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Background and Objective: Epidemiology of Visceral Leishmaniasis is affected by combination quality and interaction of Parasite-Vector-Host and environmental conditions. So, disease cycle related and eco-social factors and environmental risk factors co-analyzing, help to understanding these interactions, prognosis and orientation in disease control and treatment. This study was done to determine the role of nomadic and non-nomadic lifestyle in transmission of Kala-Azar in the Northwest of Iran. Materials and Methods: In this case-control study, firstly the prevalence of Kala-Azar among people living in selected villages were determined by both serological test (Direct Agglutination Test: DAT) and immunological test (Montenegro Skin Test: MST) in the Northwest of Iran, on the way of Shahsavan tribe travelling in summer/winter quarters villages. Then DAT was conducted on the dogs presenting in those villages during 2006. One year later Seroconversion rate was calculated through collection of the individual’s negative sera and re-analyzing them via DAT. Finally, occurrence of Visceral Leishmaniasis in relation with various involving factors like dog density/abundance and nomadic and non-nomadic lifestyle using Chi-Square test were determined. Results: Both MST and DAT were significantly higher in the nomadic lifestyle than in the non-nomadic lifestyle (P

Background and Objective: Visceral Leishmaniasis or Kala-azar is an important infectious disease in northwestern Iran. Members of the Leishmania donovani complex, L. donovani and L.infantum, are the two main parasites causing the disease in the world. In this study immunophenotype characters such as N-glycosylation, and T-cell and B-Cell epitopes of CPB gene was evaluated in the Leishmania parasites isolated from sandflies of the region. Materials and Methods: Partial of the CPB (702-741 bp) of Leishmania parasites was amplified and sequenced and used for bioinformatics analysis such as test three dimensional (3D) protein structure, N-glycosylation, and T-cell and B-Cell epitopes. Results: In 7 out of 3477 sand flies there was a positive PCR test for systeine protease B, gene. Also according to findings of this study, both agents of kala-azar L. donovani and L. infantum were bing transfered by sand flies of Phlbtomus perfiliewi transcaucasicus in North West Iran. DNA analysis of the CPB gene showed a cytosine insertion at 5’ end of the proofreading frame of the gene resulted in a stop codon (TGA) seven AA further down and hence translation is halted. This caused a short amino acid chain with only 76 AA much shorter than normal CPB peptide with 234-247 AA. This mutation has not been found in the L.infantum strain resulted in a normal CPB peptide. AA analysis showed no N-glycosylation site, T-cell and B-Cell epitopes on the short peptide of the L. donovani strain. Conclusion: This is the shortest CPB peptide chain reported for the L. donovani complex in the literature. This short peptide could have an effect on host-parasite and vector-parasite interactions. Since the CPBs genes have important implication on host–parasite and play key roles in infection and expression of the disease, further studies on the L. donovani parasite and its diminutive peptide necessitate to improve our understanding about the epidemiology of visceral leishmaniasis in Iran.

Echinops plants have received great attention for the treatment of many diseases due to pharmacological properties such as their antidiabetic, antioxidant, and anti-inflammatory characteristics. The major purpose of the present study was to investigate the cardioprotective benefits of Echinops cephalotes (Ech) against myocardial ischemia-reperfusion (MI/R) injury. Male Wistar rats were randomly allocated to three groups: sham, MI, and MI + Ech. The left coronary artery (LAD) was blocked for 30 minutes to induce MI. In the treatment group, rats were given 150 mg/kg/day of Ech extract for 28 days. Aqueous extracts were made from Echinops plants. To study heart function, fibrosis, cardiac damage indicators, and oxidative stress factors, echocardiography, Masson's trichrome staining, and biochemical tests were used. The expression of matrix metalloproteinase 2 and 9 (MMP2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP) was determined using Western blotting. Tissue damage was assessed using hematoxylin and eosin staining. MI group exhibited significantly reduced ejection fraction (EF) and fractional shortening (FS), enhanced levels of lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), cardiac Troponin I (cTnI), and malondialdehyde (MDA), as well as a decrease in the Glutathione (GSH) tissue content, reduced activity of superoxide dismutase (SOD), increasing fibrosis, upregulations of MMP-2 and MMP-9, and reduction of TIMP compared to the sham group. The findings suggest that Ech in particular, could be a promising therapeutic agent to reduce the damage in MI by targeting oxidative stress and modulating the activities of matrix metalloproteinases and their tissue inhibitors., Competing Interests: Declaration of Competing Interest We submitted a manuscript entitled “Cardioprotective effects of the aqueous extract of Echinops cephalotes on myocardial ischemia-reperfusion in rats by modulation of MMP-2, MMP-9, TIMP, and oxidative stress” for publication in the Biomedicine & Pharmacotherapy. We inform you that there were no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Maleki-Ravasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Black flies are blood-sucking insects of public health importance, and they are effective vectors of pathogens and parasites, such as filarial nematodes of the genus Onchocerca. Our previous surveys have shown that individuals of Simulium turgaicum are annoying pests of humans and livestock in the Aras River Basin of Iran. In the present study, adult black flies of S. turgaicum were trapped from different ecotopes of five villages in Khoda-Afarin County, Iran. By using a sensitive nested PCR assay and targeting the nuclear 18S rDNA-ITS1 marker, filarial infections were found in 38 (1.89%) of 2005 black flies. Homology exploration of 360 bp of the sequences indicated that the filarial worms are members of the family Onchocercidae, with maximum alignment scores of 93-95%. Phylogenetic analysis showed that two Iranian Onchocerca isolates were clustered in the O. fasciata-O. volvulus lineage and were well separated from other filarial nematodes. Both the entomological evidence (empty abdomen of the specimens) and climatologic data (adequate accumulated degree days for development) suggest that the filarial DNA was probably that of infective larvae of vertebrates. This is the first report of an infection by Onchocerca species in S. turgaicum and the first record of onchocercids in black flies in Iran; however, more research is required to demonstrate transmission of these filarial worms by black flies in nature., (© 2023. Springer Nature Limited.)

