International Journal of Medical Laboratory

Toxoplasma gondii (T. gondii) is an obligate intracellular apicomplexan parasitic protozoa that infect any nucleated cell types in many vertebrates worldwide [1, 2]. It has a widespread distribution, and almost one-third of the world’s total population is believed to be infected with toxoplasmosis [3]. Prolonged maintenance of the parasite’s infective form is the main subject for increasing live and active tachyzoites for using it in further research. Inoculation of the parasite into the mice peritoneal, embryonated egg, and cell culture media is the current method of keeping many tachyzoites in the laboratories [4]. These different methods have some complications and difficulties such as the high cost, the need to equipped laboratory and well-trained personnel, the requirement for frequent passage every 2-3 days, personal permanent attendance, and the ethical aspect of research on laboratory animals [5]. Preservation of the parasites by cryopreservation is another manner that requires the accessibility of tanks of liquid nitrogen that carries many dangers for laboratory workers [6]. Besides, because the infected mice maintained the infection for only 4–6 days, parasite maintenance through a passage in mice is considered a costly procedure; thus, cell culture is more economical for propagating tachyzoites [7]. These parasites are obligate intracellular organisms, that depending on isolate type, proliferate with a replication time of 6 to 9 hours during in vitro cultivation, and infected cells generally rupture when they reach 64 to 128 parasites/cell [2]. Diversity of cell types, like transformed cell lines (HeLa, CHO, LM, Vero, MDBK, 3T3, etc.) and culturing procedures have been used to keep tachyzoites in vitro [8, 9]. A significant limitation in performing the parasite proliferation on monolayer cell lines is that they are anchorage-dependent, and thus these cell types could not proliferate without attachment to the cell surface [10].
The purpose of this study was to develop a procedure for the culture of T. gondii tachyzoites and determine whether T. gondii tachyzoites grown in the Theileria annulata (T. annulata)-infected bovine lymphoblastoid cells.

Materials and Methods
Preparation of tachyzoites of T.gondii
Mice infection
Tachyzoites of T. gondii isolates RH were grown and increased in number by intraperitoneal injection at 5 weeks old, female BALB/c mice. The peritoneal exudates of infected mice were centrifuged for 10 min at 1000 g, and the resulted pellets were washed and resuspended in phosphate-buffered saline, pH 7.2. Tachyzoites were counted by using a Neubauer chamber slide (Haemocytometer) and their viability were assessed by trypan blue dye exclusion method. The fresh and viable tachyzoites (1×10⁶) were injected into the mice intraperitoneal, and after 3 to 4 days of infection, with appearing the symptoms of the disease, the peritoneal exudate of the infected mice was collected in phosphate-buffered saline (PBS), pH 7.2, and centrifuged at 1000g for 10 min.
T. gondii infection in monolayer Vero cell culture
Monolayer Vero cells, that were received from the cell bank of Razi Vaccine and Serum Research Institute, were used in this study. After washing the Vero cells with PBS, they were incubated with 0.25% trypsin for 5 min at room temperature and to release the cells from the bottom of the flasks, tapped sharply. Subsequently, a fresh medium, Roswell Park Memorial Institute (RPMI)-1640 (Sigma-Aldrich, USA) with 10% fetal bovine serum, was added to the trypsinized cells at 37˚C. An appropriate amount of the cells were sub-cultured every 3–4 days into flasks with fresh medium, and the cultured cells were incubated at 37˚C to make the confluent monolayer cell lines. The confluent monolayer Vero cells were infected with T.gondii tachyzoites that were obtained from the peritoneum cavity exudate of the infected mice and the infected cells were cultivated at 37˚C for 24 h. The tachyzoites from the infected cell cultures were washed twice with PBS, counted and resuspended in PBS, and were used to infect T. annulata-infected lymphoblastoids.
Infection of T. annulata-infected lympho-
blastoids with T.gondii
Bovine lymphoblastoids that were infected with attenuated merozoites of T. annulata were cultured in RPMI-1640 medium with 10% bovine serum at 37˚C. At the time of the new subculture of bovine lymphoblastoids (1×l0⁶ cells/ml), the T. gondii tachyzoites (1×10⁷ cells/ml) were obtained from the infected Vero cells, were added to the bovine lymphoblastoids suspension, and incubated at 37˚C. The interaction of tachyzoites and lymphoblastoids was studied daily using an invert microscope and the Giemsa staining method. Shape, density, and intra or extracellular positions of the tachyzoites were evaluated. At the time that was needed to reach the peak of the number of the replicated lymphoblastoids, the subculture was done by mixing the intact replicated T. annulata-infected lymphoblastoid, T. annulata-infected lymphoblastoids containing tachyzoites and the fresh RPMI-1640 medium with 10% bovine serum, each of them, one-third of the final volume. This process was carried out continuously for up to 10 passages. Each experimental procedure was repeated three times.
Determination of in vivo infectivity of cultivated parasites
During 10 passages and after every 5 passages, to determine the in vivo infectivity and pathogenicity of the tachyzoites cultivated with T.annulata-infected lymphoblastoids, the cell suspensions were inoculated to the peritoneum of female BALB/c mice (6-7 weeks). Three groups of BALB/c mice were evaluated as follows: 5 mice were infected with 1 × l0⁶ tachyzoites of the RH isolate of T.gondii maintained in T. annulata-infected lymphoblastoids, 5 mice were infected with 1 × l0⁶ parasites cultivated in Vero cells as the positive control, and 5 mice inoculated with PBS serving as the negative control. This research was performed in accordance with the recommendations in the Guide for the Care, and Use of Laboratory Animals of the Razi Vaccine and Serum Research Institute, and all animals experiments were approved by the Institutional Animal Care and Research Advisory Committee of the Razi Vaccine and Serum Research Institute based on the Specific National Ethical Guidelines for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education of Iran.
Statistical analysis
A statistical analysis by using SPSS-18 was used to perform the various comparability of the results. The student’s t-test evaluated the differences between the averages of the quantitative variables, and the p value of less than 0.05 was accepted as statistically significant.

