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Abstract Background Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. Material & methods The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. Results All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. Conclusion Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Background: The viability and persistence of engineered bacterium candidates in field conditions is one of the consid­erable challenges in the paratransgenesis approach to fighting vector-borne diseases. Methods: In this study two engineered bacterium candidates to produce paratransgenic sand flies, Serratia AS1 and Enterobacter cloacae expressing m-Cherry fluorescent were applied on the leaves of the white saxaul plant (Haloxylon persicum), sugar bait, and rodent burrow soil and their persistent time was tested in desert condition, Matin Abad Coun­ty, Isfahan, August 2022. A PBS suspension of 109 cells/ml was used for sugar bait, spraying on plant leaves (~10 cm2) and 10 cm2 of rodent burrow soil. Sand fly samples were taken daily and were plated on LB Agar and the fluorescent cells were counted after 24 hours. Results: Time course in general caused a decrease in the number of bacteria for both strains. The two strains were per­sistent in sugar bait and on plant leaves for four days and on soil for two days. Although there were slight differences between the number of the bacteria in sugar baits, which was not significant (P< 0.05). The number of E. cloacae sur­viving on plant and in soil were significantly (P< 0.0001 and P= 0.046) higher than Serratia AS1. Conclusion: This study shows that plants or sugar bait are useful routes for delivery of the transformed bacteria for the paratransgenesis approach, although, the bacteria ought to be sprayed on plants or sugar baits should be replaced with new ones in four days intervals.

Background: The saliva and salivary glands of ticks possess a wide range of immuno-pharmacologically active mole­cules that effectively modulate the activity of enzymes, antibodies, and amines that have a role in different biological processes. Derived components from saliva and salivary glands of hard ticks Ixodidae have been characterized as poten­tial natural sources for discovering promising anti-cancer drug candidates. Methods: The anti-cancer activity of salivary gland extracts (SGEs) from Hyalomma anatolicum, Hyalomma drome­darii, Hyalomma marginatum, and Hyalomma schulzei was assessed. MTT assays and flow cytometry were done on the HT-29 colorectal cancer cell line to evaluate the anti-viability and proliferative inhibition. Results: Based on the MTT assay results, the SGEs from Hy. dromedarii had the highest and lowest substantial anti-viability effects on the HT-29 cancer cell and human foreskin fibroblast (HFF) normal cell, respectively. The cytometric assessment revealed a significant increase in the apoptosis and necrosis ratio of the HT-29 cancer cells after treatment with Hy. dromedarii SGEs. Conclusion: The results demonstrated that Hy. dromedarii SGEs have significant anti-proliferative, anti-viability, and apoptotic potential. The result of this study suggests that Hy. dromedarii SGEs is an appropriate candidate for further investigations to identify and purify the mechanisms and molecules involved in the anti-cancer activity of the SGEs.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran. The nonstandard use of pesticides against pests, particularly in agriculture, indirectly has caused the de­velopment of resistance and, consequently, the threat of control measures in ZCL endemic areas. Up to 2023, several reports of resistance in Ph. papatasi have been declared in the old world. The purpose of this study was to measure the lethal time (LT50 and LT90) of Ph. papatasi sand flies in the ZCL endemic center of Esfahan to DDT and deltamethrin insecticides. Methods: Sand flies were collected in Borkhar and were tested using WHO adult mosquito test kit against DDT 4% and deltamethrin 0.0002%. The sand fly’s survival was recorded during exposure time in 225, 450|, 900, 1800, and 3600-seconds’ intervals for DDT and Deltamethrin and they were allowed to recover for 24 hours. Then LT50 and LT90 were analyzed using probit software. Phlebotomus papatasi were identified using morphological keys and other sand flies’ species were excluded from the analysis. Results: The insecticide against female Ph. papatasi revealed hundred percent mortality when exposed to DDT 4% and deltamethrin 0.0002%. The LT50 and LT90 were 19.32 and 22.74 minutes for DDT 4% and 39.92 and 51.33 minutes for deltamethrin 0.0002% respectively. Conclusion: Results of this study revealed that Ph. papatasi is still susceptible to DDT and deltamethrin. This data pro­vides valuable knowledge to implement effective control strategies against ZCL main vector and help to manage insec­ticide resistance in the region.

Background: Zoonotic cutaneous leishmaniasis is a major public health problem in Iran with the main vector of Phlebotomus papatasi. The use of entomopathogenic fungi for biological control of the vector is a potential substitute for the current methods which are being used. The purpose of the current study was to assess the virulence of two local isolates of Beauveria bassiana (OZ2 and TV) against Ph. papatasi. Methods: To perform the bioassay test, fungal suspensions were applied for every stage of the sand fly life cycle. The mortality rate, longevity, and number of eggs laid were determined. Also, the probability of fungal survival on the sur­face of rodent’s body was assessed. Results: The longevity of infected adult sand flies with both isolates of B. bassiana was significantly lower (P< 0.05) in comparison to the negative control. The estimated Lethal concentration 50 (LC50) values for adult female and male sand flies treated with OZ2 isolate were 1.4×106 and 2.2×107 conidia/ml, respectively, while they were 6.8×106 and 2.3×108 conidia/ml for TV iso­late, respectively. Both isolates of B. bassiana exhibited nonsignificant mortality rates in sand fly larvae and pupae and fecundity rate (P> 0.05). According to our findings for both isolates, the fungus continued to spread throughout the sur­face of the rodent's body for 144 hours after spraying. Conclusion: The current study demonstrated that both isolates of B. bassiana have considerable biological control ca­pacity against adult sand flies.

Abstract Vector-borne diseases, among them leishmaniasis, cause more than 700,000 deaths annually. The lack of an effective vaccination and the increasing resistance of sand flies to insecticides require the urgent development of innovative approaches to contain the disease. The use of engineered bacteria that express anti-parasite molecules (paratransgenesis) shows much promise. However, a challenge for implementation of this strategy is to devise means to introduce modified bacteria into sand flies in the field. In this study, we use rodent food bait as a delivery strategy to introduce two mCherry-fluorescent bacteria, Serratia AS1 and Enterobacter cloacae, into adult sand flies in field settings. Bacteria-infected food was provided to Rhombomys opimus rodents. These bacteria transiently pass through the rodent alimentary tract and are delivered to larval habitats with the rodent feces. The feces are ingested by sand fly larvae and, in the case of Serratia AS1, are trans-stadially transmitted to adults. This is the first report of targeting delivery of Serratia AS1 in a paratransgenic system to control transmission of leishmaniasis under field condition. This novel strategy shows promise for delivering transgenic bacteria to Leishmania vectors in the field.

Abstract Climate change will affect the distribution of species in the future. To determine the vulnerable areas relating to CL in Iran, we applied two models, MaxEnt and RF, for the projection of the future distribution of the main vectors and reservoirs of CL. The results of the models were compared in terms of performance, species distribution maps, and the gain, loss, and stable areas. The models provided a reasonable estimate of species distribution. The results showed that the Northern and Southern counties of Iran, which currently do not have a high incidence of CL may witness new foci in the future. The Western, and Southwestern regions of the Country, which currently have high habitat suitability for the presence of some vectors and reservoirs, will probably significantly decrease in the future. Furthermore, the most stable areas are for T. indica and M. hurrianae in the future. So that, this species may remain a major reservoir in areas that are present under current conditions. With more local studies in the field of identifying vulnerable areas to CL, it can be suggested that the national CL control guidelines should be revised to include a section as a climate change adaptation plan.

