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.

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

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

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

Molecular detection of leishmania infantum in naturally infected phlebotomus perfiliewi transcaucasicus in bilesavar district, Northwestern Iran

Visceral leishmaniasis is caused by Leishmania infantum, transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in Iran. To identify the vector[s], an investigation was carried out in Bilesavar District, one of the important foci of the disease in Ardebil Province in northwestern Iran, during July-September 2008. Using sticky papers, 2,110 sand flies were collected from indoors [bedroom, guestroom, toilet and stable] and outdoors [wall cracks, crevices and animal burrows] and identified morphologically. Species-specific amplification of promastigotes revealed specific PCR products of L. infantum DNA. Six sand fly species were found in the district, including: Phlebotomus perfiliewi transcaucasicus, P. papatasi, P. tobbi, P. sergenti, Sergentomyia dentata and S. sintoni. Phlebotomus perfiliewi transcaucasicus was the dominant species of the genus Phlebotomus [62.8%]. Of 270 female dissected P. perfiliewi transcuacasicus, 4 [1.5%] were found naturally infected with promastigotes. Based on natural infections of P. perfiliewi transcaucasicus with L. infantum and the fact that it was the only species found infected with L. infantum, it seems, this sand fly could be the principal vector of visceral leishmaniasis in the region

Temperature requirements of some common forensically important blow and flesh flies [diptera] under laboratory conditions

The aim of his study was to determine development time and thermal requirements of three myiasis flies including Chrysomya albiceps, Lucilia sericata, and Sarcophaga sp. Rate of development [ROD] and accumulated degree day [ADD] of three important forensic flies in Iran, Chrysomya albiceps, Lucilia sericata, and Sarcophaga sp. by rearing individuals under a single constant temperature [28°C] was calculated using specific formula for four developmental events including egg hatching, larval stages, pupation, and eclosion. Rates of development decreased step by step as the flies grew from egg to larvae and then to adult stage; however, this rate was bigger for blowflies [C. albiceps and L. sericata] in comparison with the flesh fly Sarcophaga sp. Egg hatching, larval stages, and pupation took about one fourth and half of the time of the total pre-adult development time for all of the three species. In general, the flesh fly Sarcophaga sp. required more heat for development than the blowflies. The thermal constants [K] were 130-195, 148-222, and 221-323 degree-days [DD] for egg hatching to adult stages of C. albiceps, L. sericata, and Sarcophaga sp., respectively. This is the first report on thermal requirement of three forensic flies in Iran. The data of this study provide preliminary information for forensic entomologist to establish PMI in the area of study

Detection of Borrelia persica infection in Ornithodoros tholozani using PCR targeting rrs gene and xenodiagnosis

Relapsing fever caused by Borrelia persica, is an acute tick-borne disease which is transmitted by soft ticks of Ornithodoros tholozani to human. Value of PCR and xenodiagnosis for detection of B. persica in O. tholozani ticks was compared. Sixty-four Borrelia- free ticks were fed on infected guinea pigs and used for the experiments. For xenodiagnosis, a group of 32 ticks in subsequent blood meal were fed on sterile guinea pigs and the indication of B. persica in the animal blood was tested 5-14 days later by dark-field microscopy. For PCR, all 64 ticks were subjected to PCR against B. persica rrs gene [16S-rDNA]. Also sensitivity of PCR in terms of minimum detectable number of spirochetes as well as the effects of tick sex and post digestion was tested. PCR revealed B.persica DNA in 98.4% ticks, in which B. persica were found in 25.0% by xenodiagnosis. PCR was enough sensitive to give positive results for DNA of 1 spirochete. PCR success rates were similar for male or female ticks. Course of time did not affect the efficacy of PCR and similar results were observed for ticks of immediately fed, semior completely gravid or completely digested blood ones. Our results indicate that due to very low specificity and time consuming, xenodiagnosis is not a useful method whereas PCR method has advantages for study the Borrelia prevalence in ticks

[Determination of parasite species of cutaneous leishmaniasis using nested PCR in Damghan-Iran, during 2008]

Cutaneous leishmaniases with two forms of rural and urban is the endemic diseases and as a health problem in our country. Identification of parasite species and type of disease is very important for treatment of disease as well as for planning of control program. The microscopic observations by Giemsa-stained smears is the most common laboratory test for the diagnosis of cutaneous leishmaniasis, but the determination of parasite species is impossible and utilization of other ways such as biochemical and molecular methods is required. This study was carried out to determine the parasite species caused cutaneous Leishmaniasis by Nested PCR in Damghan, Iran. This descriptive study was performed on 67 patients with dermal lesions that referred to Damghan health center laboratory in Iran during 2008. The patient's information were recorded in questionnaire. DNA of Giemsa-stained slides from patients was extracted and evaluated by specific primers of kinetoplast DNA using Nested PCR. Leishmania parasites were observed in 57 patients under light microscope. The 10 patients were infected by other dermal diseases. The PCR result showed the parasite presence in lesions of 57 patients is Leismania major. 54% of patients were male and 46% were female. 72% of the patients were lived in rural areas. 50.9% of disease was observed in over 25 years old patients. Hands were the most common region of ulcer [44.7%]. 48% of the patients had one ulcer and the other patients had two or more ulcers. High prevalence [31.6%] of disease was observed in October. This study showed that zoonotic cutaneous leishmaniasis to be prevalent in this area and Nested PCR method is a sensitive and accurate to leishmania species characterization

