Introduction of protocols for mass production of Toxoplasma gondii tachyzoites of the genotype II PRU strain

Abstract Background Few investigations of genotype II of Toxoplasma gondii, the most prevalent form of the Toxoplasma parasite in humans, have been carried out on due to the rapid conversion of tachyzoites to bradyzoites in its life cycle. The current study aimed to create animal and in vitro models for production of the tachyzoites of the Prugniaud (PRU) genotype II strain. Methods To develop an immunocompromised model and obtain tachyzoites of the PRU strain, BALB/c mice were orally treated with dexamethasone (10 mg/kg), cyclophosphamide (36 mg/kg), and cyclosporine (18 mg/kg) from 5 days prior to inoculation. Then, 10‐15 tissue cysts of PRU strain were inoculated intraperitoneally into the mice. The tachyzoites obtained from mice were then cultivated in a HeLa cell culture. The resulting yield of tachyzoites was cryopreserved in 92% fetal calf serum, 8% dimethyl sulfoxide. The infectivity of these tachyzoites was evaluated using in vivo and in vitro examinations. Results Numerous tachyzoites were observed in the peritoneal fluid of the immunosuppressed mice within 10‐15 days after inoculation, and many tachyzoites were harvested from the HeLa cell culture. Trypan Blue staining showed 80% viability of the tachyzoites recovered from cryopreservation and this was confirmed by HeLa cell culture. In addition, mice infected intraperitoneally with the recovered tachyzoites presented with cysts in the brain after 2 months. Conclusion We have developed an animal model for mass production of T. gondii tachyzoites of the PRU strain. This method can provide fresh viable tachyzoites of Toxoplasma gondii for use as and when required in future investigations.


| Parasites and cell culture
The brains were then homogenized and the number of cysts in 20 µL of the homogenized brains was counted using a light microscope.
Based on the count, a suspension containing 10-15 cysts/mL was prepared in sterile phosphate-buffered saline (PBS; 0.01 mol/L, pH 7.2), for later inoculation. In this study, the mice were provided with standard dried rodent food and water ad libitum and were housed in temperature-controlled accommodation (

| Tachyzoite production following immunosuppression of the mice
Fifteen female BALB/c mice (5-6 months old, weighing 25-30g) were orally treated with dexamethasone (10 mg/kg), cyclophosphamide (36 mg/kg), and cyclosporine (18 mg/kg) for 15 days to suppress their immune systems. After 5 days, a suspension containing 10-15 tissue cysts of PRU strain, prepared as described in the previous section, were inoculated intraperitoneally into the immunosuppressed BALB/c mice. Over consecutive days after inoculation, the mice's peritoneal exudates were stained using Giemsa staining and examined by light microscopy. Once the tachyzoites were seen in the peritoneal fluid, the peritoneal cavity of the infected mice was washed twice using 5 mL of sterile phosphate-buffered saline (PBS; 0.01 mol/L, pH 7.2) and the cellular debris was removed by centrifuging at 1200 g for 10 minutes at 4℃.

| Mass cultivation
Flasks (75 cm 2 ) were seeded with 9 × 10 5 HeLa cells in a growth medium. When 70% coverage with a HeLa cell monolayer was achieved, the cultures were infected with tachyzoites from immunosuppressed mice (tachyzoite:cell ratios were 1:10), 16 and incubated at 37°C with 5% CO 2 for 6-8 hours, during which time the active tachyzoites could penetrate the cells. The culture media containing cell debris and inactive tachyzoites were then removed and replaced with fresh culture medium. The cultures were examined by phase contrast microscopy using an inverted microscope to detect ruptured cells as well as the presence of tachyzoites. The cultures were harvested when maximally infected (>5 plaques/field at 400× magnification) and were estimated to have ≥1 × 10 5 tachyzoites/mL.
The tachyzoites and cells in the decanted supernatant were filtered out and washed twice with the maintenance medium at 1200 g for 10 min.

| Comparison of PRU strain tachyzoites with RH strain
The RH strain of T. gondii was obtained from Tehran University of Medical Sciences, Tehran, Iran. The tachyzoites of the RH strain were intraperitoneally injected into five female BALB/c mice. After 5-6 days, tachyzoites were collected by repeated flushing of the peritoneal cavity using phosphate buffered saline (PBS) at pH 7.2. Then the sizes of 100 tachyzoites of the RH strain and 100 tachyzoites of the PRU strain, obtained as described in the previous section, were measured using a light microscope equipped with a scale, and the average size of the two strains was compared.

