Genetic Population Diversity of Aedes caspius in Southern Provinces of Iran.

Aedes caspius has a wide distribution throughout the world and can transmit Chikungunya virus, West Nile Virus (WNV), Tahyna virus and the bacterium Francisella tularensis. Sequences of the mitochondrial cytochrome C-oxidase subunit 1 (COI) and cytochrome C-oxidase subunit 2 (COII) genes have been widely used to estimate phylogenetic relationships at different taxonomic levels among this species. Adult collections were carried out by human bait, Center for Deseases Control Light Traps (CDC-LT) and aspirator during February/April, June and October/December 2013-2015 from different southern provinces of Iran and then identified morphologically with reliable keys. A total of 3,570 adult mosquitoes were collected and identified as belonging to three genera, including five species of Culex, six species of Aedes and one species of Culiseta. In this study, 1,796 specimens of Aedes caspius were identified from four provinces. Based on the COI and COII sequences obtained for Ae. caspius population, 12 and 11 haplotypes were identified, respectively. The present study evidenced a high degree of intraspecific variation among these populations of Ae. caspius.

Aedes caspius largement distribué dans le monde est le vecteur du virus du chikungunya, du virus West Nile, du virus Tahyna et de la bactérie Francisella tularensis. Les séquences des gènes mitochondriaux COI et COII sont généralement utilisées pour estimer les relations phylogénétiques entre les différents niveaux taxonomiques de cette espèce. Des moustiques adultes ont été collectés sur appâts humains, à l'aide de pièges lumineux de type « CDC/light-trap ¼ et d'aspirateurs en février/avril, juin et octobre/décembre 2013­2015 dans les provinces au sud de l'Iran. Les moustiques ont ensuite été identifiés d'un point de vue morphologique. Trois mille cinq cent soixante-dix moustiques adultes ont été collectés et identifiés comme appartenant à trois genres, dont cinq espèces de Culex, six espèces d'Aedes et une espèce de Culiseta. Dans cette étude, 1 796 spécimens d'Aedes caspius ont été identifiés dans quatre provinces. Les séquences COI et COII des populations d'Ae. caspius ont permis d'identifier respectivement 12 et 11 haplotypes. Les résultats de cette étude ont démontré qu'il existe une forte variation intraspécifique dans ces populations d'Ae. caspius.

Morphological studies on adult mosquitoes (Diptera: Culicidae) and first report of the potential Zika virus vector Aedes (Stegomyia) unilineatus (Theobald, 1906) in Iran.

Beside numerous extensive studies on Anophelinae mosquitoes of Iran, little is known on Aedes species in the country and existing reports are dispersed. The objective of this study was to identify adults of Culicinae species occurring in the Sistan and Baluchestan Province, southeast of Iran, during 2012-2014. Mosquito collections were carried out three times (May-June, September, October-November) in four counties by Centers for Diseases Control (CDC) light traps and human landing catches outdoors and under bed nets baited with carbon dioxide. These trapping were carried out two consecutive nights during the field studies. Several mosquito collections were also conducted with aspirator and pyrethrum spray space catches during the day. A total of 1885 mosquitoes were collected, belonging to 10 species of genus Culex including Cx. pipiens Complex, Cx. laticinctus, Cx. sinaiticus, and Cx. tritaeniorhynchus, one species of the genus Culiseta, Cs. longiareolata, and five species of the genus Aedes, Ae. caspius, Ae. vexans, Ae. detritus, Ae. albopictus, and Ae. unilineatus. Ae. vexans was the dominant species in the area (77.7%). During the study, seven Ae. unilineatus were collected in two villages near the city of Chabahar located in a coastal area; this is the first record for Iran and identification was confirmed by Cytochrome oxidase (COI) sequences analysis. Confirmation of the presence of Ae. unilineatus in the country raises the number of species of the genus Aedes to 12. The detection of this species reveals its probable establishment in the southeast of the country, which has implications for public health such as dengue and Zika infections and requires active entomological surveillance and implementation of adapted vector control measures in the area.