Results
Qualitative evaluation of tachyzoites growth into the lymphoblastoids
One day after interaction and exposure of the cell, it was shown that the tachyzoites invaded into the lymphoblastoids and multiplied within them and, in many cases, formed clusters and caused lymphoblastoids to burst. Several parasitophorous vacuoles contained many groups of the tachyzoites that were arranged in rosettes shape due to the synchronous division were observed. In the invert microscope observed that the more than three-quarters of the lymphoblastoid were attacked by tachyzoites of T. gondii, and in the outer space of the lymphoblastoid, there were a large number of tachyzoites under multiplying and proliferation with typical rotational motions of the tachyzoites of T. gondii. Overgrowth was observed in all culture media on day 4 post-infection with many tachyzoites were floating in the culture supernatant. Parasite shapes remained in natural forms (id est. crescentic or oval, with one end pointed and the other end rounded) during the cultivation period. Figure 1 shows the multiplying tachyzoites inside and outside the T. annulata-infected lymphoblastoids in Giemsa staining after 5 days post-infection. This growth pattern was observed until the end of the 10^th passage.
Quantitative evaluation
As a quantitative point of observation, 10% of T. annulata-infected lymphoblastoids were infected with T. gondii tachyzoites on the first day after infection. The four days after infection, almost all T. annulata-infected lymphoblastoids were infected. Under the light microscope, large numbers of free extracellular tachyzoites were observed (Figure 2). The number of tachyzoites was doubled each day up to day 5 and then declined between days 6 and 7 (Figure 3). The maximum numbers of harvested tachyzoites were 2.8×10⁷ cells/ml after 5 days post-infection. In all days post-infection, when all parameters were comparable statistically, there were significant differences between tachyzoites numbers (p < 0.05).
In vivo assays
The infectivity of tachyzoites maintained in T.annulata-infected lymphoblastoids was evaluated during 10 passages. The study showed that in all processes of cultivation, the pathogenesis of parasites remained stable, and they were able to proliferate in mice and eventually lead to the death of the animals.