| Cryopreservation of the PRU strain
The tachyzoites obtained from the previous stage were poured into 1.8-ml plastic cryogenic vials. FCS (92%) and dimethyl sulfoxide (DMSO, 8%) were added to each vial and the contents were thoroughly mixed on ice. The vials were then stored at −20℃ in an ordinary refrigerator, then at −80℃, and finally they were transferred to liquid nitrogen.

| Viability evaluation
After 1 month, the cryopreserved tubes were removed from the liquid nitrogen. The cryopreserved tachyzoites were thawed and washed with RPMI 1640 at 1200 g for 5 minutes (to remove DMSO).
The viability and morphology of the recovered tachyzoites were evaluated using a light microscope. Trypan Blue exclusion (0.5% Trypan Blue solution mixed with an equal volume of the suspension) was used to test viability. Viability was also evaluated in a Hela cell culture. The tachyzoites were mixed with the growth medium and added to Hela cells as described above (Mass cultivation). The infectivity potency of the cryopreserved tachyzoites in mice was also examined by injection of 10 4 tachyzoites intraperitoneally and examining the brains of mice after two months.

| RE SULTS
In immunosuppressed mice infected intraperitoneally with tissue cysts, tachyzoites were observed in the peritoneal fluid of 10-15 days after infection. Tachyzoites of the PRU strain were smaller in size than those of the RH strain (the mean size of PRU and RH strain were 5.5 × 1.5 μm and 6.7 × 2.3 μm, respectively, P value = .1).
Moreover, mass cell cultivation of the tachyzoites from immunocompromised mice released a lot of viable tachyzoites after 4-5 days, when all of the cells were ruptured.
In addition, the viability of the cryopreservation method and the viability of the tachyzoites that were maintained in liquid nitrogen were confirmed using Trypan Blue staining and HeLa cell culture. Trypan Blue staining showed 80% viability after cryopreservation. When cell cultures were infected with the cryopreserved tachyzoites, the tachyzoites entered the HeLa cells over 2 days and a lot of viable tachyzoites were released when the all the HeLa cells ruptured after a 7-day incubation (Figure 1). The infectivity potency of the cryopreserved tachyzoites in mice was also confirmed by observation of cysts in the brain after 2 months (Figure 2).

| D ISCUSS I ON
The present study aimed to describe methods for production and maintenance of Toxoplasma gondii tachyzoites of the PRU strain in an animal model and cell culture system. Initially, the immune system of the mice was suppressed using immunosuppressive drugs and cysts of the Toxoplasma PRU strain were injected. The infection remained acute in the mice due to their weakened immune system. Ten to fifteen days later, numerous tachyzoites were removed from the peritoneal fluid. In addition, after mass cultivation and cryopreservation, the recovered tachyzoites showed more than 80% viability.
Production and maintenance of T. gondii tachyzoites are important for specific antigen preparation, immunization, and therapeutic, biochemical, genetic, and molecular research. The PRU genotype II strain of Toxoplasma is very suitable for research on latent toxoplasmosis because this strain immediately progresses to the latent phase due to conversion into bradyzoites in tissue cysts. However, it is difficult to obtain tachyzoites from the PRU strain and therefore most studies have been designed for RH strain maintenance and very few studies have been conducted on the PRU strain. 17,18 In previous studies, different methods, including trypsin digestion, purification using a 3-lm filter membrane, CF-11 cellulose purification, and Percoll solution purification, were used to separate the tachyzoites of Toxoplasma from in vivo and in vitro culture systems. 11,16,19 The trypsin digestion method showed a high salvage rate for tachyzoites purification, but the viability of the purified tachyzoites was low. This method is suitable for antigen analysis. 20 The 3-lm filter is the most common purification method for separating tachyzoites. In this low-cost method, all leukocytes or HeLa cells were removed quickly. However, the tachyzoites recovery rate was low. This method is suitable for purification of small amounts of samples for molecular research. 21 The CF-11 cellulose method can also be used for purification of tachyzoites. It removes leukocytes or HeLa cells and is suitable for polymerase chain reaction (PCR) amplification. One of the advantages of this method over the 3-lm filter is that it harvests a considerable number of tachyzoites, but it is a time-consuming process. 22 Percoll solution has been also used for separation of human mononuclear leukocytes and purification of tachyzoites. Different concentrations of Percoll solution have been used and 45% iso-osmotic Percoll solution has shown the best results. However, the tachyzoites recovery rate was low. 23 In the current study, purification by washing to eliminate the cells and extra debris yielded PRU tachyzoites with a high viability rate. Moreover, when the parasites were introduced

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset used and/or analyzed during the current study is available from the corresponding author upon reasonable request.