|Year : 2016 | Volume
| Issue : 1 | Page : 42-50
Polymerase chain reaction detection and inducible nitric-oxide synthase expression of Leishmania major in mice inoculated by two different routes
Abeer E Mahmoud1, Rasha AH Attia1, Hanan EM Eldeek1, Haiam Mohammed Mahmoud Farrag1, Rania Makboul2
1 Department of Parasitology, Faculty of Medicine, Assiut University, Assiut, Egypt
2 Department of Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
|Date of Acceptance||30-Nov-2015|
|Date of Web Publication||28-Jan-2016|
Abeer E Mahmoud
Department of Parasitology, Faculty of Medicine, Assiut University, Assiut
| Abstract|| |
Introduction: Leishmania major needs a sensitive and specific method for proper diagnosis. This study aims to study the course and histopathology of L. major in certain tissues of experimentally infected BALB/c mice after subcutaneous (sc) and intraperitoneal (ip) inoculation. Materials and Methods: After infecting BALB/c mice using sc and ip inoculation, the histopathology was studied. The kinetoplastic DNA polymerase chain reaction (PCR) for its molecular detection and detect the inducible nitric-oxide synthase (iNOS) pattern during the first 3 months of infection. Result: PCR could detect the presence of L. major in all spleens, lymph nodes, and skin ulcers by both inoculation routes while (33%) and (42%) of livers were positive after sc and ip routes, respectively. Chronic inflammatory cell infiltrates with capsulitis was found in the spleen, lymph nodes, and liver. Granulomas were found in the spleen and liver. There was a statistically significant difference in iNOS expression along the experiment in the spleen and lymph nodes by both routes and in the liver by ip only. Apart from the liver, iNOS could not be detected on the 2 nd week postinfection and was high after 1 month for both routes in all samples; a moderate decrease at 2 months and the highest decrease were detected after 3 months. Conclusions: L. major inoculation by both routes produce visceral disease in mice, and kinetoplastic DNA PCR can detect its presence from the 2 nd week up to the 3 rd month postinfection. The iNOS expression was high at 1 and 2 months and remained throughout the 3 months of the experiment; which plays an important role in the disease course and control.
Keywords: Inducible nitric-oxide synthase, intraperitoneal routes, kinetoplastic DNA polymerase chain reaction, Leishmania major, subcutaneous
|How to cite this article:|
Mahmoud AE, Attia RA, Eldeek HE, Farrag HM, Makboul R. Polymerase chain reaction detection and inducible nitric-oxide synthase expression of Leishmania major in mice inoculated by two different routes. Trop Parasitol 2016;6:42-50
|How to cite this URL:|
Mahmoud AE, Attia RA, Eldeek HE, Farrag HM, Makboul R. Polymerase chain reaction detection and inducible nitric-oxide synthase expression of Leishmania major in mice inoculated by two different routes. Trop Parasitol [serial online] 2016 [cited 2018 Nov 12];6:42-50. Available from: http://www.tropicalparasitology.org/text.asp?2016/6/1/42/175088
| Introduction|| |
Leishmaniasis is an infectious parasitic disease with a wide range of clinical manifestations affecting people in tropical and subtropical regions of the world. Over 12 million people are infected worldwide, and about 350 million people are at risk of infection. WHO includes it in the list of neglected tropical diseases.  Depending on Leishmania species, host immunity, and host genetic factors, infection leads to cutaneous, mucocutaneous, or visceral leishmaniasis.  Leishmania major is the causative agent of cutaneous leishmaniasis which is endemic in North Africa, Central Asia, and the Middle East. 
Experimental models can be used to explore the factors responsible for different clinical outcomes of the disease and the mechanisms of immune responses to eliminate the parasites. They are influenced by the developmental stage (promastigote or amastigote), dose, species, strain, and route of inoculation. More specifically, the route of infection is an important variable.  The natural route of infection in Leishmania is the skin which contains cells with potent immunomodulatory effects. L. major has the capacity to multiply at visceral and cutaneous sites at the same rate. It usually gives more potent immunity by subcutaneous (sc) than intradermal route in murine model. 
