Leishmaniasis infection is transmitted by the vector of leishmania species. The promastigote form of the protozoan, which has an extracellular flagellum, in the carrier insect called phlebotomus sand-flies. The protozoan amastigote form is infected inside human macrophages, or susceptible animals and dogs [1,2]. Leishmaniasis can be seen in various forms including cutaneous, visceral and mucosal leishmaniasis. Leishmaniasis is endemic in Asia, Africa, North-South, and Central American and Europe [3,4]. Dogs can be infected and parasite carrier between susceptible species [5,6]. More than 30 species of Leishmaniasis have been diagnosed so far [2,6]. Dogs can be infected and parasite carrier between susceptible species [5,6]. More than 30 species of Leishmaniasis have been diagnosed so far [2,6] which 10 items of them are medically important. Three species (L. major, L. tropica, L. infantum) in Iran and Asia produce cutaneous and visceral leishmaniasis. Regarding the feature of this infection, it was understood that only way to control the disease is to have a protective vaccine, which many efforts being made so far. Two major immune cells MQ and DC help to remove infection agents. Sensitivity or persistence to leishmania is begin with macrophage and dendritic cells which recognize pathogen and producing immune cytokines and lead to cleaning up of intracellular pathogens. Macrophage and neutrophils phagocytized promastigotes and digest it for terminate infection. If it deficient in digestion promastigotes change to amastigote, proliferate and induce development of leishmaniasis [6-8]. Reported of Panaro et al. showed that Lipophsphoglycan on the Leishmania promastigote’s surface can causes killing of parasites through phagocytosis killing mechanism. In leishmaniasis various factors, including host gene and internal environment of body, may help the survival of the protozoan within the macrophage and the living body. Roma et al. shown that in vitro induction of ROS production by infected peritoneal macrophages cannot help to kill micro-organisms [9]. In report of Assureuy et al. Phagocytosis cells can induce reactive oxidase after swallowing L.major and killing it [10]. In the presence of interleukin 17 nitrite oxide synthase (iNOS) can produce nitrite oxide [11]. It also stimulates production of pro inflammatory cytokines (TNF-alpha, IL-1-beta), inflammatory chemokines to elevation of phagocytosis and effect on neutrophils and macrophages [12,13]. Neutrophils in leishmaniasis are very important in remove of parasites [13], but its short life cause serve only as “Trojan Horse” to avoid destruction by immune system [14]. TLRs on surface of MQ and DC are critical for induce immune response against leishmania infection and or vaccine. First it was said that the relationship between TLRs [2,4,9] and TLRsL play an important role in the onset of leishmaniasis disease [15], but later it was observed that mice withTLR9 defects is more sensitive to leishmaniasis than mice deficient in TLRs 2 and 4 [8,16]. After recognition of leishmania by TLRs-TLRsL, signals initiated leading to production of pro-inflammatory, chemical and inflammatory peptides [17]. It seems that the preparation of an appropriate protective vaccine is the best solution for controlling the cutaneous leishmaniasis disease. The various environment and environmental factors involved in the response to leishmaniasis are not interfered with it. Therefore, it is not possible to predict the optimal immune response against this parasite, and this is an important point to be considered in the development of the vaccine. Because of factors such as antigenic characteristics of a vaccine, general condition of vaccinated individual and host genetic factors can influence the vaccine's protectiv capacity. In this study, we investigated new formulation of leishmaniasis with two different adjuvants (BCG and Teucurium pulium). Teucrium Pulium was used in Iran for abdominal pain, mal digestion, colds, and type II diabetes. It seems that this adjuvant has anti-inflammatory and antioxidant effects and has protective effects against infections, cancers and fungi [18]. For this reason, we used of alcoholic extract of Teucrium Pulium beside to BCG as an adjuvant with the new Leishmania vaccine in Balb/C mice. In fact, we also measured for the third time the survival power, spleen changes, and for the first time inflammatory cytokines (IL-23 and 17) in the susceptible animal model (Balb/C mice).

