New Data on Two Subspecies of Mesobuthus eupeus, the Most Medically Important Scorpion Species in Northwestern Iran.

Scorpions are among the most medically important arthropods in Iran, particularly northwestern areas. To date, five scorpion species, i.e. Mesobuthus eupeus, Mesobuthus caucasicus, Androctonus crassicauda, Hottentotta saulcyi, and scorpio maurus, have been identified. The family Buthidae is responsible for most cases of scorpionism in Iran. The Mesobuthus eupeus species belong to this family and is commonly distributed from Turkey to China, including Iran. Among these species, Mesobuthus eupeus is regarded as the most medically important species and responsible for most cases of envenomation in this area. Morphological differences between some species collected in the study area have been reported. The present study, thus, aimed to identify the subspecies of Mesobuthus eupeus in northwestern Iran. Scorpions were captured in the summer months from 37 localities in three northwestern provinces in Iran: West Azerbaijan, East Azerbaijan, and Ardabil. Scorpion collection was carried out using rock rolling and ultraviolet methods. A total of 376 specimens of Mesobutus eupeus (177males and 199 females) were collected and identified as Mesobuthus eupeus (98.4%) and Mesobuthus eupeus philippovitschi (1.6%). Owing to the findings of our study, M.e.philippovitschi has been added to the scorpion fauna of northwestern parts of Iran for the first time. Unlike M.e. eupeus which is widely distributed from plains to mountainous regions, M.e.philippovitschi has limited distribution and is found mostly along the borders with neighboring countries. This subspecies is the most medically important and most prevalent one in the region. The findings of the present study also provide the basis for future consideration of regional antivenom production for this medically important species.

Detection of Leishmania major DNA within wild caught Phlebotomus papatasi and species composition of sand flies in endemic focus of cutaneous leishmaniasis, in western Iran.

Cutaneous leishmaniasis is one of the most important public health problem in many developing countries. The present study was conducted to determine the vector(s), the parasite and the species composition of sand flies in the Dehloran County during May-November 2012. Sand flies were collected by sticky traps and mounted in Puri's medium for species identification. Polymerase chain reaction (PCR) techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism (RFLP) were used for identification of DNA of Leishmania parasites in infected sand flies. A total of 82443 specimens comprising 15 species of sand flies (5 Phlebotomus and 10 Sergentomyia) were collected and identified. The species of Phlebotomus papatasi was dominant in outdoor and indoor resting places. Among the 280 specimens of female P. papatasi tested by PCR of kDNA, ITS1-rDNA genes of the parasite followed by RFLP, only 5 of them (1.8 %) were positive to Leishmania major parasites. This is the first molecular detection of leishmania infection of P. papatasi to L. major in this region. The results indicated that, P. papatasi was only species found infected by L. major and the principal vector of disease agent to human.

Biodiversity of culicid mosquitoes in rural Neka township of Mazandaran province, northern Iran.

BACKGROUND & OBJECTIVES: This paper presents the results of a study on ecological parameters including diversity indices applied on populations of Culicidae mosquitoes (Diptera: Culicidae) from three rural areas (Darvishan, Chalmardi and Khairabad) of Neka township in Mazandaran province, northern Iran. METHODS: Adult and larval stages of mosquitoes were systematically sampled to obtain data required for measuring diversity of mosquitoes in these areas. Alfa and beta indices were analyzed to compare mosquito diversity. A total of 5270 specimens belonging to four genera and 14 different species were collected and identified. RESULTS: Evaluation of dominance structure of species showed that culicid mosquitoes of Neka rangelands include one eudominant species, two dominant species, two subdominant species, two rare species and eight subrare species when Heydemann classification was used. INTERPRETATION & CONCLUSION: Data analysis revealed a higher diversity of mosquitoes in Khairabad with a low degree of interspecific dominance, while Darvishan and Chalmardi recorded the lowest observed diversity and a high degree of interspecific dominance. The cluster analysis based on Jaccard's index indicated the relationship between the categories and indicates that Darvishan and Khairabad are the closest categories in their specific composition.

First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran.

OBJECTIVE: To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis. METHODS: Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer (ITS1) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Leishmania species in sand flies specimens. RESULTS: Three out of 115 females of Phlebotomus sergenti (P. sergenti) (2.6%) were positive to Leishmania tropica (L. tropica). CONCLUSIONS: This is the first report on P. sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HRM analysis. This method is rapid, sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.

Interleukin 4 (IL-4) gene promoter polymorphisms in Rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis.

