Salivary gland antigens of laboratory-bred Phlebotomus sergenti and their immunogenicity in human volunteers in laboratory condition

To investigate Phlebotomus (P.) sergenti Parrot, 1917 (Diptera: Psychodidae) salivary gland antigens and their immune response in human. Methods: Human volunteers were exposed to sand flies' bites in the laboratory, and following each exposure the size of induration was recorded. The mean protein concentration of salivary gland lysate and specific anti-P. sergenti saliva IgG was measured. Sand fly salivary proteins were separated by SDS-PAGE and their immunoreactivity was examined by Western blotting assays. Results: Individuals exposed to P. sergenti salivary gland lysate for 8 months showed both antibody and delayed type hypersensitivity responses, although exposure for one month did not provoke any immune responses. The trend of antibody fluctuated during the exposure time and dropped by the end of antigen loading. The mean protein content was (0.36?0.08) ug in each pair salivary glands. Salivary gland lysate showed 11 to 12 major protein bands and 3 to 6 of them were immunoreactive. Conclusions: Our study showed that the salivary gland components of P. sergenti provoked both cellular and humoral immune responses in human. Furthermore, there are some immunogenic proteins in P. sergenti saliva which could be subjected for further investigation as vector-based vaccine candidate/s against anthroponotic cutaneous leishmaniasis.

Cutaneous Leishmaniasis Situation and Predicting the Distribution of Phlebotomus papatasi and P. sergenti as Vectors of Leishmaniasis in Ardabil Province, Iran

Cutaneous leishmaniosis (CL) is the most common form of leishmaniasis.CL caused by L. major and L. tropica is endemic in 17 provinces of Iran. This study was carried out to elucidate situation of CL in Ardabil province and to predict distribution of Phlebotomus papatasi and Phlebotomus sergenti (Diptera: Psychodidae) as vectors of CL in the region. In this cross-sectional study, data on CL patients were collected from local health centers of Ardabil province, Iran during 2006-2018 to establish a geodatabase using ArcGIS10.3. A total of 20 CL cases were selected randomly and skin samples were collected and analyzed by PCR method. MaxEnt 3.3.3 model was used to determine ecologically suitable niches for the main vectors. A total, 309 CL human cases were reported and the highest incidence rate of disease was occurred in Bilasavar (37/100,000) and Germi (35/100,000). A total of 2,794 sand flies were collected during May to October 2018. The environmentally suitable habitats for P. papatasi and P. sergenti were predicted to be present in northern and central areas of Ardabil province. The most variable that contributed ratio in the modeling were Isothermality and slope factors. Ardabil province is possibly an endemic are for CL. The presence of P. papatasi and P. sergenti justifies local transmission while the vectors of CL are existing in the northern and central areas of the province.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Cationic immune stimulating complexes containing soluble leishmania antigens: preparation, characterization and in vivo immune response evaluation

Background: Cationic immune stimulating complexes [PLUSCOMs] are particulate antigen delivery systems. PLUSCOMs consist of cationic immunostimulatory complexes [ISCOMs] derivatives and are able to elicit in vivo T cell responses against an antigen

Objective: To evaluate the effects of PLUSCOMs containing Leishmania major antigens [SLA] on the type of immune response generated in the murine model of leishmaniasis

Methods: PLUSCOMs consisting of 1, 2-dioleoyl-3-trimethylammonium-propane [DOTAP] were used as antigen delivery system/immunoadjuvants for soluble SLA. BALB/c mice were immunized subcutaneously, three times in 2-week intervals. Footpads swellings at the site of challenge and parasite loads were assessed as a measure of protection. The immune responses were also evaluated by determination of IgG subclasses and the level of IFN- gamma and IL-4 in cultured splenocytes

Results: There was no significant difference [p<0.05] between the sizes of lesions in mice immunized with different formulations. Also, there was no significant difference in the number of parasites in the footpad or spleen of all groups compared with the control group. The highest level of IFN- gamma secretion was observed in the splenocytes of mice immunized with PLUSCOM/SLA [p<0.001] and lower amounts of IL-4 was observed in PLUSCOM group [p<0.001] as compared to negative control