Clinical features and severity of the leishmaniasis is extremely intricate and depend on several factors, especially sand fly-derived products. Bacteria in the sand fly's gut are a perpetual companion of Leishmania parasites. However, consequences of the concomitance of these bacteria and Leishmania parasite outside the midgut environment have not been investigated in the infection process. Herein, a needle infection model was designed to mimic transmission by sand flies, to examine differences in the onset and progression of L. major infection initiated by inoculation with "low" or "high" doses of Enterobacter cloacae and Bacillus subtilis bacteria. The results showed an alteration in the local expression of pro- and anti-inflammatory cytokines in mice receiving different inoculations of bacteria. Simultaneous injection of two bacteria with Leishmania parasites in the low-dose group caused greater thickness of ear pinna and enhanced tissue chronic inflammatory cells, as well as resulted in multifold increase in the expression of IL-4 and IL-1β and a decrease in the iNOS expression, without changing the L. major burden. Despite advances in scientific breakthroughs, scant survey has investigated the interaction between micro and macro levels of organization of leishmaniasis that ranges from the cellular to macro ecosystem levels, giving rise to the spread and persistence of the disease in a region. Our findings provide new insight into using the potential of the vector-derived microbiota in modulating the vertebrate immune system for the benefit of the host or recommend the use of appropriate antibiotics along with antileishmanial medicines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Amni, Maleki-Ravasan, Nateghi-Rostami, Hadighi, Karimian, Meamar, Badirzadeh and Parvizi.)

Background: Paederus fuscipes is medically the most famous rove beetle, which causes dermatitis or conjunctivitis in humans, as well as gastrointestinal toxicosis in livestock, via releasing toxic hemolymph containing pederin. Pedrin biosynthesis genes have been identified in uncultured Pseudomonas-like endosymbionts that are speculated to be acquired through a horizontal transfer. However, the composition of the P. fuscipes microbial community, especially of the gut and genital microbiome, remains unclear. This study was aimed to characterize the structure and diversity of P. fuscipes-associated bacterial communities in terms of gender, organ, and location using the Illumina HiSeq platform in the southern littorals of Caspian Sea., Results: The OTUs identified from P. fuscipes specimens were collapsed into 40 phyla, 112 classes, 249 orders, 365 families, 576 genera, and 106 species. The most abundant families were Pseudomonadaceae, Spiroplasmataceae, Weeksellaceae, Enterococcaceae, and Rhizobiaceae, respectively. Thirty top genera made up > 94% of the P. fuscipes microbiome, with predominating Pseudomonas, followed by the Spiroplasma, Apibacter, Enterococcus, Dysgonomonas, Sebaldella, Ruminococcus, and Wolbachia. Interesting dissimilarities were also discovered within and between the beetle microbiomes in terms of genders and organs. Analyses showed that Spiroplasma / Apibacter as well as Pseudomonas / Pseudomonas were the most abundant in the genitals / intestines of male and female beetles, respectively. Bacterial richness did not display any significant difference in the three provinces but was higher in male beetles than in females and more in the genitals than intestines., Conclusions: The present study identified Pseudomonas-like endobacterium as a common symbiont of P. fuscipes beetles; this bacterium begins its journey from gut and genitalia of females to reach the male rove beetles. Additionally, male and female rove beetles were characterized by distinctive microbiota in different organs, likely reflecting different functions and/or adaptation processes. Evidence of the extension of P. fuscipes microbiome from the environmental paradigm to the pathobiome was also presented herein. A comprehensive survey of P. fuscipes microbiome components may eventually lead to ecological insights into the production and utilization of defensive compound of pederin and also the management of linear dermatitis with the use of available antibiotics against bacterial pathogens released by the beetles. Video Abstract., (© 2023. The Author(s).)

Background: More than 90% of malaria cases occur in Africa where the disease is transmitted by Anopheles gambiae and Anopheles arabiensis. This study evaluated the anti-mosquito properties of Juniperus virginiana (JVO) and Pelargonium roseum (PRO) essential oils (EOs) against larvae and adults of An. gambiae sensu lato (s.l.) from East Africa in laboratory and semi-field conditions., Methods: EOs was extracted from the aerial green parts of Asian herbs by hydrodistillation. Their constituents were characterized by gas chromatography-mass spectrometry (GC-MS). Larvicidal activities of JVO, PRO, and PRO components [citronellol (CO), linalool (LO), and geraniol (GO)] were investigated against An. gambiae sensu stricto (s.s.). The percentage of knockdown effects and mortality rates of all oils were also evaluated in the adults of susceptible An. gambiae s.s. and permethrin-resistant An. arabiensis., Results: GC-MS analyses identified major constituents of JVO (sabinene, dl-limonene, β-myrcene, bornyl acetate, and terpinen-4-ol) and PRO (citronellol, citronellyl formate, L-menthone, linalool, and geraniol). Oils showed higher larvicidal activity in the laboratory than semi-field trials. The LC 50 values for JVO/PRO were computed as 10.82-2.89/7.13-0.9 ppm and 10.75-9.06/13.63-8.98 ppm in laboratory and semi-field environments, respectively at exposure time of 24-72 h. The percentage of knockdown effects of the oils were also greater in An. gambiae s.s. than in An. arabiensis. Filter papers impregnated with JVO (100 ppm) and PRO (25 ppm) displayed 100% mortality rates for An. gambiae s.s. and 3.75% and 90% mortality rates, for An. arabiensis populations, respectively. Each component of CO, LO, and GO exhibited 98.13%, 97.81%, and 87.5%, respectively, and a mixture of the PRO components indicated 94.69% adult mortality to permethrin-resistant An. arabiensis., Conclusions: The findings of this study show that PRO and its main constituents, compared to JVO, have higher anti-mosquito properties in terms of larvicidal, knockdown, and mortality when applied against susceptible laboratory and resistant wild populations of An. gambiae s.l. Consequently, these oils have the potential for the development of new, efficient, safe, and affordable agents for mosquito control., (© 2022. The Author(s).)