Background: The great gerbil (Rhombomys opimus), is widely distributed in Asia and is a natural reservoir for zoonotic cutaneous leishmaniasis in many endemic areas, as well as Iran. Materials and Methods: In this study, infection to Leishmania species was investigated by two methods, parasitological and molecular survey, in the small number of R. opimus collected from Jovain, a Zoonotic Cutaneous Leishmaniasis (ZCL) focus located in North East of Iran. Results: Parasitological observation showed infection in only one of five rodents. But, ITS2-Nested-PCR revealed Leishmania infection in three out of 5 gerbils, including the parasitological positive one. Based on the PCR amplified size, two cases of infections were Leishmania major and one Leishmania turanica, their sequences are accessible in GenBank. The results of sequence analysis were consistent with the results obtained based on the size of the PCR. Conclusion: These findings re-confirm the important role of R. opimus in the natural circulation of Leishmania spp and indicate the need to be concerned about the disease in the study area.

Abstract Background Treatment of cutaneous leishmaniasis (CL) remains a major challenge for the public health and medical community. It has been claimed that natural compounds derived from fly larvae have anti-leishmania properties against some species of Leishmania. The present study aimed at assessing the in vitro effects of larval products of Lucilia sericata against the promastigote and intracellular amastigote forms of Leishmania major. Also, the therapeutic effect of larval products on lesions induced by L. major infection was evaluated in BALB/c mice models. Methods Parasite specimens and macrophage cells were exposed to varying concentrations of larval products for 24–120 h. Lesion progression and parasite load were investigated in the models to assess the therapeutic effects of the products. Results The larval products displayed more potent cytotoxicity against L. major promastigotes. The IC50 values for larval saliva and hemolymph were 100.6 and 37.96 ug/ml, respectively. The IC50 of glucantime was 9.480 ug/ml. Also, the saliva and hemolymph of L. sericata exhibited higher cytotoxicity against the promastigotes of L. major but were less toxic to the macrophage cells. Treatment with leishmanicidal agents derived from larvae of L. sericata decreased the infection rate and the number of amastigotes per infected host cell at all concentrations. Lesion size was significantly (F (7, 38) = 8.54, P

Background: The German cockroach, Blattella germanica (Insecta: Blattodea, Blattellidae), which occurs widely in human buildings, is a small cockroach species. Cockroach control chemical pesticides are toxic to the environment, and it is sometimes impossible to prevent them. Controlling Blattella germanica through ultrasonic waves can be efficient and less dangerous for the environment. Methods: In this study, the repellency and lethal effect of ultrasonic waves on male and female German cockroaches was tested in a twin glass cubic chamber at laboratory condition. The wave frequencies tested ranged from 20 to 100kHz with 5kHz steps. A signal generator generated these frequencies, and the piezoelectric transmitter of these ultrasonic waves was positioned in the chamber's center on the upper side. Results: Fisher's test showed that there was the greatest repellency effect in both male and female at frequencies of 35 and 40kHz. According to the results of the regression test, the most lethal effect is at the frequencies of 40 and 75kHz. Conclusion: The operating ultrasonic frequencies investigated in this study can be used to repel and kill German cock­roaches as pests endangering human health and environment.

Background: Leishmaniasis is one of the most important vector-borne and neglected tropical diseases in many parts of the world. The main objective of this study was to determine the biodiversity of phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) in Khuzestan and Kermanshah Provinces of Iran. Methods: Sampling was conducted in Khuzestan and Kermanshah Provinces using sticky paper traps and CDC light traps. The samples were then stored in 96% alcohol-containing vials, mounted, and identified. Afterward, the alpha diversity (using Simpson’s, Shannon-Weiner’s diversity, evenness, Maghalef’s, Menhinick’s, Hill and indices) and beta diversity indices (using Sorensen’s and Jaccard’s coefficients) were calculated. Results: A total of 4302 sand flies were caught and identified which comprised mostly of Phlebotomus papatasi, Ph. alexandri and Ph. sergenti. It was found that among the four counties of Ahvaz, Shush, Shushtar and Dezful in Khuzestan Province, Shush had the lowest, and Shushtar, the highest amount of species diversity and evenness. In the four studied counties of Kermanshah Province, species diversity in Kermanshah County had the lowest amount and Sarpol-e-Zahab, the highest. The species richness in Kermanshah County was the lowest, while Qasr-e-Shirin County had the highest amount. Conclusion: An overview of the biodiversity of phlebotomine sand flies in Kermanshah County in Kermanshah Province, and Shush County in Khuzestan Province, showed less stability in community structure of these vectors, which can be counted as an alarm for emerging dominant vectors among the studied counties and have the potential to increase the prevalence of leishmaniasis.

Background: The primary aim of this study is to determine infection to Leishmania parasites in the wild population of Phlebotomus caucasicus and Phlebotomus mongolensis using molecular methods in some important zoonotic cutaneous leishmaniasis foci in Iran. Methods: Sand flies were collected from active colonies of rodent burrows from 16 trapping sites using sticky trap pa­per. In order to detect and identify of Leishmania parasites in females Ph. caucasicus and Ph. mongolensis, the Nested–PCR amplification of ITS2-rDNA region was performed to generate amplicon with 245bp for Leishmania major, 206bp for L. gerbilli and 141bp for L. turanica. Results: In the current study we found DNA of different gerbil parasites such as L. major and L. turanica, and mixed infection of L. major/L. turanica in Ph. caucasicus and Ph. mongolensis. It should be noted that, in Iran, natural infec­tion with Leishmania parasites is recorded for the first time in this study in Ph. mongolensis. Conclusion: Both species of Ph. caucasicus and Ph. mongolensis not only may participate in the ZCL transmission cycle between reservoir hosts, but also results of this study support the role of these species as secondary vectors in the transmission of leishmaniasis to humans.

Background: Various arthropods, including Rhipicephalus sanguineus and Ctenocephalides felis felis have been sug­gested as secondary vectors of Leishmania spp. many years ago. This study was conducted to determine zoonotic cuta­neous leishmaniasis (ZCL) PCR positivity of reservoir hosts and their ectoparasites for Leishmania spp. in Segzi plain in Esfahan Province from October 2016 to October 2017. Methods: Microscopic examination and nested PCR were used to detect and identify Leishmania spp. isolated from rodents' ear and ectoparasites, and then, the results were confirmed by two methods, PCR-restriction fragment length polymorphism and sequencing. Results: Totally, 93 rodents (92 Rhombomys opimus and one Nesokia indica) and nine different species of ectoparasites (n=527) including fleas, mites, and ticks were collected during different seasons in the study area. Fourteen R. opimus were positive for Leishmania spp. by microscopic examination while one N. indica and 77 R. opimus were positive by nested PCR. The infection rate of rodents with Leishmania major and L. turanica was 39.79% (n=37) and 15.05% (n=14), respectively. Mixed natural infections with L. major and L. turanica were seen in rodents. Moreover, 72.22% of fleas (39/54), 75.0% of mites (5/8), and 100% of tick nymph (1/1) were PCR positive for Leishmania parasites. Conclusions: The highest rate of infection with L. major and L. turanica in R. opimus populations was observed in summer and spring, respectively. It is suggested that the role of L. turanica and the probable role of ectoparasites in the epidemiology of disease should be investigated. Xenodiagnostic testing is recommended for future testing.

Background: Phlebotomine sand flies are vectors of Leishmania species, the causative agents of leishmaniasis in the world. Present study aimed to evaluate the bioecological aspects of sand flies in different ecotopes in Sari County, north of Iran. Methods: Sand flies were collected from four villages in mountainous, forest, plain and peri-urban areas monthly using sticky traps in May–October 2016. Mounted specimens were identified using valid identification keys under optical microscope. The Arc GIS 10.5 software was applied for showing the distribution of sand flies. Shannon-Weiner, Simp­son and Evenness species diversity indices were calculated. Results: Generally, 334 specimens were captured and identified, namelly Phlebotomus kandelakii, Ph. papatasi, Ph. major, Ph. sergenti, Ph. longiductus, Ph. halepensis, Ph. tobbi, Sergentomyia dentata, Se. theodori, Se. sintoni, Se. an­tennata and Se. sumbarica. The most common species was Ph. kandelakii (n= 128, 38.32 %). The highest Simpson in­dex (0.81) and abundance (N= 141) were recorded in the mountaineous area. Shannon diversity index was higher in the forest (H'= 1.53) and the highest evenness index was in the plain area (J'= 0.93). The highest richness (S= 9) and Shan­non indices (H'= 1.57) were observed in June. Conclusions: Phlebotomus kandelakii, Ph. sergenti, Ph. tobbi, Ph. longiductus, Se. theodori, Se. antennata and Se. sumbarica were recorded for the first time in the study area. Since some species are incriminated for leishmaniasis transmission, further studies are required in the northern regions of Iran to timely control measures planning.

