T. gondii purified tachyzoites were purchased from the Pasture Institute, Tehran, Iran. The tachyzoites were sonicated in PBS, centrifuged for 2 min at 600 x g at room temperature, and the supernatant containing T. gondii antigen was maintained at -20˚C. Trichomonas vaginalis (T. vaginalis) parasites maintained in liquid nitrogen from the Department of Parasitology, Isfahan University of Medical Sciences (Isfahan, Iran) were cultured in TYIS medium (produced in-house) and the antigen prepared as described in our previous work (19). Briefly, the parasites were harvested from culture medium, washed three times and sonicated, and T. vaginalis crude antigen was kept at -20˚C. Sheep lungs or livers infected with hydatid cysts were obtained from slaughtered sheep in Fasaran slaughter house in Isfahan, Iran. Hydatid cyst fluid was then aspirated and examined under a light microscope. Fluid exhibiting the presence of protoscolices was then centrifuged for 2 min at 600 x g and the supernatant containing hydatid cyst fluid antigen was stored at -20˚C. The packed protoscolices were also sonicated in PBS, centrifuged for 2 min at 600 x g and the supernatant stored as protoscolices antigen.

Mouse melanoma (B16F10) and breast (4T1) cancer cell lines were purchased from the Pasture Institute. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with a humidity of 70% and 5.0% CO₂ as previously mentioned (20). Normal lymphocytes were isolated from normal mouse spleens and prepared for further experimentation.

Rabbits and mice were used in the current study, which were purchased from the Pasture Institute. A total of 6 female Balb/c, inbred, 2-month-old mice were used to prepare spleen cells. For this purpose, the mice were euthanized by intraperitoneal injection of 250 mg/kg body weight pentobarbital (Sigma, 3636 under sterile conditions and their spleens removed. Subsequently, the spleen cells were extracted, counted and their viability checked by using trypan blue staining. To prepare cells, spleens were removed from scarified mice and transferred to a Petri Dish containing Isotonic saline. Spleens were then minced using a Scalpel blade and the mixture was passed through a four-layered gauze to remove large debris. Isotonic saline containing cells were then washed twice with isotonic saline and centrifuged at 600 x g for 2 min at room temperature. Subsequently, cells were further suspended in isotonic saline. To stain cells with Τrypan blue (Merck, 50 µl prepared cells was mixed with 50 µl Trypan blue stain at room temperature. Following 15 min, one drop of mixed cells with trypan blue was applied to a Neubauer slide (HBG, Germany) and counted using light microscope (magnification, x400).

The animals were maintained in an appropriate animal research facility and mice kept in groups of six per cage (rabbits one/cage) and fed with clean food and water. The rabbits were housed at 20-25˚C with a humidity of 70-80% with a 12 h light/dark cycle. The animal research protocols of the present study were approved by Isfahan University of Medical Sciences Ethics Committee with approval number IR.mui.REC.1394.824.

Volumes of 1 ml containing 2 mg of the different parasite antigens and sonicated breast and melanoma cancer cells were emulsified in the same volume (1 ml for each antigen) of Freund's adjuvant (Sigma-Aldrich; Merck KGaA) at room temperature and each antigen injected subcutaneously into a male 4-month-old, white New Zealand rabbit (n=5 in total). Injections were repeated fortnightly. In all rabbits, complete Freund's adjuvant was used for the first injections and incomplete Freund's adjuvant for the boosters. Following the third booster, 1 ml blood samples from the ear vein of each rabbit were collected. To prepare sera, the blood samples were centrifuged at 3,000 x g for 5 min at room temperature. The sera were then checked for the presence of specific antibodies using home-made ELISA tests as described in our previous study (20). In those rabbits with a high titer (optical density >1.5) of antibodies, the last booster was injected into each rabbit and then blood samples were collected and the sera kept at -20˚C until use. For the injection of rabbits or bleeding no anesthesia method was used. However, following final bleeding the rabbits were euthanized by intravenous injection of 150 mg/kg pentobarbital.