Great gerbils (Rhombomys opimus) are the most common gerbils in center to northeast of Iran as well as central Asia and serve as reservoirs for the zoonotic agents, including Leishmania major, the principal etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). The outcome of L. major infection in gerbils is not uniform. Among several immune-related factors including cytokine genes, the polymorphism in interleukin 4 (IL-4) promoter gene showed a great impact on outcome and pathological symptoms of L. major infection at least in mouse model. In this study gerbils' IL-4 promoter gene polymorphism is assessed. Specific primers were designed to develop a PCR-based assay to amplify IL-4 promoter gene to possibly define IL-4 promoter gene polymorphism in great gerbil populations with a range of Leishmania infection and symptoms collected from different foci of the central, north and northeast regions of Iran. The results showed that the designed primers amplify 689bp of the promoter gene. Sequence analysis of the promoter gene revealed five polymorphic sites assembly six haplotypes among the gerbil populations. Further studies are needed to assess whether or not the five polymorphisms cause different outcome phenotypes following infection with L. major in great gerbils. The data might be used to characterize the immune responses of R. opimus against L. major infection.

Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region.

Natural infection of wild caught Phlebotomus tobbi to Leishmania infantum in East Azerbaijan province, northwestern Iran.

BACKGROUND & OBJECTIVES: Zoonotic visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sandflies and is one of the most important public health problems in Iran. To detect and identify the Leishmania parasites and their corresponding vector(s), an investigation was carried out in Azarshahr County, a new and important focus of the disease in East Azerbaijan province in northwestern Iran during late April to late October 2010. METHODS: Sandflies were sampled using sticky papers (A4 white paper soaked in castor oil) from inside and outside of the houses and animal shelters, close to the vegetation and crevices. The head and three last abdomen segments of the specimens were removed and mounted in Puri's medium for species identification. The rest of body was subjected to molecular methods for detection of leishmanial parasites. RESULTS: Among 400 female sandflies tested by polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP), only 2 out of 8 Phlebotomus tobbi were positive to L. infantum parasites. CONCLUSION: The results indicated that, P. tobbi was the only species found infected by L.infantum and the principal vector of the disease agent to human.

Larval habitats and biodiversity of anopheline mosquitoes (Diptera: Culicidae) in a malarious area of southern Iran.

BACKGROUND & OBJECTIVES: Malaria is the most important mosquito-borne disease in Iran. It is endemic in south to southeastern part of the country. Knowledge about bio-ecology of vectors will support authorities for appropriate management of the disease. Bashagard district is one of the main endemic areas for malaria in south of Iran. This study was conducted to determine anopheline fauna, diversity and affinity in the area, characterization of larval habitats, and mapping their potential distribution across the district. METHODS: The potential aquatic habitats for Anopheles larvae were extracted from Indian Remote Sensing Satellite (IRS) image and digital elevation model of the area using GIS. Surface water bodies were sampled monthly during 2009­10 for anopheline larvae, while characteristics of their physical environment were recorded and water samples were analyzed. RESULTS: A total of 4511 Anopheles larvae were found during the year with the lowest and highest frequencies in February and April, respectively. Dominant species was Anopheles culicifacies. The Shanon diversity index ranged from 0.570­0.829 at fixed collection sites, while the affinity index was significant among some vector species. CONCLUSION: Riversides and riverbeds were the main breeding places which provided sandy, rocky, and clay beds for different species. The potential breeding places as well as distribution of collected species were mapped. Knowledge about ecology of malaria vectors provides information to health sector for effective control programs.

First detection of Leishmania infantum in Phlebotomus kandelakii using molecular methods in north-eastern Islamic Republic of Iran.

Shirvan district in north-eastern Islamic Republic of Iran is a new focus of visceral leishmaniasis. This study aimed to identify the vector(s), the parasite and the species composition of sand flies in the district during July-September 2009 using polymerase chain reaction techniques. In all, 2088 sand flies were collected from 3 villages. Nine sand fly species were identified: Phlebotomus kandelakii (Shchurenkova), P. major (Annandale), P. halepensis (Theodor), P. papatasi (Scopoli), P. sergenti (Parrot), P. longidactus (Parrot), P. caucasicus (Marzinovsky), Sergentomyia sintoni (Pringle) and S. sumbarica (Perfil'ev). P. sergenti and P. kandelakii were the most prevalent Phlebotomus species at 31.3% and 10.0% respectively. Of 59 female P. kandelakii, 2(3.4%) were naturally infected with L. infantum. This is the first finding of natural infection of P. kandelakii by L. infantum in this region suggesting P. kandelakii may be the vector of L. infantum in the area although it is the second most prevalent phlebotomine species.

Field evaluation of permethrin long-lasting insecticide treated nets (Olyset(®)) for malaria control in an endemic area, southeast of Iran.