Background: Among neglected zoonotic diseases, leishmaniases caused by Leishmania parasite through infected female sand fly bite, are a group of diseases found in 98 countries and territories representing a critical burden of disease worldwide. Vector management plays a crucial role in reducing the burden of vector-borne diseases by WHO’s global plan. The objective of the current study was to assess the susceptibility status of wild phlebotomine sand flies from Esfahan Province, central Iran, to the recommended insecticides by WHO. Methods: Sand flies were collected by mouth aspirator in Matin Abad desert Eco-resort and were tested using WHO adult mosquito test kit against Dichlorodiphenyltrichloroethane (DDT) 4%, Deltamethrin 0.05%, Malathion 5% and Propoxur 0.1%. The number of knockdown sand flies were recorded during exposure time in ten minutes interval for DDT and Deltamethrin and they were allowed to recover for 24 hours. Knockdown Time50 (KD50) and KD90 were generated for them using Probit software. They were mounted and identified by valid keys. Results: Among the tested insecticides against female Phlebotomus papatasi, DDT, Deltamethrin, and Malathion recorded the highest mortality rate of 100%, followed by Propoxur with 92.2% mortality for a one-hour exposure. For DDT, KD50 and KD90 were calculated 21.87 and 42.93 and for Deltamethrin, they were 23.74 and 56.50 minutes respectively. Total sand flies exposed with DDT and Deltamethrin shed their leg(s). Conclusion: It is concluded that Ph. papatasi from central Iran is susceptible to DDT, Deltamethrin, Malathion, and Propoxur.

Background: Collecting live sand flies from indoor sites is a major challenge for researchers in large cities due to the reluctance of families to survey their homes. This study was conducted to assess the efficacy of two methods for col­lecting sand flies for use in susceptibility tests in the urban area of Kerman, southeastern Iran. Methods: Sandflies were mainly collected using both baited traps and hand catch methods from outdoor and indoor sites. Susceptibility tests were separately done according to the standard World Health Organization testing protocol on Phlebotomus sergenti, including 60-minute exposure to DDT 4.0%, propoxur 0.1%, deltamethrin 0.05%, and malathion 5.0%. Results: During this research, the natural habitats and suitable indoor sites were selected to predict the density of live sand fly with perfect accuracy. The number of live Ph. sergenti caught by hand catch and baited traps methods was 42 and 361 in indoor and outdoor sites, respectively. The mortality rate of Ph. sergenti exposed to DDT 4%, deltamethrin 0.05, malathion 5%, and propoxur was 100%. Conclusion: The baited traps showed a significant efficiency compared to hand catch for collecting live Ph. sergenti for use in susceptibility tests in urban areas. The Ph. sergenti collected from both indoor and outdoor sites were suscep­tible to all insecticides.

Abstract Background Leishmaniasis is a major parasitic disease worldwide, except in Australia and Antarctica, and it poses a significant public health problem. Due to the absence of safe and effective vaccines and drugs, researchers have begun an extensive search for new drugs. The aim of the current study was to investigate the in vitro leishmanicidal activity of larval saliva and hemolymph of Lucilia sericata on Leishmania tropica. Methods The effects of different concentrations of larval products on promastigotes and intracellular amastigotes of L. tropica were investigated using the mouse cell line J774A.1 and peritoneal macrophages as host cells. The 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct observation and counting method were used to assess the inhibitory effects and cell cytotoxicity of the larval products. The effects of larval products on the amastigote form of L. tropica were quantitatively estimated by calculating the rate of macrophage infection, number of amastigotes per infected macrophage cell, parasite load and survival index. Results The 50% cytotoxicity concentration (CC50) value of both larval saliva and hemolymph was 750 µg/ml, and the 50% inhibitory concentration (IC50) values were 134 µg/ml and 60 µg/ml for larval saliva and larval hemolymph, respectively. The IC50 for Glucantime, used a positive control, was (11.65 µg/ml). Statistically significant differences in viability percentages of promastigotes were observed for different doses of both larval saliva and hemolymph when compared with the negative control (p ≤ 0.0001). Microscopic evaluation of the amastigote forms revealed that treatment with 150 µg/ml larval hemolymph and 450 µg/ml larval saliva significantly decreased the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Conclusion Larval saliva and hemolymph of L. sericata have acceptable leishmanicidal properties against L. tropica.

Background: Cutaneous leishmaniasis is an ancient endemic disease in Iran and continues to be a growing health threat to community development and the environment. This paper explains how to use the facilities of health centers for developing a laboratory network on vectors and reservoir hosts of cutaneous leishmaniasis in Iran. Methods: A literature search was performed of the relevant multiple databases to include studies on vectors and reservoirs of cutaneous leishmaniasis in Iran. A team of experienced experts was performed. After holding several meetings, field visits and organizing workshops, the activities of laboratories were determined at three levels. Results: Entomological studies showed that 5 species of the genus Phlebotomus and 10 species of the genus Sergentomyia are active in the south, 4 species of the genus Phlebotomus and one species of the genus Sergentomyia in the central part and 5 species of the genus Phlebotomus and 2 species of the genus Sergentomyia in the north east. Reservoir hosts were identified in the study areas. The tasks of laboratories were regulated at different levels. Conclusion: The Iranian Ministry of Health and Medical Education should prioritize the employment of capable persons in the field of Medical Entomology and Vector Control. The survival of this laboratory network depends on hiring and employing interested and persistent people. The universities of Medical Sciences that have the facilities to set up this network will be a very effective partner in the control of the disease in high risk areas. The results can be used in neighboring countries.

Background & objectives: Cutaneous leishmaniasis (CL) is still a public health threat in many rural areas of Iran. It causes considerable morbidity among a huge number of people in the endemic areas. Khuzestan is considered as a CL focus in Iran and recently several cases have been reported from Shush County. The aim of the current study was to determine different aspects of CL epidemiology in this County. Methods: The study was conducted in four villages in the rural district of Shush County. In order to survey the scar and acute rate of the disease, 100 inhabitants were questioned in each village. In addition, to determine the reservoir host and vector of the disease, rodents were captured using Sherman traps, and sandflies were collected from around the rodent burrows using sticky paper traps. Results: The scar and acute lesion rates were reported as 5.26 and 0.57% among inhabitants, respectively. Fifty-four rodents including Tatera indica (55.5%), Nesokia indica (33.3%), Mus musculus (9.3%) and Rattus norvegicus (1.9%) were captured and identified. Nested PCR showed 3 out of 54 rodents infected with Leishmania major. In total, 1122 sandflies were collected, and Leishmania major detected in Phlebotomus papatasi (4.35%), Sergentomyia dentata (23%), and Sergentomyia clydei (10%). Interpretation & conclusion: Zoonotic cutaneous leishmaniasis due to L. major prevailed in this area. The common and the main vector was Ph. papatasi and, T. indica seems to be the main while N. indica as a secondary reservoir host of the disease in this area.

Objective: 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) μg 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 mmunogenic proteins in P. sergenti saliva which could be subjected for further investigation as vector-based vaccine candidate/s against anthroponotic cutaneous leishmaniasis.

