Larvicidal Effects of Metabolites Extracted from Nocardia and Streptomyces Species against the Forth Larval Stage of Anopheles stephensi (Diptera: Culicidae).

Background: Larvicidal agents can be produced using microbial resources, which are environmentally friendly, biodegradable, and economical. The study's goal was to evaluate the larvicidal activity of metabolites isolated from Nocardia (N. fluminea, N. soli and N. pseudobrasiliensis) and Streptomyces (S. alboflavus) bacterial species against Anopheles stephensi. Methods: Four metabolites isolated from Nocardia and Streptomyces strains were exanimated for larvicidal activity. The experiments were performed for 24, 48, and 72 hours. 300, 350, 400, 450, 500, 550, and 600 µl of Actinobacteria metabolites were added to 100 cc of dechlorinated water. Fourth-stage larvae were placed in dechlorinated water as a control. LC50 and LC90 were calculated using toxicity data and analyzed. Results: All metabolites had a statistically significant influence on mosquito larvae (P< 0.05). At 24, 48, and 72 hours, the LC50 for N2 (N. fluminea) was 417, 386, and 370 ppm, respectively, and the LC90 was 650, 595, and 561 ppm. Moreover, LC50 for N4 (N. soli) was 389, 376, and 347 and LC90 were 591, 565, and 533 and LC50 for N5 (N. pseudobrasiliensis) was 390, 357, and 341 ppm and LC90 were 589, 532 ppm. In addition, LC50 for S921 (S. alboflavus) was 484, 416, and 382 ppm, and LC90 was 701, 612, and 574 ppm. Conclusion: The four bacterial metabolites tested in our study were found to have a notable effect on the mortality rate of Anopheles stephensi larvae, indicating their potential as natural larvicides. This is an effective technique for controlling Anopheles stephensi that has no detrimental environmental impact.

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 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.

Periplaneta americana (Blattodea: Blattidae) fungal pathogens in hospital sewer systems: molecular and phylogenetic approaches.

Cockroaches are known as mechanical vectors of some pathogens that can infect humans. The present study aims to rapidly identify Periplaneta americana fungal pathogens from sewer systems of public hospitals in Esfahan using the polymerase chain reaction (PCR) technique. A total of 55 P. americana cockroaches were randomly collected by direct trapping from sewer systems of seven hospitals and screened for fungal infectious agents using standard morphological methods and the PCR sequencing. From the American cockroach, we isolated 62 yeasts and 31 molds from the surface, hemocoel, and digestive tract of P. americana. Based on DNA sequence comparisons and other taxonomic characteristics, they were identified as more than four species of yeast and four species of mold. Yeast species including Pichia kudriavzevii, Candida glabrata, Pichia kluyveri, and Candida viswanathii, and molds such as Aspergillus niger, Penicillium italicum, Mucor plumbeus, and Rhizopus oryzae were isolated repeatedly from the surface, hemocoel, and digestive tract of P. americana. Our results show that the use of a combination of morphological, molecular techniques, and phylogenetic analysis can lead to the identification of pathogenic fungal agents in American cockroaches and also knowledge of fungal pathogens-arthropod host relationships.

Sergentomyia species identification and their screening for possible infection to Leishmania spp. in Kaleybar, East-Azerbaijan province, Iran.

Leishmaniasis is a protozoal and vector-borne disease. World health organization has considered the disease as a neglected tropical disease. Phlebotomus and Lutzumyia species (order: Diptera, family: Psychodidae) are human leishmaniasis vectors in new and old worlds. Sergentomyia spp. (Diptera, Psychodidae) are proven vectors of lizard leishmaniasis. Although some studies have identified human Leishmania parasites in Sergentomyia, their role in parasite circulation is unknown yet. Hence, the parasitological and molecular methods were used to study the possible Leishmania infection of Sergentomyia spp., in the human and canine visceral leishmaniasis endemic area in North West of Iran. Even though Sergentomyia specimens were caught in a dominant number compared to Phlebotomus spp., no Leishmania promastigote or DNA was detected in live-caught or sticky trap-caught specimens, respectively. Sergentomyia spp. are proven vectors of sauroleishmaniasis, and despite several global reports of Leishmania infection in Sergentomyia spp., such findings should be carefully interpreted to avoid false vector incriminations.

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.