Long lasting insecticide treated nets (LLINs) have been advocated as an effective tool for prevention and control of malaria. Olyset net was the first LLINs which became commercially available and obtained WHO approval. According to the national strategic plan on evaluation of Olyset net, a field trial was conducted to determine the efficacy of these nets against malaria vectors in an endemic area in the southeast of Iran. Fourteen villages with similar topographical and epidemiological situations were selected and randomly assigned to two clusters of the study: Olyset net and untreated net. Distribution of nets was carried out to cover 100% of the population in Olyset net and untreated net cluster. Anopheline mosquitoes were collected monthly using different WHO standard methods in both areas to determine their abundance, feeding pattern and resting behaviour. Human blood index was determined using ELISA test. Additionally, Olyset nets were evaluated for their biological activity using WHO cone bioassay test by susceptible colony of Anopheles stephensi (Beech strain) and then for insecticide residues by employing high performance thin layer chromatography. Malaria incidence was measured by passive and active case detection from all study population. In total 2115 adult anopheline mosquitoes were collected and identified using morphological characters. They comprised of seven species: Anopheles dthali (Liston), A. culicifacies (Giles), A. stephensi (Liston), A. superpictus (Grassi), A.fluviatilis (James), A. moghulensis (Christophers) and A. turkhudi (Liston). A. dthali, A. culicifacies and A. stephensi were most prevalent species in both areas. In the Olyset net study area, there was a significant reduction of 41.1%, 54.4%, 59.39% and 64.1% in the indoor-resting density of A. culicifacies, A. stephensi, A. dthali and A. superpictus, respectively, with an overall reduction of 39.3% in total mosquitoes in comparison with untreated net area. A significant reduction was also observed in human blood index of vector species in the Olyset net villages. Bioefficacy test results of Olyset nets showed that the median knockdown time was 1.48 and 3.25min, while the average mortality rate was 100% and 72.3%±7.07 in baseline and after 1 year of intervention, respectively. The average permethrin content reached to 68.31% (683.1mg/m(2)) of the initial insecticide dose of 937±21.69mg/m(2) (nearly 1000mg/m(2)) at the end of intervention. Malaria incidence was reduced by 96.6% and 64.8% in the village with Olyset nets and in the villages with untreated nets, respectively. During intervention period, there was a reduction of 93.2% in malaria incidence in Olyset net area as compared to the untreated area. This study indicated that Olyset nets have a major impact on malaria vectors and disease burden; therefore it could be recommended as an effective personal protection tool for malaria control in malarious areas.

Spatial analysis and mapping of malaria risk in an endemic area, south of Iran: a GIS based decision making for planning of control.

Bashagard district is one of the important malaria endemic areas in southern Iran. From this region a total of 16,199 indigenous cases have been reported in recent years. The aim of this study was to determine the situation of the disease and provide the risk map for the area. ArcGIS9.2 was used for mapping spatial distribution of malaria incidence. Hot spots were obtained using evidence-based weighting method for transmission risk. Environmental factors including temperature, relative humidity, altitude, slope and distance to rivers were combined by weighted multi criteria evaluation for mapping malaria hazard area at the district level. Similarly, risk map was developed by overlaying weighted hazard, land use/land cover, population density, malaria incidence, development factors and intervention methods. Our results reveal that the disease mainly occurs in north and east of the study area. Consequently the district is divided into three strata. Appropriate interventions are recommended for each stratum based on national malaria policy. Malaria hazard and risk map, stratification based on relevant information and data analyzing provide a useful method preparedness and early warning system for malaria control, although regular updating is required timely.

Entomological and epidemiological attributes for malaria transmission and implementation of vector control in southern Iran.

Bashagard is an important malaria endemic area in south of Iran. An epidemiological and entomological survey was performed during 2002-2010. The aim of study was to determine malaria situation, species composition of anopheline mosquitoes and susceptibility status of main vectors to insecticides/larvicides. A total of 13,490 malaria cases were recorded, Plasmodium vivax (99.64%), P. falciparum (0.35%) and mix cases (0.01%). The highest and lowest Annual Parasite Incidence (API) were observed in 2007 (145.72/1000) and 2009 (6.29/1000), respectively. Anopheles culicifacies, An. dthali, An. stephensi, An. superpictus, An. fluviatilis, An. moghulensis, An. turkhudi and An. apoci were collected from the area. Two peak activities occur in April and October. The first five species were confirmed as malaria vectors in Iran. No indication of sporozoite in mosquitoes using molecular method was performed. Susceptibility tests using diagnostic dose of insecticides and larvicides showed only resistance of An. stephensi to DDT. Tolerance in An. stephensi to deltamethrin and bendiocarb is reported. The same phenomenon was observed in An. culicifacies to DDT, propoxur and deltamethrin, and in An. dthali to malathion and deltamethrin. Larvae of vectors were susceptible to all larvicides, except for An. stephensi that exhibited tolerance to fenthion. In conclusion it should be emphasized that malaria transmission is a complex process in Bashagard. This event is attributed to five proven vectors with different behaviors which are active in the area. Regarding tolerance of vectors to deltamethrin, resistance management is suggested by using new insecticide with novel mode of action.