BALB/c mice are susceptible to L. major and sc inoculation leads to uncontrollable infection. The mice died from cachectic and anemic features with visceral infection. The susceptibility depends upon the induction of Th2 cells producing interleukin 4 (IL-4), IL-5 and IL-10, which limit the action of Th1 and result in the deactivation of macrophages and the growth of intracellular parasites, exacerbating the disease progression.  Thus, murine models are used widely for the development of vaccines against Leishmania and characterization of the immune mechanisms and organ-specific immune responses.  Moreover, the BALB/c mouse model is widely used to determine the key components of Leishmania control as nitric-oxide (NO). 
The diagnosis of cutaneous leishmaniasis is difficult because of the varied symptoms and the different species involved. The procedures for the diagnosis of Leishmania are often invasive, and isolates are frequently difficult to grow in vitro with a high risk of contamination; especially to distinguish between species; which takes several weeks.  It is diagnosed by biopsy from skin lesions and/or cultures. These techniques are highly specific but not sensitive. , The microscopic identification of amastigotes depends on experienced laboratories, correct diagnosis, and characterization for evaluating prognosis and treatment. Hence, the methods of diagnosis that are sensitive to detect low levels of parasite and can distinguish between species could be of great value in different regions.  The polymerase chain reaction (PCR) is a specific and more sensitive test for the detection of low amounts of DNA in tissues and can be directly performed on host tissues without the need for culture. Hence, it is used for Leishmania typing. 
NO is produced from amino acid L-arginine by the cytokine-inducible NO synthase (iNOS) in different cell types. NO is very labile. The expression of iNOS is used to evaluate the NO production. Activated macrophages produce NO which is required for effective resolution and control of L. major infection and for maintaining life-long control of persisting Leishmania in clinically cured host. ,
This study aims to study the course and histopathology of L. major infection in certain tissues of experimentally infected BALB/c mice after sc and intraperitoneal (ip) inoculation. Evaluate kinetoplastic DNA PCR for the molecular detection of the parasite. Study the iNOS expression in different organs of infected animals during the first 3 months of infection and discuss their relation to the course and control of Leishmania.
| Materials and Methods|| |
Animals groups, parasite inoculum, and tissue sampling
L. major (MHOM/IL/81/FEBNI) strain is obtained from Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt.
The present study was carried out on laboratory-bred, parasite free and weaning male BALB/c mice 2 months old, weighing 25 g each. They were obtained and maintained in the animal house, Faculty of Medicine, Assiut University, Assiut, Egypt. The parasite was maintained by inoculation of amastigotes from infected positive splenic homogenate. Spleens from infected mice were homogenized in sterile phosphate-buffered saline; the splenic debris and intact spleen cells were removed by multiple centrifugations. The amastigote suspension was collected, and each animal received 10 7 parasites  counted by hemocytometer in 100 ul of RPMI medium. ,,,
Animals were divided into two groups with 12 mice each. Each group was divided into 4 subgroups (three mice each). Group I was inoculated subcutaneously at the base of the tail,  and Group II was inoculated intraperitoneally.  Eight healthy mice were inoculated with 0.1 ml of 0.9% saline solution, subcutaneously into the tail base and intraperitoneally as a negative control for immunohistochemistry.
The course of the experiment was 3 months, and three mice from each group and two mice of the negative control were sacrificed at certain time points: 2 weeks, 1, 2, and 3 months postinfection. The presence of the parasite in the spleen, mesenteric lymph nodes, liver, and skin ulcers were detected using qualitative PCR and iNOS expression from positive PCR samples was evaluated.
Polymerase chain reaction
The PCR analysis for Leishmania was done on all tissue samples. DNA extraction and purification was done using the QIAamp DNA mini spin columns kit from (Millipore Corporation) and then amplified by PCR.
