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

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

High performance thin layer chromatography analysis of deltamethrin residue on the impregnated bed nets during a leishmaniasis control program in Iran

The control of leishmaniasis, a tropical neglected disease, has been concern of Iranian health authorities due to the increasing number of cases during the last two decades. The objective of this study was to determine deltamethrine residue on the impregnated bed nets using HPTLC technique in a leishmaniasis control program in Iran. During this experimental study, a total of 130 small pieces of polyester netting were sewn to top, upper, and lower sides of some bed nets and then were impregnated with deltamethrin .The treated bed nets were distributed in Isfahan and Mashhad areas in April 2003. The samples were cut randomly after impregnation intervals. Deltamethrin was extracted using acetone from samples and the extract was applied for spotting onto plates. The plates were developed with n-hexane: ethyl acetate, 90+10[v/v], as a mobile phase in a Camage chamber. The qualifying of residue was observed in UV cabinet with lambda =254 nm wavelength. Data were analyzed using SPSS version 11.5. and Stata Version 8. A three way ANOVA was used to compare the means of deltamethrin residue in each area, group and measuring time. The Kruskal-Wallis test was used to compare the means of residue for each of these factors with the control separately. The retardation factor of deltamethrin was calculated 0.50 +/- 0.02. The residues of deltamethrin persisted well on impregnated nets at least for 15 weeks after impregnation. No significant difference could be detected in the loss of residue of insecticide in comparison to measuring times and positions of sampling pieces on the bed nets in these areas. Based on the results of the present study the use of HPTLC technique is recommended instead of other chromatographic methods for analysis of insecticide residue on the impregnated bed nets

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

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

[Phlebotomus [Paraphlebotomus] alexandri, the probable vector of visceral leishmaniasis [kalaazar] in southern Iran]

Visceral leishmaniasis [kala-azar], the most dangerous form of leishmaniasis, is endemic in some parts of Iran, e.g. Ardabil, Fars, East Azerbaijan, Bushehr and Qom provinces. In recent years, the incidence of VL has increased in the Nourabad-Mamassani district in Fars Province. This study was carried out to detect VL vectors and infection rates in this region over the 2003-2004 period. Sand flies were captured in the selected villages by means of sticky traps, aspirators and CDC miniature light traps. Heads and distal abdominal segments were used for species identification and other body parts were used for DNA extraction. We employed a semi-nested PCR technique to detect Leishmania, with specific kDNA primers [LIN R4 - LIN 17 - LIN19]. Some specimens were dissected for leptomonad infection. A total of 12688 sand flies were collected. Phlebotomus [Paraphlebotomus] alexandri was the second most prevalent species [17.34%]. The anthropophilic index of this species was 32.5%. Five cases [4.17%] of L. infantum infection were detected among the 120 P. alexandri examined by PCR method. We also observed two cases of leptomonad infection among the 112 dissected specimens. High prevalence rates and anthropophilic index of P. alexandri plus its natural infection with L. infantum provide enough evidence to implicate this species as the main vector species of VL in the region and the second proven kala-azar vector in Iran. Besides, the Mahoor-Milaty district of Noorabad-Mamassani was identified as a new endemic focus

seminested PCR based detection of Leishmania infantum infection in asymptomatic dogs in a new endemic focus of visceral leishmaniasis in Iran

Visceral Leishmaniasis [Kala-azar] is a serious health problem in some northern and south western parts of Iran. The incidence of kala-azar caused by Leishmania infantum has recently increased in Nourabad-Mamassani district of Fars Province, in the south of the country. This study was designed to determine the role of asymptomatic dogs as host reservoir of L. infantum in this new formed focus and detection of prevalence of infection near them. A total of 20 asymptomatic stray and sheep dogs were randomly sampled. The Buffy coat layer of their peripheral blood was used for DNA extraction and PCR. A species specific seminested PCR was used for DNA amplification using LINR4, LIN17 and LIN19 primers. These primers amplified variable area of the minicircle kDNA of Leishmania parasites. Of the 20 sampled dogs checked for leishmanial kDNA, six [30%] were found naturally infected. It is concluded that, dogs [Canis familiaris] even if asymptomatic, is considered as the domestic host reservoir of kala-azar in this endemic focus