Molecular Identification of Leishmania infantum kDNA in Naturally Infected Dogs and Their Fleas in an Endemic Focus of Canine Visceral Leishmaniasis in Iran.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as dogs. The existence of three factors, namely a vast distribution area, different hosts, and digestive system with a specific mechanism for digesting blood has led to species of fleas who nourish from mammals be introduced as the potential vectors of diseases. The aim of this study was to assess Leishmania infantum natural infection of dog fleas in northwest Iran in 2018. Methods: A total of 20 infested domestic dogs (Canis lupus familiaris) were randomly selected from 5 villages. Fleas were collected using brushing against dog hairs and fine forceps. Then, they were morphologically identified and preserved in ethanol for molecular assay. The kinetoplast DNA of the parasite was used for detection of Leishmania infantum using a semi-nested polymerase chain reaction (PCR) assay. Results: The human flea, Pulex irritans, and the cat flea, Ctenocephalides felis were identified on 40% and 35% of dogs, respectively. The results of PCR indicated that L. infantum was found in the Ctenocephalides canis (75%) and C. felis (66.7%) collected from infected dogs. No leishmanial infection was observed in P. irritans. Conclusion: It is concluded that fleas could be infected by Leishmania infantum, but maintenance of the parasite and their vectorial competence needs to be determined.

Pulex irritans on Dogs and Cats: Morphological and Molecular Approach.

Background: The painful bite of Pulex irritans; causes wound on the host body and is a vector for Bartonella bacteria species, which can cause trench fever, Rickettsia species, Rocky Mountain spotted fever and Mediterranean spotted fever. We conducted a study to find out the hosts, abundance, as well as the molecular characteristics of this flea species in Meshkin-Shahr County, Ardabil Province, northwest of Iran. Methods: After collecting fleas from different reservoir hosts and transferring them to the laboratory, identification was done using different morphological characters as well as the internal transcribed spacer1 (ITS1) molecular marker. Results: This morphological study indicated that from the 1053 fleas, which were collected from 162 different animals, including cats and dogs, 74 specimens belonged to human flea, Pulex irritans. In addition, in molecular analysis showed a high sequence similarity (99.5%) with the P. irritans counterparts from Spain country and Zanjan of Iran available in GenBank. Conclusion: Pulex irritans species is an obligatory hematophagous ectoparasite of human and animals. Therefore, considering the relatively high frequency of this species on the body of cats and dogs, it is suggested to conduct more studies on its distribution and the possibility of being a vector of pathogens among these animals and human. The results of these studies will be used to compile and review the control programs of this vector.

Evaluation of Susceptibility Status of Phlebotomus papatasi, the Main Vector of Zoonotic Cutaneous Leishmaniasis, to Different WHO Recommended Insecticides in an Endemic Focus, Central Iran.

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.

Molecular Identification of Leishmania Species in Phlebotomus alexandri (Diptera: Psychodidae) in Western Iran.

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.

Relationship between Some Environmental and Climatic Factors on Outbreak of Whiteflies, the Human Annoying Insects.

BACKGROUND: The reports of numerous outbreaks of whiteflies from different parts of the world have increased its medical importance. The aim of this study was to determine relationship between environmental changes and climatic factors with the outbreak of the whitefly population in Tehran, the capital of Iran. METHODS: This study was carried out in urban areas of Tehran, where the increasing population of whiteflies was reported frequently during 2018. In order to entrap the whiteflies, 20 yellow sticky cards smeared with white refined grease were installed on the trunks of the trees at twice per month as trapping time intervals. The captured flies were transferred and conserved in cans containing 70% alcohol and were counted accurately under a stereomicroscope. To determine the relationship between air quality index, precipitation, air temperature and air humidity as environmental and climatic factors with the abundance of whiteflies, change point analysis and Generalized Estimating Equations (GEE) was used. RESULTS: The most density of white flies per trap was 256.6 and 155.6 in early October and late September respectively. The number moved closer to zero from November to April. The population of whiteflies was inversely correlated with the level of air quality index (p= 0.99) and precipitation (p= 0.95), and it had a direct correlation with the high temperature. Also, the population of whiteflies had a direct correlation with the level of air humidity in the first half of the year. CONCLUSION: According to these findings, during spring and summer from early May to early October.

Study on Periplaneta americana (Blattodea: Blattidae) Fungal Infections in Hospital Sewer System, Esfahan City, Iran, 2017.

