Molecular detection and conventional identification of leishmania species in reservoir hosts of zoonotic cutaneous leishmaniasis in fars province, south of Iran

The objectives of our research were to search for Leishmania species in rodents in Fars province, south of Iran, and to compare molecular with conventional methods for detecting these parasites. Rodents were captured using live traps and screened for Leishmania species using molecular and conventional methods, including the taking of smears from each ear. Nested PCR was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania [ITS1- 5.8S rRNA-ITS2] that is species-specific by DNA sequence. Totally, 122 rodents were captured. Leishmania parasites were detected using the nested PCR and three conventional methods [direct smear, NNN culture and Balb/C inoculation. 41 [33.6%] out of 122 rodents had Leishmania infections [34 Meriones lybicus and 7 M. persicus]. All PCR products of the ITS-rDNA gene were sequenced. Sequence analysis revealed that 28 out of 41 positive samples were Leishmania major. Thirteen sequences were unreadable and therefore not identified. At least two gerbil species common in Fars ZCL foci, M. lybicus and M. persicus, are acquiring infections of L. major and may be reservoir hosts of one predominant parasite haplotype. Most infections were detected molecularly not by conventional methods, because most rodents died in the traps

[Isolation and molecular typing of wolbachia pipientis from two species of phlebotomus caucasicus and phlebotomus mongolensis sandflies]

In Iran, zoonotic cutaneous leishmaniasis [ZCL] is an endemic disease in many foci in the northeastern, southern, and central parts of the country. This disease goes through the geographical distribution along with dispersion in their reservoirs [gerbils] and their vectors [sandflies]. Therefore, controlling the vectors or reservoirs has a significant role in prevention of Leishmania parasites which is transmitted by sandflies. Nowadays, because of vectors implications, the routine methods of controlling and spraying has no more useful effects on vectors and reservoirs. Consequently, in recent years maternally inherited intracellular Rickettsia like bacteria [Wolbachia] has been fascinated by many researchers. The aim of the present study was to improve our knowledge about detection of two species of Paraphlebotomus sandflies infected with W. pipientis which yet has not been reported in Iran and the world. The new surveys have been conducted in the case of Wolbachis detection in two mentioned ZCL vectors. In Turkemen Sahara within the ZCL focus, two species of Phlebotomus cauccasicus and Phlebotomus mongolensis sandflies has been frequently collected from eighteen villages. Sticky papers and CDC traps were used to sampling sandflies in rural areas. In the laboratory, sandflies were identified to species by dissecting and mounting genitalia of each sandfly. DNA from sandflies [Thorax and abdomen] was extracted, the wsp gene confirmed for the presence of Wolbachia using wsp general primers [81F/691R]. After sequencing, the data were analyzed by molecular software. We examined a total of 136 individuals [91 male and 45 males] from Phlebotomus caucasicus and Phlebotomus mongolensis species; 10 out of 44 positive [32.35%] samples had enough DNA to sequencing. Wobachia infections have been found and verified for the first time in each of two Phlebotomus caucasicus and Phlebotomus mongolensis species in Iran and the world. In this procedure, 3 haplotypes [2 common Haplotypes and 1 unique Haplotype] of 2 species of Paraphlebotomus subgenus has been recognized in 10 sand flies of Iran. Paraphlebotomus sandflies are the secondary vectors of ZCL after Phlebotomus which play a decisive role in maintaining disease of their reservoirs. Wolbachia provide a starting point for inducing changes in host sex or sexuality. By manipulating Wolbachia as a transgene, it is hoped that these bacteria may be used as controlling system for decreasing vector-borne-disease

[Identification of Leishmania using microscopic and molecular methods in suspected patients of cutaneous leishmaniasis by targeting ITS-rDNA gene, Golestan province, Iran (2009-10)]

Zoonotic Cutaneous Leishmaniasis [ZCL] is a parasitic disease which caused by a protozoan belongs to the genus Leishmania. ZCL is of great public health importance in many countries and also in endemic parts of Iran. Leishmania major is the causative agent, Phlebotomus papatasi as the main vector and Rhombomys opimus is the most important reservoir of the disease. Species identification of Leishmania in a large scale of human samples is necessary to conduct a useful program for controlling the disease outspread. This study was done to identify the Leishmania using microscopic and molecular methods in suspected patients of Cutaneous Leishmaniasis by targeting ITS-rDNA gene, Golestan province, Iran. 121 smears collected from suspected patients of ZCL, in Eastern region of Golestan province, Iran during 2009-10, stained and examined under a light microscope. DNA of parasites extracted directly from smears and ITS-rDNA gene amplified. Positive samples digested with BsuRI restriction enzyme, according to RFLP method and subsequently the parasite was identified. After sequencing the ITS-rDNA gene, Molecular software was applied for verification of RFLP results. The achieved results were definitely approved by this procedure. 113 out of 121 and 92 out of 121 samples detected as Leishmania positive using microscopic examination and molecular method respectively. All 92 molecular positive samples digested with BsuRI endonuclease and 90 individuals identified as Leishmania major. In order to final verification, 8 samples of Leishmania major sequenced and confirmed by molecular software analysis. Unfortunately, sequences of two samples which were not Leishmania major were not readable, and consequently, these could not be identified. Comparison of obtained sequences of current study with Gene Bank sequences confirmed L.major in human from Northern Iran. Other species of Leishmania were not identified in this investigation but detection of two other samples, which were not L.major, could indicate the role of other Leishmania species causing infection in human in Eastern region of Golestan province, northern Iran. These findings should be considered to improve the disease control programs, which can be led to increase the rate of public health in Golestan province

problem of mixing up of leishmania isolates in the laboratory: suggestion of ITS1 gene sequencing for verification of species