Distribution of soft ticks and their natural infection with Borrelia in a focus of relapsing fever in Iran

Tick-borne diseases such as relapsing fever and Crimean-Congo Hemorrhagic Fever [CCHF] are of public health importance in Iran. There are 471 reported cases of relapsing fever in 2003, according to the Ministry of Health of Iran. The number of cases has been increased in recent years. Its distribution is more or less prevalent in different parts of Iran. The aim of this study was to find out the fauna and natural infection of soft ticks with Borrelia in Qazvin Province, during their seasonal activity. The province covers 15821 km[2] between 48-45 to 50-50 east of Greenwich Meridian of longitude and 35-37 to 36-45 north latitude of the equator. For this purpose a field study was carried out in the region. A total of 54 villages from 19 districts were selected randomly and ticks were collected from their habitats according to the standard method. A total of 3197 Argasidae ticks were collected from human dwellings, poultry and animal shelters. They belonged to Argas and Ornithodoros genera which 36.8% were Argas persicus, 4% A. reflexus, 6.4% O. canestrini, 45.5% O. lahorensis and 7.3% O. tholozani. It should be noted that 12 ticks of O. erraticus were collected from 12 rodents borrows. We found that 8.82% of O. tholozani ticks were infected with Borrelia persica and half of the O. erraticus were infected with Borrelia microti. All the people who are involved with veterinary activities should be aware of disease transmission by the ticks. In the endemic area of the disease tick control is recommended

[Determination of reservoir[s] and vector[s] of cutaneous leishmaniasis by nested-PCR in Marvdasht District, Fars Province, Southern Iran]

Cutaneous leishmaniasis [CL] is an increasing public health problem in several parts of Iran. In southern parts, the incidence of CL has been doubled over the last decade. This epidemiological study was done for determination of reservoir[s] and vector[s] of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Iran during 2003 and 2004. A total of 126 rodents were collected from three villages using live traps and their Giemsa-stained smears were studied for leishmania infection. After DNA extraction from positive smears, Nested-PCR was used for the identification of parasite species. In another procedure, 200 sand flies were collected by aspirator and after species identification DNA extraction and PCR was done. The collected samples included Meriones libycus [75.4%], Cricetulus migratorius [14.3%] and Microtus arualis [10.3%]. Eight out of 95 Meriones libycus [8.4%] were found to be infected with Leishmania major. None of the other species were positive. Among the collected female sandflies 75% were identified to be Phlebotomus papatasi and 2.7% of them were found with L.major infection. Only 2.7% of Phlebotomus papatasi were found naturally infected with Leishmania major. This is the first report of detection of L.major by Nested-PCR in P.papatasi as a proven principal vector of zoonotic cutaneous leishmaniasis in Fars province, south of Iran

Vectors and reservoirs of cutaneous leishmaniasis in Marvdasht district, southern Islamic Republic of Iran

An epidemiological study was made of vectors and reservoirs of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Islamic Republic of Iran during 2003-04. Using live traps, 126 rodents were collected: 75.4% were Meriones libycus, 14.3% Cricetulus migratorius and 10.3% Microtus arvalis. Eight out of 95 Meriones libycus [8.4%] were found to be infected with Leishmania major, identified by nested-PCR; none of the other rodents were positive. Female sandflies were collected from indoor locations: 75% were Phlebotomus papatasi and only 2.7% were found naturally infected with L. major. This is the first report of P. papatasi as a proven vector of zoonotic cutaneous leishmaniasis in this area

[Review of dirofilariasis in Iran]

