Protection Against Toxoplasmosis in Swiss Albino Mice Immunized with Attenuated Toxoplasma Gondii

The present study aimed at investigation on developing the immune response against Toxoplasmosis in Swiss Albino mice of the species Mus musculus, BALB/c strain, which were infected, experimentally, with Toxoplasma gondii in order to render the host able to resist any infection with this disease in the future. To achieve this, Toxoplasma gondii tissue cysts, collected from placenta of aborted women, they attenuated by irradiation with two absorbed doses of X-Ray irradiation 0.4 and 0.8 KGy. Mice were injected, intraperitoneally with tissue cysts at a rate of 100 cysts/mice single or double doses. In addition, groups of mice were injected with non-irradiated tissue cysts, as a positive control groups. The criteria used in the present study was humoral immunity, represented by IgM using Enzyme–Linked Immunosorbent Assay (ELISA IgM) to demonstrate the differentiated in IgM level between mice inoculated with X-Ray irradiated tissue cysts and positive control groups, which infected with nonirradiated tissue cysts. The result shown that the immunization with X-Ray irradiated Toxoplasma gondii tissue cysts could partially protect the mice from Toxoplasma gondii infection, and produce specific IgM antibody. Introduction Toxoplasmosis is widespread in human beings and many other warmblooded animals (Dubey & Beattie, 1988). Toxoplasma gondii is an obligate intracellular parasite. The three means by which it is mainly spread are transplacental transmission, ingestion of infective tissue, and ingestion of food or water contaminated with infective feces (Dubey, 1994). Cats, including wild felidae, are the only definitive host. Cats excrete Toxoplasma gondii oocysts in their feces. Excreted oocysts are nonsporulated and, therefore are, non-infective. After defecation, sporulation requires 1 to 5 days and is dependent on environmental conditions. Oocysts can survive for several months to a year during unfavorable environmental conditions (Dubey & Beattie, 1988). Infection consists of a transient acute phase caused by proliferative tachyzoites followed by the formation of dormant tissue cysts containing bradyzoites. The disease can be serious if acquired congenitally (Wong & Remington, 1994) or in immunosuppressed individuals, particularly patients with Acquired Immunodeficiency Scientific Studies, vol.7, No.1, 2012 – Journal of Kirkuk University 35 Syndrome (AIDS) (Luft et al., 1993). Toxoplasmosis, in an immunocompetent host, leads to the induction of a lifelong protective immunity against illness due to re-infection. These privileged protective antigens are candidates for the development of a vaccine strategy (Alexander et al., 1993). Vaccination attempts with live, attenuated, killed or lysed parasites, as well as, different antigenic fractions of the parasite, have been conducted with varying success (Araujo, 1994). Vaccines with live organisms are currently in use (Buxton, 1993; Dubey et al., 1994). The development of vaccines in the future will not only have to take into account all life-cycle stages that need to be targeted but will also have to consider which immune responses need to be generated and in which tissue sites (Elsaid et al., 1999). As a consequence, there has been an interest in defining how the immune system controls these organisms, with the hope that this would lead to the design of effective vaccines. In the last 30 years, there has been significant progress in defining the role of different immune cell types in resistance or susceptibility to many of these parasites (Donald & Roos, 1993; Soldati & Boothroyd, 1993). Humoral immunity was studied because administration of Toxoplasma antigens enhanced survival after parenteral Toxoplasma challenge in the presence of specific Toxoplasma antibody (Krahenbuhl et al., 1972; Sharma et al., 1984). Attenuated Toxoplasma gondii with different source has been used in number of studies (Duarte et al., 2002). On the other hand irradiated Toxoplasma gondii with cobalt 60 irradiation has been used to vaccinate cats and mice against Toxoplasma infection (Omata et al., 1996). Killing of