This study was conducted in accordance with the Helsinki Declaration. The protocol is supported by both the research fellows of the School of Medicine and Deputy of Research of Tehran University of Medical Sciences. Tehran, Iran.

Culture and isolation of leismania parasites

Leishmania parasites and promastigote antigens from the L. major, WHO strain prepared from the Pasteur Institute. It was cultured and grown in mediuNNN (Novy-MacNealNicoll) medium supplemented with Hemin, Homa, RPMI 1640, normal saline, and/or 5–10% heat inactivated fetal calf serum.

The harvested parasites were washed three times with normal saline (0.9%) or phosphate/salt serum. The parasites were counted using a Neubar chamber and then kept at 80°C until use. The collected parasites then reached the concentration of 5.92 × 10¹⁰, and were divided into five equal volume tubes. The first group was killed by pasteurization in water at 56°C for 30 min. The second bottle was autoclaved at 121°C for 30 min. The third batch was merthiolated for 30 min at a dilution of 1:10,000. The fourth batch was freeze-thawed three times and the fifth batch was left innate.

Vaccine preparation

The five batches were mixed and centrifuged, and the sediments were dispensed into sterile vials. Leishmania components Vaccines were tested for complete parasitic infections .The culture was carried out on a plate of blood agar and injected into the footpads Balb/c mice.

Detailed procedures have already been described [9]. In short, just before the injection, the BCG vaccine “SSI” was suspended in a suspension of SST and 0.1 mg BCG (first dose) or 0.01 mg BCG (consecutive dose) was added to each bottle containing promastigote. Based on previous studies, 100 mg/0.1 ml or 200 mg/0.1 ml of leishmania protein was selected at each dose of the temporary vaccine to formulate and prepare the vaccine. The protein content of each dose was estimated by the Lowry method [19]. The vaccine was kept at 4°C until injected. BCG adjuvant for each injection dose is included 2 × 10⁵ CFU/0.1 ml. To prepare Teucrium adjuvant Palm, 400 mg of Teucrium polium alcoholic extract in 1 ml distilled water without endotoxin deionized, 205 mg/0.1 ml was used for each of the doses of the leishmania antigens given above, and two injection doses containing 100 μg/0.1 ml antigens or 200 μg/0.1 ml containing adjuvants [20-22].

Animal model

They included 40 Balb/c mice kept at the Faculty of Medicine's animal House. For detailed procedures please refer to Latifynia and collaborations [20-25]. In brief, Balb/C and traditional white laboratory mice (n=40) were obtained at three months old from the Pasture institute.

Vaccination

All doses of vaccine antigens of Leishmania and the bold reminder to the tail (or legs) of mice were intra dermally injected.

Dosages (100, 200 μg protein) were used as follow: Group LB (100, 200) received 100, 200 μg/0.1 ml antigen combined with BCG Group LT (100, 200) received 100, 200 μg/ 0.1 ml antigen combined with BCG alcoholic extract of Teucrium polium.

Group LBT (100, 200) received 100, 200 μg/0.1 ml antigen combined with BCG and alcoholic extract of Teucrium polium.

The control group did not receive any injections.

Hematocylin and Eosin staining

All groups were injected subcutaneously with vaccine preparation at the foot of the tail and increased one week later using similar doses.

Twenty five days after the booster injection, all animals, including the control group, were weighed and Blood sampling was performed and then the spleens were harvested. All spleens were cut in equal length and width and fixed in 10% formaldehyde buffer solution. Stable protein tissue was processed in tissue processing. Paraffin blocks were made and tissue sections of 4 to 5 microns were prepared and stained with Hariis Hematoccillin and Eosin. The fixed spleen was stained in paraffin blocks and stained in a tissue processor (Tissue sections (5–6 μm thick) and stained with hematoxylin and eosin.

The number and diameter of the white pulp of the spleen (lymphoid spleen follicles) were examined using a microscope of light with the fragment of the eye. The diameter of the SWP sections was measured and compared with each other and with the controls.

