Characterization of bacteria expectorated during forced salivation of the Phlebotomus papatasi: A neglected component of sand fly infectious inoculums.

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors.

Geographical and Molecular Analysis of Haplotype Variations in Leishmania major Among Infected Iranian Phlebotomus papatasi.

PURPOSE: Leishmania major is main causative agent and Phlebotomus papatasi is only proven vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in Iran. Human leishmaniasis is mostly susceptible to climatic conditions and molecular variations of Leishmania parasites within sandflies. METHODS: L. major was analyzed based on geographical, environmental, climatic changes and haplotype variations within P. papatasi. Molecular tools and different geographical aspects were employed using Arc-GIS software for mapping the geographic distribution of samples and other statistics tests. Fragments of ITS-rDNA, k-DNA, and microsatellite genes of Leishmania were used for PCR, RFLP, sequencing, and phylogenetic analyses. RESULTS: Totally 81 out of 1083 female P. papatasi were detected with Leishmania parasites: 70 and five were L. major and L. turanica, respectively. Golestan and Fars provinces had the highest (13.64%) and lowest (4.55%) infection rates, respectively. The infection rate among female P. papatasi collected from gerbil burrows was significantly higher (15.15%) than animal shelters, yards, and inside houses (4.48%) (P < 0.0%). Microsatellite was more sensitive (22.72%) than k-DNA (18.8%) and ITS-rDNA (7.48%). More molecular variations of L. major were found in Isfahan province. CONCLUSIONS: Arc-GIS software and other statistics tests were employed to find Leishmania positive and haplotype variations among sand flies. Geographical situations, altitude, climate, precipitation, humidity, temperature, urbanization, migrations, regional divergences, deforestation, global warming, genome instability, ecology, and biology of the sand flies intrinsically, and the reservoir hosts and neighboring infected locations could be reasons for increasing or decreasing the rate of Leishmania infection and haplotype variations.

CRISPR-Cas Technology as a Revolutionary Genome Editing tool: Mechanisms and Biomedical Applications.

Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.

Low genetic heterogeneity of Leishmania major in different geographical regions of Iran.

To examine the genetic diversity of Leishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019-2021. The Leishmania ITS-rDNA gene was amplified and Leishmania sp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity of L. major in human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity of L. major was observed in Southwest/Southeast Iran (Hd: 0.104-0.286). The statistically Fst value indicated that L. major is not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.

Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice.

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

Corrigendum: Co-infection of Phlebotomus papatasi (Diptera: Psychodidae) gut bacteria with Leishmania major exacerbates the pathological responses of BALB/c mice.

[This corrects the article DOI: 10.3389/fcimb.2023.1115542.].

Evaluation of expression variations in virulence-related genes of Leishmania major after several culture passages compared with Phlebotomus papatasi isolated promastigotes.

Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycoprotein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×106 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10th to 15th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5th to 20th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.

Co-infection of Phlebotomus papatasi (Diptera: Psychodidae) gut bacteria with Leishmania major exacerbates the pathological responses of BALB/c mice.

Clinical features and severity of the leishmaniasis is extremely intricate and depend on several factors, especially sand fly-derived products. Bacteria in the sand fly's gut are a perpetual companion of Leishmania parasites. However, consequences of the concomitance of these bacteria and Leishmania parasite outside the midgut environment have not been investigated in the infection process. Herein, a needle infection model was designed to mimic transmission by sand flies, to examine differences in the onset and progression of L. major infection initiated by inoculation with "low" or "high" doses of Enterobacter cloacae and Bacillus subtilis bacteria. The results showed an alteration in the local expression of pro- and anti-inflammatory cytokines in mice receiving different inoculations of bacteria. Simultaneous injection of two bacteria with Leishmania parasites in the low-dose group caused greater thickness of ear pinna and enhanced tissue chronic inflammatory cells, as well as resulted in multifold increase in the expression of IL-4 and IL-1ß and a decrease in the iNOS expression, without changing the L. major burden. Despite advances in scientific breakthroughs, scant survey has investigated the interaction between micro and macro levels of organization of leishmaniasis that ranges from the cellular to macro ecosystem levels, giving rise to the spread and persistence of the disease in a region. Our findings provide new insight into using the potential of the vector-derived microbiota in modulating the vertebrate immune system for the benefit of the host or recommend the use of appropriate antibiotics along with antileishmanial medicines.

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR.