Background: Anopheles stephensi is an important malaria vector mosquito in Iran and other western Asian countries. In many human communities, plant products have been used traditionally instead of synthetic pesticides for mosquito control due to their minimal hazardous effects. Teucrium polium , known popularly as felty germander, has been introduced in Persian Medicine (PM) as an insect repellent from a long time ago., Methods: The present study was undertaken to evaluate repellent and larvicidal activity of dichloromethane (DCMETP) and ethanolic extracts (EE-TP) of T. polium against An. stephensi under laboratory conditions. The possible chemical components of the extracts were also investigated through gas chromatography/mass spectrometry (GC-MS) technique., Results: Based on the results, DCME-TP showed better repellent activity than EE-TP with 56.67 and 28.33 % protection, respectively. Larvicidal activity of DCME-TP with 49.41% mortality was also higher than EE-TP (20.24%). The main identified constituents of DCME-TP were long chain alkanes, phenol, aromatic ester, oxaspiro and triterpenoid. While phenolic and aliphatic acid were only the identified components in EE-TP. It is notable that lupeol was detected in DCME of T. polium for the first time., Conclusion: DCME-TP can be considered as a new herbal candidate to control An. stephensi mosquitoes. Further studies are required on this extract for the fractionation and identification of the active compounds, and the evaluation of their bioactivity in the laboratory and field., Competing Interests: Conflict of interest statement Authors declare that there is no conflict of interest., (Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences.)

Background: Updated information on the vectorial capacity of vectors is required in each malarious areas as well in Iran and its neighboring countries such as Afghanistan. The aims of this study were to investigate the potential infection of about 800 specimens collected from malarious areas of Afghanistan and Iran, and to differentiate biological forms of Anopheles stephensi., Method: Two molecular markers, 18S RNA gene subunit and AsteObp1 intron I, were used respectively for investigation Plasmodium infection and identifying the biological forms of An. stephensi., Results: Plasmodium infection was detected in 4 pools of Afghanistan specimens, including An. stephensi, collected from Nangarhar. Individually examination showed infection in 5 An. stephensi (infection rate: 1.25), to P. falciparum (2), P. vivax (2) and a mix infection. Out of five infected specimens, three were intermediate forms and two were mysorensis. No infection was found in specimens collected from Iran (Chabahar County), probably due to the active malaria control program in south-east of Iran., Conclusion: The key role of An. stephensi, as a known Asian malaria vector, was re-emphasized in Afghanistan by the results achieved here. The fauna of vectors and the pattern of biological forms of An. stephensi are similar in both countries that urge regional investigations to provide evidence-based and applied data for decision-maker in malaria control., (© 2022 Vahideh M., et al.)

Background: Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. Paratransgenesis using symbiotic bacteria offers a sustainable and environmentally friendly strategy to combat this disease. In the study reported here, we evaluated the disruption of malaria transmission in the Anopheles stephensi-Plasmodium berghei assemblage using the wild-type (WT) and three modified strains of the insect gut bacterium, Enterobacter cloacae., Methods: The assay was carried out using the E. cloacae dissolvens WT and three engineered strains (expressing green fluorescent protein-defensin (GFP-D), scorpine-HasA (S-HasA) and HasA only, respectively). Cotton wool soaked in a solution of 5% (wt/vol) fructose + red dye (1/50 ml) laced with one of the bacterial strains (1 × 10 9 cells/ml) was placed overnight in cages containing female An. stephensi mosquitoes (age: 3-5 days). Each group of sugar-fed mosquitoes was then starved for 4-6 h, following which time they were allowed to blood-feed on P. berghei-infected mice for 20 min in the dark at 17-20 °C. The blood-fed mosquitoes were kept at 19 ± 1 °C and 80 ± 5% relative humidity, and parasite infection was measured by midgut dissection and oocyst counting 10 days post-infection (dpi)., Results: Exposure to both WT and genetically modified E. cloacae dissolvens strains significantly (P < 0.0001) disrupted P. berghei development in the midgut of An. stephensi, in comparison with the control group. The mean parasite inhibition of E. cloacae WT , E. cloacae HasA , E. cloacae S-HasA and E. cloacae GFP-D was measured as 72, 86, 92.5 and 92.8 respectively., Conclusions: The WT and modified strains of E. cloacae have the potential to abolish oocyst development by providing a physical barrier or through the excretion of intrinsic effector molecules. These findings reinforce the case for the use of either WT or genetically modified strains of E. cloacae bacteria as a powerful tool to combat malaria., (© 2022. The Author(s).)