Toxoplasma gondii oocysts (Dubey et al., 1996) and tissue cysts (Dubey and Thayer, 1994) by 137 Cs irradiation and protective immunity induced by vaccination with irradiated Toxoplasma gondii (Lin et al., 1999). The purpose of the present study was to administrate X-ray irradiated Toxoplasma tissue cysts, which activates IgM antibody to inhibit or kill Toxoplasma gondii, contribute to protection against any infection in the future. Materials and methods Mice: Six eight weeks old female were divided in ten groups of thirty animals. Parasites: Tissue cysts were isolated from the placenta tissue of aborted women (Sharma & Dubey, 1981; Dubey et al., 1986. Irradiation: X-Ray source was used to irradiation Toxoplasma tissue cysts (Dubey et al., 1986 and Song et al., 1993) to absorbed doses of 0.4 and 0.8 KGy (Dubey et al., 1998). Scientific Studies, vol.7, No.1, 2012 – Journal of Kirkuk University 36 Experiment Design: Animals were injected intraperitoneally (Derouin et al., 1987; Freyre, 1995 and Freyre et al., 1999) with one two inoculations at 15 days intervals using non-irradiated and irradiated Toxoplasma tissue cysts to absorbed doses of 0.4 and 0.8 KGy with X-ray irradiation source(Omata et al., 1996) as following: Group 1: 30 mice were inoculated only once with irradiated tissue cysts at 0.4 KGy. Group 2: 30 mice were inoculated twice with irradiated tissue cysts at 0.4 KGy, at 15 days intervals. Group 3: 30 mice were inoculated firstly with non-irradiated tissue cysts, and after 15 days inoculated with irradiated tissue cysts at 0.4 KGy. Group 4: 30 mice were inoculated with irradiated tissue cysts at 0.4 KGy, and after 15 days infected with non-irradiated tissue cysts as a challenge dose. Group 5: 30 mice were inoculated only once with irradiated tissue cysts at 0.8 KGy. Group 6: 30 mice were inoculated twice with irradiated tissue cysts at 0.8 KGy, at 15 days intervals. Group 7: 30 mice were inoculated firstly with non-irradiated tissue cysts, and after 15 days inoculated with irradiated tissue cysts at 0.8 KGy. Group 8: 30 mice were inoculated with irradiated tissue cysts at 0.8 KGy, and after 15 days infected with non-irradiated tissue cysts as a challenge dose. Group 9: 30 mice were infected only once with non-irradiated tissue cysts (positive control group 1). Group 10: 30 mice were infected twice with non-irradiated tissue cysts at 15 days intervals (positive control group 2). Immune Response in Mice: All animals surviving were bled 3, 15 and 30 days after the single injection and 3, 15 and 30 days after challenge injection (Omata et al., 1996) and IgM antibody was determined by ELISA (Acebes et al., 1994). Statistical Analysis: The survival rate was evaluated by the Complete Randomized Design (CRD). The means of absorbance of ELISA were examined using the Duncan's Multiple Range Test (Duncan, 1955), at 0.05% level of probability. Scientific Studies, vol.7, No.1, 2012 – Journal of Kirkuk University 37 Results and Discussion Animal Surviving: All non-immunized (non-inoculated with irradiated tissue cysts), 30 mice (100%) infected with non-irradiated Toxoplasma tissue cysts single dose (positive control group 1), and 30 mice (100%) infected with nonirradiated Toxoplasma tissue cysts double dose (positive control group 2) become ill starting 2-4 days. In this study, 14 (46.7%) out of 30 mice of positive control group 1 died starting 20-28 days post infection. Whereas, 21 mice (70%) of positive control group 2 died starting 8-16 days post challenge. All immunized 120 mice (100%), inoculated with X-ray irradiated Toxoplasma tissue cysts at 0.4 and 0.8 KGy single or double dose were survived. Experimentally group which infected with nonirradiated Toxoplasma tissue cysts first dose, and with irradiated tissue cysts at 0.4 KGy second dose only 20 out of 30 mice (66.7%) were survived. Whereas, 21 mice (70%) injected with non-irradiated tissue cysts first dose, and with irradiated tissue cysts at 0.8 KGy second doses were survived. Finally, all 60 mice (100%) immunized with irradiated Toxoplasma tissue cysts at 0.4 or 0.8 KGy, and after 15 days challenged with nonirradiated tissue cysts were survived. It has been explained that X-ray