BACKGROUND: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. METHODS: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. RESULTS: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. CONCLUSIONS: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

Immunoinformatics Evaluation of a Fusion Protein Composed of Leishmania infantum LiHyV and Phlebotomus kandelakii Apyrase as a Vaccine Candidate against Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is a lethal parasitic disease, transmitted by sand fly vectors. Immunomodulatory properties of sand fly saliva proteins and their protective effects against Leishmania infection in pre-exposed animals suggest that a combination of an antigenic salivary protein along with a Leishmania antigen can be considered for designing a vaccine against leishmaniasis. Methods: Three different fusion forms of L. infantum hypothetical protein (LiHyV) in combination with Phlebotomus kandelakii salivary apyrase (PkanAp) were subjected to insilico analyses. Major Histocompatibility Complex (MHC) class I and II epitopes in both humans and BALB/c mice were predicted. Antigenicity, immunogenicity, epitope conservancy, toxicity, and population coverage were also evaluated. Results: Highly antigenic promiscuous epitopes consisting of truncated LiHyV (10-285) and full-length PkanAp (21-329) were identified in human and was named Model 1. This model contained 25 MHC-I and 141 MHC-II antigenic peptides which among them, MPANSDIRI and AQSLFDFSGLALDSN were fully conserved. LALDSNATV, RCSSALVSI, ALVSINVPL, SAVESGALF of MHC-I epitopes, and 28 MHC-II binding epitopes showed 60% conservancy among various clades. A population coverage with a rate of >75% in the Iranian population and >70% in the whole world was also identified. Conclusion: Based on this in-silico approach, the predicted Model 1 could potentially be used as a vaccine candidate against VL.

Accurate Identification of Leishmania Parasites in Sand Flies by Polymorphism Analysis of Cytochrome Oxidase Subunit 2 Gene Using Polymerase Chain Reaction and Quantitative PCR-High Resolution Melting Techniques in Iranian Border with Iraq.

Background: Firmly identification of Leishmania in Phlebotomus papatasi and understanding of natural transmission cycles of parasites in sand flies are important for treatment and local control. Methods: Modified and developed method of High Resolution Melting (HRM) as a preferable technique was employed to accurate identification of Leishmania in sand flies from Iranian border with Iraq, by targeting cytochrome oxidase II (COII) gene and designing suitable primers. PCR products cloned into pTG19-T vector, then purified plasmid concentration was measured at 260 and 280nm wavelength. The melting curve plots were generated and DNA sequences were analyzed using Sequencher 3.1.1, CLC Main Workbench 5.5, MEGA 6, DnaSP5.10.01 and MedCalc® version 13.3.3 soft wares. Results: Among about 3000 collected sand flies, 89 female Ph. papatasi were identified and two with L. major. In amplified fragment of COII gene among 611bp, 452bp had no genetic variations with low polymorphic sites (P= 0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). Leishmania major was discriminated in Ph. papatasi with 0.84 °C melting temperature (Tm) and unique curve based on thermodynamic differences was an important criterion using HRM technique. Conclusion: Subsequent war in Iraq made a high risk habitat for parasites transmission. It is important to discover accurate diagnostic procedures for leishmaniasis control.

An overview of the sand fly salivary proteins in vaccine development against leishmaniases.

Leishmaniases are a group of vector-borne parasitic diseases transmitted through the infected sand flies. Leishmania parasites are inoculated into the host skin along with sand fly saliva. The sand fly saliva consists of biologically active molecules with anticoagulant, anti-inflammatory, and immunomodulatory properties. Such properties help the parasite circumvent the host's immune responses. The salivary compounds support the survival and multiplication of the parasite and facilitate the disease progression. It is documented that frequent exposure to uninfected sand fly bites produces neutralizing antibodies against specific salivary proteins and further activates the cellular mechanisms to prevent the establishment of the disease. The immune responses due to sand fly saliva are highly specific and depend on the composition of the salivary molecules. Hence, thorough knowledge of these compounds in different sand fly species and information about their antigenicity are paramount to designing an effective vaccine. Herein, we review the composition of the sand fly saliva, immunomodulatory properties of some of its components, immune responses to its proteins, and potential vaccine candidates against leishmaniases.

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

ABSTRACT Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran.

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2-2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Species diversity and spatial distribution of CL/VL vectors: assessing bioclimatic effect on expression plasticity of genes possessing vaccine properties isolated from wild-collected sand flies in endemic areas of Iran.