Upwind responses of Anopheles stephensi to carbon dioxide and L-lactic acid: an olfactometer study.

Excretion of carbon dioxide and L-lactic acid through exhalation and perspiration provides olfactory signals to mosquitoes which allow them to find and bite humans; however, mosquito species differ in this regard. This study investigated upwind responses of Anopheles stephensi, mysorensis form, an important malaria vector in Asia, to carbon dioxide and L-lactic acid under laboratory conditions. While a minimal dose of carbon dioxide (90 ppm) activated the mosquitoes, 10 times this amount suppressed them. L-lactic acid alone did not produce a significant effect by itself, but addition of 6 microg/min of L-lactic acid to a range of 90 to 410 ppm carbon dioxide resulted in attraction. The results provide further support for the hypothesis that CO2 plays an important role in the host-seeking behaviour of zoophilic mosquitoes, and suggests that L-lactic acid might play a more critical role than CO2 in the attraction of An. stephensi.

Upwind responses of Anopheles stephensi to carbon dioxide and L-lactic acid: an olfactometer study

Excretion of carbon dioxide and L-lactic acid through exhalation and perspiration provides olfactory signals to mosquitoes which allow them to find and bite humans; however, mosquito species differ in this regard. This study investigated upwind responses of Anopheles stephensi, mysorensis form, an important malaria vector in Asia, to carbon dioxide and L-lactic acid under laboratory conditions. While a minimal dose of carbon dioxide [90 ppm] activated the mosquitoes, 10 times this amount suppressed them. L-lactic acid alone did not produce a significant effect by itself, but addition of 6 microg/min of L-lactic acid to a range of 90 to 410 ppm carbon dioxide resulted in attraction. The results provide further support for the hypothesis that CO2 plays an important role in the host-seeking behaviour of zoophilic mosquitoes, and suggests that L-lactic acid might play a more critical role than CO2 in the attraction of An. stephensi

First detection of Leishmania infantum in Phlebotomus kandelakii using molecular methods in North-Eastern Islamic Republic of Iran

Shirvan district in north-eastern Islamic Republic of Iran is a new focus of visceral leishmaniasis. This study aimed to identify the vector [s], the parasite and the species composition of sand flies in the district during July-September 2009 using polymerase chain reaction techniques. In all, 2088 sand flies were collected from 3 villages. Nine sand fly species were identified: Phlebotomus kandeiakii [Shchurenkova], P. major [Annandale], P. halepensis [Theodor], P. popatasi [Scopoli], P. sergentl [Parrot], P. longidoctus [Parrot], P. caucasicus [Marzinovsky], Sergentomyia sintoni [Pringle] and S. sumbarica [Perfii'ev]. P. sergentl and P. kandetakii were the most prevalent Phlebotomus species at 31.3% and 10.0% respectively. Of 59 female P. kandelokll, 2 [3.4%] were naturally infected with L infantum. This is the first finding of natural infection of P. kandelakii by L infantum in this region suggesting P. kandelakii may be the vector of L infantum in the area although it is the second most prevalent phlebotomine species

Ecology of malaria vector Anopheles culicifacies in a malarious area of Sistan va Baluchestan province, south-east Islamic Republic of Iran.

A study was done of the bionomics, insecticide susceptibility and irritability status of Anopheles culicifacies in Sistan va Baluchestan province. Sampling was performed to determine the following parameters: species identification, seasonal activity, adult and larval susceptibility tests, irritability tests, anthropophily index and sporozoite rate. An. culicfacies adults were susceptible to all tested pyrethroid insecticides. An. culicfacies started to appear indoors in late May, showing 2 peaks in June and September. Fenitrothion, cyfluthrin and permethrin had the least irritancy effect and DDT the highest. Only 2/860 females tested were infected with Plasmodium spp. (sporozoite rate: 0.25%). ELISA testing of 250 blood meals derived from night-biting collections of female mosquitoes from humans and cows revealed that only 12.5% were human-fed.

Knowledge, attitudes and practices regarding malaria control in an endemic area of southern Iran.

