Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

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.

Epidemiological survey on Cutaneous Leishmaniasis in southwestern Iran.

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.

Susceptibility status of wild population of Phlebotomus sergenti (Diptera: Psychodidae) to different imagicides in a endemic focus of cutaneous leishmaniasis in northeast of Iran.

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.

Anti Leishmania activity of Lucilia sericata and Calliphora vicina maggots in laboratory models.

Use of sterile fly larvae (maggots) of blow flies for the treatment of many different types of skin and soft tissue wounds is called Maggot debridement therapy (MDT). The larvae of blow flies secrete a broad spectrum of compounds with diverse mechanisms of action in the gut and salivary glands called excretion/secretion (ES) products which showed to have antimicrobial activities against Gram negative and positive bacteria. Cutaneous leishmaniasis (CL) which is the common form of leishmaniasis is difficult to treat. In this study, the effect of ES from 2nd and 3rd stages of L. sericata and C. vicina larvae on in vitro Leishmania major amastigote growth in macrophage was evaluated. The effect of ES on Leishmania growth was estimated by assessing the rate of macrophage infection and the number of amastigotes per infected macrophages. In addition, the anti Leishmania activities of larval and ES of L. sericata and C. vicina on the skin lesion induced by L. major infection was evaluated in susceptible BALB/c mice. The results showed that ES of both flies reduced the number of infected macrophages; 2.6 and 1.5-fold using L. sericata ES and C. vicina ES, respectively, and inhibited amastigotes growth in macrophages; 2.03 and 1.36-fold by L. sericata ES and C. vicina ES, respectively as compared to the control group. The results showed that L. sericata ES was significantly more effective than C. vicina ES to inhibit in vitro L. major amastigotes growth, The size of lesion was significantly smaller in BALB/c mice treated with L. sericata ES than treated with C. vicina ES. The results of in vivo experiments suggested that pre-treatment with ES derived from L. sericata may have some protective effects on the development of L. major lesion. Therefore, it seems that maggot ES might be considered as a possible candidate for the treatment of cutaneous leishmaniasis.

Bioassay evaluation of residual activity of attractive toxic sugar-treated barrier fence in the control of Phlebotomus papatasi (Diptera: Psychodidae).

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.

Modeling the Distribution of Cutaneous Leishmaniasis Vectors (Psychodidae: Phlebotominae) in Iran: A Potential Transmission in Disease Prone Areas.

Cutaneous leishmaniasis (CL) is now the main vector-borne disease in Iran. Two forms of the disease exist in the country, transmitted by Phlebotomus papatasi and Phlebotomus sergenti s.l. Modeling distribution of the vector species is beneficial for preparedness and planning to interrupt the transmission cycle. Data on sand fly distribution during 1990-2013 were used to predict the niche suitability. MaxEnt algorithm model was used for prediction using bioclimatic and environmental variables (precipitation, temperature, altitude, slope, and aspect). Regularized training, area under the curve, and unregularized training gains were 0.916, 0.915, and 1.503, respectively, for Ph. papatasi. These values were calculated as 0.987, 0.923, and 1.588 for Ph. sergenti s.l. The jackknife test showed that the environmental variable with the highest gain when used in isolation has the mean temperature of the wettest quarter for both species, while slope decreases the gain the most when it is omitted from the model. Classification of probability of presence for two studied species was performed on five classes using equal intervals in ArcGIS. More than 60% probability of presence was considered as areas with high potential of CL transmission. These areas include arid and semiarid climates, mainly located in central part of the country. Mean of altitude, annual precipitation, and temperature in these areas were calculated 990 and 1,235 m, 273 and 226 mm, and 17.5 and 16.4°C for Ph. papatasi and Ph. sergenti s.l., respectively. These findings can be used in the prediction of CL transmission potential, as well as for planning the disease control interventions.

Association Between Wolbachia Infection and Susceptibility to Deltamethrin Insecticide in Phlebotomus papatasi (Diptera: Psychodidae), the Main Vector of Zoonotic Cutaneous Leishmaniasis.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups. Result: The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi. Conclusion: Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management.

The therapeutic effect of larval saliva and hemolymph of Lucilia sericata on the treatment of Leishmania major lesion in BALB/c mice946.

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 < 0.0001) smaller in the treated mice compared with the untreated control group. The average parasite burden in the treated mice groups (1.81 ± 0.74, 1.03 ± 0.45 and 3.37 ± 0.41) was similar to the group treated with a daily injection of glucantime (1.77 ± 0.99) and significantly lower (F (7, 16) = 66.39, P < 0.0001) than in the untreated control group (6.72 ± 2.37). CONCLUSIONS: The results suggest that the larval products of L. sericata were effective against L. major parasites both in vivo and in vitro. However, more clinical trial studies are recommended to evaluate the effects of these larval products on human subjects.

Evaluation of Repellency and Lethal Effects of Ultrasonic Waves on the Blattella germanica (Blattodea: Blattellidae).

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.

In Vitro Assessment of the Anti-Proliferative and Anti-Viability Effects of Salivary Gland Extracts from Hyalomma ticks (Acari: Ixodidae) on Human Colorectal Cancer Cells.

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.

Predicting current and future high-risk areas for vectors and reservoirs of cutaneous leishmaniasis in Iran.

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.

Rodents as vehicle for delivery of transgenic bacteria to make paratransgenic sand fly vectors of cutaneous leishmaniasis in field condition.

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.

Evaluation of the Entomopathogenic Fungus Beauveria bassiana on Different Stages of Phlebotomus papatasi (Diptera: Psychodidae), Vector of Zoonotic Cutaneous Leishmaniasis in Iran.