Susceptibility status of phlebotomus papatasi and P. sergenti [diptera: psychodidae] to DDT and deltamethrin in a focus of cutaneous leishmaniasis after earthquake strike in Bam, Iran

The cutaneous leishmaniasis [CL] has been occurred in Dehbakri County, located 46 km of Bam District, Kerman Province since 2004-2005. Phlebotomus papatasi is an important vector of zoonotic cutaneous leishmanisis [ZCL] as well as sand fly fever and P. sergenti is considered as main vector of anthroponotic cutaneous leishmaniasis [ACL] in Iran. There are several measures for vector control with emphasizing on insecticides. The objective of this study was to determine the baseline susceptibility of leishmaniasis vectors to the DDT and deltamethrin in an endemic focus of CL in southern Iran. Baseline susceptibility tests were carried out on field collected strains of P. papatasi and P. sergenti and tested with WHO impregnated papers with DDT 4.0% and deltamethrin 0.05% in the focus of disease in Dehbakri County during summer 2010. The values of LT[50] and LT[90]0 were determined using probit analysis and regression lines. The LT[50] value of DDT 4.0% and deltamethrin 0.05% against P. papatasi was 20.6 and 13.6 minutes respectively. The same data for P. sergenti were ranged between 21.8 and 17.7 minutes. The results of tests will provide a guideline for implementation of vector control using pesticides such as impregnated bed nets, indoor residual spraying and fogging

Utility of filter paper for preserving insects, bacteria, and host reservoir DNA for molecular testing

Appropriate methodology for storage biological materials, extraction of DNA, and proper DNA preservation is vital for studies involving genetic analysis of insects, bacteria, and reservoir hosts as well as for molecular diagnostics of pathogens carried by vectors and reservoirs. Here we tried to evaluate the utility of a simple filter paper-based for storage of insects, bacteria, rodent, and human DNAs using PCR assays. Total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. Extracted DNA of five different bacteria species as well as blood specimens of human and great gerbil Rhombomys opimus was pipetted directly onto filter paper. The papers were stored in room temperature up to 12 months during 2009 until 2011. At monthly intervals, PCR was conducted using a 1-mm disk from the DNA impregnated filter paper as target DNA. PCR amplification was performed against different target genes of the organisms including the ITS2-rDNA of mosquitoes, mtDNA-COI of the sand flies and cockroaches, 16SrRNA gene of the bacteria, and the mtDNA-CytB of the vertebrates. Successful PCR amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. The PCR amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. Time of storage did not affect the amplification up to one year. The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis

Study on presence of borrelia persica in soft ticks in Western Iran

A molecular survey was conducted to investigate the presence of pathogenic Borrelia persica species causing the tick borne relapsing fever [TBRF] in Takistan district Qazvin Province, western Iran. A number of 1021 soft ticks were collected from 31 villages including previously reported infected and none-infected TBRF cases and individually examined for the presence of B. persica DNA by conventional PCR targeting the 16SrRNA. A total of 1021 soft ticks of three species of Ornithodouros tholozani [120: 11.75%]. O. lahorensis [461: 45.15%] and Argaspersicus [440: 43.1%] were collected and tested against Borrelia infection. Soft ticks were more prevalent [67%] in infected areas than none infected areas. The rate O. tholozani in infected areas was much greater [29 times] than none infected areas. Ninety seven percent of soft ticks in none infected areas were of O. tholozani. Sixteen [16.7%] ticks of tested [n=95] O, tholozani were infected with B. persica. Three [1.3%] out of 205 soft ticks of O. lahorensis were positive for Borrelia sp., and no infection was observed in A persicus. Taq/ RFLP analysis and sequence analysis of the positive PCR products showed the presence of B. persica. The RFLP analysis showed that the positive ticks of O. lahorensis were infected with unknown Borrelia species. This study showed that although there were no TBRF cases in Takisan. but still infected O. tholozani, the known vector of TBRF. presented in the region. Control measures needs to be fulfilled in Thakisan

Species composition and diversity of mosquitoes in neka county, mazandaran province, Northern Iran