BACKGROUND: Leishmaniasis is one of the ten most important neglected tropical diseases worldwide. Understanding the distribution of vectors of visceral and cutaneous leishmaniasis (VL/CL) is one of the significant strategic frameworks to control leishmaniasis. In this study, the extent of the bioclimatic variability was investigated to recognize a rigorous cartographic of the spatial distribution of VL/CL vectors as risk-maps using ArcGIS modeling system. Moreover, the effect of bioclimatic diversity on the fold change expression of genes possessing vaccine traits (SP15 and LeIF) was evaluated in each bioclimatic region using real-time PCR analysis. METHODS: The Inverse Distance Weighting interpolation method was used to obtain accurate geography map in closely-related distances. Bioclimatic indices were computed and vectors spatial distribution was analyzed in ArcGIS10.3.1 system. Species biodiversity was calculated based on Shannon diversity index using Rv.3.5.3. Expression fold change of SP15 and LeIF genes was evaluated using cDNA synthesis and RT-qPCR analysis. RESULTS: Frequency of Phlebotomus papatasi was predominant in plains areas of Mountainous bioclimate covering the CL hot spots. Mediterranean region was recognized as an important bioclimate harboring prevalent patterns of VL vectors. Semi-arid bioclimate was identified as a major contributing factor to up-regulate salivary-SP15 gene expression (P = 0.0050, P < 0.05). Also, Mediterranean bioclimate had considerable effect on up-regulation of Leishmania-LeIF gene in gravid and semi-gravid P. papatasi population (P = 0.0109, P < 0.05). CONCLUSIONS: The diversity and spatial distribution of CL/VL vectors associated with bioclimatic regionalization obtained in our research provide epidemiological risk maps and establish more effectively control measures against leishmaniasis. Oscillations in gene expression indicate that each gene has its own features, which are profoundly affected by bioclimatic characteristics and physiological status of sand flies. Given the efficacy of species-specific antigens for vaccine production, it is essential to consider bioclimatic factors that have a fundamental role in affecting the regulatory regions of environmentally responsive loci for genes used in vaccine design.

High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran.

BACKGROUND AND OBJECTIVES: The three old world Leishmania species i.e., L. major, L. tropica, and L. infantum are considered as potential etiological agents of the various clinical forms of leishmaniasis in Iran. Different species co-exist in some areas. Accurate differentiation between the species is essential for choosing an appropriate therapy. Conventional and gold standard methods for the detection and characterization of parasites are time-consuming, laborious, and have low sensitivity. A polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis has been employed for detection and species identification. Most of the studies suffer from the use of multiple targets and/or requiring more than one reaction to identify a single sample. The present study aimed to design a PCR method based on the amplification of kinetoplast DNA minicircles (kDNA) and HRM analysis of the amplicons for rapid discrimination of the three mentioned species. MATERIALS AND METHODS: DNA from reference strains including L. major, L. tropica, and L. infantum and fifty-eight strains subjected to PCR-HRM analysis targeting kDNA. All the samples were also analyzed by conventional kDNA-PCR. RESULTS: The PCR-HRM analysis allowed discrimination between the three Old World species. The normalized HRM curves for the amplicons of kDNA indicated a unique and repeatable melting plot for each species, even in combination with human and mouse genomic DNA. Conventional kDNA-PCR could not properly discriminate L. tropica from L. infantum. CONCLUSION: PCR-HRM analysis of kDNA proved to be fast and accurate for discrimination of L. major, L. tropica, and L. infantum.

Comparative evaluation of salivary glands proteomes from wild Phlebotomus papatasi-proven vector of zoonotic cutaneous leishmaniasis in Iran.

BACKGROUND: Zoonotic Cutaneous Leishmaniasis is increasing in the world and Phlebotomus papatasi as a proven vector was considered in different aspects for disease control. Sandfly saliva contains proteins which provoke host immune system. These proteins are candidates for developing vaccines. OBJECTIVES: The main purpose of this research was comparing evaluation of salivary glands proteomes from wild P. papatasi. Extracting these proteins and purifying of original SP15 as inducer agent in vector salivary glands from endemic leishmaniasis foci were other objectives. METHODS: Adult sandflies were sampled using aspirators and funnel traps from three endemic foci in 2017-2018. Each pair of salivary glands of unfed females was dissected and proteins were extracted using thermal shocking and sonication methods. Purification was performed through RP-HPLC. All equivalent fractions were added together in order to reach sufficient protein concentration. Protein content and profile determination were examined with SDS-PAGE. RESULTS: The protein concentration of whole-salivary glands of specimens was determined approximately 1.6 µg/µl (Isfahan) and 1 µg/µl (Varamin and Kashan). SDS-PAGE revealed 10 distinct bands between 10 and 63 kDa. Analysis of proteomes showed some similarities and differences in the chromatograms of different foci. SDS-PAGE of all collected fractions revealed SP15-like proteins were isolated in 24 min from Varamin, 26 to 30 min from Kashan and 29.4 min from Isfahan and were around 15 kDa. CONCLUSIONS: Isolation of salivary components of Iranian wild P. papatasi is very important for finding potential proteins in vaccine development and measuring control strategy of zoonotic cutaneous leishmaniasis in Iran and this could be concluded elsewhere in the world.

Cloning, high-level gene expression and bioinformatics analysis of SP15 and LeIF from Leishmania major and Iranian Phlebotomus papatasi saliva as single and novel fusion proteins: a potential vaccine candidate against leishmaniasis.