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.

First Report of Natural Infection of Phlebotomus mongolensis to Leishmania major and Leishmania turanica in the Endemic Foci of Zoonotic Cutaneous Leishmaniasis in Iran.

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.

PCR Positivity of Gerbils and Their Ectoparasites for Leishmania Spp. in a Hyperendemic Focus of Zoonotic Cutaneous Leishmaniasis in Central Iran.

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' ears 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 Leishmania 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. A Xenodiagnostic test is recommended for future study.

An outbreak of cutaneous leishmaniasis due to Leishmania major in an endemic focus in central Iran.

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major is an important endemic disease and a major public health concern in Iran. Following an outbreak of leishmaniasis in 2013 in one of the important foci in Iran, the researchers were prompted to determine the underlying causes of the epidemic. Adult sand flies were collected using sticky traps and aspirating tubes and infection with Leishmania parasites was evaluated. Also, rodents were captured using Sherman live traps and stray dogs were hunted and were tested for the presence of leishmanial infection. Active case detection was also conducted and data related to each household were recorded using a researcher-designed form. Nested PCR and PCR-RLFP techniques were employed to determine Leishmania infection in the samples. Phlebotomus papatasi was the most dominant species among the 7 different species of sand flies collected in this study. Sergentomyia clydei, Sergentomyia theodori, and Sergentomyia grekovi were identified for the first time in the study area. 20% of collected Ph. papatasi species and one Meriones libycus were infected with Leishmania major. Stray dogs demonstrated no lesions on different parts of their body. The relative frequency of active lesions and scars on the dogs were respectively 5.49% and 1.23% in 2013 and 5.82% and 0.56% in 2014. The highest number of ZCL cases due to L. major was recorded in Ardestan city. Phlebotomus papatasi is the main vector and M. libycus seems to be the primary reservoir host of ZCL in the suburbs of this city. Furthermore, due to the low density of rodent reservoir hosts in the study area, we support the hypothesis that humans may have had a role in maintaining the parasite cycle in the population.

Molecular and Biochemical Detection of Insecticide Resistance in the Leishmania Vector, Phlebotomus papatasi (Diptera: Psychodidae) to Dichlorodiphenyltrichloroethane and Pyrethroids, in Central Iran.

The aim of the present study was to explore resistance markers and possible biochemical resistance mechanisms in the Phlebotomine sand fly Phlebotomus papatasi in Esfahan Province, central Iran. Homogenous resistant strains of sand flies were obtained by exposing P. papatasi collected from Esfahan to a single diagnostic dose of DDT. The adults from the colony were tested with papers impregnated with four pyrethroid insecticides: Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambdacyhalothrin 0.05% to determine levels of cross-resistance. To discover the presence of mutations, a 440 base pair fragment of the voltage gated sodium channel (VGSC) gene was amplified and sequenced in both directions for the susceptible and resistant colonies. We also assayed the amount of four enzymes that play a key role in insecticide detoxification in the resistant colonies. A resistance ratio (RR) of 2.52 folds was achieved during the selection of resistant strains. Sequence analysis revealed no knockdown resistance (kdr) mutations in the VGSC gene. Enzyme activity ratio of the resistant candidate and susceptible colonies were calculated for α-esterases (3.78), ß-esterases (3.72), mixed function oxidases (MFO) (3.21), and glutathione-S-transferases (GST) (1.59). No cross-resistance to the four pyrethroids insecticides was observed in the DDT resistant colony. The absence of kdr mutations in the VGSC gene suggests that alterations in esterase and MFO enzymes are responsible for the resistant of P. papatasi to DDT in central Iran. This information could have significant predictive utility in managing insecticide resistant in this Leishmania vector.

A Comparative Study on the Biodiversity and Species Richness of Phlebotomine Sand Flies (Diptera: Psychodidae) in Kermanshah and Khuzestan Provinces of Iran.

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 N 1 and N 2 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.

Bioecological Study on the Sand Flies (Diptera: Psychodidae, Phlebotominae) in Sari County, North of Iran.

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.

Climate change and its effect on the vulnerability to zoonotic cutaneous leishmaniasis in Iran.

Zoonotic cutaneous leishmaniasis (ZCL) is an important vector-borne disease with an incidence of 15.8 cases per 100,000 people in Iran in 2019. Despite all efforts to control the disease, ZCL has expanded into new areas during the last decades. The aim of this study was to predict the best ecological niches for both vectors and reservoirs of ZCL under climate change scenarios in Iran. Several online scientific databases were searched. In this study, various scientific sources (Google Scholar, PubMed, SID, Ovid Medline, Web of Science, Irandoc, Magiran) were searched. The inclusion criteria for this study included all records with spatial information about vectors and reservoirs of ZCL which were published between 1980 and 2019. The bioclimatic data were downloaded from online databases. MaxEnt model was used to predict the ecological niches for each species under two climate change scenarios in two periods: the 2030s and 2050s. The results obtained from the model were analysed in ArcMap to find the vulnerability of different provinces for the establishment of ZCL foci. The area under the curve (AUC) for all models was >0.8, which suggests the models are able to make an accurate prediction. The distribution of all studied species in different climatic conditions showed changes. The variables affecting each of the studied species are introduced in the article. The predicted maps show that by 2050 there will be more suitable areas for the co-occurrence of vector and reservoir(s) of ZCL in Iran compared to the current climate condition and RCP2.6 scenario. An area in the northwest of Iran is predicted to have suitable environmental conditions for both vectors and reservoirs of ZCL, although the disease has not yet been reported in this area. These areas should be considered for field studies to confirm these results and to prevent the establishment of new ZCL foci in Iran.