Cytokine assay

ELISA and sandwich methods were used to evaluate the IL-17 and IL-23 in animal serum and their serum levels were measured using an auto-reader at 405 nm.

Statistical analysis

Data were obtained using statistical software (SPSS Inc., Chicago, IL, USA). The means were analyzed using standard variance analysis/simple factorial test with two-way Student–Newman–Keuls methods. The correlation coefficient was determined using a Pearson bivariate, two tests.

More than one month after vaccination and twenty-five days later to all the injected groups (LB, LT and LBT) and doses (100 and 200 μg/0.1 ml) that had been injected with the vaccine, booster dose was administered again.

At the end of the experiment, the survival rates were 100%. In the autopsy, no microscopic and macroscopic pathological changes were seen in the internal, visceral and external organs, and were normal in all respects. The results of cytokines and also the microscopic and macroscopic spleen are as follows:

IL-23 of serum: The highest was for group LT 100 μg/0.1 ml and (29.55 pg/ml) and was the lowest for LBT-200 μg/0.1 ml group (16.36 pg/ml) (Figures 1, 2 and 15) (Table 1).

Figure 1: Mean of IL-23 for three ijection groups: LB, LT, LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 2: Mean of IL-23 (IL-23) for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

Table 1: Effects of provisional Leishmania Vaccine on spleen parameters, IL-17, IL-23 and Survival Rates of the Female Balb/C Mice in mean of injection groups: LB, LT, LBT and control group and injection doses 100 and 200 μg/0.1 ml. Mean within column with no common superscripts are significantly different (P<0.005)

The levels of serum IL-23 in all groups were as follow: LT-100 (29.55 pg/ml), LBT-100 (20.50), LB-100 (18.48), LT-200 (18.22), LB 200 (18.15), control (16.96) and LBT-200 (16.36 pg/ml) (Table 1). Differences between groups were not statistically significant (P=o.268) IL-17 of Serum: The highest was for control group (24.56 pg/ml), and was the lowest for LBT-200 (9.86 pg/ml).

The levels of Il-17 in all groups were as follow: Control (24 pg/ ml), LBT 100 μg/0.1 ml (22.90), LT 100 (19.67), LB 200 (14.32), LT 200 (13.8), LB 100 (11.77) and LBT 200 μg/0.1 ml (9.86 pg/ml) (Figures 3, 4 and 15) (Table 1). Statistic differences between all groups were not significant (P=0.344). Mean body weight (MBW): The highest for group LT-200 μg/0.1 ml (21.103 g) and was the lowest for LBT 100 μg/0.1 ml (18.223 g).

Figure 3: Mean of IL-17 for three ijection groups: LB, LT, LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 4: Mean of IL-17 (IL-17) for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

The MBW in all groups were as follow: LB-200[0] μg/0.1 ml (21.312 g) LT 200 (21.103 g) control (20.993 g) LT 100 (20.881 g) LB 200 (20.538 g) LBT 200 (20.44 g) and LT-100 μg/0.1 ml (18.233 g) (Figures 5, 6 and 15) (Table 1). Differences between groups were not significant (P=0880).

Figure 5: Mean of mouse weight for three ijection groups: LB, LT, LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 6: Mean of mouse Weight (MV) for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

The mean of spleen weight (MSW): The highest level seen in LT 100 μg/0.1 ml (0.391 g) and lowest related to LBT 100 (0.102 g) (MSW) were as follow: LT-100 μg/0.1 ml (0.391 g), LB 100 (0.264) LB 100 (0.264) LB 200 (0.247) LT 200 (0.116) control (0.108) LBT 200 (106) and LBT 100 μg/0.1 ml (0.102 g) (Figures 7, 8 and 15) (Table 1). There was not significant differences between animal groups (p=0.398) (Table 2).

Figure 7: Mean of spleen (SPW) weight for three ijection groups: LB, LT, LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 8: Mean of spleen (SPW) weight for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

Table 2: Effects of provisional Leishmania Vaccine on spleen parameters, IL-17, IL-23 and Survival Rates of the Female Balb/C Mice in mean of injection groups: LB, LT, LBT and control group and injection doses 100 and 200 μg/0.1 ml.