Objective: To determine the diversity of sand flies in different biotopes of mountainous and plain areas of Bam County as the most infected focus of anthroponotic cutaneous leishmaniasis in southeast Iran, and synanthropic index of Phlebotomus sergenti Parrot, and Phlebotomus papatasi Scopoli as the main vectors of cutaneous leishmaniasis in Iran. Methods: Sand flies were captured once a month using sticky traps in domestic, peri-domestic, agricultural, and sylvatic biotopes in the plain and mountainous areas. Alpha diversity indices, including richness, evenness, Shannon-Wiener; beta diversity indices (Jaccard’s and Sorensen’s similarity indices) and synanthropic index were calculated. Results: A total of 2 664 specimens of 9 sand fly species were collected from mountainous (47%) and plain (53%) areas. Species richness, species evenness, and Shannon-Wiener indices were obtained as 9, 0.637, and 1.399, respectively in the mountainous area. Phlebotomus sergenti and Phlebotomus papatasi were constant species with the synanthropic index of -18.463 and -29.412, respectively. In addition, species richness, species evenness, and Shannon-Wiener indices were 4, 0.690, and 0.956, respectively in the plain area. Phlebotomus sergenti and Phlebotomus papatasi were dominant species with the synanthropic index of +9.695 and +36.207, respectively. Similarity indices were low among different biotopes of plain and mountainous areas. Conclusions: A basic knowledge about the diversity of sand flies in various biotopes is essential to design sound control programs. Biodiversity and synanthropic indices of sand flies are different in plain and mountainous areas due to the difference in biotic and abiotic factors between the two areas.

Abstract Background and purpose: This study was done from October 2016 to October 2017 to determine funa and distribution of ectoparasites of zoonotic cutaneous leishmaniasis reservoirs to prevent and control of zoonotic diseases in the hyperendemic area (Segzi area), in Esfahan Province in the future. Materials and methods: Rodents were collected by Sherman's Living Traps during different seasons and were identified by the standard morphological keys and comparison of morphometric characteristics with standard specimens. The collected rodents were anesthetized and ectoparasites were removed and conserved in 96% Alcohol. Then the ectoparasites were identified by the standard keys. Results: Ninety-two Rhombomys opimus and 1 Nesokia indica were captured. 50 out of 93 rodents (53.88%) were infested by the ectoparasites and a total of 527 ectoparasites were collected from them. The 9 species of identified ectoparasites were included 1 tick (0.19% Rhipicephalus sanguineus), 1 sucking lice (0.19% Polyplax spp.), 5 fleas (75.71% Xenopsylla nuttalli, 8.73% Echidnophaga oschanini, 0.95% Nosopsyllus ziarus, 0.95% Coptopsylla mesghalii and 0.19% Nosopsyllus turkmenicus turkmenicus) and 2 mites (7.78% Dermanyssus sanguineus and 5.31% Hirstionyssus sp.). Conclusion: The current study showed that R. opimus rats are found in large numbers in Segzi area and the X. nuttalli fleas were the most frequency. It is suggested to prevent the prevalence of vector- and rodent-borne diseases, the population of rodents and their ectoparasites should be controlled at the same time.

Objective: To delineate reliable morphological characteristics for identifying and separating female Phlebotomus caucasicus and Phlebotomus mongolensis which exist sympatrically in the main foci of zoonotic cutaneous leishmaniasis in Iran. Methods: Sand flies were collected using sticky trap papers from active colonies of rodent burrows installed from 16 catching sites. Morphometric measurements were analyzed of 87 Phlebotomus caucasicus and 156 Phlebotomus mongolensis. Univariate and multivariate analysis were carried out to determine significant morphometric variables for discrimination of the two species. Finally, seven morphological characteristics of 65 female Phlebotomus caucasicus and 124 female Phlebotomus mongolensis were described. Results: Univariate and multivariate analyses of 10 morphometric variables via Discriminant Function Analysis (DFA) and Principal Component Analysis (PCA) showed that five morphometric variables had an accuracy of 100% for discriminating female Phlebotomus caucasicus and Phlebotomus mongolensis. Moreover, PCA revealed that the five morphometric variables with the highest loadings separated these two species. Morphological studies on antennal flagellum (and its associated structures) and mouth-parts of female specimens demonstrated significant differences in several structures. Conclusions: The results show that morphological and morphometrical features can be used to discriminate two female isomorphic species, Phlebotomus caucasicus and Phlebotomus mongolensis accurately.

Background and purpose: The leishmaniases are a group of diseases that appear as cutaneous, mucocutaneous, and visceral leishmaniasis, transmitted by different species of sand flies. Cutaneous leishmaniasis (CL) is considered as a major public health problem in Iran. The current study was carried out to determine the spatial and temporal distribution of CL in Mazandaran Province, Iran according to Geographical Information System (GIS). Materials and methods: This retrospective descriptive study was carried out according to demographic and epidemiologic data collected from patients diagnosed with CL during 2009-2017 recorded in Health deputy of Mazandaran University of Medical Sciences. The spatial and temporal distribution of CL in different cities of Mazandaran Province was mapped using ArcGIS 10.4 software. Results: The number of patients with CL was 378 during the years studied. The highest and lowest numbers of CL patients were seen in Sari and Abbas Abad, respectively (n=71, n=2, respectively). Most of the cases (n= 182) were reported in east of Mazandaran and the least number of cases (n= 73) were observed in the west of province. The incidence rate in the west of the province was lower than that of the center and east of the province. Conclusion: Distribution of CL in Iran, agricultural activities, livestock breeding, and migratory status in Mazandaran Province call for appropriate planning to control this disease.

Background: Leishmaniasis is an expanding neglected tropical disease in the world reporting from 98 countries including Iran. This study focused on eco-epidemiological determinants of the disease following a rapid and unexpected increase of leishmaniasis incidence in a strategic residential district in North-East of Isfahan County, Iran. Methods: This study was accomplished from Apr 2012 to Jan 2014 in a strategic residential zone in North-East of Isfahan County, Esfahan, Iran. Monthly activity, parity, Leishmania infection and susceptibility tests, were determined on sand flies. Some portion of inhabitants and school children were surveyed to find active or passive cases of leishmaniasis and also wild rodents were collected to determine reservoir host. Results: Totally 5223 sand flies belonging to Phlebotomus and Sergentomyia genus were collected and identified; Ph. papatasi was the dominant species and started to appear in May and disappeared in Oct. The majority of living dissected sand flies were unfed and parous. Ph. papatasi showed 4.6% Leishmania infection through direct examination and 39.54% by nested-PCR respectively. Phlebotomus papatasi was susceptible against deltametrin 0.05%. Totally 2149 people were surveyed and incidence and prevalence of zoonotic cutaneous leishmaniasis estimated as 45.39 and 314.40 per 1000 population. Rodents showed 73.91% and 80% Leishmania infection by direct examination and nested-PCR respectively. Conclusion: Cutaneous leishmaniasis due to L. major has been established in this area. Rodent control operation and personal protection are highly recommended to control the disease in this focus.

Background: Esfahan Province is considered as one of the main focus of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Recently, ZCL distribution is expanding through this province leading to report of new cases in non-endemic areas. In the current study epidemiological aspects of ZCL has been investigated in Naein County in Esfahan Prov­ince. Methods: Adult sand flies were collected from beginning to the end of their seasonal activity. Rodents were caught by Sherman live traps once a month for one year. To active case detection, a hundred households in each selected village were visited in November and December 2016. Nested-PCR was employed to detect Leishmania parasite in the vector, reservoir and human. Results: Totally 1562 sand flies including Phlebotomus sergenti, Phlebotomus papatasi, Sergentomyia sintoni and Sergentomyia mervinae were collected and identified. No Leishmania infection was detected in the collected sand flies. All of the 30 collected rodents were identified as Rhombomys opimus and of these 3.3% and 26.7% were in­fected by Leishmania major using microscopic and molecular technique respectively. Totally, 914 individuals were investigated and the ulcer and scar rate of ZCL calculated to be at 1.1 and 15.3per 1000 population, respectively. Molecular results confirmed L. major infection in human and reservoir samples. Conclusion: It is concluded that ZCL is established in the area in low endemicity, and it is extrapolated the disease will not be a serious increasing health problem in near future in this region.