The amount of tissue samples were determined (25 mg for skin, lymph nodes and liver tissue, 10 mg for spleen) and ground thoroughly with a mortar and pestle and tissue powder was decanted into 1.5 ml micro-centrifuge tube. Homogenates were mixed with 180 μl of buffer ATL and 20 μl of proteinase K and incubated at 56°C overnight until the tissue homogenates were completely lysed. 200 μl ethanol (96-100%) was added and mixed to each sample by pulse vortexing for 15 s. The mixture was centrifuged at 8000 rpm for 1 min. Then 500 μl washing buffer AW1 was added and centrifuged at 8000 rpm for 1 min. After that, 500 μl buffer AW2 was added and centrifuged at 14,000 rpm for 3 min. Finally, 200 μl buffer AE was added and incubated at room temperature for 1 min and then centrifuged at 8000 rpm for 1 min. The extracted DNA was stored at −20°C until it could be tested.
Polymerase chain reaction amplification
The PCR is used to amplify the variable area of the minicircle kinetic plastic DNA contained in the conserved sequence blocks 3 and 2, respectively of Leishmania in the tissue samples.  The forward LINR4 (5′-GGG GTT GGT GTA AAA TAG GG-3′) and reverse LIN17 (5′-TTT GAA CGG GAT TTC TG-3′) primers used have been designed within the conserved area of the minicircle kinetic plastic contained the conserved sequence blocks 3 and 2, respectively 2.5 μl of extracted DNA was added to 2 μl of forward and reveres primer mix with 8 μl of DNA se free water, and 12.5 μl of 2x green PCR Master Mix to prepare a final volume of 25 μl reaction mixture (The final concentration of DNA extract is 250 ng, 1 μM LINR4, 1 μM LIN17 Primers, and [1x] Go Taq Green Master mix).
The PCR was performed with an automated thermocycler; the annealing temperature was optimized to 50°C. A hot start procedure was used to increase specificity. After an initial denaturation (94°C for 5 min), 30 cycles (denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min) were carried out and PCR was terminated by a final extension at 72°C for 10 min, after which the reaction was stopped, and the amplified product was detected by electrophoresis on 1% agarose gel containing ethidium bromide and visualized on an ultraviolet transilluminator. Gel photographs were taken and specific amplified bands at 100 bp were looked. PCR positive control sample is positive splenic homogenate (taken from L. major infected mice obtained from Cairo). The negative control for PCR was by using distilled water instead of template DNA in the reaction. 
Sections were taken from the spleen, lymph nodes, skin ulcers, and liver. The samples were fixed in 10% formalin for 24 h at room temperature, embedded in paraffin and sectioned at 5 μm for conventional histopathological examination. Sections were stained with hematoxylin and eosin and Giemsa stains for a demonstration of Leishmania.
The presence of iNOS protein was analyzed by immunohistochemical staining using the avidin-biotin immunoperoxidase complex technique (Ultravision plus detection system anti-polyvalent HRP/DAB, ready to use, Thermo scientific Corporation Fremont, CA, USA). Immunohistochemistry was performed as a manufacturing protocol. Tissue sections (4-μm thick) of formalin-fixed, paraffin embedded specimens were deparaffinized, rehydrated in graded alcohol, and then endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in methanol for 5 min. For antigen retrieval, the slides were immersed in a citrate puffer and put in the microwave for 8 min. The samples then were incubated for 1 h at room temperature with iNOS (rabbit polyclonal antibody) (Thermo Scientific) at a dilution of 1:100. After the application of a secondary antibody, 3,3′-diaminobenzidine chromogen was added, and then sections were counterstained with hematoxylin. Negative control slides were done by omitting the primary antibody. Sections from the lung were stained as a positive control.