causes many changes in the parasite which is ranged between decreasing its ability to cause infection and retaining its antigen effect to killing this parasite by using high doses of irradiation (Dubey & Thayer, 1994; Dubey et al., 1996; Omata et al., 1996; Dubey et al., 1998 and Lin et al., 1999). Antibody Response: When ELISA test was used, a significant increase was noticed in IgM level for the injected mice with one dose (figure 1 D1 D2, figure 2), two doses (figure 1 E2 E3, figure 2) of non-irradiated Toxoplasma tissue cysts (positive control groups 1 & 2). It is shown in figure 1 (G1 A2), that the IgM level was higher at day 15 post injection and then started to decline. On the other hand, injection with two doses of non-irradiated Toxoplasma tissue cysts led to a significant increase in IgM level since the third day after the second injection (figure 1 E2 B3) and this increase continued till day 15 and then declined (figure 1 C3 E3, figure 2). It has been shown that giving a second dose of non-irradiated Toxoplasma tissue cysts caused an increase in the IgM level, as well as, its availability in the body (Buxton & Innes, 1995; Lunden, 1995 and Wastling et al., 1995). Its appearance as a high level at day 3 post challenged and remained in the peak at day 15 post challenged. On the other words, it Scientific Studies, vol.7, No.1, 2012 – Journal of Kirkuk University 38 remained in the high level till day 30 post challenged which indicated to the activation of humoral immune response for the mice injected with two doses of non-irradiated Toxoplasma tissue cysts (Barriga, 1981; Remington & McLeod, 1981; Dubey, 1986; Buxton et al., 1989; Denkers & Gazzin


Introduction
Toxoplasmosis is widespread in human beings and many other warmblooded animals (Dubey & Beattie, 1988). Toxoplasma gondii is an obligate intracellular parasite. The three means by which it is mainly spread are transplacental transmission, ingestion of infective tissue, and ingestion of food or water contaminated with infective feces (Dubey, 1994).
Cats, including wild felidae, are the only definitive host. Cats excrete Toxoplasma gondii oocysts in their feces. Excreted oocysts are nonsporulated and, therefore are, non-infective. After defecation, sporulation requires 1 to 5 days and is dependent on environmental conditions. Oocysts can survive for several months to a year during unfavorable environmental conditions (Dubey & Beattie, 1988). Infection consists of a transient acute phase caused by proliferative tachyzoites followed by the formation of dormant tissue cysts containing bradyzoites. The disease can be serious if acquired congenitally (Wong & Remington, 1994) or in immunosuppressed individuals, particularly patients with Acquired Immunodeficiency Syndrome (AIDS) (Luft et al., 1993).
Toxoplasmosis, in an immunocompetent host, leads to the induction of a life-long protective immunity against illness due to re-infection. These privileged protective antigens are candidates for the development of a vaccine strategy (Alexander et al., 1993).
Vaccination attempts with live, attenuated, killed or lysed parasites, as well as, different antigenic fractions of the parasite, have been conducted with varying success (Araujo, 1994). Vaccines with live organisms are currently in use (Buxton, 1993;Dubey et al., 1994). The development of vaccines in the future will not only have to take into account all life-cycle stages that need to be targeted but will also have to consider which immune responses need to be generated and in which tissue sites (Elsaid et al., 1999). As a consequence, there has been an interest in defining how the immune system controls these organisms, with the hope that this would lead to the design of effective vaccines. In the last 30 years, there has been significant progress in defining the role of different immune cell types in resistance or susceptibility to many of these parasites (Donald & Roos, 1993;Soldati & Boothroyd, 1993). Humoral immunity was studied because administration of Toxoplasma antigens enhanced survival after parenteral Toxoplasma challenge in the presence of specific Toxoplasma antibody (Krahenbuhl et al., 1972;Sharma et al., 1984).