Background: The attraction of phlebotomine sand flies to plant and animal hosts is due to the produced chemical compounds, affecting the olfactory receptors of the insects. Therefore, novel and effective methods, such as Attrac­tive Toxic Sugar Baits (ATSB) and Attractive Toxic Baits (ATB), are based on the effective materials that attract sand flies toward the host. The present study was designed to identify the attractive materials in plants and animals for using in ATSB and ATB. Methods: This cross-sectional study was carried out in July 2018 on endemic areas of leishmaniasis in Iran. Differ­ent baits, including mango, nectarine, grape, banana, melon and watermelon, defibrinated blood of cattle, sheep, goat and chicken, urine of cattle, sheep, goat and ultimately, simple and complex chemicals, such as CO2, 1-octanol, lactic acid and human sweat were placed inside the traps, and the rate of the sand flies attraction to these materials was studied. Furthermore, data were analyzed using the Kruskal-Wallis test and Mann Whitney U test. Results: There was a significant difference in the sand flies attraction between the traps containing watermelon, urine of cattle, and sheep, and chemicals such as CO2 and human sweat and the control trap (p< 0.05). Conclusion: This study showed that watermelon and CO2 are the potential candidates for using in ATSB and ATB, respectively.

Background: Leishmaniasis represents the important public health problem in the world. One of the main objectives of World Health organization is capacity building of managers and authorities who are involved with diseases control ac­tivities. Methods: The course was conducted in Esfahan Health Research and Training Center (E.H.R.T.C) in summer 2005 and 2009. The course carried out jointly by the Ministry of Health and Medical Education (MOH) of Iran, World Health Or­ganization-Eastern Mediterranean Regional Office (WHO-EMRO) and School of Public health, Tehran University of Med­ical Sciences (SPH-TUMS) and designed for medical officers, senior technicians and managers involved in leish­mania­sis control. Prior to initiate the course, pre-test evaluations including different subjects on leishmaniasis and its control were carried out. The examinations include multiple choice questions. The duration of the course was 3 weeks. A total of 206 contact hours were taught. The main subjects were Basic epidemiology, Leishmaniasis parasitology, Leishmaniasis en­tomology, control of vectors and reservoirs, principles of integrated pest management, Field work and Planning. Dif­ferent methods of teaching including lecture, laboratory, workshop, team work, field exercise and presen­tation were used. Requirement for achievement of the course was to have at least 60% of the total mark for awarding the diploma certificate. Results: A total of 45 participants from Iraq, Afghanistan and Iran graduated from this course. Conclusion: The course is providing the skill for managers, how to combat against disease in their country and is paral­lel to the policy of the leishmaniasis control for capacity building in endemic areas of their countries.

Background & objectives: Phlebotomine sandflies (Diptera: Psychodidae) transmit several important zoonotic diseases to humans and leishmaniasis is one of them. Two types of leishmaniasis, viz. visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are endemic in Iran. The main vector of anthroponotic cutaneous leishmaniasis (ACL) is Phlebotomus sergenti. The aim of the present study was to determine the susceptibility status of wild strain of P. sergenti to different imagicides of DDT, bendiocarb and permethrin at the median lethal time, LT50 level. Methods: Sandflies were collected from selected village in North Khorasan Province, northeast of Iran from indoors using CDC light-traps. Susceptibility test was carried out against DDT (4%), bendiocarb (0.1%) and permethrin (0.75%) for all the females according to WHO method, and mortality was calculated. Species identification was carried out using the morphological keys. Data were analysed using probit regression analysis to determine the LT50 and LT90 values. Results: In total, 851 female P. sergenti sandflies were tested. LT50 values to DDT (4%), Bendiocarb (0.1%) and permethrin (0.75%) were 15.4, 19.2 and 6.3 min respectively. The values for LT90 were 51.1, 47.4 and 18.6 min respectively. The mortality rates for 1 h exposure time to DDT, bendiocarb and permethrin were 89.8 ± 1.4; 93.6 ± 1.4; and 95.6 ± 1.7%, respectively. Interpretation & conclusion: The suscesptibility studies revealed development of resistance against DDT (4%) in the wild strain of P. sergenti population. Monitoring and mapping of insecticide resistance in the region is recommended for vector control.

Background: We evaluated the effect of the ethanolic extract of Scrophularia striata on the Iranian strain of Leishmania major (MRHO/IR/75/ER) both in vitro and in vivo conditionsMethods: The effective dose (ED) of ethanolic extract of S. striata were determined using MTT assay on the growth of promastigote forms of L. major in axenic culture media. Then, the ED50 of S. striata on mice peritoneal macrophages was determined using calculation of amastigote forms on mice peritoneal macrophages. For in vivo experiments, the therapeutic effects of various concentrations of S. striata on infected BALB/c mice was studied. A total of 75 infected mice were randomly divided into five groups: two groups (10% and 50% of S. striata) as experimental and three as control (ethanol 50%, Glucantime® and no treatment). The efficacy were determined by comparing the diameters of lesions and the microscopically examinations.Results: The effect of S. striata extract (0/625%, 1/25%, 2/5%, 5%, 10%, 20% and 50%) on peritoneal macrophages of Balb/c mice infected with L.major in tissue-culture slides was assessed. S. striata extract (10%) removed the L.major amastigotes-infected macrophages significantly after 24 h (P < 0.05 ). The higher concentrations of S. striata ethanolic extract (20%, and 50%) had highly toxic effects on macrophages, resulted in the disintegration of the cytoplasm of macrophages after 48 and 72 h. In concentration 10% of S. striata, more than 85% of L. major amastigotes-infected macrophages were damaged without cytotoxicity effects on macrophages. The higher concentrations had toxic effects on cultured macrophages. Conclusion: S. striata ethanolic extract 10% had anti leishmanial effects in both in vivo and in vitro.

Background: Leishmaniasis diseases are known to be one of the most important public health problems in World and Iran. Visceral leishmaniasis is considered to be the most serious form and transmitted by sand flies species. The aim of this study was to investigate the dynamic activities of sandflies in northwestern Iran. Methods: This crass-sectional study was conducted from April to December 2018 in Meshkinshahr County, Ardabil Province. Sticky traps have been used to collect sand flies. They are stored in 70% alcohol and finally identified using valid keys. The aspects of the synoptic information were inquired from the Meshkin Shahr weather department and results analyzed for SPSS24. Results: Totally 259 sandflies were collected during study period. From collected samples 78.7% were male and 21.3% female. There were 8 different species. Phlebotomus kandelakii was the most prevalent one (30.8%). The average tem­perature, relative humidity, and average wind speed was 13.5 °C, 84%, and 2 meters per hour on the onset of sandflies’ activity, respectively. These values were 18.3 °C, 85% and 1.5 meters per hour at the peak of their activity and 16 °C, 62% and 5 meters per hour at the final stage of their activity. Sand flies had one peak in July which is strongly influ­enced by temperature and humidity conditions. Two species of Ph. kandelakii and Phlebotomus perfilewi transcaucasi­cus, had the highest activity in this endemic area. Conclusion: The results of current study will provide a guideline for control of diseases in the country.