Ethnopharmacological Relevance: Trehala manna (TM), the edible cocoons of several weevil species, e.g. Larinus hedenborgi Boheman, 1845 (Coleoptera: Curculionidae) and their host plant, i.e. Echinops cephalotes DC. (EC) (Asteraceae), are traditionally used to treat pain, inflammation, infectious diseases, as well as respiratory, renal, reproductive and metabolic disorders., Aim of the Study: This study investigated the metabolic effects of aqueous extracts from TM and EC on diabetic male Wistar albino rats., Materials and Methods: Animals were orally gavaged with the extracts (75, 150, and 300 mg/kg), normal saline, and glibenclamide (Glbn), for 28 days. The serum levels of glucose, insulin, lipid profile, and hepatic enzymes, plus the body weight of rats were measured at the beginning and the end of study. The proximate composition of the extracts was determined, additionally. The antioxidant and cytotoxic potency of the extracts were evaluated by radical scavenging/ferric reducing and viability assays, respectively., Results: Treatment of diabetic rats with the extracts significantly altered metabolic biomarkers compared with diabetic, control and Glbn-treated groups, but not in a dose-dependent manner. However, the antihyperglycemic effects of TM 75 /EC 300 , the antiobesity effects of EC 150 , and the hepatoprotective effects of TM 150 /EC 150 were even stronger than those of Glbn. TM/EC-treated groups represented normal cell architecture in the pancreatic and renal tissues. Nutrient analysis displayed that TM is rich in sugar and magnesium, whereas EC is abundant in protein, sodium, potassium, and calcium. The extracts showed no antioxidant and cytotoxic effects, as compared to the control groups., Conclusions: The findings suggest that active ingredients in the extracts evaluated are responsible for the metabolic effects by lowering blood sugar and restoring the damaged islets of Langerhans. The close trophic relationship of the TM-producing beetle with the host thistle justifies the overlaps of the bioactivity of the TM and EC extracts., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Scuttle flies (Diptera: Phoridae) are mega-diverse and often synanthropic insects that play superb roles in various ecosystems. Identification of this group of insects is challenging due to their small size, morphological identification difficulties, niche diversity, and lack of taxonomic keys. To pave the way, an in-depth investigation was directed toward the scuttle flies in Iran using morphological and molecular data. A dichotomous key was also developed to identify the genus and species of the phorids reported in the country. The faunistic findings revealed the presence of about 22,000 (13,903 male and 8,097 female) phorid materials organized into 11 genera. Megaselia species (n = 13768), made up about 99% of the specimens studied. Moreover, 71 morphologically defined species belonging to nine genera were molecularly characterized using COI, 28S rRNA, and Arginine kinase datasets. Excluding four Megaselia Rondani, 1856 species, our results specified that morphologically delimited species were in agreement with the molecular analyses inferred from the COI/28S rRNA and COI/Arginine kinase sequences with genetic distances and phylogenetic trees. According to the results of the present study and previously published data, the Phoridae recorded for Iran are a total of 97 species that are ordered in 13 genera and three subfamilies, including Chonocephalinae, Metopininae and Phorinae. By comparing the known world phorid genera, a new monotypic genus of scuttle flies, Mahabadphora aesthesphora gen. nov., sp. nov., was identified based on its morphological and molecular characteristics and included in an updated key. Our results could comprehensively determine the taxonomic status of scuttle flies in Iran, scrutinize their phylogenetic structures and facilitate their identification., Competing Interests: The authors have declared that no competing interests exis.

Anopheles stephensi with three different biotypes is a major vector of malaria in Asia. It breeds in a wide range of habitats. Therefore, safer and more sustainable methods are needed to control its immature stages rather than chemical pesticides. The larvicidal and antibacterial properties of the Pelargonium roseum essential oil (PREO) formulations were investigated against mysorensis and intermediate forms of An. stephensi in laboratory conditions. A series of nanoemulsions containing different amounts of PREO, equivalent to the calculated LC50 values for each An. stephensi form, and various quantities of surfactants and co-surfactants were developed. The physical and morphological properties of the most lethal formulations were also determined. PREO and its major components, i.e. citronellol (21.34%), L-menthone (6.41%), linalool (4.214%), and geraniol (2.19%), showed potent larvicidal activity against the studied mosquitoes. The LC50/90 values for mysorensis and intermediate forms were computed as 11.44/42.42 ppm and 12.55/47.69 ppm, respectively. The F48/F44 nanoformulations with 94% and 88% lethality for the mysorensis and intermediate forms were designated as optimized formulations. The droplet size, polydispersity index, and zeta-potential for F48/F44 were determined as 172.8/90.95 nm, 0.123/0.183, and -1.08/-2.08 mV, respectively. These results were also confirmed by TEM analysis. Prepared formulations displayed antibacterial activity against larval gut bacteria in the following order of decreasing inhibitory: LC90, optimized nanoemulsions, and LC50. PREO-based formulations were more effective against mysorensis than intermediate. Compared to the crude PREO, the overall larvicidal activity of all nanoformulations boosted by 20% and the optimized formulations by 50%. The sensitivity of insect gut bacteria may be a crucial factor in determining the outcome of the effect of toxins on target insects. The formulations designed in the present study may be a good option as a potent and selective larvicide for An. stephensi., Competing Interests: The authors have declared that no competing interests exist.