Regarding to the significant of the possibility of the malaria epidemic and nuisance of mosquitoes during the active season, the fauna and some ecological activities of mosquitoes in related to tree holes were investigated from April to December 2009 in Neka county of Mazandaran Province, northern Iran. Larval collection was carried out from natural, artificial breeding places, and tree holes inside the forest in Neka County, Mazandaran Province in 2009. In addition, human bait net trap collection was conducted using suction tube several times during this investigation. Four genera and five species were found in tree holes. Anopheles plumbeus, Culiseta annulata Culex pipiens, and Ochlerotatus geniculatus were collected by larval collection whereas, Ochlerotatus pulcritarsis was found by adult collection. Overall Cx.pipiens.44.6%., Oc. Geniculattus 32.6%. An. Plumbeus 22.5% and Cs. annulata 0.3% were collected by larval collection. During the bait net collection five specie were identified including: Oc. Geniculatustts 55.8%. Oc. Echinus 1.33%, Qs.pulcritarsis 8.8%, Cx.pipiens 33.8% and An plumbeus 0.2%. Cs. annulata larvae was detected for the first time with a low abundance in tree cavity. Tree holes were found the main habitat for the species of Oc. geniculatus. The species of Cs. annulata was found in tree holes

Tick infestation rate of sheep and their distribution in abdanan county, ham province, Iran, 2007-2008

Ticks are hematophagous arthropod belonging to the Class of Arachnids. Ticks are also one of the major vectors of pathogens to animal and human. This study was conducted to determine tick infestation rate of sheep in Abdanan during 2007-2008. Sampling was performed seasonally in 19 villages during spring 2007 until winter 2008. A total of 1095 sheep were selected and tested for tick infestation. After collection, all ticks were transported to laboratory of Medical Entomology and were identified with appropriate identification keys. Totally, 864 hard ticks were collected. The ticks were classified into two genera and 5 species including: Hyalomma marginalum [44.67%], Hy. anatolicum [43.17%], Hy.asiaticwn [6.37%], Hy. dromedarii [5.55%], Hea-maphysalis sulcata [0.24%]. The highest seasonal activity was observed in spring [36.46%] and the lowest seasonal was in winter [11.57%]. The rate of tick frequency in mountainous region was 48.15% and it was 51.85% in plateau regions. In this study, tick infestation of sheep was 11.41%. Hy.marginatum has the more frequent density in the study area

Differential responses of anopheles stephensi [diptera: culicidae] to skin emanations of a man, a cow, and a guinea pig in the olfactometer

Biting habit of mosquitoes plays an important role in the epidemiology of mosquito-borne diseases. Mosquitoes use a set of elaborate sensory modalities to find their preferred hosts by exploiting cues emanating from a nearby host. It has been suggested that the chemical profile of skin can provide further support for anthropophilic mosquito species to find their suitable hosts. This study aimed at revealing the value of skin emanation for a zoophilic species like Anopheles stephensi as a model. Skin emanations of a man, a cow and a Guinea pig were collected by ethanol soaked cottons. Upwind responses of mosquitoes to 100 and 200 micro L of filtered skin materials were non-competitively explored in a dual-choice olfactometer. L-lactic acid and other chemical content of the skin samples were identified by an enzymatic kit and GC-MS, respectively. Unexpectedly, only human skin emanation was resulted in the statistically significant activation and attraction responses of Anopheles. stephensi in the wind tunnel. L-lactic acid content of this skin sample was 10 and 29 times more than the cow and the Guinea pig, respectively. The possible role of lactic acid and a few other identified compounds have been discussed here. An. stephensi showed higher and more specific upwind responses to human skin emanation in the olfactometer. Undoubtedly, the thorough explanation of this unexpected finding needs further investigation. But, if new data verify this result, then, it may be necessary to reconsider the role of skin emanation and thence the human blood index and vectorial capacity of this zoophilic mosquito

Hard ticks on domestic ruminants and their seasonal population dynamics in Yazd Province, Iran

Ticks are the main vectors for transmission of different pathogens to human and animals. This survey was carried out distribution of ticks, which infested the domestic ruminants in Yazd Province, central Iran during year 2008-2009. A total number of 30 villages form both mountainous and [20%] plateau[80%] regions of the province were selected randomly. Ticks were colleted from the body of infested animals and transported to the Laboratory of Medical Entomology, School of Public Health, Tehran university of Medical Sciences and then were identified using valid identification key. A total of 583 hard ticks were collected. The ticks were classified into three genera and 7 species including: Hyalomma dromedarii [55.92%], Hyalomma marginatum [13.20%], Hy. anatolicum [9.78%], Hy. detritum [4.98%], Hyalomma asiaticum [3.94%] Rhipicephalus sanguineus [11.84%] and Dermacentor marginatus [0.34%]. The highest seasonal activities occurred in summer. The prevalence of the Ixodidae ticks was more evident in plateaus area in Yazd Province. Among the hosts including: cow, goat, sheep and camel, the ticks that collected from camel was more prevalent. The ratio of male was more than female ticks. Hyalomma dromedarii was the predominant tick species and accounted for 55.92% of the ticks. Some of the collected ticks may play an important role for transmission of vector borne disease to human; therefore, the results of this study will provide a clue for vectors of tick-borne diseases in the region for local authorities for implementation of disease control

