Análises computacionais para o estudo da fumarato hidratase como potencial alvo para o desenvolvimento de fármacos leishmanicidas / Computational analysis for the study of fumarate hydratase as a potential target for the development of leishmanicidal drugs

A leishmaniose é um problema de saúde pública em diversas partes do mundo, devido a sua ampla distribuição e alta prevalência. Os principais fatores de risco resultantes de processos sociais, econômicos e ambientais facilitam a transmissão e dificultam seu controle. A infecção causada por parasitas do gênero Leishmania pode causar no ser humano um conjunto de sintomas. O tratamento atualmente empregado é extremamente tóxico, pode produzir resistência e possui alto custo para o paciente, o que limita a sua utilização em áreas endêmicas. Desta forma, é de grande relevância a identificação de novos alvos terapêuticos com importância crítica na sobrevivência do parasito visando ao desenvolvimento de novos fármacos mais eficazes e menos agressivos para os seres humanos. Neste contexto, a enzima fumarato hidratase (FH) surge como um alvo molecular promissor, uma vez que a FH de L. major e de H. sapiens são consideradas análogas funcionais

Estudos recentes mostram a importância desta enzima para a viabilidade dos parasitos, o que a torna um alvo potencial para o planejamento de compostos com ação leishmanicida. O mecanismo de ação dessa enzima no parasito é diferente da atividade no organismo humano e, sua inibição prejudicaria os processos essenciais para a sobrevivência do parasito. A comparação entre as sequências da fumarato hidratase de classe I entre algumas espécies do gênero Leishmania apontou que os resíduos do sítio catalítico permanecem totalmente conservados, o que sugere a possível inibição da enzima para várias espécies desse gênero. A análise da estrutura da enzima do parasita e do hospedeiro mostrou diferenças nos resíduos catalíticos envolvidos na reação, reforçando a ideia de uma possível inibição específica. (AU)

N-Myristoyltransferases as antileishmanial targets: a piggyback approach with benzoheterocyclic analogues

Leishmaniasis is one of the neglected diseases that remain in need for pharmacological alternatives. In this context, N-Myristoyltransferases (NMT) arise as interesting targets to explore since they are involved in the co/post-translational processing of peptides which are responsible for host cell invasion. Studies that consider these enzymes as targets point out the potential of benzoheterocyclic compounds as inhibitors of Candida albicans's N-myristoyltransferase. Here we applied a combination of comparative binding site analysis and molecular docking studies based on a Piggyback approach in the search for new Leishmania major NMT ligands. Our results revealed that NMT enzymes from both pathogens present enough structural similarity to allow extrapolation of the knowledge available from C. albicans studies to develop new L. major NMT inhibitors. Molecular docking studies with benzoheterocyclic analogues indicate the potential of benzothiazole derivatives as L. major NMT ligands, giving rise to a completely new class of chemical compounds to be explored in the development of antileishmanial drugs.

Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran

ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania) major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis

BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

Evaluation of the Interventional Approaches in the Management of Cutaneous Leishmaniasis in Jazan: An Observational Study

BACKGROUND: Cutaneous leishmaniasis is a tropical infection of public health importance. Numerous treatment approaches are in practice with variable degree of success however its management has no universal consensus or practice guidelines to follow. OBJECTIVE: Analyze the management of cutaneous leishmaniasis retrospectively at a central hospital of Jazan Province, Kingdom of Saudi Arabia to identify the current treatment pattern and compare the outcomes. METHODS: This cross-sectional study was conducted based on the hospital records of patients who attended the dermatology clinic for cutaneous leishmaniasis during the year 2012 to 2015. RESULTS: Forty three patients were included in the study. There was a male preponderance (65.1%) among the patients and 60.5% of them were of pediatric age group. Monotherapy was the initial choice for 58.1% of the patients. Intralesional sodium stibogluconate (SS-IL) was the most preferred treatment for initial therapy, as monotherapy and as part of combination therapy. A complete response was achieved in 22 patients (51.2%) with initial therapy. Among the different treatment groups, SS-IL+itraconazole showed significantly higher complete response rate compared to other treatments offered as initial therapy (p<0.01). Initial SS-IL monotherapy provided complete response in 41.2% patients receiving it, while itraconazole monotherapy provided complete response in 75% and 90.9% of the patients receiving initial itraconazole+SS-IL combination therapy with achieved complete response. CONCLUSION: The findings and observations suggest that initial combination therapy with SS-IL+itraconazole significantly improved the complete response rates and thus reduced the need for additional or prolonged therapies.