Background: Blackflies (Diptera: Simuliidae) are known as effective vectors of human and animal pathogens, worldwide. We have already indicated that some individuals in the Simulium turgaicum complex are annoying pests of humans and livestock in the Aras River Basin, Iran. However, there is no evidence of host preference and their possible vectorial role in the region. This study was conducted to capture the S. turgaicum (s.l.), to identify their host blood-meals, and to examine their potential involvement in the circulation of zoonotic microfilariae in the study areas., Methods: Adult blackflies of the S. turgaicum complex were bimonthly trapped with insect net in four ecotopes (humans/animals outdoors, irrigation canals, lands along the river, as well as rice and alfalfa farms) of ten villages (Gholibaiglou, Gungormaz, Hamrahlou, Hasanlou, Khetay, Khomarlou, Larijan, Mohammad Salehlou, Parvizkhanlou and Qarloujeh) of the Aras River Basin. A highly sensitive and specific nested PCR assay was used for detection of filarial nematodes in S. turgaicum (s.l.), using nuclear 18S rDNA-ITS1 markers. The sources of blood meals of engorged specimens were determined using multiplex and conventional cytb PCR assays., Results: A total of 2754 females of S. turgaicum (s.l.) were collected. The DNA of filarial parasites was detected in 6 (0.62%) of 960 randomly examined individuals. Sequence analysis of 420 base pairs of 18S rDNA-ITS1 genes identified Dirofilaria spp. including 5 D. immitis and 1 D. repens. Importantly, all filarial positive specimens have been captured from humans and animals outdoors. Cytb-PCR assays showed that in all ecotypes studied, members of the S. turgaicum complex had preferably fed on humans, dogs, bovids, and birds, respectively., Conclusions: To the best of our knowledge, this is the first report of D. immitis/D. repens detection in blackflies. Results showed that S. turgaicum (s.l.) was the most abundant (97%) and anthropophilic (45%) blackfly in all studied ecotypes/villages and that DNA of Dirofilaria spp. was detected in the flies taken from six villages. Dirofilariasis is a common zoonosis between humans and carnivores, with mosquitoes (Culicidae) as the principal vectors. Further investigations are needed to demonstrate that blackflies are actual vectors of Dirofilaria in the studied region.

Black flies are insects of medical, veterinary, and environmental significance. Historically, they have attacked humans and caused simuliotoxicosis in livestock in the Aras River Basin in northwest Iran. However, information on the species and their bionomics is limited in the region. Adult flies were collected from diverse ecotopes of the Aras River Basin. After morphological identification, representative specimens of each morphological group were subjected to mtDNA COI gene sequence analysis for species diagnosis and to infer relationships. Flies also were examined for pollinia. A total of 1880 specimens representing 12 morphotaxa in two genera (Simulium and Metacnephia) were identified: Simulium turgaicum (n=1834), S. kiritshenkoi (n=12), S. bezzii (n=7), S. brevitarse (n=7), S. pseudequinum (n=5), S. aureum species group (n=4), S. vernum species group (n=3), S. transcaspicum (n=1), three unidentified species of the subgenus Simulium (n=5), and Metacnephia possibly persica (n=2). Fifty two haplotypes were detected for the 65 COI sequences analyzed. Intraspecific genetic divergence was 0.19-8.83%, whereas the mean interspecific genetic distances among the morphotaxa were 1.41-19.58%. Molecular analyses recovered three well-supported lineages within S. turgaicum. One lineage included black flies collected from agricultural fields, a second lineage involved black flies captured from animals, and a third lineage included specimens that had visited flowers, as evidenced by presence of pollinia. The relative abundance (97%) and observations of the S. turgaicum complex biting humans are important epidemiological factors. Future studies are needed to define the potential epidemiological risk of simulids in Khoda-Afarin County of Iran., Competing Interests: Declaration of Competing Interests All authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Resistance of Plasmodium falciparum against common anti-malarial drugs emphasizes the need of alternative and more effective drugs. Synthetic derivatives of 1-(heteroaryl)-2-((5-nitroheteroaryl)methylene) hydrazine have showed in vitro anti-plasmodial activities. The present study aimed to evaluate the molecular binding and anti-plasmodial activity of synthetic compounds in vivo., Methods: The molecular docking was used to study the binding of compounds to haem and Plasmodium falciparum lactate dehydrogenase (PfLDH). Acute toxicity of the synthetic compounds was evaluated based on the modified up & down method. The anti-plasmodial activity of the compounds was conducted by the two standard tests of Peters' and of Rane, using chloroquine-sensitive Plasmodium berghei in mice. Also, the toxicity to the internal organs of mice was evaluated on the seventh day after the treatment in addition to the histopathology of their liver. Compound 3 that showed high activity in the lowest dose was selected for further pharmacodynamic studies., Results: According to the docking studies, the active site of PfLDH had at least four common residues, including Ala98, Ile54, Gly29, and Tyr97 to bind the compounds with the affinity, ranging from - 8.0 to - 8.4 kcal/mol. The binding mode of ligands to haem revealed an effective binding affinity, ranging from - 5.1 to - 5.5 kcal/mol. Compound 2 showed the highest  % suppression of parasitaemia (99.09%) at the dose of 125 mg/kg/day in Peters' test. Compound 3, with 79.42% suppression, was the best in Rane's test at the lowest dose (31 mg/kg/day). Compound 3 was confirmed by the pharmacodynamic study to have faster initial parasite elimination in the lowest concentration. The histopathology of the livers of mice did not reveal any focal necrosis of hepatocytes in the studied compounds., Conclusions: The docking studies verified Pf LDH inhibition and the inhibitory effect on the haemozoin formation for the studied compounds. Accordingly, some compounds may provide new avenues for the development of anti-malarial drugs without liver toxicity, although further studies are required to optimize their anti-plasmodial activity.