Attenuated Toxoplasma gondii with different source has been used in number of studies (Duarte et al., 2002). On the other hand irradiated Toxoplasma gondii with cobalt 60 irradiation has been used to vaccinate cats and mice against Toxoplasma infection . Killing of Toxoplasma gondii oocysts (Dubey et al., 1996) and tissue cysts (Dubey and Thayer, 1994) by 137 Cs irradiation and protective immunity induced by vaccination with irradiated Toxoplasma gondii (Lin et al., 1999).
The purpose of the present study was to administrate X-ray irradiated Toxoplasma tissue cysts, which activates IgM antibody to inhibit or kill Toxoplasma gondii, contribute to protection against any infection in the future.

Materials and methods
Mice: Six -eight weeks old female were divided in ten groups of thirty animals. Parasites: Tissue cysts were isolated from the placenta tissue of aborted women (Sharma & Dubey, 1981;Dubey et al., 1986. Irradiation: X-Ray source was used to irradiation Toxoplasma tissue cysts (Dubey et al., 1986 andSong et al., 1993) to absorbed doses of 0.4 and 0.8 KGy .

Results and Discussion
Animal Surviving: All non-immunized (non-inoculated with irradiated tissue cysts), 30 mice (100%) infected with non-irradiated Toxoplasma tissue cysts single dose (positive control group 1), and 30 mice (100%) infected with nonirradiated Toxoplasma tissue cysts double dose (positive control group 2) become ill starting 2-4 days. In this study, 14 (46.7%) out of 30 mice of positive control group 1 died starting 20-28 days post infection. Whereas, 21 mice (70%) of positive control group 2 died starting 8-16 days post challenge. All immunized 120 mice (100%), inoculated with X-ray irradiated Toxoplasma tissue cysts at 0.4 and 0.8 KGy single or double dose were survived. Experimentally group which infected with nonirradiated Toxoplasma tissue cysts first dose, and with irradiated tissue cysts at 0.4 KGy second dose only 20 out of 30 mice (66.7%) were survived. Whereas, 21 mice (70%) injected with non-irradiated tissue cysts first dose, and with irradiated tissue cysts at 0.8 KGy second doses were survived.
Finally, all 60 mice (100%) immunized with irradiated Toxoplasma tissue cysts at 0.4 or 0.8 KGy, and after 15 days challenged with nonirradiated tissue cysts were survived.
It has been explained that X-ray causes many changes in the parasite which is ranged between decreasing its ability to cause infection and retaining its antigen effect to killing this parasite by using high doses of irradiation (Dubey & Thayer, 1994;Dubey et al., 1996;Lin et al., 1999).

Antibody Response:
When ELISA test was used, a significant increase was noticed in IgM level for the injected mice with one dose (figure 1 D1 -D2, figure 2), two doses (figure 1 E2 -E3, figure 2) of non-irradiated Toxoplasma tissue cysts (positive control groups 1 & 2). It is shown in figure 1 (G1 -A2), that the IgM level was higher at day 15 post injection and then started to decline. On the other hand, injection with two doses of non-irradiated Toxoplasma tissue cysts led to a significant increase in IgM level since the third day after the second injection (figure 1 E2 -B3) and this increase continued till day 15 and then declined (figure 1 C3 -E3, figure 2).
It has been shown that giving a second dose of non-irradiated Toxoplasma tissue cysts caused an increase in the IgM level, as well as, its availability in the body (Buxton & Innes, 1995;Lunden, 1995 andWastling et al., 1995). Its appearance as a high level at day 3 post challenged and remained in the peak at day 15 post challenged. On the other words, it remained in the high level till day 30 post challenged which indicated to the activation of humoral immune response for the mice injected with two doses of non-irradiated Toxoplasma tissue cysts (Barriga, 1981;Remington & McLeod, 1981;Dubey, 1986;Buxton et al., 1989;Denkers &Gazzinelli, 1998 andDunn et al., 1999).