Epidemiological Aspects of Cutaneous Leishmaniasis during 2009–2016 in Kashan City, Central Iran

Cutaneous leishmaniasis (CL) can be seen in 2 forms, zoonotic and anthroponotic, in Iran. In this study, epidemiological aspects of CL were studied during an 8-year period (2009–2016) in city of Kashan, central Iran. The demographic and epidemiological data, including age, sex, occupation, number and site of the lesions, treatment regimen, past history of CL, and season of all patients were gathered from the health centers. Descriptive statistics were used to describe features of the study data. Total 2,676 people with CL were identified. The highest annual incidence was estimated to be 182 per 100,000 population in 2009 and the least was in 2016 (47 per 100,000 population). The highest frequency affected age groups were observed in 20–29 year-old patients (20.9%). More than 51% of the patients were under 30 years old. The maximum frequency of the disease, 1,134 (43.3%), was seen in autumn. The most common location of lesions was hands (61.4%). Most of the patients (81.6%) were treated by systemic glucantime regimen. In the city of Kashan, the incidence rate of the CL disease is significantly higher than many other regions of Iran. To reduce the risk of disease, control of reservoir hosts and vectors of disease, and education of individual protection are strongly recommended.

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.

Imported cutaneous leishmaniasis caused by Leishmania major in a Chinese laborer who worked in Saudi Arabia

Abstract: We report an imported case of cutaneous leishmaniasis in a 37-year-old man from Saudi Arabia caused by Leishmania major. He presented with non-healing nodulo-ulcerative lesions with a "volcanic crater" on the lower limbs. It was clearly cutaneous leishmaniasis - a rare disease in China - as reflected by the patient's clinical history, the lesions' morphology, histopathological examination, culture and PCR analysis of the lesions. The patient was completely cured after two cycles of sodium stibogluconate treatment. This case report demonstrates that dermatologists should be aware of sporadic cutaneous leishmaniasis cases in non-endemic areas.

Cutaneous Leishmaniasis of the Eyelids: A Case Series with Molecular Identification and Literature Review

Cutaneous leishmaniasis (CL) is a protozoan disease which is endemic in Iran. It is transmitted by the Phlebotomus sand fly. The eyelid is rarely involved possibly because the movement of the lids impedes the sand fly from biting the skin in this region. Here, we report 6 rare cases of eyelid CL. The patients were diagnosed by skin scraping, culture, and PCR from the lesions. Skin scraping examination showed Leishmania spp. amastigotes in the cytoplasm of macrophages. Culture examination was positive for Leishmania spp. PCR was positive for Leishmania major and Leishmania tropica. The lesions were disguised as basal cell carcinoma, chalazion, hordeolum, and impetigo. The patients were treated with intramuscular meglumine antimoniate (20 mg/kg/day) for at least 3 weeks. They showed a dramatic response, and the lesions almost completely disappeared. We emphasized the importance of clinical and diagnostic features of lesions, characterized the phylogenetic relationship of isolated parasites, and reviewed the literature on ocular leishmaniasis.

Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 µg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 µg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-gamma/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

The existence of only one haplotype of Leishmania major in the main and potential reservoir hosts of zoonotic cutaneous leishmaniasis using different molecular markers in a focal area in Iran

Introduction Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species. Methods All rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification. Results Of 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmania infections (9/55) were found. In addition, eight Meriones libycus and two Tatera indica were sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculus were sampled with no infection. Conclusions The results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major (GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations. .