Because of the nutritional ecology of dung- and carrion-feeding, bacteria are the integral part of Lucilia sericata life cycle. Nevertheless, the disinfected larvae of the blowfly are applied to treat human chronic wounds in a biosurgery named maggot debridement therapy (MDT). To realize the effects of location/diet on the gut bacteria, to infer the role of bacteria in the blowfly ecology plus in the MDT process, and to disclose bacteria circulating horizontally in and vertically between generations, bacterial communities associated with L. sericata specimens from various sources were investigated using culture-based and culture-independent methods. In total, 265 bacteria, including 20 families, 28 genera, and 40 species, were identified in many sources of the L. sericata . Culture-dependent method identified a number of 144 bacterial isolates, including 21 species, in flies reared in an insectary; specimens were collected from the field, and third-instar larvae retrieved from chronic wounds of patients. Metagenetic approach exposed the occurrences of 121 operational taxonomic units comprising of 32 bacterial species from immature and adult stages of L. sericata . Gammaproteobacteria was distinguished as the dominant class of bacteria by both methods. Bacteria came into the life cycle of L. sericata over the foods and transovarially infected eggs. Enterococcus faecalis , Myroides phaeus , Proteus species, Providencia vermicola , and Serratia marcescens were exchanged among individuals via transstadial transmission. Factors, including diets, feeding status, identification tool, gut compartment, and life stage, governed the bacteria species. Herein, we reemphasized that L. sericata is thoroughly connected to the bacteria both in numerous gut compartments and in different life stages. Among all, transstadially transmitted bacteria are underlined, indicating the lack of antagonistic effect of the larval excretions/secretions on these resident bacteria. While the culture-dependent method generated useful data on the viable aerobic gut bacteria, metagenomic method enabled us to identify bacteria directly from the tissues without any need for cultivation and to facilitate the identification of anaerobic and unculturable bacteria. These findings are planned to pave the way for further research to determine the role of each bacterial species/strain in the insect ecology, as well as in antimicrobial, antibiofilm, anti-inflammatory, and wound healing activities., (Copyright © 2020 Maleki-Ravasan, Ahmadi, Soroushzadeh, Raz, Zakeri and Dinparast Djadid.)

The following 21 new species from Iran are described, 7 from East Azerbaijan province Megaselia ajabshirensis, M. chicheckliensis, M. exkaleybar, M. kaleybarensis, M. qurigolensis, M. shabestarensis, M. zonuzensis. and 14 from West Azerbaijan province, M. evogliensis, M. farshbafi, M. ghalateshahensis, M. haddadi, M. hejazii, M. khaghaniniai Namaki Disney, M. khoyensis, M. ledzona, M. mahabadensis, M. miandoabensis, M. namakiae Khaghaninia Disney, M. pereensis, M. yaseri, M. zarghanii.

Background: The impact of environmental factors and host on Hyalomma spp. community structure and abundance in the main Crimean-Congo haemorrhagic fever (CCHF) foci of Iran is largely unknown., Methods: Biotic and abiotic factors, including host, temperature, humidity, altitude, Köppen-Geiger climate types, season, and precipitation on Hyalomma spp. community structure and abundances in 11 provinces of Iran were investigated. Additionally, the possible infection of ticks with CCHF virus was evaluated using reverse transcription PCR technique., Results: Species analyses demonstrated the presence of Hyalomma anatolicum , H. marginatum , H. dromedarii , H. asiaticum , H. detritum and H. schulzei in the study area. Hyalomma anatolicum was the dominant species in the southern and northern parts, whereas H. dromedarii was distributed mostly in central parts of the country. The highest tick infestation was recognized in hot season. Spatial variation in tick relative density was observed between habitat types where more ticks were collected in deserts, semi-deserts, and Mediterranean habitats. Except for H. dromedarii , which was more prevalent on camel (P= 0.044), there were no significant variations in the frequencies of other Hyalomma species on different hosts. Hyalomma anatolicum , H. dromedarii frequencies had significant positive and negative association with temperature and precipitation respectively. Also humidity has positive impact on H. asiaticum frequency., Conclusion: Data presented here will help improve ecological models to forecast the distribution of Hyalomma spp. ticks, to evaluate the risk of CCHF and other tick-borne diseases, and to design proper vector control measures to suppress Hyalomma populations in Iran., (Copyright© Iranian Society of Medical Entomology & Tehran University of Medical Sciences.)

The dual occurrence of Pseudomonas-like and Wolbachia endobacteria has not been investigated in the Pederus beetles yet. We investigated pederin-producing bacteria (PPB) infection in Paederus fuscipes specimens from the southern margins of the Caspian Sea by designed genus-specific (OprF) and species-specific (16S rRNA) primers. Wolbachia infection was studied through a nested-PCR assay of Wolbachia surface protein (wsp) gene. Of the 125 analyzed beetles, 42 females (82.35%) and 15 males (20.27%) were positive to PPB infection; this is the first study reporting male P. fuscipes infection to PPB. Wolbachia infection was found in 45 female (88.23%) and 50 male (67.57%) analyzed beetles. Surprisingly, a number of 36 females (70.59%) and 13 males (17.57%) were found to be infected with both PPB and Wolbachia endosymbionts. In general, population infection rates to PPB and Wolbachia were determined to be 45.6% and 76%, respectively. The infection rates of female beetles to PPB and PPB-Wolbachia were significantly higher than males. In Paederus species, only female beetles shelter PPB and the discovery of this bacterium in adult males may reflect their cannibalistic behavior on the contaminated stages. Phylogenetic analysis showed that the sequences of OprF gene were unique among Pseudomonas spp.; however, sequences of 16S rRNA gene were related to the PPB of Pederus species. The co-occurrence and random distribution of these endobacteria may imply putative tripartite interactions among PPB, Wolbachia, and Paederus. In order to elucidate these possible tripartite interactions, further studies are required even at gender level., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Background: Leishmaniasis is caused by Leishmania parasites and is transmitted to humans through the bite of infected sand flies. Development of Leishmania to infective metacyclic promastigotes occurs within the sand fly gut where the gut microbiota influences development of the parasite. Paratransgenesis is a new control method in which symbiotic bacteria are isolated, transformed and reintroduced into the gut through their diet to express anti-parasitic molecules. In the present study, the midgut microbiota of three sand fly species from a steppe and a mountainous region of northern Iran, where zoonotic visceral leishmaniasis (ZVL) is endemic, was investigated., Methods: Briefly, adult female sand flies was collected during summer 2015 and, after dissection, the bacterial composition of the guts were analyzed using a culture-dependent method. Bacterial DNA from purified colonies was extracted to amplify the 16S rRNA gene which was then sequenced., Results: Three ZVL sand fly vectors including Phlebotomus major, P. kandelakii and P. halepensis were found in the highlighted regions. In total, 39 distinct aerobic bacterial species were found in the sand fly midguts. The sand fly microbiota was dominated by Proteobacteria (56.4%) and Firmicutes (43.6%). Bacterial richness was significantly higher in the steppe region than in the mountainous region (32 vs 7 species). Phlebotomus kandelakii, the most important ZVL vector in the study area, had the highest bacterial richness among the three species. Bacillus subtilis and Pantoea agglomerans were isolated from the guts of the sand flies; these are already used for the paratransgenesis of sand flies and mosquitoes, respectively., Conclusions: The existence of B. subtilis and P. agglomerans in the ZVL vectors and other sand fly species studied so far suggests that these two bacterial species are potential candidates for paratransgenic approach to prevent ZVL transmission. Further research needs to test the possible relationship between the gut microbiome richness and the vector competence of the ZVL vectors.