Evaluation of immunohistochemistry
Cytoplasmic iNOS expression was evaluated by the H-score. The stained slides were examined to identify the cellular localization of iNOS immunoreactivity and scored for intensity (0-3) and the percentage of positive-stained cells and values of iNOS intensity were multiplied by the percentage to provide a single iNOS score for each slide.
The collected data were analyzed by the program (SPSS; Statistical Package for Social Sciences) version 20 (IBM Enterprize, Armonk, New-York, USA). All values were expressed as mean ± standard deviation. The significance of differences between the groups was calculated using Kruskal-Wallis test. All reported P values were two-sided. P values were significant at (≤0.05) and highly significant at (P ≤ 0.001).
The experimental animals were held under specific pathogen-free conditions, in accordance with the international valid guidelines. They were maintained under convenient conditions in our institute.
| Results|| |
Evaluation of Leishmania major dissemination by polymerase chain reaction in murine model
In the first group (Group I), in which mice were inoculated subcutaneously at the base of the tail, the kinetoplastic DNA PCR technique detected the presence of L. major DNA at the edge of ulcers developed at the site of sc inoculation in 4 out of 12 mice (33%). All spleens, lymph nodes (100%), and 4 out of 12 (33%) of the examined livers were also positive by PCR [Table 1]. The first detectable sc lesion was around the 2 nd week as mice had tissue necrosis and loss, and then an open ulcer that progressed to a non-healing one over the course of infection (3 months) [Figure 1].
|Figure 1: BALB/c mice inoculated subcutaneously with Leishmania major, noteskin ulcers at the base of the tail|
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|Table 1: PCR detection of Leishmania major DNA in different tissues of experimentally infected mice by two different routes of inoculation|
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In the second group (Group II), in which mice were inoculated intraperitoneally, the kinetoplastic DNA PCR detected the presence of L. major DNA in all spleens, mesenteric lymph nodes (100%), and in 5 out of 12 (42%) of the examined livers [Table 1]. No skin lesions were detected. L. major was considered positive by PCR at 650 bp [Figure 2].
|Figure 2: Polymerase chain reaction amplification of kinetoplast DNA, the bands shown on 1.5% agarose gel stained with ethidium bromide. Lane 1 ladder, Lane 2 positive control, Lane 3 negative control, Lane 4, 5 spleen, Lane 6, 7 lymph node, Lane 8, 9 liver, Lane 10, 11 skin after subcutaneous and intraperitoneal inoculation respectively; positive samples at 650 bp|
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Microscopic examination of tissue sections from the spleen of both routes of inoculation showed capsulitis and thickening of splenic capsule. There was extensive infiltration of the red pulp and the interfollicular areas of the white pulp by chronic inflammatory cells in the form of histiocytes and plasma cells in addition to megakaryocytes and lymphoblasts that are caused by extramedullary hematopoiesis. Granulomas could be detected as aggregates of four or more epithelioid macrophages. Late in the course of the disease, the specimens showed low lymphoid follicle density with the absence of germinal centers and splenic architecture disruption [Figure 3]a and b. Microscopic examination of lymph nodes also showed capsulitis. There were follicular hyperplasia and infiltration of the cortex and medulla by plasma cells, and macrophages. Late in the course of infection, there was effacement of the lymph node by the mixed inflammatory cell infiltrate [Figure 3]c.
|Figure 3: (a) H and E staining of spleen showing capsulitis and extensive disorganization of splenic tissue (×200). (b) Showing infiltration of the spleen by macrophages, plasma cells and megakaryocytes (arrow) (×400). (c) H and E staining of lymph node showing capsulitis (arrow) and infiltration by plasma cells and histiocytes (×400). (d) H and E staining of the liver showing the presence of well-formed granuloma formed mainly of macrophages. Also hypertrophy/hyperplasia of kupffer cells (arrows) (×400)|
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Microscopic examination of liver sections showed evidence of inflammation of hepatic capsule. Interlobular granulomas were dispersed throughout the hepatic lobules. Intense hyperplasia/hypertrophy of hepatic kupffer cells were seen together with moderate periportal inflammatory cell infiltrate composed mainly of plasma cells, macrophages, and lymphocytes [Figure 3]d. Microscopic examination of skin sections after subcutaneously inoculation showed the presence of dermal perivascular and periadnexal chronic inflammatory cell infiltrate in the form of lymphocytes, plasma cells, and histiocytes.