Percentage of mean spleen weight/mean body weight (%MSW/ MBW): The highest level seen in three groups of LB 100 μg/0.1 ml, LBT 100 and LT 200 (0.55%) and LBT 200 μg/0.1 ml (0.52%) had lowest levels (Figures 10 and 15) (Table 1).The percentages were as follow: LB 100 μg/0.1 ml=LB 200=LT 200 (0.55%) LT 100 = LT 200 (0.53) LBT 200 (0.52) (Figures 9, 10 and 15) (Table 1).

Figure 9: Mean of percent spleen weight/mouse weight (PSM) for three ijection groups: LB, LT, LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 10: Mean of percent spleen weight/mouse weight (PSM) for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

No significant differences of %MSW/BW was observed for all groups (Table 2). Number of Spleen White Pulp Unit (NPU): The highest mean seen in LB 200 μg/0.1 ml (41.67) and lowest was for LT 100 μg/0.1 ml (29.5). (NPU) were as follow: LB 200 μg/0.1 ml (41.67) LBT 200 (41.2) LT 200 (39.50) LB 100 (35.50) control (35.00) LBT 100 (33.50) Figures 11, 12 and 15) (Table 1). ANOVA test for NPU indicated a significant difference (P=0.001) (Table 2).

Figure 11: Mean of number of Pulp splen (NPU) for three ijection groups: LB, LT ,LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 12: Mean of Num Pulp spleen (NPU) for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

Mean of spleen white pulp size or diameter (MSWPS): The highest was related to LT 200 μg/0.1 ml (2022 micron) and lowest was to LT 100 (1697.52 micron). (MSWPS) was as follow: LT 200 μg/0.1 ml (2022 micron) control (1897.22) LB 200 (1895.82) LBT 200 (1800.98) LB 100 (1785.50) LBT 100 (1777.8) and LT 100 (1697.52 micron) (Figures 13-15) (Table 1). ANOVA test for (MSWPS) did not showed significant (P=0.152) (Table 2).

Figure 13: Mean of Pulp spleen size (MPS) for three ijection groups: LB, LT, LBT with normal group in serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 14: Mean of Pulp spleen size (MPS) for two ijection doses: 100 and 200 microgram/0.01 ml with normal group 0.00 micro/ml serum of Balb/c mice after vaccination with new leishmania vaccine.

Figure 15: The effects of the treatment of crude cocktail L. major antigen preparation. On, mean mouse weight, Spleen Pulp Weight, Number of Pulp Unit and mean Pulp Size/10, IL-23 and IL-17 in two injection dose (100, 200 μg/0.1 ml) and three injection groups (LT, LB and LBT ) compared to control group.