Fig. 2: IgM antibody level after different times (days) in mice inoculated with single and double dose of non-irradiated Toxoplasma tissue cysts (positive control 1&2) by using ELISA
Mice injection with X-ray irradiated Toxoplasma tissue cysts at 0.4 and 0.8 KGy revealed a significant reduction in IgM level for all animals under investigation at the days 3, 15 and 30 after injection compared with positive control groups 1 and 2 ( figure 3, figure 4). This reduction was inversely proportional with the increase of irradiation dose. The above results were confirmed by other investigators (Casaratt, 1968;Assmer et al., 1999 andAgwan, 2005). Mice injection with one dose of X-ray irradiated Toxoplasma tissue cysts at 0.4 KGy, led to a significant decrease in IgM level (figure 1 F3 -F4) compared with positive control group 1 (which was received one dose of non-irradiated tissue cysts) ( figure 1 D1 -D2). Also, it noticed in figure 1 (A4 -C4) that the IgM level was higher at day 15 after injection then started to decline. Injection of mice by two doses of X-ray irradiated Toxoplasma tissue cysts at 0.4 KGy ( figure 1 G4 -G5, figure 3) enhanced a little increase of IgM level since day 3 after second injection (figure 1 G4 -A5) compared with its analogous for the one dose ( figure 1 F3 -F4). IgM level remained low compared with positive control group 2 (which was received two doses of non-irradiated tissue cysts). Generally, the IgM level was higher at day 15 after second injection (figure1 B5 -D5) and then started to decline at day 30 ( figure 1 E5 -G5). The above results were confirmed by other investigators (Remington et al., 1995 andAgwan, 2005).
The use of X-ray irradiated tissue cysts at 0.8 KGy one dose and two doses revealed a significant decrease in IgM level compared with their analogous at 0.4 KGy till day 15 after injection. This decrease stayed to be significantly at day 30 after injection ( figure 3, figure 4). This can be confirmed by comparing figure 5 (F3 -F4) with its analogous in figure 1 (F3 -F4), for the injection with one dose of X-ray irradiated tissue cysts. However, the injection with two doses showed no differences compared to one dose, where the same decrease in IgM level was noticed. This can be detected when comparing the figure 5 (G4 -G5) with the figure 1 (G4 -G5). The above result was confirmed by .
The immune response was lower for the mice injected with X-ray irradiated tissue cysts at 0.4 and 0.8 KGy (one or two doses). It looks as, there is a reversible relationship between the intensity of immune response and the irradiation dose. It can be explained that irradiation effect on parasite antigen on one way or another for inducing humoral immune response which led to a decrease in its ability. This result was previously noticed by Agwan (2005) which mentioned that different physical treatments (ultrasonic waves, irradiation with laser or microwaves, ect) which affected in parasite antigens and weakened its ability to induce humeral immune response. Mice injection with two doses of Toxoplasma tissue cysts (the first dose non-irradiated and the second one was irradiated with X-ray at 0.4 KGy) revealed a significant decrease in IgM level at day 3 after the second injection ( figure 1 H5 -H6, figure 6) compared with the positive control group 2 (which was received two doses of non-irradiated tissue cysts). This decrease was continued till day 30 after second injection. When comparing the IgM level with that of its analogous at previous experiment (mice inoculated with two doses of X-ray irradiated tissue cysts at 0.4 KGy), it can be noticed that IgM level was increased at the second case compared with its analogous at the first case. IgM level reached its maximum peak at the first 3 days after second injection, then started to be declined. Figure 1, demonstrates the above results, where the IgM level appeared to be higher at the first 3 days (figure 1 H -B6) compared with it's analogous at day 15 after second injection (figure 1 C6-E6) which was higher than it's analogous at day 30 after second injection ( figure 1 F6-H6).