Leishmania major inducible nitric-oxide synthase profile by immunohistochemistry
L. major iNOS expression was studied for all PCR positive samples (spleens, lymph nodes, and livers). In the negative control tissues, iNOS expression was not found. The iNOS showed cytoplasmic immunoreactivity in T lymphocytes, plasma cells, macrophages, and hepatocytes. However, B lymphocytes showed negative expression as evident by negative iNOS expression in the lymphoid follicles of spleen and lymph nodes.
iNOS expression could not be detected at 2 weeks postinfection in the spleens and lymph nodes, but it can be detected in positive PCR liver samples of mice inoculated by both routes. The mean of expression was high after 1 month for both routes in all samples taken from the spleens, lymph nodes, and livers, followed by a moderate decrease at 2 months while the lowest detected expression was after 3 months. Skin samples taken from the ulcers showed positive cytoplasmic iNOS expression in the dermal chronic inflammatory cells. Positive iNOS expression in the spleen, lymph nodes, and liver cells is illustrated in [Figure 4]a-c. There was a statistically significant difference in iNOS expression during the course of the experiment in the spleen and lymph nodes samples taken from both routes of inoculation (P = 0.017). However, there was a statistically significant difference in iNOS expression in hepatocytes during the course of the experiment in the ip route only (P = 0.035), but not in the sc route (P = 0.067) [Table 2]. Giemsa stain could demonstrate Leishmania amastigotes in and outside the macrophages in samples taken from the skin, spleen, lymph nodes, and liver [Figure 4]d.
|Figure 4: (a) Immunohistochemical expression of inducible nitric-oxide synthase in the spleen showing strong expression in the interfollicular area containing T-lymphocytes, plasma cells and macrophages and negative lymphoid follicle B lymphocytes (×400). (b) Lymph node showing strong inducible nitric-oxide synthase expression in lymphocytes, plasma cells and histiocytes (×400). (c) Strong inducible nitric-oxide synthase expression in hepatocytes (×400) with periportal inflammation (arrow). (d) Giemsastain of spleen showing macrophages contain Leishmania amastigotes (arrows) (×1000)|
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|Table 2: iNOS expression in different organs of experimentally infected mice by two different routes of inoculation|
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| Discussion|| |
The results obtained in our study proved that sc and ip routes were able to produce visceral leishmaniasis from the 2 nd week postinfection. As certain Leishmania strains possess the capacity to multiply in cutaneous and viscera in a susceptible mouse.  L. major and Leishmania infantum multiplies at the sites of inoculation and results in progressive visceral disease that indicate blood dissemination. , On the other hand, sc injection of Leishmania donovani amastigotes in BALB/c mice developed swelling in the popliteal lymph nodes but without splenomegaly. This difference may be due to different species.  sc inoculation in our study lead to nonhealing skin ulcers. 
In our study, the use of a high dose (10 7 ) of L. major amastigotes resulted in visceral dissemination. The parasite inoculum is critical in determining the direction of the immune response in mice and delayed or rapid development of the disease, as the use of (10 7 ) of Leishmania chagasi leads to a rise in parasite load and Th2 cytokine responses. On the other hand, a low dose (10 3 ) of the same parasite leads to a minimum infection with the increase of postinoculation days. 
In the present study, we used the amastigotes for inoculation. They are more infectious compared to promastigotes and the difference in infectivity is obvious as early as the first 24 h post inoculation. , We used mice of a single sex (male) and similar age (2 months) to minimize variability by host factors as males have a higher susceptibility to parasite dissemination and this susceptibility decreases slightly with age. 
