzoites with extreme purity. The Journal of Parasitology 79, 613–615. Dösßkaya, M., Caner, A., Ajzenberg, D., Deg˘irmenci, A., Dardé, M.L., Can, H., Erdog˘an, D.D., Korkmaz, M., Üner, A., Güngör, Ç., Altıntasß, K., Gürüz. Y., 2009. Türkiye’de _ saptanan Toxoplasma gondii Isolatlarının Genotiplendirilmesi (Genotyping Toxoplasma gondii isolates detected in Turkey). Abstract book of 16th National Parasitology Congress held in Adana, Turkey, p. 211. Dösßkaya, M., Deg˘irmenci, A., Çiçek, C., Ak, M., Korkmaz, M., Gürüz, Y., Üner, A., 2006. Behaviour of Toxoplasma gondii RH Ankara strain tachyzoites during continuous production in various cell lines. Parasitology 132, 315–319. Ekmen, H., Altay, G., Altıntasß, K., 1974. Konjenital toxoplasmosizli bir bebekten Toxoplasma gondii izolmanı (Isolation of Toxoplasma gondii from a newborn with congenital toxoplasmosis). Abstract book of 16th Turkish Microbiology Congress held in Izmir, Turkey, pp. 291–294. Engvell, E., Perlman, P., 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874. Evans, R., Chatterton, J.M., Ashburn, D., Joss, A.W., Ho-Yen, D.O., 1999. Cell-culture system for continuous production of Toxoplasma gondii tachyzoites. European Journal of Clinical Microbiology & Infectious Diseases 18, 879–884. Francis, J.M., Payne, R.A., Joynson, D.H.M., 1988. Rapid indirect enzyme-linked immunosorbent assay (ELISA) for detecting antitoxoplasma IgG: comparison with the dye test. Journal of Clinical Pathology 41, 802–805. Franck, J., Garin, Y.J., Dumon, H., 2008. LDBio-Toxo II immunoglobulin G Western blot confirmatory test for anti-toxoplasma antibody detection. Journal of Clinical Microbiology 46, 2334–2338. Fulton, J.D., Voller, A., 1964. Evaluation of immunofluorescent and direct agglutination methods for detection of specific Toxoplasma antibodies. British Medical Journal 2, 1173–1175. Gallego-Marín, C., Henao, A.C., Gómez-Marín, J.E., 2006. Clinical validation of a western blot assay for congenital toxoplasmosis and newborn screening in a hospital in Armenia (Quindio) Colombia. Journal of Tropical Pediatrics 52, 107– 112. Grimwood, B.G., Hechemy, K., Stevens, R.W., 1979. Toxoplasma gondii: purification of trophozoites propagated in cell culture. Experimental Parasitology 48, 282– 286. Guo, Z.G., Gross, U., Johnson, A.M., 1997. Toxoplasma gondii virulence markers identified by random amplified polymorphic DNA polymerase chain reaction. Parasitology Research 83, 458–463. Gürüz, A.Y., Ok, U.Z., Korkmaz, M., 1996. Assessment of latex indirect agglutination test (Toxolatex Fumouze) for the detection of Toxoplasma specific antibodies in human sera in Turkey. Journal of the Egyptian Society of Parasitology 26, 367– 374. Hassl, A., Aspöck, H., 1990. A rapid and simple method of purification of Toxoplasma gondii trophozoites originating from tissue culture for use in the indirect immunofluorescent antibody test. Zentralblatt für Bakteriologie 272, 509– 513. Howe, D.K., Sibley, L.D., 1994. Toxoplasma gondii: analysis of different laboratory stocks of the RH strain reveals genetic heterogeneity. Experimental Parasitology 78, 242–245. Ho-Yen, D.O., Annual Reports, 2005–2009, Scottish Toxoplasma Reference Laboratory. http://www.hps.scot.nhs.uk/reflab/RefLabDetail.aspx?id=21 (Active, 2 August 2010). Hughes, H.P., Hudson, L., Fleck, D.G., 1986. In vitro culture of Toxoplasma gondii in primary and established cell lines. International Journal for Parasitology 16, 317–322. Klein, H., Anduleit, M., Bornhak, M., Fischer, M., Gross, U., Löschner, B., Nicol, S., Reiter-Orwona, I.I., Zyto, N., Montag-Lessing, T., 1998. Characteristics of Toxoplasma gondii tachyzoites from different culture systems. ALTEX 15, 37–39. Mavin, S., Joss, A.W., Ball, J., Ho-Yen, D.O., 2004. Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage? Journal of Clinical Pathology 57, 609–611.
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A. Deg˘irmenci et al. / Experimental Parasitology 128 (2011) 1–8
Naot, Y., Barnett, E.V., Remington, J.S., 1981. Method for avoiding false-positive results occurring in immunoglobulin M enzyme-linked immunosorbent assays due to presence of both rheumatoid factor and antinuclear antibodies. Journal of Clinical Microbiology 14, 73–78. Payne, R.A., Joynson, D.H., Balfour, A.H., Harford, J.P., Fleck, D.G., Mythen, M., Saunders, R.J., 1987. Public health laboratory service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody. Journal of Clinical Pathology 40, 276–281. Remington, J.S., Thulliez, P., Montoya, J.G., 2004. Recent developments for diagnosis of toxoplasmosis. Journal of Clinical Microbiology 42, 941–945. Robert-Gangneux, F., Amrein, C., Lavarde, V., Botterel, F., Dupouy-Camet, J., 2000. Neosynthesized IgG detected by western blotting in Toxoplasma-seropositive heart or lung transplant recipients. Transplant International 13, 448–452. Rodgers, L., Wang, X., Wen, X., Dunford, B., Miller, R., Suzuki, Y., 2005. Strains of Toxoplasma gondii used for tachyzoite antigens to stimulate spleen cells of
infected mice in vitro affect cytokine responses of the cells in the culture. Parasitology Research 97, 332–335. Sibley, L.D., Boothroyd, J.C., 1992. Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 359, 82–85. Valkoun, A., 1983. Continual cultivation of Toxoplasma gondii on HeLa cells. Folia Parasitologica 30, 289–294. van Loon, A.M., van der Logt, J.T., Heessen, F.W., van der Veen, J., 1983. Enzymelinked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: comparison with indirect immunofluorescence and double-sandwich enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 17, 997–1004. Voller, A., 1964. Fluorescent antibody methods and their use in malaria research. Bulletin of the World Health Organization 30, 343–354. Voller, A., Bidwell, D.E., Bartlett, A., Fleck, D.G., Perkins, M., Oladehin, B., 1976. A microplate enzyme-immunoassay for toxoplasma antibody. Journal of Clinical Pathology 29, 150–153.