Leishmaniasis is endemic in Iran. Different species of Leishmania [L.] parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 [ITS1] sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species. Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis confirmed the confirmation of the results of ITS1. ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences. ITS1 Sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species

[Leishmania infections in rodents, reservoir hosts of zoonotic cutaneous leismaniasis in turkemen sahara, gonbad, golastan province]

Zoonotic Cutaneous Leishmaniasis [ZCL] is a tropical diseases caused by Leishmania major, which sandflies are vectors and rodents are reservoirs host. Turkmen Sahara after Isfahan, is the most important endemic disease focus in Iran. Determination of Leishmania Infections in Rodents, Reservoir Hosts of Zoonotic Cutaneous Leismaniasis in Turkemen Sahara, Gonbad, Golastan Province. Rodents were captured by live-capture tarps. Cucumber and sometimes date were used for bating. A part of rodents ears was used to have serousite. Serosite transferred into NNN media, a part of rodents ears was used for sliding and staining for amastigote detection and other parts were used for inoculation to susceptible animal [Balb/c] in suspension form and biopsy was used. 40 rodents were captured, 27 were Rhombomis opimus, 12 were Meriones libycus and one was Meriones persicus. Of 40 rodents captured, 5 were positive in routine laboratory methods which 3 positive were M. libycus, one was R. opimns and one was M. persicus Besides these new findings in R. opimus as well as in M. libycus and M. persicus.These two rodents should be considered as reservoirs of ZCL in region

Molecular typing and phylogenetic analysis of some species belonging to phlebotomus [larroussius] and phlebotomus [adlerius] subgenera [diptera: psychodidae] from two locations in Iran

Haematophagous females of some phlebotomine sandflies are the only natural vectors of Leishmania species, the causative agents of leishmaniasis in many parts of the tropics and subtropics, including Iran. We report the presence of Phlebotomus [Larroussius] major and Phlebotomus [Adlerius] halepensis in Tonekabon [Ma-zanderan Province] and Phlebotomus [Larroussius] tobbi in Pakdasht [Tehran Province]. It is the first report of these species, known as potential vectors of zoonotic visceral leishmaniasis in Iran, are identified in these areas. In 2006-2007 individual wild-caught sandflies were characterized by both morphological features and sequence analysis of their mitochondrial genes [Cytochrome b]. The analyses were based on a fragment of 494 bp at the 3' end of the Cyt b gene [Cyt b 3' fragment] and a fragment of 382 bp CB3 at the 5' end of the Cyt b gene [Cyt b 5' fragment]. We also analysed the Cyt b Long fragment, which is located on the last 717 bp of the Cyt b gene, followed by 20 bp of intergenic spacer and the transfer RNA ser [TCN] gene. Twenty-seven P. halepensis and four P. major from Dohezar, Tonekabon, Mazanderan province and 8 P. tobbi from Packdasht, Tehran Province were identified by morphological and molecular characters. Cyt b 5 and Cyt b 3 fragment sequences were obtained from 15 and 9 flies, respectively. Cyt b long fragment sequences were obtained from 8 out of 27 P. halepensis. Parsimony analyses [using heuristic searches] of the DNA sequences of Cyt b always showed mono-phyletic clades of subgenera and each species did form a monophyletic group

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

Nuclear elongation factor-I alpha gene a molecular marker for Iranian sandfly identification

Elongation factor-1 alpha, a conserved nuclear protein coding gene was used to identify Iranian sandfly species. The phlebotomine sand flies are the vectors of the parasitic protozoan Leishmania, the causative agents of leishmaniasis, in Iran. Seven sets of primers were tried. PCR amplification of elongation factor-l alpha was successfully achieved for all 14 species of Iranian sandflies that we caught, but different primers had to be used. The aligned DNA sequences of 454 bp [without primers] of the gene had the most similarity to a coding region of the elongation factor-l alpha genes of D. melanogaster, as identified by a BLAST search of GenBank. Each Iranian species, except Phlebotomus caucasicus and P. mongolensis, had a unique combination of nucleotides, i.e. each had a diagnostic sequence. There were no diagnostic sequences for different geographical populations of the species in Iran. We found only a single copy of Ef-l alpha gene in most individual sandflies. However EF-l alpha gene was successfully amplified by PCR but, unfortunately, phylogenetic analysis showed that it might be multicopy in sandflies and so the markers could not be trusted. More highly polymorphic nuclear loci, like microsatellites, might be needed to distinguish morphologically indistinguishable females of the subgenus Paraphlebotomus, e.g. P. caucasiscus from P. mongolensis, in order to resolve their roles as vectors of Leishmania species in gerbils