Our results showed that regardless of the inoculation route (sc or ip), kinetoplastic DNA PCR could detect L. major in 100% of the spleens, mesenteric lymph nodes, skin ulcers, and 33% of the livers after the sc inoculation and 42% after the ip inoculation.  Moreover, the inoculation of BALB/c mice by the ip route with high parasite dose resulted in parasite loads close to that obtained by the endovenous route.  On the other hand, our results are in contrast to Oliveira et al.  who reported that the sc route was able to induce a quicker infection with high parasite load than the intravenous route. sc route of infection was the slowest route of infection in the BALB/c mice.  The ip route of inoculation results in an accuracy of infection more than the endovenous route, so this route should be used in experimental infections for immune studies. 
Diagnosis of cutaneous leishmaniasis was based primarily on clinical symptoms, microscopic examination of stained tissue smears, and/or culture from the tissue. These techniques are highly specific but not sensitive and insufficient to diagnose all cases, and limited by access to specialized laboratories. Due to the wide clinical spectrum of Leishmania and the variety of responses to treatment according to the parasite species, there is a need to find a highly sensitive method for disease diagnosis especially in the endemic regions for evaluating prognosis and appropriate treatment.  The use of PCR has become the preferred way and a "gold standard" diagnostic test for Leishmania.  Many researchers have reported 100% specificity with sensitivity 92-98% and 100% sensitivity, respectively. , It detects low amounts of DNA even in cases missed by either microscopic examination or culture. ,
The kinetoplastic DNA PCR is considered to be the most sensitive method for diagnosing cutaneous leishmaniasis including L. major in patients.  Moreover, the forward LINR4 and reverse LIN17 primers that were used in the present study can differentiate between three species of Leishmania according to the band site, Leishmania tropica at (760 bp), L. major at (650 bp), and L. infantum at (720 bp), respectively.  The ability to identify species is important in the prognosis and therapy especially in regions where more than one species and disease are seen by clinicians.  The period of this experiment was 3 months as PCR positivity of spleens decreases by 50% after 3 months.  The animals control infection in the spleen in the 5 th months postinfection with 40% reduction of the splenic parasite burden. 
In our study, the spleen and lymph nodes showed infiltration by macrophages and plasma cells mainly in the red pulp and interfollicular areas of the spleen and the medulla and interfollicular areas of lymph nodes. While late in the course of infection there was disorganization of splenic and lymph nodal architecture with the absence of lymphoid follicle germinal centers. During early infection of Leishmania, parasites localize in the marginal zone macrophages (MZMs) of the splenic white pulp. After a week, parasites are found in the macrophage-rich red pulp while late in L. donovani infection, the splenic architecture is disrupted with the loss of MZMs with progressive destruction of follicular dendritic cells and eventual loss of germinal centers. 
In our study, all infected groups after the first 2 weeks showed increase in the plasma cell population replacing other cell populations in areas of the red pulp of the spleen and medulla of lymph nodes. This is due to well-characterized polyclonal B-cell activation and production of cytokines and chemokines which may contribute to plasma cell differentiation and retention.  In our study, extramedullary hematopoiesis was evident in the spleen, as the infection increases the production of myeloid progenitors to produce a large number of phagocytic cells and antigen presenting cells to enhance the immune response. 
In the present study, the histopathology of liver sections after 1 month showed periportal plasmocyte/histiocyte/lymphocyte infiltration which reflects the polyclonal activation of the B cells characterizing visceral leishmaniasis.  Sections taken after 2 and 3 months revealed the presence of many granulomas in liver parenchyma. The resolution of disease in the livers of mice infected with L. donovani or L. chagasi correlates with the local formation of granulomas which are normally formed during disease regression and are poorly formed in immune-deficient murine and human hosts.  Such granulomas are formed by epithelioid cells, a small number of plasma cells and lymphocytes, and rare granulocytes, possibly containing amastigote forms inside the macrophages. 