First report of human nasal myiasis caused by eristalis tenax in Iran [diptera: syrphidae]

We report a case of human nasal myiasis caused by flower fly larva in a 14-year-old rural girl in Central Province of Iran. Entomological studies on larva showed the larva as Eristalis tenax which is a rarely cause of nasal myiasis. This is the first reported case of E. tenax larva causing human nasal myiasis in Iran

Blood meal identification in field-captured sand flies: comparison of PCR-RFLP and ELISA assays

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. 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. 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%]. 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

Study on ectoparasites of rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis in endemic foci in Iran

Ectoparasites of mammalian hosts play an important role for transmission of diseases from the host reservoirs to human. The aim of this study was to determine the species composition and infestation parameters for parasitic arthropods associated with Rhombomys opimus. Ectoparasites of R. opimus were collected from seven endemic district of ZCL in Iran including Shirvan and Sfaraysen in North Khorasan Province, Kalaleh in Golestan Province, Damghan and Shahrood in Semnan Province, and Badrood and Habibabad in Isfahan Province. The areas of study were mainly desert and plain. Rodents were captured using Sherman life traps during active seasons from May to November 2008. Captured rodents were transported to laboratory and their ectoparasites were picked up using brushing against the fur of the rodents. Ectoparasites were stored in 70% ethanol for their preservation and then identified based on morphological characters. Ectoparasites belonged to one flea species of Xenopsylla nuttalli and one mite species of Ornithonussus bacoti. The flea species with 75.3% was more common than the mite. O. bacoti might play an important role in transmission of rat mite dermatitis among R. opimus colony. Results will provide an essential clue for combating zoonotic diseases in the region

Identification of sand flies of the subgenus Larroussius based on molecular and morphological characters in North Western Iran

The adult female sand flies [Diptera: Psychodidae] of the subgenus Larroussius are important vectors of Leishmania infantum [Kinetoplastida: Tripanosomatidae] in Meshkinshahr district, Northwest of Iran. Four Phlebotomus [Larroussius] species are present in this area, i.e. Phlebotomus [Larroussius] kandelakii, P. [La] major, P. [La] perfiliewi and P. [La] tobbi. The objective of the present study was to identify and distinguish the females of P. perfiliewi, P. major and P.tobbi, in this district. Adult sand flies were collected with sticky papers, CDC light traps, and aspirator in 2006. Individual sand flies of this four species from thirty different locations were characterized morphologically and by comparative DNA sequences analyses of a fragment of mitochondrial gene Cytochrome b [Cyt b] and nuclear gene Elongation Factor 1- alpha [EF -1 alpha]. PCR amplification was carried out for all three species P. major, P. perfiliewi, and P. tobbi in the sub-genus Larroussius. Phylogenetic analyses of P. major populations in this study displayed two different populations and genetic diversity. Spermathecal segment number, pharyngeal armature and other morphological characters of these three species were examined and found to present consistent interspecific differences. According to our findings, the phylogeny of Cyt b and EF-1 alpha haplotypes confirms the relationships between P. major, P. tobbi and P. perfiliewi as already defined by their morphological similarities

Molecular detection of leishmania major in the vectors and reservoir hosts of cutaneous leishmaniasis in Kakaleh district, Golestan province, Iran

An epidemiological study was carried out on the vector[s] and reservoir[s] of cutaneous leishmaniasis in rural areas of Kalaleh District, Golestan Province during 2006 - 2007. Totally 4900 sand flies were collected using sticky papers and were subjected to molecular methods for detection of leishmanial parasite. Phlebotomus papatasi was the common species in outdoor and indoor resting places. Employing PCR technique showed only 1 out of 372 P. papatasi [0.3%] was positive to parasite due Leishmania major. Sixteen rodent reservoir hosts were captured by Sherman traps and identified as Rhombomys opimus. Microscopic investigation on blood smear of the animals for amastigote parasites revealed 6[37.5%] infected rodents. Infection of these animals to L. major was then confirmed by PCR against rDNA loci of the parasite. This is the first molecular report of parasite infection of both vector [P. papatas] and reservoir [R. opimus] to L. major. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and R. opimus was the most important host reservoir for maintenance of the parasite source in the area