 

 

In the infected mice, the typical characteristics of the T. gondii infection, including hispid hair, low physical activity, and apathy, were presented. 3-4 days post-infection of inoculated mice, free tachyzoites were presented in the peritoneal exudate of infected animals. The high parasitemia and mortality rates observed in mice infected with tachyzoites cultivated with the T.annulata-infected lymphoblastoids, which were similar to the control group that were infected with parasites cultivated in Vero cells in the same periods.

Discussion
Toxoplasma as an opportunistic parasite, could not continue its life without a host cell, thus, we are obligated to use different host cells to support its growth and maintenance. As a parasitic disease, toxoplasmosis is one of the significant causes of morbidity and mortality, especially in immune-compromised patients and the unborn fetus, and causes very significant economic losses to the livestock industry worldwide [11-13].
The different techniques of cells culture are approaches to make attempt to prepare complicated habitat situations of living organisms to develop our knowledge about their behavior and find appropriate methods, like an effective vaccine, to prevent adverse side effects of them [14]. The significant factors that affect the in vitro cultivation of T. gondii tachyzoites are the suitability and sufficiency of the nutritional requirements in the culture medium, the growth and behavioral characteristic of the host cell, and the multiplication rate of the parasite isolate and host cell. Optimizing a mass production technique means balancing factors such as the multiplication rate of the host cells and parasites, as well as, their generation periods under given culture conditions [15].
In some of the laboratories, the tachyzoites of T. gondii maintained by serial passages in the peritoneal cavity of mice. However, this method is ethically undesirable [16-19], thus, because cell cultures from different origins provide regular harvests of fresh viable tachyzoites, as an alternative method, many different mammalian cells such as HFF [20, 21], Hep-2 [6, 15], Vero [6, 8, 22, 23]; and HeLa [15, 16, 24, 25] that are sensitive to the T. gondii attack, are used for cultivation of this parasite [9, 15, 26]. In some researches have been indicated that a suspension culture of Hela cell has been a suitable host for in vitro cultivation of T. gondii tachyzoites [27], and other studies have been shown that a non-adherent cell line derived from TG 180 murine sarcoma cells could infect by tachyzoites of T. gondii and increased intracellularly [28]. Suspension culture could be used for many research applications and does not need enzymatic or mechanical separation, and is easier to passage and used for mass production and harvesting [29, 30].
Many years ago, as a congress oral presentation, we introduced a novel method for long-term maintenance of T. gondii tachyzoites in Theileria anuulata- infected and transplanted bovine lymphoblastoids [31]. After that presentation, in another study, researchers found that Neospora caninum tachyzoites could invade and proliferated well in the T. annulata-infected lymphoblastoids, suggest that a suspension culture of non-adherent cell lines of T. annulata-infected lymphoblastoids are appropriate to host cells for in vitro cultivation of N. caninum tachyzoites and are offered, as a viable alternative to the cultivation of the parasite in vitro. They indicated that,  their work is the first report of suspension culture of N. caninum tachyzoites with T. annulata-infected cell line [10].
An important point in our research was that bovine serum was very suitable for the nutritional requirement of T. gondii and
was a comparatively simply available and inexpensive serum that could be replaced in the media that require fetal calf serum enhancement and indicated to a potent new medium that did not need expensive fetal calf serum and could be used in long-term in vitro cultivation of T. gondii [31]. It should be mentioned that another advantage of our cultivation method is that Theileria anuulata- infected and transplanted bovine lymphoblastoids, as the non-adhesive cell lines, could be easily cultivated in fermenters that enable us to cultivate parasite in large scales that could be leading to mass production of probable vaccines. We must indicate that using this novel cultivation system, also accompanied by some little difficulties such as adaptation obstacles of the parasite to new serum and cell line. However, this issue takes place in any medium change, and the important point is the final adaptation of parasites to the novel culture system.

Conclusion
This research has introduced an alternative novel cultivation system that could be used for the usual and mass cultivation of T. gondii that enables us to reach suitable amounts of parasites for our different purpose. Further biochemical, physiological, and molecular studies need to show the details of the mechanism of this type of host-parasite interaction that could lead us to find new ways of controlled cultivation of the parasite in this type of cell.

Conflict of Interest
We have no conflict of interest to declare.

Acknowledgment
The author would like to thank all those who helped in carrying out this research.
 

 
References