For ELISA, 96 wells plates were coated with the antigens (T. gondii, T. vaginalis or hydatid cyst protoscolices crude antigens) diluted 1:20 with carbonate buffer. Following overnight incubation at 37˚C, the plates were blocked with 1% bovine albumin (Merck KGaA) and then washed with sodium chloride buffer containing 0.05% Tween-20, and then diluted (1:100) antisera were added and incubated at 37˚C for 1 h. The plates were then washed and a secondary antibody (A6154; Sigma-Aldrich was added and incubated at 37˚C for 1 h. Finally, following washing, the plates were incubated with the chromogenic substrate and the optical density of wells was read at 450 nm using an ELISA reader (Kbiokit-ELx800).

Mouse cancer cells were harvested from culture medium and normal lymphocytes obtained from normal mouse spleens. All the cells were washed with PBS and incubated for 1hour at 37˚C with different antisera (1:100) which raised in rabbits against different mentioned antigens, namely anti-T. gondii, anti-T. vaginalis, anti-hydatid cyst fluid, anti-protoscolices antigens, anti-melanoma cells, anti-breast cancer cells or normal rabbit serum primary antibodies. After 1 h, the cells were washed with sodium chloride buffer containing 0.05% Tween-20, and secondary antibodies conjugated with fluorescein isothiocyanate (Donkey anti-rabbit IgG BioLegend, Inc., San Diego, CA, USA; 406403; 1:1,000) were applied in the dark at 4˚C for 1 h. Following the last washing of the test tubes, cells were analyzed using a flow cytometer. BD Cell Quest Pro software version 6 (BD Biosciences, San Jose, CA, USA) was used for analysis.

The experiments were repeated three times and data are presented as the mean ± standard deviation. A linear mixed model test was used for statistical analysis. In comparison of means, P<0.05 was considered to indicate significance.

The reaction of antiserum against T. gondii and normal rabbit serum with the surface of mouse melanoma cells was investigated. Anti-T. gondii antiserum exhibited a marked reaction with melanoma cells, while the reaction of normal rabbit serum was weak. In this experiment, anti-melanoma cell antiserum was used as a positive control and normal rabbit serum as a negative control (Fig. 1 and Table I). Analysis of experimental repeats indicated that the difference in M2 (reactive) cells between the anti-T. gondii antiserum and normal rabbit serum groups was statistically significant (P<0.05).

The reaction of antisera against T. gondii and normal rabbit serum with the surface of mouse breast cancer cells was investigated. Again, T. gondii antiserum exhibited a strong reaction with breast cancer cells while the reaction of normal rabbit serum was weak. Anti-breast cancer cell antisera were used as a positive control and normal rabbit serum as a negative control (Fig. 2 and Table II). When the experiment was repeated, the difference in M2 cells between the anti-T. gondii antiserum and normal rabbit serum groups was statistically significant (P<0.05).

The reaction of anti-T. gondii antiserum and normal rabbit serum with the surface of mouse normal spleen lymphocytes was investigated. The results revealed that the reaction of anti-Toxoplasma antibodies with melanoma cells was greater than the reaction with mouse normal lymphocytes. However, the reaction of normal rabbit serum with mouse normal lymphocytes was greater than the reaction with melanoma cells (Fig. 3 and Tables I and II).

vaginalis and anti-protoscolices antibodies with surface of melanoma, breast cancer or mouse normal spleen lymphocytes. To determine if antisera against different parasites exhibited a reaction similar to that of anti-T. gondii antiserum, the reactions of antisera against T. vaginalis and hydatid cyst crude protoscolices antigen with the surface of breast cancer cells, melanoma cells and normal mouse spleen lymphocytes were investigated. The results revealed no statistically significant differences in the reactions of the sera with cancer cells compared with spleen lymphocytes (Table III).