Conclusion: Our results indicated that SLA in different formulations generated an immune response with mixed Th1/Th2 response that was not protective enough despite the activation of CD4+ T cells with secreting IFN- gamma in groups which received PLUSCOM with antigen

Molecular identification of Leishmania tropica infections in patients with cutaneous leishmaniasis from an endemic central of Iran

The most common form of the disease is cutaneous leishmaniasis (CL) which is a public health and social problem in many countries especially Iran. In endemic areas where other diseases with similar clinical symptoms occur, definitive diagnosis of CL is very important. The detection and identification of Leishmania in infected patients is crucial for achieving a correct treatment and prognosis. To our knowledge, this is the first comprehensive study in terms of geographical distribution and molecular identification of Leishmania tropica isolates in central of Iran. This study was performed between 2010 and 2011, during which 218 CL suspected patients referred to Shahid Sadoughi University of Medical Sciences in Yazd, Iran for confirmation were examined. After microscopic analysis, DNA extraction was performed for identification. The molecular target region was ITS1 gene. Results showed that out of 218 isolates, 102 (46.8%) samples were positive for Leishman body using molecular assay. After PCR-RFLP, analysis identified 50 (49.01%) samples as L. major and 52 (50.98%) as L. tropica. Two samples showed a different pattern that were reported as unknown. Among L. tropica, six different isolates were identified in this endemic area. Finally, this study showed heterozygosity among L. tropica isolates in this endemic area such as some other studies from the world. This heterozygosity among the strains may suggest a sexual recombination or genetic exchange between strains.

P-glycoprotein a gene expression in glucantime-resistant and sensitive leishmania major [MRHO/IR/75/ER]

Leishmaniasis is a parasitic disease caused by different species of Leishmania parasites with a wide range of clinical manifestations. Antimonial compounds such as meglumine antimoniate [glucantime] are the first line drugs for the treat-ment of leishmaniasis. However, according to reports of the drug resistance of parasites, the efficacy of antimonial compounds is low. The ATP-binding cassette [ABC] proteins are present in all organisms and mediate the transport of vital elements through biological membranes. One of the important mechanisms of resistance in Leishmania parasites is the overexpression of ABC efflux pumps. P-glycoprotein A [pgpA] is a related gene for ABC transporter in Leishmania species. The aim of this study was to compare the pgpA expression in laboratory-induced resistant L. major [MRHO/IR/75/ER] and sensitive parasites. RNA extraction of promastigotes of sensitive and resistant clones was performed and total RNA was reverse transcribed. The real-time quantitative polymerase chain reaction [PCR] was used to assess RNA expression profiles and the expression levels were calculated using 2-deltaCt method. The mean expression level of pgpA mRNA was 2.70 +/- 0.51 in in sensitive Leishmania clone and 6.08 +/- 1.50 in resistant Leishmania clone [P = 0.021]. The expression of pgpA gene in resistant strains of L. major was almost fivefold higher than those in susceptible strains. Therefore, this can be used in field isolates, i.e. overexpression of the gene can prove resistance in wild type field isolates

Co-existence of cutaneous leishmaniasis with pleural effusion: a case report from Iran

Herein, a 12-year-old Afghan boy with chronic cutaneous leishmaniasis on the face and verrucous lesions on the body and pleural effusion suspected of having co-existent tuberculosis has been presented. The cutaneous lesions were appeared for five years before his admission. Leishman-Donovan bodies were seen in H and E [Hematoxylin and eosin] slide of skin lesion specimens. The pathogenic species was proved to be Leishmania tropica using Polymerase Chain Reaction [PCR] method. Purified Protein Derivative [PPD] and Leishmanin Skin Test [LST] were strongly positive. The patient was treated with systemic and intralesional meglumine antimoniate [Glucantime] for cutaneous leishmaniasis and then with anti-tuberculosis drugs for pleural effusion. Afterwards, pleural effusion was disappeared and cutaneous leishmaniasis cured

First molecular identification of Leishmania species in a new endemic area of cutaneous leishmaniasis in Lorestan, Iran

