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

Abstract 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

Morphological method for sexing anopheline larvae

Background & objectives: Most of autocidal control of malaria vectors relies on the rearing andrelease of large numbers of sterile male into a wild population and it would be crucial to separate themales from females before release. This could result in enormous economic benefits in the massrearing and raise the efficiency of the field operations. The development of genetic sexing ofmosquitoes, enabling the release of males only, but impairing the overall fitness of the releasedinsect has been considered greatly. Here we report on a morphological sexing method for thepreferential diagnosis and separation of males in late III and IV instar larvae for the mosquitoesAnopheles stephensi Liston and An. culicifacies s.l. (Diptera: Culicidae), the principal vectors ofhuman malaria in Asia and Indian subcontinent.Methods: Male mosquitoes are identified by their tube like organ at the 9th abdomen segment whichoriginates from segment parallel to the spiracles. Length and width of this organ is measured as66.66 ± 9.5 and 14.3 ± 1.5 μm respectively. The whole length of the organ is 201.63 ± 23.4 μm. Twofried eggs in the anterior portion of the segment are apparent in males. The length of tube in femaleis shorter than the male (almost half of the length–37.95 ± 4.0 μm), its width is slightly stout andwider than the male (16.72 ± 1.4 μm). Two fried eggs in the anterior portion of the segment areabsent. After separation of live male larvae by those characteristics, they were transferred into thetrays 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 clubshaped palpi, whereas all the female larvae developed into adult females.Interpretation & conclusion: The sex separation at the larval stage will provide a clue for embryonicorigin of sex organs, insecticide selection at the larval stage, sex related genes, male sterility andother measures

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

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

Background: A molecular survey was conducted to investigatethe 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 presenceof B. persica DNA by conventional PCR target­ing the 16SrRNA. 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 analysisand 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.

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 LC50 and LC90 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

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

Molecular Detection of Leishmania major in the Vectors and Reservoir Hosts of Cutaneous Leishmaniasis in Kalaleh District, Golestan Province, Iran

Background: 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. Methods: Totally 4900 sand flies were collected using sticky papers and were subjected to molecular methods for de­tection of leishmanial parasite. Results: 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. Conclusion: 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

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

Determination of parasite species of cutaneous leishmaniasis using Nested PCR in Damghan – Iran, during 2008

Background and Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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

Phlebotomus perfiliewi transcaucasicus is circulating both Leishmania donovani and L. infantum in northwest Iran

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general