Background: Visceral and cutaneous leishmaniasis are common in some areas of Iran and consider as health problems. Phlebotomus alexandri has been incriminated as a suspected vector for the both form of leishmaniasis. Methods: This study was carried out in 4 western provinces of Iran. Sand flies were collected using sticky traps and light traps from indoor and outdoor resting places. Nested PCR was employed to detect Leishmania parasites among collected sand flies. Results: Seven hundred and twenty two P. alexandri females were collected and pooled in 179 batches. Results of nested PCR showed, out of 9 samples from East Azerbaijan Province, only one sample was infected by Leishmania infantum. Of 34 individual and pooled samples from Kermanshah Province, only one pooled sample was infected with Leishmania major and among 30 individual and pooled samples in Fars Province, five specimens were infected by L. major, L. infantum, Leishmania donovani and Leishmania tropica. Furthermore, out of 108 individual and pooled samples from Khuzestan Province, 10 samples showed infection with L. major and L. infantum. Conclusion: The results of this study showed that P. alexandri is more active in hot zones than in moderate zones and this species may be considered as a permissive species.

Background: Phlebotominae sand flies (Diptera: Psychodidae) are the vectors of leishmaniasis. There are different methods for sand fly collection with different performance. The purpose of the current study was to compare the effect of different traps for collection of Phlebotominae sand flies in three endemic leishmaniasis foci in North Khorasan Prov­ince, northeast of Iran. Methods: Sand flies were collected using seven different traps from three villages, three times each twenty days during peak periods of seasonal activity in 2016. Results: A total of 7253 sand flies were collected. The specimens belonged to19 species. Phlebotomus sergenti was the most predominant species in the study area. Light trap baited with Carbon dioxide (CLT) and sticky paper trap (SPT) caught 22.6% and 22.3% of sand flies respectively. Animal baited trap (ABT) and white Shannon trap (WST) caught significantly fewer sand flies than the other traps. The sex ratio was different by phlebotominae sand fly species and collection methods. The sex ratio was highest in SPT and lowest in black Shannon trap (BST). Species diversity and species richness in SPT were more than other traps. Conclusion: Our findings confirm that CLT and SPT are the most efficient sand fly collection methods. CLT is higher attractive for females and Phlebotomus genus and is an ideal method for monitoring the population of Phlebotomus ge­nus during surveillance. SPT is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities and provide a more realistic estimation of sand flies biodiversity.

Background: Cutaneous leishmaniasis due to Leishmania major is an important public health problem in the world. Khuzestan Province is one of the main foci of zoonotic cutaneous leishmaniasis (ZCL) in the southwest of Iran. We aimed to predict the spatial distribution of the vector and reservoir(s) of ZCL using decision-making tool and to pre­pare risk map of the disease using integrative GIS, RS and AHP methods in endemic foci in Shush (plain area) and Khorramshahr (coastal area) counties of Khuzestan Province, southern Iran from Mar 2012 to Jan 2013. Methods: Thirteen criteria including temperature, relative humidity, rainfall, soil texture, soil organic matter, soil pH, soil moisture, altitude, land cover, land use, underground water depth, distance from river, slope and distance from human dwelling with the highest chance of the presence of the main vector and reservoir of the disease were chosen for this study. Weights of the criteria classes were determined using the Expert choice 11 software. The pres­ence proba­bility maps of the vector and reservoir of the disease were prepared with the combination of AHP method and Arc GIS 9.3. Results: Based on the maps derived from the AHP model, in Khorramshahr study area, the highest probability of ZCL is predicted in Gharb Karoon rural district. The presence probability of ZCL was high in Hossein Abad and Benmoala rural districts in the northeast of Shush. Conclusion: Prediction maps of ZCL distribution pattern provide valuable information which can guide policy mak­ers and health authorities to be precise in making appropriate decisions before occurrence of a possible disease out­break.

Background: In domestic and per domestic area,insecticides such as DDT, malathion, fenitrothion, propoxur and, more recently, synthetic pyrethroids such as deltamethrin and lambda-cyhalothrin, have been successfully used to control sand flies in many countries. The present study reports the results of time-mortality bioassay to DDT 4%, lambda-cyhalothrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and deltamethrin 0.05% in recently colonized Phlebotomus papatasi populations in Iran. Methods: The insecticide susceptibility status of P. papatasi laboratory population was assessed during 2016–2017, following the standard WHO technique for mosquito (WHO, 2013) based on diagnostic dose. Sand flies collected from rural area of Badrood (Matin Abad), Natanz County, Esfahan Province, using aspirator. Results: Susceptibility test to DDT and pyrethroids was assessed on 3534 laboratory-reared P. papatasi (1746 females and 1788 males). The LT50 and LT90 values were measured using probit analysis and regression lines. The test results against males of P. papatasi revealed that LT50 values to DDT 4%, Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 439.28, 108.90, 97.75, 5.00 and 57.84 seconds. The figures for females were 641.62, 136.15, 146.44, 8.71 and 72.69 seconds , respectively. Conclusion: According to presented results, the reared population of sand flies collected from a hyper-endemic region of Esfahan Province is still susceptible to prethroids and Resistance candidate to DDT 4%.

Background: Cutaneous leishmaniasis (CL) remains a serious public health concern in some parts of Iraq. The aims of this study to report of CL in some parts of Iraq, by different parasitological, cultural, and molecular methods and evaluate sex differences among infected patients. This is the first study conducted to characterize Leishmania species causing CL among Iraqi patients using the sequence analysis of Internal Transcribed Spacer1 (ITS1). Methods: A total of 700 cases of suspected CL who were referred to the Iraqi's clinics and health centers and they checked for Leishmania amastigote using Giemsa-stained , however the NovyMacneal Nicolle (NNN) culture led to the growth of promastigotes in all samples, then the samples were examined using PCR and RFLP-PCR methods. Results: The present study indicated that the prevalence of CL in eight Iraqi provinces as follow: Diwaniya 88(15.1%) , Wasit 85(14.5%),Najaf 79(13.6%), Thi-Qar 76(13.1%), Basrah 67(11.5%), Baghdad 65(11.2%), Diyala 63(10.8%) and Salah-Adin province which recorded the lower infection 60(10.3%), and it appeared 83.3% by using Giemsa-smeared in eight Iraqi provinces. The highest infection (100 %) appeared using PCR while the lowest infection (68%) appeared by culture on NNN media. The present study was revealed that the highest infection (60 %) caused by L.major than L.tropica (40 %) . Our results showed that 368 (52.6 %) of CL patients were had single lesion and 215 (30.7 %) had multiple lesions, and the ulcerative wet type lesions were present in 49.6% , while the nodule dry type lesions were present in 33.7 %. The overall prevalence of cutaneous leishmaniasis in the study area was very high (83.3%) having a statistical significant association with sex; males are more prone (56.4%) to Cutaneous Leishmaniasis (CL) as compared to females(43.6%). Conclusions: A clear and reliable bias toward males exists in some tropical diseases, such as leishmaniasis. Cutaneous leishmaniasis is a major health problem in Iraq and CL caused by many countries including Iraq. Health authorities should be aware of the fact that war and terrorist activities induce expansion of the disease and increase incidence rate in the situation that access to medical treatment is not easy especially in poor conditions of leishmaniasis endemic areas.

Background: Due to strict ethical rules, the risk of accidental disease transmission and the most importantly, inconven­ience regarding using of live animals, artificial feeding apparatus has been developed for colonization of haematophagous insects. Rearing of sandfly is more difficult than other haematophagous insects. Methods: In the current study, a new apparatus for membrane feeding of Phlebotomus papatasi was designed, made and compared with available apparatus in Sand Fly Insectary, Tehran University of Medical Sciences, Tehran, Iran, in 2014. Results: In comparison to other apparatus designed for artificial feeding of other arthropods, our designed apparatus had the highest performance which after up to 1h, the majority of sand flies landed and took blood and among tested membranes, chicken skin was proved the most efficient membrane. Conclusion: Sand fly artificial feeding apparatus can be used at least for rearing of Ph. papatasi.

An amendment to this paper has been published and can be accessed via the original article.