BACKGROUND: Early exacerbation of cutaneous leishmaniasis is mainly affected by both the salivary and Leishmania parasite components. Little is known of the vaccine combination made by immunogenic proteins of sandfly saliva (SP15) with Leishmania parasites (LeIF) as a single prophylactic vaccine, namely SaLeish. Also, there are no data available to determine the species-specific sequence of SP15 isolated from the Iranian Phlebotomus papatasi. METHODS: Integrated bioinformatics and genetic engineering methods were employed to design, optimize and obtain a vector-parasite-based vaccine formulation in a whole-length fusion form of LeIF-SP15 against leishmaniasis. Holistic gene optimization was initially performed to obtain a high yield of pure 'whole-SaLeish' expression using bioinformatics analyses. Genomic and salivary gland RNAs of wild-caught P. papatasi were extracted and their complementary DNA was amplified and cloned into pJET vector. RESULTS: The new chimeric protein of whole-SaLeish and randomly selected transcripts of native PpIRSP15 (GenBank accession nos. MT025054 and MN938854, MN938855 and MN938856) were successfully expressed, purified and validated by immunoblotting assay. Furthermore, despite the single amino acid polymorphisms of PpIRSP15 found at positions Y23 and E73 within the population of wild Iranian sandflies, antigenicity and conservancy of PpIRSP15 epitopes remained constant to activate T cells. CONCLUSIONS: The SaLeish vaccine strategy takes advantage of a plethora of vector-parasite immunogenic proteins with potential protective efficacy to stimulate both the innate and specific cellular immune responses against Leishmania parasites.

Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis.

BACKGROUND: The characterization of Leishmania species is important for clinical management of the diseases and the epidemiological control of the parasite distribution. Most of the published polymerase chain reaction (PCR) amplification methods lack the ability to identify all species complexes, have low performance and usually need post-PCR procedures. There is a need for improving the diagnosis of CL by development of an accurate affordable PCR method to identify all Leishmania species in clinical specimens. METHODS: We designed an optimized a PCR amplifying the internal transcribed spacer 2 sequence of the ribosomal RNA gene (ITS2) aligned from different strains of CL-causing Leishmania species in the Old World. The performance of the method was evaluated on lesion samples from several CL suspected patients and the limit of detection (LOD) was determined on DNA of promastigotes from reference strains. RESULTS: The universal PCR enabled simultaneous discrimination of L. major, L. tropica and L. infantum using one primer pair in one reaction. Stained smear microscopy and Novy-MacNeal-Nicolle (NNN) medium culture alone detected 77.27% (17/22) and 72.73% (16/22) of the positive CL samples, respectively. The PCR assay showed 100% sensitivity (22/22) (95% CI: 84.56-100%) and 100% specificity (3/3) (95% CI: 29.24-100%) for species identification on isolates from lesion scraping/exudate and 100% sensitivity (13/13) (95% CI: 75.29-100%) and 100% specificity (11/11) (95% CI: 71.51-100%) for species identification on biopsy samples of CL patients, while being capable to successfully amplify as little as 0.01-0.1 pg of Leishmania DNA from cultured promastigotes. CONCLUSIONS: We present a validated easy-to-use affordable universal PCR assay to identify the most common Old World Leishmania species causing CL. This PCR assay could be used as a sensitive/specific technique to diagnose CL-causing Leishmania species in clinical samples with high accuracy.

Designing and developing a high-resolution melting technique for accurate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real-time PCR and in silico genetic evaluation.

Discrimination, accurate identification, and reliable techniques are required for accurate identification of Leishmania parasites. High-resolution melting (HRM) is recognized as an authentic and exact method. The main objective of this research was optimizing HRM analysis for detecting and screening Leishmania major, Leishmania tropica and mix infections. Thirty-six DNA samples of Leishmania parasite were prepared and analyzed. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were targeted and six pairs of specific new primers were designed. Bioinformatics analysis was employed to predict DNA temperature resolution for each species and compared with in-vitro results. The genetic diversity of the selected gene regions was analyzed using PCR-sequencing method and DnaSP 5.10.01 software. They were submitted in GenBank (KU680818- KU680821 and KY041643- KY041649). The haplotype diversity for both AAP3 and COII genes was 96% and 87%, respectively. Tajima's D index was 0.65 for AAP3 and 0.36 for COII. CLC Genomics Workbench 11 software predictions were significant and close to these findings. The designed primers could be able to identify at least two Leishmania species. Temperature variations in HRM technique separated Iranian Leishmania parasites of L. major, L. tropica and mix infections. The target genes and our modified HRM method proved this technique could be useful in both clinical and experimental settings. Also, it can be effective for detecting Leishmania parasites in different hosts such as humans, reservoir hosts and vectors. Indeed, HRM can be used as a technique in Leishmania identification as well as for ecological and epidemiological research.