In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves / 中国天然药物

AIM@#The anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves against extracellular (promastigote) and intracellular (amastigote) forms of Leishmania major were evaluated in this study.@*METHOD@#In the first stage, promastigote forms of L. major, were treated with different doses of the plant extracts in a 96-well tissue-culture microplate and IC50 values for each extract were measured with colorimetric MTT assay. In the second stage, macrophage cells were infected with L. major promastigotes. Infected macrophages were treated with plant extracts. Then the macrophages were stained with Gimsa and the number of infected macrophages and amastigotes were counted with a light microscope.@*RESULTS@#The results indicated that the plant extracts inhibited the growth of promastigotes and amastigotes of L. major. Inhibitory concentrations (IC50) for promastigote assay were 108.19, 155.15, and 184.32 μgmL(-1) for C. officinalis flowers, D. stramonium seeds and S. officinalis, respectively. The extracts also reduced the number of amastigotes in macrophage cells from 264 for control group to 88, 97, and 102 for test groups. Although the anti-leishmanial activity of the extracts were not comparable with the standard drug, miltefosine; but they showed significant efficiency in reducing the number of amastigotes in macrophages, in comparison with the control group (P < 0.001). These plant extracts had lower toxicity compared with miltefosine.@*CONCLUSION@#This study demonstrates the potential efficacy of the methanolic extracts of C. officinalis flowers, D. stramonium seeds, and S. officinalis leaves to control of cutaneous leishmaniasis.

Experimental effect of feeding on ricinus communis and bougainvillea glabra on the development of the sand fly phlebotomus papatasi [diptera: psychodidae] from Egypt

Plants are promising sources of agents useful for the control of vectors of human diseases including leishmaniasis. The effect of Ricinus communis [Euphorbiaceae] and Bougainvillea glabra [Nyctaginaceae] on transmission of leishmaniasis was investigated using them as diets for Phlebotomus papalasi to monitor their effect on life-history traits. P. papatasi were allowed to feed separately on both plants then offered a blood-meal. Fed-females were observed daily for egg-laying and subsequent developmental stages. P. papatasi was able to feed on B. glabra [29.41% females and 46.30% males] and R. communis [5.80% females and 10.43% males]. 34.28% of females died within 24-48 hours post-feeding on R. communis, whereas, it was 16.5% in females fed on B. glabra. Overall fecundity of surviving females was reduced compared to controls, reared on standard laboratory diet; however there was no effect on the sex ratio of progeny. Female P. papatasi in the control group had significantly longer life span compared to plant-fed group. Feeding on these plants not only decreased sand fly survival rates but incurred negative effects on fecundity. Findings indicate that planting high densities of R. communis and B. glabra in sand flies-endemic areas will reduce population sizes and reduce the risk of Leishmania major infections

Avaliação do papel do receptor marco na infecção de macrófagos murinos por Leishmania major / Evaluation of the role of the receptor in March infection of murine macrophages with Leishmania major

Camundongos CBA são resistentes à infecção por Leishmania major e permissivos à infecção por L. amazonensis. Adicionalmente, macrófagos de camundongos CBA controlam à infecção por L. major, mas não por L. mazonensis in vitro. Em estudo comparativo realizado por nosso grupo foi demonstrado que o receptor scavenger MARCO teve expressão aumentada em resposta à infecção por L. major, mas não na infecção por L. amazonensis. Ainda, o bloqueio do receptor com o anticorpo específico reduziu a infecção por L. major em 30%, indicando que esta proteína tem participação no reconhecimento de promastigotas de L. major em macrófagos de CBA. Assim, nossa hipótese é que o receptor MARCO participa do reconhecimento e fagocitose de L. major por macrófagos, direcionando o curso da infecção. O objetivo do presente estudo consistiu em evidenciar o papel do receptor MARCO na infecção de macrófagos por L. major. Inicialmente, células J774 foram transfectadas com os vetores pcDNA3.1-MARCO (J774-MARCO) ou pcDNA3.1 (J774-MOCK)...