Introduction: Dirofilariasis or dirofilariosis is a mosquito [Diptera: Culicidae] borne metazoonotic disease, caused by different species of the nematod genus Dirofilaria [Spirurida: Onchocercidae]. Its reservoirs are mostly canids and it is cosmopolitan. Human dirofilariasis is now classified as an emerging zoonosis

Objective: This article reviewed all previous records and investigations in Iran and the world in relation to dirofilariasis in order to provide a foundation for forthcoming studies

Materials and Methods: From the first report of dirofilariasis in Iran in 1969 up to 2005, all human and animal cases and important data such as: the pathogen and reservoir species, distribution, frequency, and human cases, as retrospective study, were investigated and analyzed. Also, the natural history of parasite, pathogenicity, the disease diagnosis, treatment, and control, and the last status of the disease in the world were briefly mentioned

Results: Two species of the genus Dirofilaria; D. immitis [canine heartworm] and D. repens are found in Iran. By now, nine human cases have been formally reported including four subcutaneous and two ocular cases of D. repens, a rare case of D. immitis in testicular hydrocele, and two pulmonary cases [most probably D. immitis]. As a whole, human and animal dirofilariasis has been recorded in eleven provinces of Iran. The species D. immitis has been reported in East Azerbaijan, West Azerbaijan, Ardebil, Tehran, Khorasan, Khuzistan, Fars, Golestan, Mazandaran, and Hormozgan Provinces and D. repens in Tehran, Khorasan, Guilan, and Mazandaran Provinces. Different investigators have reported D. immitis in dogs [with the frequency of 0.95 to 36.8%], jackals [7.5-57.1%], foxes [5.7-50%], wolves [20-50%], and cats [0.8%] and D. repens in dogs [1.4-60.8%] and jackals [10%] in the different areas of the country

Conclusion: Data on dirofilariasis in Iran are to some extent scattered as regard to distribution or other aspects of the disease. More studies need to be done on dirofilariasis in the country, especially in places where no investigation has been carried out. There is no information about the vectors of dirofilariasis in Iran

Meriones libycus is the main reservoir of zoonotic cutaneous leishmaniasis in south Islamic Republic of Iran

A study was made in rural regions around Neiriz city, Fars province in the south of the Islamic Republic of Iran during 2002-03 to further investigate a new focus of zoonotic cutaneous leishmaniasis. Meriones libycus was the dominant rodent [100% of 65 rodents collected] and 4.6% were naturally infected with the amastigote form of Leishmania spp. Promasigotes were identified as L. major. This confirms M. libycus as the main reservoir host of zoonotic cutaneous leishmaniasis in southern parts of 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

Investigation on zoonotic cutaneous leishmaniasis, Southern Iran

Since occurrence of an epidemic of cutaneous leishmaniasis with high incidence of disease [8.9/1000] in Arsanjan county, Fars province, southern Iran, an attempt was made to investigate an epidemiological study during 2001-2002. Symptomatic investigation among 270 schoolchildren over one year of study revealed that 11.9% of population had active lesion with 28.1% scar rate. Parallel study showed that among 7968 people which comprise 1572 households, 6.18% were positive for scars and 1.2% for ulcer. In our study three species of rodents, including Meriones libycus [71%], Cricetulus migratorius [22%] and Microtus arvalis [6%] were captured. By employing Nested-PCR technique, 6.8% [4 of 56] of M.libycus were found infected with Leishmania major. About 83% of indoor collected sandflies comprised, Phlebotomus papatasi. From its density it can be concluded that this species plays an important role in leishmania transmission in the area. The seasonal activity of the sandflies starts at early April and ended in early November

First Report on Natural Promastigote Infection of Phlebotomus caucasicus in a New Focus of Visceral Leishmaniasis in North West of Iran