OBJECTIVE@#To identify Leishmania using PCR.@*METHODS@#This study was conducted from April 2009 to March 2011 in order to identify Leishmania species in a new endemic area of CL in Lorestan, Iran. Samples were taken from 62 patients that referred to the health centers in different cities of Lorestan province, the presence of Leishmania was confirmed using direct smear and then grown in NNN media and mass cultured in RPMI 1 640 medium supplemented with 10% heat-inactivated fetal bovine serum. DNA was extracted from cultured promastigotes and used in ITS-PCR.@*RESULTS@#45(72.6%) samples out of 62 showed a band in the range of 485 bp and 17 (27.4%) with a band in the range of 626 bp which were similar to standard strains of Leishmania tropica(L. tropica) and Leishmania major(L. major), respectively. 50 (65.80%) of samples were collected from people with no history of travel in at least a year prior to the onset which shows that indigenous source of infection.@*CONCLUSIONS@#Since the vector and reservoir of the two species are different, so precise and extensive control and prevention methods should be designed and carried out.

Evaluation of infection course in mice induced by L. major in presence of positively charged liposomes containing CpG ODN

An inoculation of virulent Leishmania major is known as leishmanization [LZ] which is proven to be the most effective control measure against Cutaneous Leishmaniasis [CL]. However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides [CpG ODN] as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously [SC] with L. major plus empty DSPC, DSPC [CpG ODN], DSPC [Non CpG ODN], empty DMPC, DMPC [CpG ODN], DMPC [Non CpG ODN] or HEPES buffer. The results showed that group of mice received DMPC [CpG ODN] nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC [CpG ODN] liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC [CpG] induced IFN-gamma in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC [CpG ODN] nanoliposomes might be a practical approach to improve the safety of LZ

Evaluation of a possible synergistic effect of meglumine antimoniate with paromomycin, miltefosine or allopurinol on in vitro susceptibility of leishmania tropica resistant isolate

Pentavalent antimonials are still the first choice treatment for leishmaniasis, but with low efficacy and resistance is emerging. In the present study, the effect of meglumine antimoniate [MA, Glucantime] combined with paromomycin, miltefosine or allopurinol on in vitro susceptibility of Leishmania tropica resistant isolate was evaluated. The drugs were obtained from commercial sources and diluents of each drug in medium were prepared on the day of experiment. J774 A.1 murine macro-phage cell lines were attached to the cultured on slide and incubated at 37 [degree sign]C with 5% CO[2] for 24 h. Then the stationary phase promastigotes were added to the cells and after 4 hrs of incubation different concentrations of MA, paromomycin, miltefosine or allopurinol were added and incubated for an additional of 72 h. Then the slides were dried and fixed with methanol, stained by Giemsa and studied under a light microscope. Drug activity was evaluated by assessing the macrophage infection rate and the number of amastigotes per infected macrophage was done by examining 100 macrophages. The experiment was done in triplicates. Various concentrations of MA along with paromomycin, miltefosine or allopurinol significantly inhibited [P<0.01] the proliferation of L. tropica amastigote stage in the macrophage cell line as compared with MA alone or positive control. Combination of Glucantime with paromomycin, miltefosine or allopurinol showed a synergistic effect on the clinical isolate of L. tropica in vitro. Use of combination therapy is a new hope and a logical basis for therapy of the patients with cutaneous leishmaniasis. Further investigations are needed to evaluate the therapeutic effects of these drugs on the CL patients

Cutaneous leishmaniasis in suspected patients referred to the center for research and training in skin diseases and Leprosy, Tehran, Iran from 2008 to 2011