Fig. 6: IgM antibody level after different times (days) in mice inoculated with different doses of Toxoplasma tissue cysts (first dose non-irradiated, second dose irradiated and first dose irradiated, second dose non-irradiated) irradiated with X-ray at 0.4 KGy by using ELISA
Mice injection with two doses of Toxoplasma tissue cysts (the first dose was non-irradiated and the second dose was irradiated with X-ray at 0.8 KGy) led to decrease in IgM level which was higher than that of 0.4 KGy, since the third day of the second injection. This decrease was significant since day 15 after the second injection. It looks as, inversely relationship between IgM level and irradiated dose (figure 7). The above observation can be confirmed when comparing figure 5 (H5 -H6) with it's analogous in figure 1 (H5 -H6). This result was previously confirmed by Agwan (2005).

Fig. 7: IgM antibody level after different times (days) in mice inoculated with different doses of Toxoplasma tissue cysts (first dose
non-irradiated, second dose irradiated and first dose irradiated, second dose non-irradiated) irradiated with X-ray at 0.8 KGy by using ELISA Similar results were obtained when mice received two doses of Toxoplasma tissue cysts (the first was irradiated with X-ray at 0.4 KGy and the second one was non-irradiated) (figure 6). A significant reduction was noticed in IgM level compared with its analogous positive control group 2 which was received two doses of non-irradiated tissue cysts. The IgM level reached its peak at day 15 after second injection. Generally, the IgM level was higher than its analogous when using two doses of X-ray irradiated tissue cysts at 0.4 KGy, and two doses of Toxoplasma tissue cysts (the first was non -irradiated and the second one was irradiated with X-ray at 0.4 KGy). This was obvious on figure 1 (A7-A8), since this appeared at day 15 after the second injection (D7-F7) which was approximately similar to its analogous at day 3 after the second injection (A7-C7) except a little difference which could not be seen in the figure.
A significant reduction was observed when mice were injected with two doses of tissue cysts (the first one was irradiated with X-ray at 0.8 KGy and the second one was non-irradiated) compared with a positive control group 2 which was received two doses of non-irradiated tissue cysts, since day 3 after the second injection. On the other hand, the reduction in IgM level at 0.8 KGy was significantly higher than that of 0.4 KGy since day 3 after the second injection, but it was not significant at day 30 after the second injection. When comparing the foregoing results with these analogous for mice injected with two doses of X-ray irradiated tissue cysts at 0.8 KGy, one can noticed that the IgM level was higher for the three periods (3, 15 and 30 days after the second injection). A significant reduction on IgM level was noticed only at day 3 after the second injection compared with its analogous which was received two doses of tissue cysts; the first one was non-irradiated and the second was irradiated with X-ray at 0.8 KGy. However, the IgM level was higher than that of mice received two doses of tissue cysts; the first one was irradiated at 0.8 KGy and the second non-irradiated compared with its analogous for the injected mice with two doses; first one was non-irradiated and the second one was irradiated with 0.8 KGy, since the day 15 after the second injection.
It has been known that the challenging dose usually increase the immune response for the host, this result was previously noticed by , but the present results indicated that it is preferably that the challenging dose in non-irradiated parasites to preserve parasite antigen in a healthy state and non-effected by any physical treatment.
A positive results were obtained when ELISA-IgM used were applied through the injection of mice by X-ray irradiated Toxoplasma tissue cysts at 0.4 and 0.8 KGy although the mice stayed a life. This can be reasoned that bradyzoites inside the tissue cysts lost their abilities for reproduction at the same time, they retain their metabolic functions. These results are in accordance with that reported by (Hiramoto et al., 2002) when they observed that Toxoplasma gondii RH strain failed to reproduce when exposed to γ-ray at 200 Gy invitro and invivo.

Conclusions
1. IgM level was lower in mice infected with irradiated tissue cysts, with no consideration to irradiation dose, compared with those infected with non-irradiated tissue cysts. 2. IgM level was higher in mice infected with tissue cysts irradiated with X-ray at 0.4 KGy compared with those infected with tissue cysts irradiated with X-ray at 0.8 KGy. 3. Irradiation with 0.4 and 0.8 KGy of X-ray resulted in attenuating tissue cysts as to evoke immunological response without causing infection. 4. IgM level was higher in mice infected first with irradiated and then with non-irradiated tissue cysts than those infected first with non-irradiated and then with irradiated tissue cysts.