Background & objectives: Phlebotomus papatasi is the main vector of the zoonotic cutaneous leishmaniasis (ZCL) in Qom Province and many other provinces of Iran. Attractive toxic sugar baits (ATSB) treated barrier fence is one of the new methods for controlling the vectors such as sandflies. The present study was designed to evaluate the residual activity of ATSB-treated barrier fence that was used in control of P. papatasi. Methods: Following the selection of villages in Markazi district of Qom Province, central Iran during 2015 for ATSB and ASB (bait containing no active ingredient) methods; barrier fences on the ground in front of the rodent′s colony were installed. A total of four conical tubes were installed and fixed on surfaces of treated barrier net of dimension 25 Χ 25 cm at biweekly interval. In each conical tube, 10 sand flies were released and after 3 min of exposure they were transferred to sterile cups. After 24 h, the obtained results were recorded according to the survival and mortality rate of sandflies. These tests were carried out five days after the installation of barrier fences, and repeated every 15 days until the mortality rate decreased to 60-65%. Results: The bioassay tests results showed that the mortality rate of P. papatasi on ATSB-treated barrier fence for 5, 15, 30 and 45 days after spraying was 100, 95.83, 88.18 and 66.67% respectively, which decreased to 50.83% after 60 days. Interpretation & conclusion: Persistence and residual activity of the active ingredient of the bait in the hot and dry climatic conditions of Qom Province remained significantly effective for at most 45 days, which subsequently decreased at a high rate. Hence, every 45 days barrier fences need to be impregnated with ATSB bait. The method also appeared cost-effective and could be practical in implementation of vector control programmes against ZCL.

Background and Objective: Cutaneous leishmaniasis (CL) remains a serious public health concern in some parts of Iraq. The aims of this study to diagnose the Leishmania sp. causative agent of CL in some parts of Iraq, by different parasitological, cultural, and molecular methods. It was carried during the period October 2014 to February 2015. Materials and Methods: One hundred-sixty one of skin samples were examined by direct Giemsa-smeared, culture on NNN medium and Nested – PCR methods in different age groups. Results: The results of our study showed that 110 (68.3%) gave positives by Giemsa-smeared, 104 (64.6%) by culture and 67 (100%) by Nested – PCR. Our results appeared that there was slight gender predilection ; CL cases were more frequent in males (62.1%) than females (37.9%). Also, the type of infection showed that disease was in wet type 133(82.6%) more than in dry type 28 (17.4%). It was found that 98 (60.9%) of CL cases were in contact with rodents while 63(39.1%) of cases were not. Conclusions: CL disease is endemic in many parts of Iraq with high incidence and expanding to new foci that is considered a public health threat which needs special attention.

Background: We aimed to determine the species composition, richness and diversity indices of the Phlebotomine sand flies at different topographic condition in visceral (VL) and cutaneous leishmaniasis (CL) foci in the northeast of Iran. Methods: This cross-sectional study was conducted during 2016 in North Khorasan Province. The sampling was focused on rural regions, where human cases of VL and/or CL were diagnosed and reported during last 5 years. Sand flies were collected three times each twenty days during peak periods of seasonal activity. Seven collection methods were used. Some Alpha and Beta diversity indices were calculated. Results: Overall, 7253 sand flies were collected and identified. They were from 19 species of Phlebotominae sand­flies. Phlebotomus sergenti and Ph. papatasi were the most prevalent (84.9%) species in the study area. Species rich­ness (S) was very different in three areas and were18, 8, and 4 respectively but Evenness (E) were 0.357, 0.345, and 0.380, so evenness was almost equal in the study areas. Shannon Index (H ) and Margalef Richness Index were cal­culated 1.033, 0.718, 0.527 and 2.117, 0.8998, 0.4006 respectively. Conclusion: The sand fly fauna in North Khorasan Province was very rich and often included some of the most im­portant proven or suspected vectors of leishmaniasis. Species diversity indices (Shannon index, and Simpson’s in­dex) were not high due to decreasing in evenness. The Margalef richness index could accurately reflect the biodiver­sity of sand flies between three subtidal locations.

Background: Leishmaniasis is a re-emerging serious international public health problem, and both visceral and cu­taneous types of leishmaniasis became important endemic diseases in Iran. In this study, the relationships between environmental factors (vegetation and elevation) and the prevalence of diseases have been investigated. Methods: All international and national online databases were searched by terms such as leishmaniasis, incidence, prevalence and other related words attributed to Iran and published until first quarter of 2015. The developed data­base in Excel, later imported to the ArcMap for spatial analyst and mapping. Afterwards, the software was used for modeling the relationship between the prevalence/incidence and environmental variables (vegetation and elevation) by both linear and nonlinear regression. Results: After mapping the prevalence data from 144 studies, considering non-parametric ANOVA, the tendency of zoonotic visceral leishmaniasis to presence in high elevation and high vegetation was more than Anthroponotic and zoonotic cutaneous leishmaniasis. While linear regression showed weaker results for modeling, however, additive nonparametric regression analysis suggested that 10km buffers for elevation, and 10 as well as 50km buffers for veg­etation could contribute in better fitness in modeling of these variables. Conclusion: The detailed maps for distribution of disease concluded. The nonlinear regression is a reliable predictor of the relationship between environmental factors and disease incidence, although more and wide researchers are needed to confirm it.

Background: Establishment of sand flies laboratory colonies is essential to understand various biological aspects of Phlebotominae sand flies. The aims of the current study were to establish the colony of Phlebotomus sergenti Parrot (1917), the main vector of anthroponotic cutaneous leishmaniasis in old world, and to study biological parameters of this species. Methods: The sand flies were reared at 26–28 °C temperature, 14:10 (light: dark) photoperiod and 70–80% relative humidity. Larval diet was a composted mixture of rabbit faces and rabbit pellets which is prepared through a special process. First to fifth generations of P. sergenti were used to define biological parameters. Results: Results showed that, P. sergenti blood feeding percentage were 42% on chicken, 21% on BALB/c and 37% on golden hamster. Average time of blood digestion, egg incubation, 1st instar larva, pupa and adult emerging was recorded at 3.4, 8.7, 15, 33.3 and 41.2 days after blood feeding, respectively. Mean number of laid eggs was 55.1 and retained eggs were 35 per a female. Fecundity and production rate were 61.6%, and 42.2% respectively. Average longevity recorded at 15.2 days for females and 14.8 days for males. Conclusion: Colony of P. sergenti has been established for the first time in Iran. Average interval time from egg to adult of this species was 32.5 days. Chicken and golden hamster were recommended as a blood source for colony initiation and routine blood feeding, respectively.

Background: Cutaneous leishmaniasis (CL) is one of the most important health problems in many areas of Iran. There are two forms of the disease in Iran, anthroponotic and zoonotic CL. This study conducted to assess the epi­demiological situation of CL in an endemic area of Qom Province, central Iran from Apr to Nov 2015. Methods: The sticky paper traps and aspirating tubes were used for collecting adult sand flies. Sherman traps and small insect nets were used to capture rodents and small mammals. Giemsa staining was used for preparing the ex­panded smear and followed by PCR for identifying the causative agent in human, vectors, and reservoirs. In this study, relative frequency of CL was also calculated. Results: Fourteen species of Phlebotomine sand flies were collected. Phlebotomus papatasi (61.74%) was the pre­dominant species through the period of activity. Overall, 62 Meriones libycus, 8 Nesokia indica, 4 Mus musculus, 16 Allactaga elater and 2 Hemiechinus auritis were caught. PCR technique showed 6 out of 150 P. papatasi (2%), two out of 62 M. libycus (3.23%) and all of suspected human's skin tissue samples (100%) were infected with Leishmania major. The relative frequency of CL was 0.30%. Conclusion: This is the first detection of L. major within P. papatasi, M. libycus and human in Kahak District in Qom Province of Iran. Zoonotic cycle of CL exists in this area, L. major is the causative agent, P. papatasi is the main vector and M. libycus is the main reservoir of the disease.