Iran is in the malaria pre-elimination stage. In this situation community based strategies are important for malaria control. In order to intensify elimination activities we need to have a better understanding of the knowledge, attitudes and practices (KAP) of malaria prevention in the community. We carried out a KAP study in one malaria endemic district in southern Iran in 2009 using a structured questionnaire. Five villages with indigenous malaria cases during the previous year and a high potential for anopheline mosquito breeding were selected. All the households in each village were visited and the head of the household or other responsible adult was interviewed. Data were analyzed using SPSS 11.5. A total of 494 respondents from the five villages participated. More than 33% of people in the study area were living in sheds with poor facilities. The illiteracy level of the studied population was high (44.2%) and significantly affected the knowledge and practices of the respondents about malaria control (p<0.05). Descriptive analysis showed significant differences between sex, job and history of malaria infection of the respondents and their knowledge and practices about malaria control (p<0.05). Knowledge of the respondents about the route of transmission in malaria was good; 72.1% knew mosquitoes were the vector. Most people knew at least one symptom of the disease. The practices of respondents were 59.5% for bednet use and 9.3% for screening windows. The knowledge, attitudes and practices of respondents in this survey about malaria control were good in some cases, but in general needed to be improved with educational programs.

Anopheline species composition in borderline of Iran-Azerbaijan.

Malaria is still one of the most important health-problems in the world and is endemic in Iran. Since 1994, after collapse of former Soviet Union, a new threat of malaria importation emerged from those countries into the northern Iran. This work was carried out to provide further evidence on the status of anopheline species composition, the malaria parasite species, and natural infectivity of mosquitoes distributed in Pars-Abad district, on the borderline of Azerbaijan in northwestern Iran. Mosquitoes were collected from May to December 2008 in anopheline seasonal activity and were identified at the species level. The genus- and species-specific primers against Plasmodium ssrDNA gene were used for specific amplification on female mosquito head+thorax. Members of the Anoheles maculipennis complex were identified by sequence analysis of the ribosomal internal transcribed spacer II (ITS2-rDNA). Morphological character-based identification showed that out of 1455 anopheline female specimens, 1121 (77%) were of A. maculipennis s.l. and 334 (23%) were of Anoheles hyrcanus. Molecular analysis of the species complex indicated the presence of Anoheles sacharovi 984 (67.6%) and A. maculipennis 137 (9.4%) in the region. None of themosquito's head-thorax was found to be naturally infected by malaria parasite. Results of this study, particularly high dominance of A. sacharovi, suggest a potential risk of malaria epidemic in the region, and the need for a continuous epidemiological surveillance.

First report on Anopheles fluviatilis U in southeastern Iran.

Anopheles fluviatilis James, one of the malaria vectors in Iran, is a complex of at least three cryptic species provisionally designated as species S, T and U. These species are morphologically indistinguishable at any stage of their life cycle and can be identified only by the examination of species-specific fixed inversions in the polytene chromosomes. Recently, sequence analysis of 28S D3 and second internal transcribed spacer (ITS2) regions of ribosomal DNA has revealed 7 haplotypes of S, U, T1, T2, Y, X and V within the complex. Identification of the cryptic species of the complex is of paramount importance in a disease control program due to contrasting differences in their vectorial efficiency, preference for feeding on humans and resting behavior. In this study we analyzed the sequence of 28S D3- and ITS2-rDNA loci to identify the species composition of the An. fluviatilis complex in Jiroft and Chabahar districts, two of the most important endemic malaria foci in southeastern corner of Iran. The ITS2 sequence analysis revealed that all of the An. fluviatilis specimens were identical to the Y/T2 haplotype of An. fluviatilis T, whereas D3 sequence analysis revealed presence of species T in Jiroft and species U in Chabahar district. It is the first report of species U in Iran.

Ecology of malaria vector Anopheles culicifacies in a malarious area of Sistan va Baluchestan province, south-east Islamic Republic of Iran

A study was done of the bionomics, insecticide susceptibility and irritability status of Anopheles culicifacies in Sistan va Baluchestan province. Sampling was performed to determine the following parameters: species identification, seasonal activity, adult and larval susceptibility tests, irritability tests, anthropophily index and sporozoite rate. An. culicifacies adults were susceptible to all tested pyrethroid insecticides. An. culicifacies started to appear indoors in late May, showing 2 peaks in June and September. Fenitrothion, cyfluthrin and permethrin had the least irritancy effect and DDT the highest. Only 2/860 females tested were infected with Plasmodium spp. [sporozoite rate: 0.25%]. EL1SA testing of 250 blood meals derived from night-biting collections of female mosquitoes from humans and cows revealed that only 12.5% were human-fed