Cutaneous leishmaniasis [CL] is a major health problem in many parts of Iran, although diagnosis of CL especially in the endemic area is easy, but treatment and management of the disease is a global dilemma. Diagnosis of CL in non-endemic area is not as simple as in endemic foci. In this study, the status and the proportions of CL induced by Leishmania major and L. tropica among CL suspected patients referred to the Center for Research and Training in Skin Diseases and Leprosy, [CRTSDL] during 2008 to 2011 are described. CL patients with suspected lesions were clinically examined. History of trip to zoonotic CL and/or anthroponotic CL endemic areas and the characteristics of their lesion[s] were recorded. Diagnosis of the lesion was done using direct smear microscopy, culture and conventional polymerase chain reaction [PCR]. A total of 404 [M=256, F=148] patients with 776 lesions were recruited and parasitologically examined. The results showed that 255 of the patients with 613 lesions; patients with lesion[s] induced by L. major=147 [M=63, 43%, F=84, 57%] and lesion[s] induced by L. tropica=108 [M=35, 32%, F=73, 68%]. History of travel to endemic area was not always correlated with isolated Leishmania species. Although travel history to endemic area is an important factor to be considered for diagnosis, but parasitological confirmation is necessary initiation of treatment

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Antioxidants and proinflamatory cytokines in the sera of patients with cutaneous leishmaniasis

Leishmania is a significant health problem in many parts of the world. Tumor necrosis factor [TNF] plays an essential role in Leishmania major infections. To study the pro-inflammatory cytokines and antioxidants in four groups of cutaneous leishmaniasis patients. 39 patients were divided into four groups of: 1] active [acute phase of treatment]; 2] non-healing [received treatment for almost two years without recovery]; 3] healing [recovered upon treatment]; and 4] healed [previously received treatment and achieved complete remission] patients. Serum levels of pro-inflammatory cytokines [IL-1B, TNF-alpha, IL-6] and serum antioxidant levels were measured by ELISA and FRAP assays, respectively. While serum antioxidant levels were elevated in the non -healing group, there was no difference among other groups of patients and healthy controls in this regard. Interleukin-1beta showed the highest level in the non-healing group followed by the other groups of patients. The mean serum IL-6 level was highest in the non-healing group, but showed no significant change in the other groups. TNF-alpha and IL-1beta levels were non-significantly elevated in the sera of active and non-healing patients. Pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 maybe related to the progression of leishmaniasis. Serum antioxidant levels maybe correlated with patient response to drug treatment.

[Comparison of efficacy of intralesional injection of meglumine anti-moniate once-weekly with twice-weekly in the treatment of anthroponotic cutaneous leishmaniasis in Mashhad: a randomized clinical trial]

Cutaneous leishmaniasis [CL] is endemic in Iran, where it is one of the most important health problems. Both anthroponotic CL [ACL] caused by L. tropica and zoonnotic CL [ZCL] caused by L. major are reported. Antimoniate derivatives as the standard therapy for CL need multiple injections and are not easy to tolerate for the patients. This study was conducted in Mashhad to compare the efficacy of weekly versus twice a week intralesional injections of meglumine antimoniate [MA] in the treatment of ACL. This randomized controlled trial was performed during 2006 to 2008 in Mashhad, Iran. Using computerized sequence of random numbers, participants were randomly allocated in the two arms of the study: one receiving weekly and the other receiving twice-a-week intralesional injections of MA. The lesion size, induration and healing rate were assessed, recorded and compared. Healing was defined as complete re-epithelialisation and disappearance of duration. A total of 252 suspected CL patients with 372 lesions were screened. 82 parasitologically proven cases with 121 lesions caused by L. tropica were included and 74 patients with 113 lesions completed the study. At 12[th] week after initiation of treatment, complete healing was observed in 38 out of 44 lesions [86.4%] in the group which received weekly intralesional MA injection. The median time-to-heal in this group was 36 days [95% confidence interval [CI]: 32.0-39.9]. Complete healing was recorded in 60 out of 69 lesions [86.9%] in the group which received twice a week intralesional injections of MA with a median time-to-heal of 25 days [95% CI:20.9-29.1]. While no significant difference was observed between the two groups in terms of complete healing rate [P=0.999], time-to-heal was significantly different between the 2 groups [P=0.003]. It seems that the effectiveness of twice-weekly intralesional injections of MA is similar to once-weekly regimen while the former regimen causes more rapid healing of lesions

[Identification of Malassezia species associated with pityriasis versicolor using PCR-RFLP]