BACKGROUND: American cockroaches contaminated with pathogens inside hospital manholes can be one of the major problems that health care systems face. OBJECTIVES: The aim of this study was to investigate the fungal infections of American cockroaches in the Esfahan hospital sewage network. The principle goal of the study was about the roaches as a vector of fungi and other pathogens. METHOD: The type of study was descriptive-analytical. A total of 55 American cockroach specimens from the manhole walls of the sewerage system of 7 large hospitals were captured. Samples were taken from the surface of the body, digestive tract, and haemocoel of cockroaches. The specimens were then cultured on Sabouraud dextrose agar separately, and fungi were identified according to the macroscopic and microscopic characteristics. RESULTS: All cockroaches collected from hospitals were infected with fungi. Among the 24 (13 infected and 11 noninfected) (44%) female cockroaches and 31 (18 infected and 13 noninfected) (56%) male cockroaches, it was identified that 40.00% was infected with Aspergillus niger, 3.64% with Rhizopus, 7.27% with Penicillium, and 5.45% with Mucor. 6 cockroaches had no yeast contamination. 17 (30.91%) cockroaches were contaminated with Candida glabrata, 23 (41.82%) cockroaches were contaminated with Candida krusei, and 22 (40%) cockroaches were contaminated with other yeast species. The results of this study showed that Candida krusei had the highest prevalence among the isolated fungi with 35.37% of the digestive system and Aspergillus niger with 70.97% of the surface of the cockroach body. CONCLUSION: The results emphasized the role played by cockroaches as potential pathogenic vectors in hospital environments. Therefore, suitable management is needed for controlling this insect to prevent disease transmission in hospitals.

Human immune response to Phlebotomus sergenti salivary gland antigens in a leishmaniasis-endemic focus in Iran.

Salivary proteins specific antibodies have been shown to be useful biomarkers of exposure to sand fly bites. This study aimed to investigate the level, duration, and dynamics of the human immune response against the SGL of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae), and to assess the immunoreactivity of human sera with SGL components in an endemic area of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The study was carried out in 2-phase; longitudinal and cross-sectional. Sand flies were collected monthly from indoors and outdoors. In the longitudinal study, sera from healthy volunteers were collected monthly, and in the cross-sectional study, sera from healthy volunteers and patients with ACL lesion/s, were collected for immunoassay studies. The level of anti-P. sergenti saliva IgG was detected using the ELISA. Immunoreactivity of individual human sera with saliva components was also assessed by western blotting. Phlebotomus sergenti was the predominant sand fly species in the study area. The maximum and minimum percentages of IgG responses were seen in October (66%) and March (29%), respectively. Additionally, the cross-sectional study showed that 59.3% of the healthy volunteers and 80% of the patients were IgG positive. The antibody response against P. sergenti salivary gland was high during the sand fly active season and declined by the end of the activity of the vectors.  Antibody response against the SGL components of P. sergenti was transient and individual-specific. Some individuals shared a strong reaction against certain individual antigens, which could be considered as vector exposure markers for further investigation. LIST OF ABBREVIATIONS: ELISA: Enzyme-Linked Immunosorbent Assay; SDS PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; SGL: Salivary Gland Lysate; ACL: Anthroponotic Cutaneous Leishmaniasis; PBS: Phosphate Buffered Saline; BCA: Bicinchoninic Acid; PBS-T: Phosphate Buffered Saline Tween; FBS: Fetal Bovine Serum; HRP: Horseradish Peroxidase; TMB: 3,3',5,5'-Tetramethylbenzidine; PVDF: Polyvinylidene Difluoride; SGA: Salivary Gland Antigens; OD: Optical Density; KDa: Kilodalton; VL: Visceral Leishmaniasis; CL: Cutaneous Leishmaniasis; SGs: Salivary glands.

Effectiveness of plant-based repellents against different Anopheles species: a systematic review.

