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.

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.

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.

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.

Effect of Leishmania gerbilli injection on mice immunization against cutaneous leishmaniasis (CL) caused by Leishmania major.

In the present study Leishmania gerbilli were used to immunize BALB/c mice against pathogenic strains of leishmania to determine whether injection of L. gerbilli in mice could protect them against later L. major inoculation. Eighty female BALB/c mice were divided by random in eight groups. Promastigotes of L. major and L. gerbilli were used. Mice were inoculated with three different doses of L. gerbilli (3 x 10(6), 2 x 10(7) and 5 x 10(7)) via subcutaneous (SC) in the base of their tails or interpretoen (IP). Forty days after the first injection, all mice received the same doses as a booster. Two control groups received PBS (SC or IP) only. All BALB/c mice were inoculated subcutaneously with 2 x 10(6). Promastigotes of L. major in the base of their tails after 75 days of the first injection of L. gerbilli. When leishmania lesion developed (35 days after challenge), the size was measured and continued once a week for 12 weeks. Meanwhile, the liver and spleen samples of dead mice moved to culture media and examined for the parasite. Delayed Type Hypersensitivity (DTH) and immunoflurecent tests were used to determine results of immunization. Compared with the control group and the other groups that received different doses of L. gerbilli via IP, an evident decrease in lesion size was observed in group that received 2 x 10(7) promastigotes (p < 0.05). By contrast, in those groups received L. gerbilli subcutaneously, no difference was observed through the different doses of inoculated parasite. Comparison of the inoculation styles showed that IP method caused smaller lesions than SC (p < 0.05).