Malassezia is a lipophilic and dimorphic fungus which has different species. Some of them can be found as natural flora on skin and in some conditions may cause pityriasis versicolor. The aim of this study was to identify Malassezia species associated with pityriasis versicolor in Iranian patients, using PCR-RFLP. In this study out of 65 patients with pityriasis versicolor to have pityriasis versicolor,isolates of 60 patients were positive. Malassezia species. using by PCR-RFLP. The Internal Transcribed Spacer 2 [ITS2] region was amplified by PCR employing the ITS3 and ITS4 primers and the restriction endonucleases AluI, BanI and MspAI were selected for producing distinct RFLP patterns. M. furfur [36.7%], M. globosa [30.0%], M. sympodialis [20.0%], M. slooffiae [8.3%], M. restricta [3.3%] and M. obtusa [1.7%] were the microorganisms responsible for the infection among participants. The M. sympodialis infection was strongly correlated with the female gender [P=0.02]. Our findings suggest that, the most common Malassezia species associated with pityriasis versicolor was M. furfur, followed by M. globosa

[Phenotyping of peripheral memory T cell subsets in cutaneous leishmaniasis]

The heterogenous population of memory T lymphocytes is distinguished based on surface markers and effector functions such as cytokine secretion. Recently, two subsets of memory T cells are defined by expression of chemokine receptor CCR7 and CD45RA designating as "central memory" T cells [TCM] and "effector memory" T cells [TEM]. The objective of this study was to evaluate the phenotype and function of these lymphocytes in healed cases of cutaneous leishmaniasis. The phenotype of lymphocytes were determined in blood samples of 13 volunteers with history of self healing cutaneous leishmaniasis [HCL] and in 6 healthy controls. No significant difference was found in memory T cell subsets between HCL volunteers and healthy controls using flow cytometry. However, following sorting of different memory subsets, a significantly higher proliferation was seen in cells of HCL volunteers comparing to the control group. A significantly higher IFN-gamma response in TEM and a significantly higher IL-2 response in TCM were observed in cell culture of HCL volunteers comparing controls. The responses were elicited when the cells were stimulate with SLA in vitro, it is concluded Leishunania-specific TEM and Leishmania-specific TEM subsets exist in HCL volunteers and since the volunteers with history of CL presumed to be protected against reinfection, it seems that both TCM and TEM play role in the protection against Leishmania infection in these individuals

[Comparison of the efficacy of weekly vs. twice a week intralesional injections of meglumine antimoniate in the treatment of anthroponotic cutaneous leishmaniasis: a randomized clinical trial]

Treatment of cutaneous leishmaniasis, especially when caused by L. tropica, is challenging. Meglumine antimoniate [Glucantime[R]] is used as the standard treatment, but multiple injectiond are necessary. The objective of this study was to compare the efficacy of weekly intralesional injections with twice weekly injections of Glucantime for the treatment of anthroponotic cutaneous leishmaniasis [ACL]. This randomized open clinical trial was conducted, in Bam, Kerman province, Iran. 96 eligible patients according to inclusion and exclusion criteria who were willing to participate were included. The included patients were randomly assigned into two groups, one group treated with weekly intralesional injections of Glucantime[R] and the other group treated with intralesional Glucantime[R] twice a week. Type and size of each lesion [induration, ulcer and scar] were recorded weekly. Complete healing was defined as complete re-epithelialization and absence of induration in all lesions and was considered as the primary outcome measure. A total of 48 patients completed the study; complete cure was seen in 24 of 27 [89%] patients who received weekly intralesional MA with a mean duration of healing equals to 70 +/- 10 days. Complete cure was seen in 24 of 31 [77%] patients who received intralesional MA twice a week, the mean duration of healing in the latter group was 58 +/- 5 days. There was no significant difference between the two groups [P=0.23]. It seems that the efficacy of intralesional injections of Glucantime[R] once a week is similar to efficacy of twice a week Glucantime[R] injections

[Efficacy of paromomycin ointment prepared in Iran [Paromo-U] against cutaneous leishmaniasis caused by leishmania major in mouse model]

Background: Considering the difficulties, adverse effects and unsuitable response to commonly used drugs; it is essential to find an alternative, particularly local treatment for cutaneous leishmaniasis [CL]

Objective: Determination of efficacy of topical paromomycin [Paromo-U ointment] against CL caused by L.major in mouse model