Some Epidemiological Aspects of Cutaneous Leishmaniasis in a New Focus, Central Iran.

Following the epidemic of cutaneous leishmaniasis in Khatam County, Yazd Province, this study was carried out to determine vector, and animal reservoir host(s) and investigate the human infection during 2005-2006. Four rural districts where the disease had higher prevalence were selected. Sticky paper traps were used to collect sand flies during April to November, biweekly. Meanwhile rodents were captured using Sherman traps from August to November. Households and primary schools were visited and examined for human infection in February 2006. The parasite was detected by RAPD-PCR method. The rate of ulcers and scars among the inhabitants was 4.8% and 9.8%, respectively. Three rodent species were captured during the study: Meriones libycus, Rhombomys opimus, and Tatera indica. Six sand fly species were also collected and identified; among them Phlebotomus papatasi had the highest frequency. Leishmania major was detected as the agent of the disease in the area. It was detected from R. opimus and native people.

Predicted Distribution of Visceral Leishmaniasis Vectors (Diptera: Psychodidae; Phlebotominae) in Iran: A Niche Model Study.

Visceral leishmaniasis (VL) is an important vector-borne disease in Iran. Till now, Leishmania infantum has been detected from five species of sand flies in the country including Phlebotomus kandelakii, Phlebotomus major s.l., Phlebotomus perfiliewi, Phlebotomus alexandri and Phlebotomus tobbi. Also, Phlebotomus keshishiani was found to be infected with Leishmania parasites. This study aimed at predicting the probable niches and distribution of vectors of visceral leishmaniasis in Iran. Data on spatial distribution studies of sand flies were obtained from Iranian database on sand flies. Sample points were included in data from faunistic studies on sand flies conducted during 1995-2013. MaxEnt software was used to predict the appropriate ecological niches for given species, using climatic and topographical data. Distribution maps were prepared and classified in ArcGIS to find main ecological niches of the vectors and hot spots for VL transmission in Iran. Phlebotomus kandelakii, Ph. major s.l. and Ph. alexandri seem to have played a more important role in VL transmission in Iran, so this study focuses on them. Representations of MaxEnt model for probability of distribution of the studied sand flies showed high contribution of climatological and topographical variables to predict the potential distribution of three vector species. Isothermality was found to be an environmental variable with the highest gain when used in isolation for Ph. kandelakii and Ph. major s.l., while for Ph. alexandri, the most effective variable was precipitation of the coldest quarter. The results of this study present the first prediction on distribution of sand fly vectors of VL in Iran. The predicted distributions were matched with the disease-endemic areas in the country, while it was found that there were some unaffected areas with the potential transmission. More comprehensive studies are recommended on the ecology and vector competence of VL vectors in the country.

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.

Field evaluation of phostoxin and zinc phosphide for the control of zoonotic cutaneous leishmaniasis in a hyperendemic area, central Iran.

BACKGROUND & OBJECTIVES: ZCL is a growing threat in many rural areas of Iran which involves 17 out of 31 provinces. This study was conducted from April to November 2011 for evaluation of the efficacy of phostoxin and zinc phosphide against rodents. METHODS: Rodent control operations were carried out using phostoxin and zinc phosphide. To evaluate the effect of rodent control operation on the main vector density, an entomological survey was carried out. The effects of the operation on the disease incidence were also evaluated. RESULTS: After intervention, the reduction rate of rodent burrows was 32.68% in the village treated with phostoxin and 58.14% in the village treated with zinc phosphide. The number of rodent holes in the control area showed 6.66-fold increase at the end of the study. The incidence of the disease decreased to 19.23 and 11.40 in areas treated with phostoxin and zinc phosphide, respectively. A total of 4243 adult sandflies were collected and identified. The most common and dominant species was Phlebotomus papatasi. In the village treated with phostoxin, the density of P. papatasi in outdoors was lower than indoors. Nevertheless, the density of P. papatasi in the village treated with zinc phosphide was higher in outdoors. INTERPRETATION & CONCLUSION: It is concluded that phostoxin is less effective and has low safety in comparison with zinc phosphide, so that this rodenticide can be used only in special situations such as lack or ineffective rodenticides and only in the colonies far from human and animal dwelling places in small scales.