Plant-based repellents have been applied for generations in traditional practice as a personal protection approach against different species of Anopheles. Knowledge of traditional repellent plants is a significant resource for the development of new natural products as an alternative to chemical repellents. Many studies have reported evidence of repellant activities of plant extracts or essential oils against malaria vectors worldwide. This systematic review aimed to assess the effectiveness of plant-based repellents against Anopheles mosquitoes. All eligible studies on the repellency effects of plants against Anopheles mosquitoes published up to July 2018 were systematically searched through PubMed/Medline, Scopus and Google scholar databases. Outcomes measures were percentage repellency and protection time. A total of 62 trials met the inclusion criteria. The highest repellency effect was identified from Ligusticum sinense extract, followed by citronella, pine, Dalbergia sissoo, peppermint and Rhizophora mucronata oils with complete protection time ranging from 9.1 to 11.5 h. Furthermore, essential oils from plants such as lavender, camphor, catnip, geranium, jasmine, broad-leaved eucalyptus, lemongrass, lemon-scented eucalyptus, amyris, narrow-leaved eucalyptus, carotin, cedarwood, chamomile, cinnamon oil, juniper, cajeput, soya bean, rosemary, niaouli, olive, tagetes, violet, sandalwood, litsea, galbanum, and Curcuma longa also showed good repellency with 8 h complete repellency against different species of Anopheles. Essential oils and extracts of some plants could be formulated for the development of eco-friendly repellents against Anopheles species. Plant oils may serve as suitable alternatives to synthetic repellents in the future as they are relatively safe, inexpensive, and are readily available in many parts of the world.

Molecular Characterization of Anopheles sacharovi Based on Sequences of ITS2-rDNA Region and COI Gene in North of Iran.

BACKGROUND: Malaria is an important mosquito-borne disease considered as one of the public health concerns across many countries. Anopheles mosquitoes are the main vectors of Plasmodium parasites, which cause malaria. Some of these vectors such as Anopheles maculipennis s.l. and Anopheles sacharovi are considered as complex of sibling species distributed in north of Iran. METHODS: This study was conducted in north and northwest of Iran including East Azerbaijan, West Azerbaijan, Ardabil, Golestan and North Khorasan provinces with emphasis on the northern borders of the country during 2015-2016. Adult specimens were collected and subjected to morphological identification as well as molecular analysis. RESULTS: Overall, 10405 mosquitoes were collected comprising 21 species. Culex pipiens and Cx. theileri were found as the most frequent species in whole study area. Morphological identification showed that out of 1455 female Anopheles specimens, 77% belonged to An. maculipennis Group. Out of the identified species, ITS2 region and COI gene sequences of 8 An. maculipennis s.s. and 31 An. sacharovi representing all provinces were obtained and submitted to GenBank. The COI sequences for An. sacharovi revealed the presence of 9 haplotypes with similarity of 98.17-100%. CONCLUSION: Some investigations have reported An. martinius as a member of sibling species of An. sacharovi among Iranian Anopheles genus; while based on our study, there was no evidence of the presence of this species in north and northwest of Iran.

The Potential Role of Humans in the Transmission Cycle of Leishmania major (Kinetoplastida: Trypanosomatidae), the Causative Agent of the Old World Zoonotic Cutaneous Leishmaniasis.

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major (Yakimoff and Schokhor, 1914) is known as a neglected tropical disease, transmitted by Phlebotomus papatasi (Scopoli, 1786) in North Africa and the Middle East. The main reservoirs of ZCL are desert gerbils and the role of humans as a reservoir host of the disease is not clearly defined and is therefore investigated in this study. In order to achieve this objective, Ph. papatasi sand flies were allowed to take blood through indirect blood sources (human, Rhombomys opimus (Lichtenstein, 1823) (great gerbil), and BALB/c mice) using artificial feeding or direct xenodiagnoses from ZCL patients. To detect Leishmania promastigotes inside digestive canal, blood-fed sand flies were dissected and examined under light microscope and then confirmed by nested-PCR. In indirect xenodiagnoses, promastigote forms of Leishmania parasite were observed in 7.1%, 12%, and 13.6% of sand flies which fed on Rh. opimus, BALB/c mice, or human blood sources, respectively. In direct xenodiagnoses, among 93 female lab-reared sand flies which fed on active L. major lesion(s), 2.1% Leishmania infection was subsequently observed in the sand flies. No infection was detected in those sand flies that fed on nonlesioned skin of CL patients. Humans can serve as a reservoir of ZCL since the data indicate that Ph. papatasi is able to acquire L. major parasites from an active lesion of ZCL patients and the parasites can complete metacyclogenesis in the sand fly.

Some epidemiological aspects of cutaneous leishmaniasis with emphasis on vectors and reservoirs of disease in the borderline of Iran and Iraq.