Abstract Background Identification of the microflora of the sand fly gut and the environmental distribution of these bacteria are important components for paratransgenic control of Leishmania transmission by sand flies. Methods Biotic and abiotic bacterial communities of four compartments of a hyper-endemic focus of Zoonotic Cutaneous Leishmaniasis (ZCL) were investigated using 16S ribosomal DNA sequencing and phylogenetic tree construction. These compartments include Phlebotomus papatasi’s gut, skin and intestinal tract of great gerbil Rhombomys opimus, the gerbil nest supplies, and plant food sources of the vectors and reservoirs. Results Sequence homology analysis using nine available 16S rDNA data bases revealed 40, 24, 15 and 14 aerobic bacterial species from the vector guts, the gerbil bodies, the gerbil nests, and the plants, respectively. The isolated bacteria belong to wide ranges including aerobic to facultative anaerobic, pathogen to commensals, sand fly oviposition inducers, land to air and ocean habitats, animal and human probiotics, and plant growth-promoting rhizobacteria. Matching data analysis suggested that the adult P. papatasi gut bacteria could be acquired from three routes, adult sugar feeding on the plant saps, adult blood feeding on the animal host, and larval feeding from nest supplies. However, our laboratory experiment showed that none of the bacteria of the reservoir skin was transmitted to female sand fly guts via blood feeding. The microflora of sand fly guts were associated with the sand fly environment in which the predominant bacteria were Microbacterium, Pseudomonas, and Staphylococcus in human dwellings, cattle farms, and rodent colonies, respectively. Staphylococcus aureus was the most common bacterium in sand fly guts. Presence of some sand fly ovipoisition inducers such Bacillus spp. and Staphylococcus saprophyticus support association between gut flora and oviposition induction. Conclusions Results of this study showed that Bacillus subtilis and Enterobacter cloacae particularly subsp. dissolvens are circulated among the sand fly guts, the plants, and the sand fly larval breeding places and hence are possible candidates for a paratransgenic approach to reduce Leishmania transmission.

Background: Aquatic insects are very abundant and divers groups of insects that are associated with an aquatic or semiaquatic environment in one or more of their life stages. These insects have been, in some cases, well studied because they are vectors of several diseases. This is the first comprehensive faunistic study of aquatic insects from Babol County. The results may provide basic data for further taxonomic and ecological studies of aquatic insects as biological control agent or classification of water quality for the country. Methods: The specimens were collected using different methods including: D-frame net collector, standard mos­quito dipper (350ml), Sweep-Netting and plastic pipette. Sampling carried out in different part of breading places in several times. Results: During this study a total of 196 aquatic specimens were collected from different habitats and were mor­phologically identified including 18 families classified in 6 orders: Diptera, Trichoptera, Ephemeroptera, Plecoptera, Hemiptera and Odonata. Babol and Amol district in Mazandaran Province are located in humid climate regions with suitable ecological factors of humidity, moderate temperature and the variety of plant species. There are different species of aquatic insects in different habitats. Conclusion: The results will provide information for biodeveristy, species richness, their role for biological control as well as calcification of rivers based on abundance of aquatic insects. Therefore the understanding of ecological specifications of aquatic insects could provide a clue for further Arthropod-borne disease control. Additionally aquatic insect could be used for classification of water bodies

Background: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease prevailed in many rural areas of 17 out of 31 provinces in Iran. The main vector of the disease is Phlebotomus papatasi and the causative agent is Leishmania major in ZCL foci of Iran. In the current study we investigated the validity of accessory glands secre­tions as an indicator to recognize parous from nulliparous Ph. papatasi females under laboratory conditions. Methods: Over 235 laboratory-reared females of Ph. papatasi were dissected in 6 groups including: newly emerged, one hour, one day, two days, and three days after blood feeding and also after oviposition under stereo microscope for their parity in 2014–2015. Results: Transparent glands were compatible with nulliparous only in newly emerged sand flies. In sand flies dis­sected after oviposition, accessory glands were rather large as a result of oviposition though they were transparent. Conclusion: The accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of Ph. papatasi females.

Attractive Toxic Sugar Baits (ATSB) is a new vector control method that meets Integrated Vector Management (IVM) goals. In an experimental design, this study aimed to determine effects of ATSB on control of Phlebotomus papatasi, as a main vector of Zoonotic Cutaneous Leishmaniasis (ZCL), in Qom Province, center of Iran.In a cross-sectional design, boric acid was mixed with brown sugar solution and tested as toxic baits for P. papatasi. Two methods were utilized to use the baits: (a) spraying ATSB on vegetation, bushes, and shrubs; and (b) setting ATSB-treated barrier fences in front of colonies at 500 m distance from the houses in outskirts of villages. In order to examine the residual efficacy rate of ATSB-treated barrier fences, the bioassay test was used. Density of P. papatasi sandflies was measured using sticky and light traps biweekly. For data analysis, Mann-Whitney U Test and Kruskal-Wallis were used. Results ATSB-treated barrier fences led to 3 times reduction in P. papatasi population. Besides that, ATSB spraying on plants led to more than 5 times reduction in P. papatasi population.Comparing the incidence of leishmaniasis in treated villages before and after the study showed that the incidence was statistically reduced. Therefore, ATSB is an effective method to control vectors and prevent leishmaniasis.

Background: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected disease with public health importance that is common in many rural areas of Iran. In recent years, behavioral resistance and/or bait shyness against the common rodenticide among reservoir hosts of ZCL have been reported. The aim of this study was to evaluate the effectiveness of Klerat® and zinc phosphide against natural reservoir of ZCL. Methods: This survey was carried out in four villages located 45 to 95 km far from Esfahan City Esfahan province, central Iran from April to November 2011. The rodent burrows were counted destroyed and reopened holes baited around all villages. Effect of rodent control operation on the main vector density and incidence of ZCL were evaluated. Results: The reduction rate of rodent burrows after intervention calculated to be at 62.8% in Klerat® and 58.15% in zinc phosphide treated areas. Statistical analysis showed no difference between the densities of the vector in indoors and outdoors in intervention and control areas. The incidence of the disease between treated and control areas after intervention was statistically different (P< 0.05). Conclusion: Klerat® could be a suitable alternative for zinc phosphide in a specific condition such as behavior resistance or occurrence of bait shyness.

INTRODUCTION:Cutaneous leishmaniasis (CL) is among the top 10 infectious disease priorities in the world, and the leading cause of morbidity in Iran. The present study was conducted to assess the risk of CL, and to determine some epidemiological features of the disease in endemic areas of Qom Province in Central Iran during 2009 to 2013. METHODS:Data regarding human cases of the disease were obtained from the Qom Province Health Center, prepared and stored in a spatial database created in ArcGIS10.3. A total of 9 out of 212 Leishmania spp. positive slides taken in 2013 from patients residing in Qom city were examined using molecular methods and the species of Leishmania was identified by PCR-RFLP. Those 9 patients had no history of travel outside the city. Spatial analysis and clustering methods were applied to find major hot spots and susceptible areas for the establishment of novel foci of the disease. Transmission patterns were examined for spatial autocorrelation using the Moran's I statistical application, and for the clustering of high or low values using the Getis-Ord Gi* statistics. RESULTS:During the period of study, a total of 1767 CL cases were passively reported in the area, out of which were 65% males and 35% females. The highest and lowest numbers of cases were reported in 2010 and 2013, respectively. Importantly, 979 cases were reported from urban areas, while the remainder came from rural areas. Leishmania major was detected as the causative agent of CL in the city of Qom. Remarkably, most patients recorded in Qom city were associated with a history of travel to the endemic areas of CL within the province, or to other endemic areas of the disease in Iran. Spatial distribution of CL cases revealed northeastern and southwestern quarters of the city were the major hot spots of the disease (P