Baseline insecticide susceptibility data of Phlebotomus papatasi in Iran.

BACKGROUND & OBJECTIVES: Phlebotomine sandflies (Diptera: Psychodidae) play main role in the transmission of different forms of leishmaniasis in the world. Phlebotomus papatasi is the main vector of zoonotic cutaneous leishmaniasis (ZCL) in Iran. There are several control measures for vector control using different classes of insecticides. The aim of this study was to breed the sandflies which were collected from a hyperendemic focus of the disease in central Iran in the laboratory condition and to determine its baseline susceptibility to commonly used insecticides. METHODS: Sandflies were collected from the field and were reared in insectary. Susceptibility tests were carried out on their generation. Baseline susceptibility of sandflies to DDT and pyrethroids was evaluated based on LT50 values. A total of 1305 specimens were tested using different time intervals. The LT50 and LT90 values were measured according to the WHO standard tests. The results were plotted using probit analysis and regression lines. RESULTS: The results against female sandflies revealed the LT50 values of 1312.66, 253.66, 36.04, 9.38 and 6 sec to DDT (4%), permethrin (0.75%), deltamethrin (0.1%), cyfluthrin (0.15%) and lambda-cyhalothrin (0.05%), respectively. The figures for male sandflies were 1200.97, 310.10, 18.63, 6.08 and 0.77 sec respectively to the above insecticides. CONCLUSION: The results of this study could help to provide a clue for implementation of currently used insecticides. Furthermore, a specific guideline is needed for monitoring and evaluation of insecticide susceptibility test against sandflies.

Dynamics of Leishmania infection rates in Rhombomys opimus (Rodentia: Gerbillinae) population of an endemic focus of zoonotic cutaneous leishmaniasis in Iran.

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is a great public health problem in the Old World. Leishmania major is widely distributed in populations of rodents in arid and savannah regions. In this study, seasonal variation of natural infection with Leishmania parasites in Rhombomys opimus (Rodentia: Gerbillinae) population of an endemic focus of ZCL in Iran was monitored. The study was conducted from October 2007 to October 2008 in the central part of the country. Nested polymerase chain reaction (PCR) assay was used for the detection and identification of Leishmania parasites, and the results were confirmed by PCR-restriction fragment length polymorphism (RFLP). The results showed that Leishmania infection rate was 55.8% (29 out of 52 gerbils) using nested PCR. The highest and lowest Leishmania infection rates were observed in fall and summer, respectively. Gerbils that were found to be infected only with L. major were 5.8%, and that with Leishmania turanica were 23.1%. A mixed natural infection was seen in the rodents with L. major and L. turanica (21.2%), with L. major and L. gerbilli (1.9%), and with all the three species (3.9%). Leishmania major infection alone was seen in fall and winter whereas mixed infection of L. major and L. turanica was observed in all seasons except in summer. Leishmania turanica infection was observed throughout the year. It is concluded that L. major, L. gerbilli, and L. turanica circulate in the population of R. opimus in central part of Iran. Leishmania major infection is usually accompanied by L. turanica in naturally infected gerbils with the highest rate in fall. It is recommended that the role of L. turanica in the epidemiology and transmission of ZCL should be reconsidered.

Distribution and ecology of mosquitoes in a focus of dirofilariasis in northwestern Iran, with the first finding of filarial larvae in naturally infected local mosquitoes.