Cutaneous leishmaniasis (CL) is endemic and a major health problem in 17 provinces out of 31 in Iran. This study aimed to determine vectors and reservoirs of the disease using molecular techniques in the borderline of Iran and Iraq. Sand flies and rodents were sampled using sticky paper traps and metal wire live traps, respectively, in the selected villages. About 10% of archived confirmed human positive slides was randomly checked for Leishmania by PCR-RFLP assay. The female sand flies were dissected in alcohol 96% in a sterile condition, the head and two segments of the abdomen end permanently mounted for identification and the remaining of body used for DNA extraction. The direct parasitological tests were carried out on the stained slides of rodents for Leishmania as well as PCR-RFLP assay used for molecular detection of parasite. A total of 2050 sand flies were identified comprising of Phlebotomus papatasi, Sergentomyia sintoni, Se. clydei, Se. mervynae, Se. theodori, Se. dentate and Se. iranica. The Ph. papatasi was ranked as a prevailing sand fly species. Molecular tests on female sand flies revealed infection of Ph. papatasi to Leishmania major. Direct parasitology and molecular tests confirmed of 20% infection to L. major among the sole rodents species "Tatera indica". Due to wide dispersion of rodents colonies in the area and long favorite climate condition for sand flies, the CL foci will be provided the health risk for the religious tourists.

Geographic and ecological features of phlebotomine sand flies (Diptera: Psychodidae) as leishmaniasis in Central Iran.

Phlebotomine sand flies occur throughout the tropics and subtropics, as well as in temperate regions of the world. They are vectors of human and canine leishmaniasis and sand fly fevers caused by phleboviruses. This study was aimed to determine the geographic and ecological characteristics of phlebotomine sand flies as vectors of leishmaniasis and to prepare a checklist of phlebotomine sand flies. The study was conducted in Qom province, central Iran, between April and November 2016. Qom is located in latitude 34.6399°N and longitude 50.8759°E with average annual minimum and maximum temperatures of 16.5 and 49 °C, annual rainfall of 150 mm and relative humidity of 84 and 28%, respectively. Sand flies were collected by sticky paper traps from Qom city and its six districts. The sand flies collected were separated from the sticky paper traps using an insulin syringe and kept in 70% ethanol for species identification using taxonomic keys. Also, a literature review was performed using all published reports on phlebotomine sand flies in this province during 1999-2015. A total of 28,410 sand flies from two genera and 14 species were collected. Phlebotomus papatasi, the main vector of zoonotic cutaneous leishmaniasis and arboviruses, and Phlebotomus sergenti, the vector of anthroponotic cutaneous leishmaniasis, were the predominant species followed by Phlebotomus kandelakii, P. major and P. alexandri. Fourteen species from two genera mostly from wet and mountainous areas were identified in the study area. Kahak and Markazi districts were identified as high-risk foci with numerous leishmaniasis vectors species; we recommend intensifying and strengthening of vector control programme in the area of study.

Assessing the insecticide susceptibility status of field population of Phlebotomus papatasi (Diptera: Psychodidae) in a hyperendemic area of zoonotic cutaneous leishmaniasis in Esfahan Province, Central Iran.

Leishmaniasis is a Neglected Tropical Disease (NTD) and emerging parasitic infection that affect mainly poor regions around the world. This study aimed to determine the baseline susceptibility of Phlebotomus papatasi to commonly used insecticides in a hyper endemic area using WHO standard procedure in central Iran. A total of 4-5 replicates containing 120-200 sand flies were used for each insecticide. Baseline susceptibility to DDT and pyrethroids was assessed on 5326 specimens collected from the study area. The LT50 and LT90 values were measured according to the World Health Organisation test using probit analysis and regression lines. The test results against males P. papatasi revealed that LT50 values to DDT 4%,Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 564.07, 38.08, 1.95, 0.60 and 9.78s and the figures for females were 584.44, 110.10, 11.64, 1.53 and 16.91s, respectively. Our results indicated that P. papatasi as the main cutaneous leishmaniasis vector was susceptible to Cyfluthrin 0.15%, Lambdacyhalothrin 0.05%, Permethrin 0.75% and Deltamethrin 0.05% and tolerant to DDT 4%. This study was carried out in one out of many Leishmaniasis foci in Iran. We recommend that future studies incorporate other regions and use the same procedure for monitoring and evaluating sand fly resistance. Also, WHO can provide a specific guideline and create a test kit for sand fly resistance monitoring and for applying susceptibility test because the tubes prepared for mosquitoes are not actually fit for sand flies.