An investigation was carried out in Ahar district, [a new focus of visceral leishmaniasis], Eastern Azerbaijan, North-West of Iran in order to determine the vector[s] of the disease, during the summer of 2003 s and -flies were collected using sticky papers, CDC light traps and aspirators. All of the collected s and -flies were dissected in a drop of normal saline for surveying of promastigotes. Head and two last abdomen segments of-s and flies were mounted in a drop of Puri's Medium for species identification. Totally 215 s and flies were collected including: Phlebotomus k and elakii [57.2%], P.perfiliewi [18.1%], P.caucasicus [11.2%], P. sergenti [7.9%], P. papatasi [3.3%] and P.[adlerius] sp.[2.3%]. Only 1 of 24 P. caucasicus species [4.2%] was found naturally infected with parasite. This is the first infection report of P. caucasicus in this region. Some behavioral aspects of the mentioned s and fly have been discussed in details

susceptibility to 4 % DDT and host preference of the probable vectors of visceral leishmaniasis in north west of Iran

This study was carried out on Phlebotomus kandelakii and Ph.perfiliewi, the probable vectors of visceral leishmaniosis in the north west of Iran during the summer of 1994. The results of the susceptibility test showed that the mortality rate with 60-min exposure to 4% DDT was 100% for each species, while with 30-min exposure the average mortality rates were 93.3% and 94.4% for P. Kandelakii and P.perfiliewi, respectively, indicating the sensitivity of the two probable vectors to DDT in Ardabil Province. The blood meals of 116 engorged sand-flies were collected from 3 villages of Meshkin-shahr County, including P.papatasi, P.caucasicus and P. kandelaki, and eighty one of them belonged to P. kandelakii. All of the blood meals were positive with human and 11.1% with dog antisera, indicating a strong preference to man

Confirmation of Tatera indica [rodentia: gerbillidae] as the main reservoir host of zoonotic cutaneous leishmaniasis in the west of Iran

During our study [summer of 1994] twenty two Tatera indica were collected and two out of them [9.09%] were naturally infected with amastigote. Parasites were cultured in NNN+LIT medium and isolated promastigotes were sent to the Medical University of Montpellier in France for identification. The result of isoenzyme showed, the parasite was Leishmania major zymodem MON 26-[=LON 1], therefore Tatera Indica was known the main reservoir host of zoonotic cutaneous leishmaniasis in the west of Iran. This is the first report on the isolation and identification of L.major zymodem MON 26-[=LON 1] from this species of rodent. In the meantime, Rhombomys opimus was absent in these areas

experience of leishmanization in the Islamic Republic of Iran

Leishmanization programmes in a hyperendemic area [Isfahan] and a high-risk group [army recruits and the Revolutionary Guard] are described and their effectiveness and complications are discussed. A trial of a non-living crude vaccine is outlined. More than two million people underwent leishmanization and it was found to reduce the incidence of the disease between one-sixth and one-eighth of its original level. The procedure is recommended whenever people are at a very high risk of contracting the disease

Sand-flies of ilam province, west of iran with description of two new species from the genus phlebotomus: ph ilami and ph nadimi

During the period of April to November 1994, an investigation was carried out on fauna of Phlebotominac in Ilam province, west of Iran. About 12000 sandflies were caught by sticky trap, light trap and aspirator from different ecological regions and were identified. These sandflies consist of 22 species [11 species from the genus Phlebotomus: Ph. paptasi, Ph.sergenti, Ph.alexandri, Ph.rnajor, Ph.tobbi, Ph.kandelakii, ph.perfiliewi. Ph.balcanicus and Ph.halepensis plus two new species of Phlebotomus and 11 species from the genus Sergentomyia: S. sintoni, S.dentata, S.antennata, S.theodori, S.mervynae, S.pawlowskyi, S.africana, S.clydei, S.tiberiadis, S.iranica and S.squatnipleurs]. The two new species from genus Phlebotomus [Ph.nadimi and Ph.ilami] found in this study have been described in detail