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

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

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

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

Detection of Borrelia persica Infection in Ornithodoros tholozani Using PCR Targeting rrs Gene and Xenodiagnosis.

BACKGROUND: Relapsing fever caused by Borrelia persica, is an acute tick-borne disease which is transmitted by soft ticks of Ornithodoros tholozani to human. METHODS: 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. RESULTS: 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, semi- or completely gravid or completely digested blood ones. CONCLUSION: 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.

Molecular Detection of Leishmania infantum in Naturally Infected Phlebotomus perfiliewi transcaucasicus in Bilesavar District, Northwestern Iran.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis.

Larvicidal Activity of Essential Oils of Apiaceae Plants against Malaria Vector, Anopheles stephensi.

BACKGROUND: Plant extracts and oils may act as alternatives to conventional pesticides for malaria vector control. The aim of this study was to evaluate the larvicidal activity of essential oils of three plants of Apiaceae family against Anopheles stephensi, the main malaria vector in Iran. METHODS: Essential oils from Heracleum persicum, Foeniculum vulgare and Coriandrum sativum seeds were hydro distillated, then their larvicidal activity were evaluated against laboratory-reared larvae of An. stephensi according to standard method of WHO. After susceptibility test, results were analysis using Probit program. RESULTS: Essential oils were separated from H. persicum, F. vulgare and C. sativum plants and their larvicidal activities were tested. Result of this study showed that F. vulgare oil was the most effective against An. stephensi with LC(50) and LC(90) values of 20.10 and 44.51 ppm, respectively. CONCLUSION: All three plants essential oil can serve as a natural larvicide against An. stephensi. F. vulgare oil exhibited more larvicidal properties.

Study on Presence of Borrelia persica in Soft Ticks in Western Iran.

BACKGROUND: 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. METHODS: 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 16S rRNA. RESULTS: A total of 1021 soft ticks of three species of Ornithodouros tholozani (120: 11.75%), O. lahorensis (461: 45.15%) and Argas persicus (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. TaqI 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. CONCLUSION: 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.

Blood Meal Identification in Field-Captured Sand flies: Comparison of PCR-RFLP and ELISA Assays.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%). CONCLUSION: 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.

Comparative performance of imagicides on Anopheles stephensi, main malaria vector in a malarious area, southern Iran.

BACKGROUND & OBJECTIVES: Jiroft district has subtropical climate and prone to seasonal malaria transmission with annual parasite index (API) 4.2 per 1000 in 2006. Anopheles stephensi Liston is a dominant malaria vector. The monitoring of insecticide susceptibility and irritability was conducted using discriminative dose as described by WHO. METHODS: The IV instar larvae were collected from different larval breeding places and transported to the temporary insectary, fed with Bemax and then 2-3 days-old emerged and sugar-fed adults were used for susceptibility and irritability tests employing WHO methods and kits to organochlorine (OC) and pyrethroid (PY) insecticides. RESULTS: Mortality rates of field strain of An. stephensi were 91.3 +/- 0.14 and 90 +/- 0.47% to DDT and dieldrin, respectively at one hour exposure time but was susceptible to all pyrethroids tested. The average number of take-offs per min per adult was 2.09 +/- 0.13 for DDT, 0.581 +/- 0.05 for dieldrin, 1.85 +/- 0.08 for permethrin, 1.87 +/- 0.21 for lambda-cyhalothrin, 1.53 +/- 0.13 for cyfluthrin, and 1.23 +/- 0.1 for deltamethrin. INTERPRETATION & CONCLUSION: Currently, deltamethrin is being used for indoor residual spraying against malaria vectors in the endemic areas of Iran. The findings revealed that the main malaria species is susceptible to all pyrethroids including deltamethrin, permethrin, cyfluthrin and lambda-cyhalothrin but was tolerant to DDT and dieldrin. This report and the finding are coincided with results of previous studies carried out during 1957-61 in the same area. Irritability tests to OC and PY insecticides revealed the moderate level of irritability to DDT compared to pyrethroids and dieldrin. Monitoring for possible cross-resistance between OC and PY insecticides should come into consideration for malaria control programme.