CBA mice are resistant to Leishmania major yet permissive to L. amazonensis infection. In addition, CBA macrophages control L. major, but not L. amazonensis infection in vitro. In a comparative study performed by our group increase in expression of the scavenger receptor MARCO has been detected in response to L. major, but not to L. amazonensis infection. Moreover, ED31 monoclonal antibody against MARCO reduced by 30% L. major infection in CBA macrophages. These findings indicate that MARCO plays a role in L. major recognition by CBA macrophages. We hypothesized that MARCO receptor participates in the recognition and phagocytosis of L. major by macrophages, directing the outcome of infection. In the present study, we aimed to further disclose the role MARCO plays in L. major infection of murine macrophages. First J774 cells were transfected with pcDNA3.1-MARCO vector (MARCO-J774) or pcDNA3.1 vector (MOCK-J774)...

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.

The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

Cutaneous leishmaniasis caused by Leishmania major in Morocco: still a topical question

In the past decade in Morocco, there has been a reactivation of zoonotic cutaneous leishmaniasis [ZCL] foci with an unusual long outbreak episode. The aims of this review article were to update our knowledge of this disease in Morocco, to compare the situation with that prevailing in the other countries of the Maghreb region and to highlight factors that could be responsible for the current situation. The data indicate a global increase of ZCL incidence in the other Maghreb countries. Several factors could be influencing this increase but most of them will depend on human activities. On the other hand, ZCL is particularly sensitive to climate changes acting on the vector and the reservoir. An analysis of prevention strategies against the disease in Morocco and the other Maghreb countries highlights the need for integrated regional control measures in all the countries of the region

Monarch-1 activation in murine macrophage cell line [j774 a.1] infected with Iranian strain of leishmania major

Leishmania major is an intracellular parasite transmitted through the bite of the female phlebotomine sand flies. Leishmania major is able to escape the host immune defense and survive within macrophages. Modulation of the NF-kappaB [Nuclear Factor-Kappa B] activation and suppression of the pro-inflammatory cytokines by L. major are the main evasion mechanisms that remain to be explored. This study aims to examine the expression level of the Monarch-1 in L. major-infected macrophages, as a negative regulator of the NF-kappaB activation. Murine macrophage cell line [J774 A.1] was infected by metacyclic form of Leishmania promastigotes at macrophage/parasite ratio of 1:10. After harvesting infected cells at different times, total RNA was extracted and converted to cDNA. Semi-quantitative RT-PCR was performed for Monarch-1 by specific primers. Hypoxanthine Phospho-Ribosyl Transferase [HPRT] was used as an internal control to adjust the amount of mRNA in each sample. Semiquantitive analysis of Monarch-1 mRNA expression level showed a significant expression increase within 6 to 30 hours after L. major infection of macrophages when compared to the control macrophages. Monarch-1 expression level reveals a significant increase in the early phase of macrophage infection with L. major, which in turn may suppress IL-12 production in Leishmania infected macrophages and deeply influence the relationship between host and parasite.

Molecular detection and conventional identification of leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in fars province, south of Iran

The objectives of our research were to search for Leishmania species in rodents in Fars province, south of Iran, and to compare molecular with conventional methods for detecting these parasites. Rodents were captured using live traps and screened for Leishmania species using molecular and conventional methods, including the taking of smears from each ear. Nested PCR was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania [ITS1- 5.8S rRNA-ITS2] that is species-specific by DNA sequence. Totally, 122 rodents were captured. Leishmania parasites were detected using the nested PCR and three conventional methods [direct smear, NNN culture and Balb/C inoculation. 41 [33.6%] out of 122 rodents had Leishmania infections [34 Meriones lybicus and 7 M. persicus]. All PCR products of the ITS-rDNA gene were sequenced. Sequence analysis revealed that 28 out of 41 positive samples were Leishmania major. Thirteen sequences were unreadable and therefore not identified. At least two gerbil species common in Fars ZCL foci, M. lybicus and M. persicus, are acquiring infections of L. major and may be reservoir hosts of one predominant parasite haplotype. Most infections were detected molecularly not by conventional methods, because most rodents died in the traps