A study of the distribution and ecology of mosquitoes (Diptera: Culicidae) in Ardebil Province of northwestern Iran, with special reference to the known dirofilariasis focus in Meshkinshahr County, was carried out in July-August 2005 and April-October 2006. In total, 17,533 adult mosquitoes and 3090 third- and fourth-instar larvae were collected on 14 occasions and identified using morphological characters and DNA sequence data. Twenty species belonging to seven genera were found: Anopheles claviger (Meigen), An. hyrcanus (Pallas), An. maculipennis Meigen, An. pseudopictus Grassi*, An. sacharovi Favre, An. superpictus Grassi, Aedes vexans (Meigen)*, Coquillettidia richiardii (Ficalbi)*, Culex hortensis Ficalbi, Cx. modestus Ficalbi, Cx. pipiens Linnaeus, Cx. theileri Theobald, Cx. torrentium Martini*, Cx. tritaeniorhynchus Giles, Culiseta longiareolata (Macquart), Cs. annulata (Schrank)*, Cs. subochrea (Edwards), Ochlerotatus caspius (Pallas) s.l.* (= Aedes caspius sensu auctorum), Oc. geniculatus (Olivier)* (= Aedes geniculatus sensu auctorum) and Uranotaenia unguiculata Edwards (asterisks indicate new occurrence records for the province). The most prevalent species in adult catches were An. maculipennis (52%), Cx. theileri (45%) and Cx. hortensis (1%); the most prevalent species caught as larvae were Cx. theileri (27%), Cx. hortensis (21%) and An. maculipennis (19%). Anopheles maculipennis, Cx. pipiens and Cx. theileri were most widely distributed in the province. The occurrence of Cx. torrentium in Iran is verified based on differential characters of fourth-instar larvae. Anopheles maculipennis and An. sacharovi of the Maculipennis Group were identified from their diagnostic ITS2 sequences. For the first time, cytochrome c oxidase I (COI) sequences were obtained from Iranian specimens of An. hyrcanus, An. pseudopictus, Cx. theileri and Oc. caspiuss.l. Culex theileri and An. maculipennis were found naturally infected with third-stage (infective) larvae of Dirofilaria immitis (Leidy) and Setaria labiatopapillosa (Alessandrini) (Spirurida: Onchocercidae), respectively, for the first time in Iran.

Population structure and geographical subdivision of the Leishmania major vector Phlebotomus papatasi as revealed by microsatellite variation.

Multi-locus microsatellite typing (MLMT) has been employed to infer the population structure of Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) sandflies and assign individuals to populations. Phlebotomus papatasi sandflies were collected from 35 sites in 15 countries. A total of 188 P. papatasi individuals were typed using five microsatellite loci, resulting in 113 different genotypes. Unique microsatellite signatures were observed for some of the populations analysed. Comparable results were obtained when the data were analysed with Bayesian model and distance-based methods. Bayesian statistic-based analyses split the dataset into two distinct genetic clusters, A and B, with further substructuring within each. Population A consisted of five subpopulations representing large numbers of alleles that were correlated with the geographical origins of the sandflies. Cluster B comprised individuals collected in the Middle East and the northern Mediterranean area. The subpopulations B1 and B2 did not, however, show any further correlation to geographical origin. The genetic differentiation between subpopulations was supported by F statistics showing statistically significant (Bonferroni-corrected P < 0.005) values of 0.221 between B2 and B1 and 0.816 between A5 and A4. Identification of the genetic structure of P. papatasi populations is important for understanding the patterns of dispersal of this species and to developing strategies for sandfly control.

First detection of Leishmania infantum in Phlebotomus (Larroussius) major (Diptera: Psychodidae) from Iran.