Morphological method for sexing anopheline larvae.

BACKGROUND & OBJECTIVES: Most of autocidal control of malaria vectors relies on the rearing and release of large numbers of sterile male into a wild population and it would be crucial to separate the males from females before release. This could result in enormous economic benefits in the mass rearing and raise the efficiency of the field operations. The development of genetic sexing of mosquitoes, enabling the release of males only, but impairing the overall fitness of the released insect has been considered greatly. Here we report on a morphological sexing method for the preferential diagnosis and separation of males in late III and IV instar larvae for the mosquitoes Anopheles stephensi Liston and An. culicifacies s.l. (Diptera: Culicidae), the principal vectors of human malaria in Asia and Indian subcontinent. METHODS: Male mosquitoes are identified by their tube like organ at the 9th abdomen segment which originates from segment parallel to the spiracles. Length and width of this organ is measured as 66.66 +/- 9.5 and 14.3 +/- 1.5 microm respectively. The whole length of the organ is 201.63 +/- 23.4 microm. Two fried eggs in the anterior portion of the segment are apparent in males. The length of tube in female is shorter than the male (almost half of the length--37.95 +/- 4.0 microm), its width is slightly stout and wider than the male (16.72 +/- 1.4 microm). Two fried eggs in the anterior portion of the segment are absent. After separation of live male larvae by those characteristics, they were transferred into the trays and emerged adults were identified to ascertain correct identification of sex. RESULTS: All the larvae with male organs developed into male adults with hairy antennae and club shaped palpi, whereas all the female larvae developed into adult females. INTERPRETATION & CONCLUSION: The sex separation at the larval stage will provide a clue for embryonic origin of sex organs, insecticide selection at the larval stage, sex related genes, male sterility and other measures.

Genetic structure of the malaria vector Anopheles superpictus in Iran using mitochondrial cytochrome oxidase (COI and COII) and morphologic markers: a new species complex?

Anopheles superpictus has been implicated as the most widespread malaria vector in Iran. We collected adult specimens from eight provinces across the country and subjected them to a morphological investigation as well as molecular analysis of mitochondrial DNA COI-COII region, using PCR-RFLP and analysis of DNA sequence alignment for 708bp of the COI locus. Two distinct morphological forms (A and B) of the species were found sympatric in all areas of study. PCR-RFLP using AluI separated the specimens into at least three genotypes (X, Y, and Z), and alignment of DNA sequences revealed a 12.3% variation in the COI region between the genotypes. However, the sequence variation does not correspond to the morphological forms. Our observations suggest that A. superpictus in Iran is likely a group species. However, further ecological, molecular, cytological, and epidemiological studies are necessary to clear the status of the taxon and the potential role of each putative species in the transmission of malaria.

Implication of ESR signals from ceruloplasmin (Cu(2+)) and transferrin (Fe(3+)) in pleural effusion of lung diseases.

Pleural effusions of seven lung cancer patients (mean age; 58) and seven non-cancer patients (mean age; 49) were examined and Cu(2+) was measured in ceruloplasmin and Fe(3+) in transferrin signals by electron spin resonance (ESR) method. The variations of total Fe and Cu ions, ceruloplasmin and transferrin, proteins, neutrophil cell counts, LDH and nitrite/nitrate were also examined. The Cu(2+) peak was decreased and the Fe(3+) peak was increased in the cancer group. The interrelationship among Cu(2+), total Cu and ceruloplasmin, and among Fe(3+), total Fe and transferrin clarified that Cu(2+) and Fe(3+) are not a representative of ceruloplasmin and transferrin, respectively. The ratio of Cu(2+)/Fe(3+) in pleural effusion distinguished lung cancer from benign inflammation as a cause. The ratio of total Cu/total Fe measured by the chemical analysis method also distinguished these, but the ratio of ceruloplasmin/transferrin was unable to distinguish the cancer. In conclusion, the simple and rapid measurement of Cu(2+)/Fe(3+) by ESR effectively abstracts the variation of total ion concentrations caused by malignant disease.