Sand flies of Phlebotomus papatasi and P. sergenti are the main vectors of cutaneous leishmanisis (CL) in the old world. We aimed to screen Iranian P. papatasi and P. sergenti for their natural infections with Wolbachia and to determine their phylogenetic association with other species. Wolbachia surface protein (wsp) gene was PCR amplified from DNA extracted from Phlebotomus species, sequenced, and were analysed in combination with wsp sequences related to Phelebtominae and other insects. All Wolbachia-infecting Iranian sand flies of P. papatasi and P. sergenti were classified in the Supergroup A., Wolbachia isolated from P. sergenti were clustered in a new subgroup within Supergroup A so-called wSreg. The Wolbachia strains identified from the P. papatasi clustered mainly in the subgroup wPap and partly in wSerg. Multiple Wholbachia infection within a single population of P.papatasi warrants investigation on existence and intensity of cytoplasmic incompatibility between the wPap and wSerg subgroups.

Background: Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium within An. stephensi and its introduction methods to the mosquitoes have not yet been explored., Methods: Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium., Results: The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadially from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time., Conclusion: Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

Background: Malarious areas in Iran are close to Afghanistan and Pakistan that urge the researchers to extend their knowledge on malaria epidemiology to the neighboring countries as well. Vectorial capacity differs at species or even at population level, the first essential step is accurate identification of vectors. This study aimed to identify Anopheles species composition in selected malarious areas of Afghanistan and Iran, providing further applied data for other research in two countries., Methods: Adults Anopheles spp. were collected from four provinces in Afghanistan (Badakhshan, Herat, Kunduz, Nangarhar) by pyrethrum spray catch, hand collection methods through WHO/EMRO coordination and from Chabahar County in Iran by pyrethrum spray catch method. Identification was performed using reliable identification key., Results: Totally, 800 female Anopheles mosquitos, 400 from each country were identified at species level. Anopheles composition in Afghanistan was An. superpictus , An. stephensi and An. hyrcanus . Most prevalent species in Badakhshan and Kunduz were An. superpictus , whereas An. stephensi and An. hyrcanus were respectively found in Nangarhar and Heart. Anopheles species in Chabahar County of Iran were An. stephensi , An. fluviatilis , An. culicifacies and An. sergentii . The most prevalent species was An. stephensi., Conclusion: Current study provides a basis for future research such as detection of Plasmodium infection in collected samples which is on process by the authors, also for effective implementation of evidence-based malaria vector intervention strategies.

Background: Flea-borne diseases have a wide distribution in the world. Studies on the identity, abundance, distribution and seasonality of the potential vectors of pathogenic agents (e.g. Yersinia pestis, Francisella tularensis, and Rickettsia felis) are necessary tools for controlling and preventing such diseases outbreaks. The improvements of diagnostic tools are partly responsible for an easier detection of otherwise unnoticed agents in the ectoparasitic fauna and as such a good taxonomical knowledge of the potential vectors is crucial. The aims of this study were to make an exhaustive inventory of the literature on the fleas (Siphonaptera) and range of associated hosts in Iran, present their known distribution, and discuss their medical importance., Methodology/principal Findings: The data were obtained by an extensive literature review related to medically significant fleas in Iran published before 31st August 2016. The flea-host specificity was then determined using a family and subfamily-oriented criteria to further realize and quantify the shared and exclusive vertebrate hosts of fleas among Iran fleas. The locations sampled and reported in the literature were primarily from human habitation, livestock farms, poultry, and rodents' burrows of the 31 provinces of the country. The flea fauna were dominated by seven families, namely the Ceratophyllidae, Leptopsyllidae, Pulicidae, Ctenophthalmidae, Coptopsyllidae, Ischnopsyllidae and Vermipsyllidae. The hosts associated with Iran fleas ranged from the small and large mammals to the birds. Pulicidae were associated with 73% (56/77) of identified host species. Flea-host association analysis indicates that rodents are the common hosts of 5 flea families but some sampling bias results in the reduced number of bird host sampled. Analyses of flea-host relationships at the subfamily level showed that most vertebrates hosted fleas belgonging to 3 subfamilies namely Xenopsyllinae (n = 43), Ctenophthalminae (n = 20) and Amphipsyllinae (n = 17). Meriones persicus was infested by 11 flea subfamilies in the arid, rocky, mountainous regions and Xenopsyllinae were hosted by at least 43 mammal species. These findings place the Persian jird (M. persicus) and the Xenopsyllinae as the major vertebrate and vector hosts of flea-borne diseases in Iran including Yersinia pestis, the etiological agent of plague. We found records of at least seven vector-borne pathogenic agents that can potentially be transmitted by the 117 flea species (or subspecies) of Iran., Conclusions/significance: Herein, we performed a thorough inventary of the flea species and their associated hosts, their medical importance and geographic distribution throughout Iran. This exercise allowed assessing the diversity of flea species with the potential flea-borne agents transmission risk in the country by arranging published data on flea-host associations. This information is a first step for issuing public health policies and rodent-flea control campaigns in Iran as well as those interested in the ecology/epidemiology of flea-borne disease., Competing Interests: The authors have declared that no competing interests exist.