In the present study, regardless of the inoculation route, the iNOS expression begins earlier in the liver (2 nd week) than in the spleen and lymph node and remains lower than them throughout the experiment (3 months). This indicates that the parasite burden is higher in the liver than in the spleen and lymph node, which represent the acute phase of infection. The liver is an indicator of the multiplication of parasites during the acute phase of infection. Parasites multiply in large numbers, but once the infection becomes chronic, hepatic parasite load decreases and the parasites in the spleen tend to increase slowly and can persist throughout the animal life. , NO production by activated macrophages is required for effective destruction of a wide range of pathogens such as viruses, bacteria, protozoa, fungi, and helminths.  Hence, we use PCR positive samples to make sure that this expression is due to L. major infection.
In our study, we detected iNOS expression in macrophages, plasma cells, lymphocytes, and hepatocytes only in the liver after 2 weeks and the strongest expression pattern was detected after 1 month and then decreased until the end of the experiment in all examined organs. sc infection of BALB/c mice with L. infantum, the hepatic parasite load reaches a peak between 20 and 30 days postinfection. Later on the parasites were cleared from this organ, indicating the existence of immune response mechanisms which lead to their elimination.  There is a variable degree of iNOS/NO-dependent control of Leishmania in the liver during the acute phase of infection. , This strong iNOS expression at 1 and 2 months indicates that the inflammatory cells are able to destroy and prevent multiplication of amastigotes to control infection. On the other hand, iNOS expression was absent in the first 32 weeks and low in the 3 rd month in the lymph node and spleen; this indicates that there may be numerous numbers of Leishmania amastigotes. NO produced by iNOS plays an important role in controlling the dissemination of the infection. Its failure in production facilitates multiplication and dissemination of Leishmania leading to severe visceral leishmaniasis.  The above-mentioned data proved that NO which is produced by iNOS plays an important role in controlling the dissemination of such infection.  Negative control cells in the present study showed no iNOS expression, suggesting that these cells were not activated. ,
In the present study, the iNOS expression in the spleen and lymph nodes was detected in the interfollicular cells which represent T cells. The iNOS expression was dependent on several factors including CD4 + T cells. , Moreover, the immunity to Leishmania is probably expressed by mononuclear phagocytes activated by lymphokines released from sensitized T lymphocytes. 
In our study, the iNOS expression was more prominent in the spleens, lymph nodes, than in the liver by both routes of inoculation.  On the other hand, early in L. major infected mice the iNOS was the most prominent in the skin, somewhat less impressive in the lymph node, and very weak in the spleen , and in Leishmania braziliensis infected mice. 
Sustained expression of (iNOS) was detected until the end of the experiment (3 months). iNOS expression still presents up to 12 months which indicate that mice are efficient reservoirs for transmission to sand flies even in the absence of reinfection.  In contrast, the iNOS expression was not present on 2 months and increased on 4 months in postinfected mice.  This difference is due to different route of inoculation.
Data herein clarify the usefulness of a drug or natural element that increases iNOS especially during the first 3 months of infection for proper control and elimination of this disease. The failure to maintain iNOS activity reactivates latent infection in immune suppression. Moreover, the administration of a potent inhibitor of iNOS in L. major-infected mice which have cured their cutaneous lesions led to a 10 4 -10 5 times increase of the parasite burden and caused clinical recrudescence of the disease.  iNOS should receive particular attention during further clinical studies, which will lead to new ideas for a new drug or vaccine targeting Leishmania, as there is still no vaccine against it. 
| Conclusion|| |
sc and ip routes of L. major inoculation were able to produce visceral leishmaniasis in mice. The kinetoplastic DNA PCR can be used for the detection of L. major in experimentally infected BALB/c mice from the 2 nd week up to the 3 rd month postinfection. The iNOS expression was strong at 1 and 2 months and remained throughout the 3 months of the experiment which plays an important role in the disease course and control.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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