The components of the effective immune system at the site of entry of the parasite are potentially important in the immune response and the elimination of microorganisms. Macrophage activity by killed agents includes PRR (unnecessary receptors), local tissue matrix and intrinsic immunity, including T helper cells, DCs and tissue macrophages that provide effective and useful cytokine networks that are relevant before and after leishmanial parasitic infiltration to the host cells (macrophages). In this regard, optimal production of local cytokines such as IFN-γ, TNFα, IL12, IL17 and IL23, IL-8 in the immune response against parasites of the entrance to the site is important. In the present study, vaccines and mice were evaluated for more than one month after vaccination and 25 days after booster dose. IL-17, IL-23 and white spleen measured in 6 groups. According to the data in Tables 1 and 2 and Figures 1-15, new vaccines for vaccinated animals create immunity and provide satisfactory reactions (safety and toxicity). As shown in Tables 1 and 2 and Figures 1-15, the vaccine can induce systemic immune responses. The microscopic changes of the spleen in the vaccinated groups of Balb/c mice showed that the vaccine activates the secondary lymphoid tissues (white pulp). It is suggested that the spleen lymphoid tissue can respond effectively to the anti-leishmaniasis response, which includes the following: when the parasite is systematically (visceral) or local (skin or cutaneous, mucocutaneous). In this connection, According to the references appeared in the literature, spleen lymphoid cells including B cell can produce IL- 10 which suppress the cytokines production required to activate CMI which damaging to leishmenia parasites [26,27]. B10 cells have also been identified in mesenteric lymph nodes, peritoneal cavity and the peripheral blood and lymph nodes of mice; however the spleen is the predominant location for B10 cells [28]. These B10 cells are capable of producing IL-10 which acts as a potent regulatory cytokine [27]. IFNgamma and TNF-alpha and absence of IL-10 is associated with tissue destruction and development of the lesions observed in cutaneous and mucosal leishmaniasis despite low parasites number in the lesion [27]. Incubation of splenic B cells with LPS,CPG or apoptotic cells induced significantly elevated IL-10 secretion indicating that B cells are primed to respond to mitogenic stimulate [28]. Also, in [29] proposed the role of B cell regulation of immune response in tuberculosis via suppression of delayed type hypersensivity. They concluded that the suppression is immunologically specific and is dependent on B cells. At a glance a double functions could be proposed when induction of immune responses against leishmania parasite is the principle interest: firstly, the immune responses must provide the body with a well harmonized cytokines–chemokines networks, reasonable cellular activation and well-conditioned tissue matrix capable to confront with pathogenic leishmania parasite, secondly the induced immune responses must either be harmless, or if produce harm must be mild and well tolerated to the naïve subjects or in the patient during the course of infection. In essence, induction or fortification of a satisfactory immune response against leishmania parasites requires a potent and harmless vaccine which could be used to immunized susceptible species. According to our present and previous experiments on Balb/C mice [20-24], the vaccine seem to be safe and effective; 100% viability was recorded in all groups vaccinated with different doses of antigen (100 μg/0.1 ml or 200 μg/0.1 ml) with BCG or alcoholic extract of T. polium and or both as adjuvant. The provisional vaccine induced satisfactory cytokine responses in such a way that are not only safe to the vaccinated animals (Balb/C mice), could protect vaccinated subjects against challenging with live leishmania parasite as shown previously [30,31]. There are some suggestions related to the vaccine: The immune responses against organism leading to intracellular destruction or blocking some unknown ligands necessary to the course of the life cycle continuity or survival of organisms within the vertebrate host.

Nevertheless, the parasite maybe protected from hostile extracellular environment, helping survival of the organisms. These host- parasite interactions are basically complicated and may be affected by various parameter related to host (genetic, nutrition, body physiological conditions, the body immunological potency at the same time when the organisms penetrate the vertebrate host, and the standard of living, etc.), and parasite (virulence, or ability of organism to survive within vertebrate host). Following vaccination, the quality and quantity of immune components and the biochemical conditions of local microenvironment in which the parasites penetrate, are important to confer immunity against leishmaniasis. An ideal vaccine which enable the vaccinated subjects to deal with the pathogen, must provide local innate immune responses which detrimental to leishmania organisms at the time of entry. Also the vaccine could mount an effective acquired immune response against the penetrated organisms in such a condition that if the organisms evade innate immune responses, would face effective acquired immune responses which could destroy intracellular organism, via activation of effective cell mediated immunity orchestrated by IL12, IL17, IL23 and IFN-γ cytokines. The present findings indicate that the provisional vaccine could be administrated safety at least in mice. Beside the safety of the vaccine, it may induce production of a series of protective immune components which could be transferred via the blood stream to the cutaneous leishmania lesions resulted in activation of anti-leishmania macrophages response or could destroy metacyclic promastigotes released from the raptured parasite-laden phagocytes (macrophages or neutrophils) present within cutaneous perhaps mucocutaneous ulcers. It is postulated that, the Teucrium adjuvant could solely or together with leishmania antigens could provide body with effective anti-leishmania immune components. The percentage of survival in the present experiment was 100% at 25- 30 days post-vaccination which is in the favor of our previous findings [20,32,33] Regards to our results related to the body weight and spleen parameters showed that with the exception of mean number of spleen white pulp (lymphoid follicles), the differences between the groups were not significant post vaccination. The findings can be summarized as follow: [mean mouse weight (P=0.68), mean of spleen weight (P=0.398), mean of percentage of spleen weight/mouse weight (P=0.694), and mean of spleen white pulp size (P=0.152)], however when mean number of spleen white pulp (follicles) in seven groups of mice were compared, the differences between groups were highly significant (P=0.001). These findings were showed that our new leishmania vaccine could affect the spleen tissue; however the effect was moderate as shown previously. The interesting point is that the group LB which received 100 or 200 μg/0.1 ml had lowest mean of spleen white pulp size, but they had the highest mean number of spleen white pulp and thus had highest levels of white pulp expansion (71191.45 micron).The results confirmed our previous theory about regulation of homeostasis in vivo in different situations [34] but injection group LT for both injection doses 100 and 200 μg/0.1 ml had lowest levels of spleen white pulp expansion (64923. 0).