Background: Wolbachia are common intracellular bacteria that infect different groups of arthropods including mosquitoes. These bacteria modify host biology and may induce feminization, parthenogenesis, male killing and cytoplasmic incompatibility (CI). Recently Wolbachia is being nominated as a bio-agent and paratransgenic candidate to control mosquito borne diseases., Methods: Here we report the results of a survey for presence, frequency, and phylogenetic congruence of these endosymbiont bacteria in Culex pipiens populations in Northern, Central, and Southern parts of Iran using nested-PCR amplification of wsp gene., Results: Wolbachia DNA were found in 227 (87.3%) out of 260 wild-caught mosquitoes. The rate of infection in adult females ranged from 61.5% to 100%, while in males were from 80% to 100%. The Blast search and phylogenetic analysis of the wsp gene sequence revealed that the Wolbachia strain from Iranian Cx. pipiens was identical to the Wolbachia strains of supergroup B previously reported in members of the Cx. pipiens complex. They had also identical sequence homology with the Wolbachia strains from a group of distinct arthropods including lepidopteran, wasps, flies, damselfly, thrips, and mites from remote geographical areas of the world., Conclusion: It is suggested that Wolbachia strains horizontally transfer between unrelated host organisms over evolutionary time. Also results of this study indicates that Wolbachia infections were highly prevalent infecting all Cx. pipiens populations throughout the country, however further study needs to define Wolbachia inter-population reproductive incompatibility pattern and its usefulness as a bio-agent control measure.

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: Microbes particularly bacteria presenting in the gut of haematophagous insects may have an important role in the epidemiology of human infectious disease., Methods: The microbial flora of gut and surrounding environmental of a laboratory strain of Phlebotomus papatasi, the main vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in the old world, was investigated. Biochemical reactions and 16s rDNA sequencing of the isolated bacteria against 24 sugars and amino acids were used for bacteria species identification. Common mycological media used for fungi identification as well., Results: Most isolates belonged to the Enterobacteriaceae, a large, heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. Enterobacteriaceae groups included Edwardsiella, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leminorella, Pantoea, Proteus, Providencia, Rahnella, Serratia, Shigella, Tatumella, and Yersinia and non Enterobacteriaceae groups included Bacillus, Staphylococcus and Pseudomonas. The most prevalent isolates were Proteus mirabilis and P. vulgaris. These saprophytic and swarming motile bacteria were isolated from all immature, pupae, and mature fed or unfed male or female sand flies as well as from larval and adult food sources. Five fungi species were also isolated from sand flies, their food sources and colonization materials where Candida sp. was common in all mentioned sources., Conclusion: Midgut microbiota are increasingly seen as an important factor for modulating vector competence in insect vectors so their possible effects of the mirobiota on the biology of P. papatasi and their roles in the sandfly-Leishmania interaction are discussed.

Background: Great gerbils, Rhombomys opimus, are the main reservoir host of zoonootic cutaneous leishmaniasis (ZCL) in Iran and neighboring countries. Based on morphological traits two subspecies R. opimus sodalis and R. opimus sargadensis have reported in the country. However, variation in infection rate and signs to Leishmania parasites, phenotype, size, and sexual polymorphisms demand more details to elucidate clearly the role of great gerbils in ZCL epidemiology., Methods: PCR-RFLP and PCR-direct sequencing were used to analyze mitochondrial DNA cytochrome B (mtDNA-cytB) gene structure of R. opimus collected from Golestan and Khorasan-e-Razavi Provinces in 2011 that are neighbor to Turkmenistan Country where ZCL is endemic in both sides of the borderline., Results: All of the specimens (n= 61) were morphologically or genetically similar to the typical R. opimus sodalis. However, there were 9 (1.5%) DNA substitutions throughout the 583 bp of the Cyt b gene of the samples sequenced comprising six DNA haplotypes. Maximum likelihood or neighbor joining phylogenetic trees inferred from the sequences could resolve the populations according to their subspecies as well as geographical origins., Discussion: The DNA polymorphisms in the great gerbils may correspond to the signs and infection rate in the animal. However, further studies are needed to match these six haplotypes with different signs and parasite sustaining following infection with L. major in the great gerbils.

Background: We aimed to develop a PCR-RFLP assay based on available sequences of putative vertebrate hosts to identify blood meals ingested by field female sand fly in the northwest of Iran. In addition, the utility of PCR-RFLP was compared with ELISA as a standard method., Methods: This experimental study was performed in the Insect Molecular Biology Laboratory of School of Public Health, Tehran University of Medical Sciences, Iran in 2006-2007. For PCR-RFLP a set of conserved vertebrate primers were used to amplify a part of the host mitochondrial cytochrome b (cyt b) gene followed by digestion of the PCR products by Hae III enzyme., Results: The PCR-RFLP and ELISA assays revealed that 34% and 27% of field-collected sand flies had fed on humans, respectively. Additionally, PCR-RFLP assays could reveal specific host DNA as well as the components of mixed blood meals. Results of PCR-RFLP assay showed that the sand flies had fed on cow (54%), human (10%), dog (4%), human and cow (21%), dog and cow (14%), and human and dog (3%)., Conclusion: The results can provide a novel method for rapid diagnosis of blood meal taken by sandflies. The advantages and limitations of PCR and ELISA assays are discussed.