Ghir-Karzin district is one of the most important endemic foci of visceral leishmaniaisis (VL) in Fars province of southern Iran. To identify the vector(s) of Leishmania in this focus, a total of 2,539 sand flies were collected during June to September 2005. Eleven species (six Phlebotomus and five Sergentomyia) were identified, of which Phlebotomus papatasi Scopoli and Phlebotomus major Annandale were the first (37.4%) and third (11.2%) most common species, respectively. Natural leptomonad infections were observed in two P. papatasi (4.25%) and three P. major (6.65%) specimens on dissection and microscopic examination. Using a Leishmania genus-specific standard polymerase chain reaction (PCR; primers RV1-RV2) and a species-specific nested-PCR (primers LINR4, LIN17, and LIN 19), Leishmania infantum and Leishmania major kinetoplast minicircle DNA was detected in 6 of 72 P. major (8.3%) and 4 of 65 P. papatasi (6.1%), respectively. This is the first detection of L. infantum in P. major, implicating this sand fly as a probable vector of VL in Iran.

Geographical variation in populations of Phlebotomus (Paraphlebotomus) caucasicus (Diptera: Psychodidae) in Iran.

A comparative morphological and molecular study was carried out on 11 different populations of Phlebotomus (Paraphlebotomus) caucasicus Marzinovsky 1917 caught in 7 provinces in Iran (2004-2005). Differences in the implantation level of the two distal spines of the style, the number of setae of the basal lobe of coxite, and the length of the third antennal segment, revealed the existence of two morphotypes within P. (Pa) caucasicus, a species having a confused history if we take into account an unclear synonymisation with Phlebotomus (Paraphlebotomus) grimmi Porchinsky 1874. Sequencing of mtDNA (a fragment of cytchromeB gene, tRNA for serine gene and a fragment of NADH1 gene) and Neighbour-Joining analysis showed a partial correlation between morphotypes and haplotypes. We also found a correlation between the latter and the geographical origin of the specimens. These results need further studies in order to appreciate the role of each morphotype/haplotype in the transmission of Leishmania major.

Evaluation of deltamethrin-impregnated bed nets and curtains for control of zoonotic cutaneous leishmaniasis in a hyperendemic area of Iran.

In the study carried out in the rural district of Borkhar (Isfahan, Iran) from March 2003 to March 2004 efficacy of deltamethrin impregnated bed nets (IBs) and curtains (ICs), polyester mesh size 156 holes/ inch2, (25 holes/cm2) were compared to that of non-impregnated bed nets (NIBs) and curtains (NICs), in relation to zoonotic cutaneous leishmaniasis (ZCL) control. Deltamethrin impregnated bed nets and curtains at 25 mg a.i./m2 were distributed among 168 households in one of the villages and NIBs plus NICs among the same number of households in another village. In the third village similar numbers of households were used as control. Health education messages were disseminated to ensure the population's compliance with the proper use of bed nets and curtains in the two intervention areas. Entomological surveys using sticky paper traps were carried out in ten households in each village for the collection of sand flies from indoors and outdoors, every two weeks to assess the impact of insecticide impregnated bed nets and curtains on the density of Phlebotomus papatasi. Deltamethrin susceptibility tests and also bioassay tests were carried out on this species by WHO standard method. Case findings were done by house-to-house visits once a season and all the inhabitants of the selected households in each village were examined. The results showed that IBs and ICs can provide a definite personal protection against sand fly bites and interrupted the transmission of ZCL in the intervention village. NIBs and NICs did not provide any protection against the disease. There was no significant difference in monthly density of P. papatasi in indoors and outdoors among the villages (P > 0.05). Susceptibility tests showed that this species was susceptible to deltamethrin in the field population in the area. Bioassays confirmed that the treated nets with deltamethrin remain effective for more than three months and was an excellent mean of individual protection. It is recommended that IBs and ICs can be used widely in the control of ZCL in other similar foci such as hyperendemic and mesoendemic areas of Iran.

Phlebotomus (Paraphlebotomus) alexandri: a probable vector of Leishmania infantum in Iran.