Lowest space occupied by the white pulp lymphoid follicle was related to injection dose 100 μg/0.1 ml (57669.7 micron) and the highest was related to injection dose 200 μg/0.1 ml (77869.7micron). Lowest levels for IL-17 related to injection group LB (13.05 pg) and injection dose 200 (12.66 pg.), and highest related to injection group LT (16.74 pg) and injection dose 100 (18.11 pg.) (Figures 3 and 4) (Table 1). IL-23 had lowest level in group LB (18.33 pg.) was highest in LT group (23.89 pg.), and similarity had lowest level in dose 200 (17.58 pg.) and highest level in dose 100 μg/0.1 ml (22.84 pg) (Table 1) (Figures 1 and 2).

In a summary it can be concluded that: 1-LB group had, highest mean number spleen white pulp (38.5), highest mean spleen white pulp expansion (71191.45 micron), lowest mean IL-17 (13.05 pg.), lowest mean IL-23 (18.33 pg), 2-LT group had lowest mean number spleen white pulp (34.5), lowest mean spleen white pulp expansion (64923 micron), highest mean IL-17 (16.74 pg.), highest mean IL-23 (23.89 pg.), highest mean of body weight (20.99 g), 3-LBT group had highest mean IL-17 (16.38 pg.), lowest mean IL-23 (18.45 pg), lowest mean of body weight (19.33 g), 4-Injection dose 100 μg/0.1 ml had lowest mean expansion of spleen white pulp (57669.7 micron), highest mean IL-17 (18.11 pg.), highest mean IL-23 (22.84 pg.), lowest mean of body weight (20.14 g), 5-Injection dose 200 μg/0.1 ml had highest mean expansion of spleen white pulp (77869.7 micron) , lowest mean IL-17 (12.66 pg), lowest mean IL-23 (17.58 pg.) and highest mean of body weight (20.69 g).

Our results from post vaccination and pre challenging are desirable and as it was demonstrated in the present experiment, the best vaccinated group was LT group and the best injection dose was 100 μg/0.1 ml nominated LT100 which had lowest spleen white pulp expansion and highest levels of IL-17 and IL-23. These finding confirmed our previous results concerning expansion of spleen white pulp [35] and the levels of serum IL17 and IL-23 in the vaccinated animals [36-40,41]. This is a satisfactory results for this new leishmania Vaccine, since IL-23 and IL-17 axis and activation of JAK-STAT is essential for immunity against Chronic Mucocutaneous Candidiasis (CMCC) and CMCC also is Mendelian disease that leishmaniasis is one of its Complications. Fortunately our vaccine was safe and its survival rate following vaccination was 100%. Also that no complications or skin nodule formation at the site of the vaccine injection were noticed following vaccination. Besides that, It was safe, harmless, without any complications and clinical pathological macroscopic and microscopic dangerous in animal models. The experience with the vaccine was repeated for the third time and the results were satisfactory and confirmed the previous experience.

The research work was supported financially by the vice chancellor of Tehran University of Medical Sciences and health Services registered under No. 15886- 30-02-91.