Patients and Methods: Skin lesions were created 1 to 3 months after inoculation of L.major promastigotes to the base of tail of small, white mice [Outbreed]. Then the mice were randomly divided to three groups including interventional [Treatment by Paromo-U], control 1 [Treatment by urea] and control 2 [Treatment by distilled water] groups. The mice were treated topically twice a day for 8 weeks

Results: The mean lesion diameter of the lesions prior to treatment in interventional and control groups [Urea and distilled water] were measured 10.9, 5.9 and 6.0 mm respectively and changed to 4.0, 12.7 and 14.3 mm 8 weeks after treatment. No Leishman bodies were observed within the lesions of interventional group, whereas they were seen in all control groups

Conclusion: Paromo-U ointment was effective in the treatment CL caused by L.major in mouse model

Effects of a nitric oxide releasing cream on cutaneous leishmaniasis in animal model

Leishmaniasis is a common disease endemic in some parts of Iran. Chemical or physical treatments or a combination of both are used for treatment of the disease. Nitric oxide [NO] is important for healing of leishmaniasis in human and animal. This study was designed to evaluate the benificial effects of a NO releasing cream on cutaneous leishmaniasis in an animal model. Balb/c mice were infected with Leishmania major by injecting promastigotes into the base of tails of mice to induce the lesion. Then the animals were divided into 3 groups [control, placebo and treatment]. Mice were treated with the drugs one time daily. The diameter of lesions were measured on days 0, 5, 10, 15, 20, 25 and 30 after the appearance of the lesions. Data were analyzed using one-way ANOVA test and p<0.05 considered as significant. The diameter of lesions were significantly reduced in 15, 20, 25 and 30 days in NO cream treated animals compared to control and placebo groups [P<0.05]. NO releasing compounds may be effective in the treatment of leishmaniasis

Evaluation of protectivity and immunogenicity of alum precipitated autoclaved Leishmania major vaccine mixed with Mycobacterium vaccae in the murine model of leishmaniasis

Leishmaniases represent a group of diseases caused by protozoan parasite of the genus Leishmania. Control strategies are not always effective, it seems that the sole control measure is to search for an effective vaccine. The objective of this study was evaluation of the rate of protection and immune response induction in Balb/c mice immunized with alum precipitated autoclaved Leishmania major [Alum-ALM] vaccine mixed with M. vaccae. Eleven groups of female 8-10 week old Balb/c mice were immunized subcutaneously [SC] three times, 21 days apart, with different doses of Alum-ALM mixed with different doses of either M. vaccae or BCG. The immunized animals and control group were challenged with 1 x 10 [6] L. major. Development and progression of leishmania infection were assessed by footpad swelling measurement at the site of challenge and parasite burden in lymph nodes. The immune responses of vaccinated animals were evaluated in vivo by leishmanin skin test [LST] and in vitro by measurement of cytokine [IFN-lamda and IL-4] levels in mononuclear cell culture] supernatants and titration of serum anti-leishmania antibodies [IgG and its sub-classes]. Footpad thickness measurment after challenge with live L. major showed no significant difference between immunized groups and control group. However, there were some prominent exceptional cases in the parasite burden titration in groups 1, 4, 6, and 8. Immunization with low dose of Alum-ALM mixed with M. vaccae or BCG induced IFN-lamda production, and diminished IL-4 level [in vitro], and caused a stronger LST response in a group that received BCG as an adjuvant. Mice that were immunized with high doses of Alum-ALM mixed with high doses of M. vaccae showed an increase in footpad thickness at the site of challenge and higher levels of IL-4 and IgGl. It seems that immunization of mice with a low dose of Alum-ALM mixed with M. vaccae as an adjuvant might induce a Thl type response. M.vaccae mixed with low dose of Alum-ALM has an inhibitory influence on the parasite burden in the infected tissues of mice. Also, BCG mixed with different doses of Alum-ALM induces a Thl immune response in Balb/c mice. Apparently, there is no significant difference in adjuvancity of BCG and M. vaccae. High dose of Alum-ALM mixed with high dose of M. vaccae induces a Th2 response