The incidence of human visceral leishmaniasis (VL) caused by Leishmania infantum, which is endemic in several parts of Iran, has recently increased in the Nourabad-Mamassani district of Fars province, in the south of the country. Between 2003 and 2005, 12,688 sandflies were caught in this focus, using CDC miniature light traps, sticky traps and aspirators, in an attempt to identify the main vector of L. infantum. When 120 of the parous, female Phlebotomus (Paraphlebotomus) alexandri caught were individually checked for Leishmania infection, in an assay based on PCR and kinetoplast minicircle primers (LINR4 and LIN17), five (4.2%) of the flies were found to be infected with L. infantum. Thirty-nine (32.5%) of the 120 blood-fed Ph. alexandri checked in an ELISA were found positive for human blood. Since Ph. alexandri was the third-most common species caught (representing over 17% of the total catch), was clearly anthropophilic, and was not infrequently infected with L. infantum, it is probably an important vector in the Nourabad-Mamassani focus. Although other sandfly species have been found naturally infected with promastigotes in Iran, Ph. alexandri is the first that has been proven to harbour L. infantum in the country.

Characterization of Leishmania infection in rodents from endemic areas of the Islamic Republic of Iran.

Between 1991-2000, Leishmania species were isolated and characterized by isoenzyme and molecular analysis from rodents caught in various parts of the Islamic Republic of Iran. In areas endemic for cutaneous leishmaniasis, parasites were observed by direct microscopy in smears from 18.6% of 566 specimens. L. major was isolated from 4 species: Rhombomys opimus, Meriones libycus, Tatera indica and Mer. hurrianae. L. turanica was isolated from R. opimus for the first time in this country. In endemic areas of visceral leishmaniasis, parasites were observed in liver and spleen from 13.7% of 504 rodents. Two species were positive on culture; promastigotes isolated from Mer. persicus were characterized as L. donovani zymodeme LON50 and from Mesocricetus auratus as L. infantum LON49.

Characterization of Leishmania infection in rodents from endemic areas of the Islamic Republic of Iran

Between 1991-2000, Leishmania species were isolated and characterized by isoenzyme and molecular analysis from rodents caught in various parts of the Islamic Republic of Iran. In areas endemic for cutaneous leishmaniasis, parasites were observed by direct microscopy in smears from 18.6% of 566 specimens. L. major was isolated from 4 species: Rhombomys opimus, Meriones libycus, Tatera indica and Mer. hurrianae. L. turanica was isolated from R. opimus for the first time in this country. In endemic areas of visceral leishmaniasis, parasites were observed in liver and spleen from 13.7% of 504 rodents. Two species were positive on culture; promastigotes isolated from Mer. persicus were characterized as L. donovani zymodeme LON50 and from Mesocricetus auratus as L. infantum LON49

Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran.

An outbreak of cutaneous leishmaniasis (CL) in Sabzevar county prompted this study of the epidemiology and the ecology of vectors and reservoirs. Examination of 541 schoolchildren showed rates of 9.4% for scars and 5.9% for ulcers. Among 807 inhabitants of 4 villages, 10.4% had scars and 3.0% had active lesions. The most highly infected age group was 0-4 years with a rate of 5.9%. A total of 12 849 sandflies representing 7 species were collected in the study area. Leptomonad infection was found in Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni. Parasites from man, P. papatasi and Rhombomys opimus, were isolated and characterized as Leishmania major. Based on this survey, this is an epidemic of zoonotic CL, with R. opimus the main reservoir host, and P. papatasi the main vector.

Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran

An outbreak of cutaneous leishmaniasis [CL] in Sabzevar county prompted this study of the epidemiology and the ecology of vectors and reservoirs. Examination of 541 schoolchildren showed rates of 9.4% for scars and 5.9% for ulcers. Among 807 inhabitants of 4 villages, 10.4% had scars and 3.0% had active lesions. The most highly infected age group was 0-4 years with a rate of 5.9%. A total of 12 849 sandflies representing 7 species were collected in the study area. Leptomonad infection was found in Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni. Parasites from man, P. papatasi and Rhombomys opimus, were isolated and characterized as Leishmania major. Based on this survey, this is an epidemic of zoonotic CL, with R. opimus the main reservoir host, and P. papatasi the main vector