Resistance status of main malaria vector, Anopheles stephensi Liston (Diptera: Culicidae) to insecticides in a malaria Endemic Area, Southern Iran

Objective: To evaluate the susceptibility of Anopheles stephensi (An. stephensi) Liston, the main malaria vector in southern Iran, to WHO recommended insecticides. Methods: Larvae of An. stephensi were collected from three different larval habitats in both urban and rural area of Bandar Abbas city and one rural area in Rudan county southern Iran. WHO standard method was used for evaluation of adult and larval mosquito susceptibility. Bendiocarb, permethrin, lambda-cyhalothrin, deltamethrin as insecticide and temephos and chlorpyriphos as larvicide were used at the diagnostic dosages recommended by WHO. Results: Findings of this study showed all larval populations of An. stephensi were completely susceptible to temephos and candidate for resistance to chlorpyriphos. Adult mosquitoes in rural areas of Bandar Abbas city were resistant to pyrethroid and carbamate insecticides. Conclusion: Comparison of the results of this survey with previous studies indicates that the resistance to pyrethroids and carbamates in this malaria endemic region is increasing. Wide use of pesticides in agriculture is certainly effective in increasing resistance. The inter-sectoral coordination and collaboration in health and agriculture seem to be necessary to manage insecticide resistance in malaria vectors.

Molecular characterization, biological forms and sporozoite rate of Anopheles stephensi in southern Iran

<p><b>OBJECTIVE</b>To identify the biological forms, sporozoite rate and molecular characterization of the Anopheles stephensi (An. stephensi) in Hormozgan and Sistan-Baluchistan provinces, the most important malarious areas in Iran.</p><p><b>METHODS</b>Wild live An. stephensi samples were collected from different malarious areas in southern Iran. The biological forms were identified based on number of egg-ridges. Molecular characterization of biological forms was verified by analysis of the mitochondrial cytochrome oxidase subunit I and II (mtDNA-COI/COII). The Plasmodium infection was examined in the wild female specimens by species-specific nested-PCR method.</p><p><b>RESULTS</b>Results showed that all three biological forms including mysorensis, intermediate and type are present in the study areas. Molecular investigations revealed no genetic variation between mtDNA COI/COII sequences of the biological forms and no Plasmodium parasites was detected in the collected mosquito samples.</p><p><b>CONCLUSIONS</b>Presence of three biological forms with identical sequences showed that the known biological forms belong to a single taxon and the various vectorial capacities reported for these forms are more likely corresponded to other epidemiological factors than to the morphotype of the populations. Lack of malaria parasite infection in An. stephensi, the most important vector of malaria, may be partly due to the success and achievement of ongoing active malaria control program in the region.</p>

Determination of leishmania parasite species of cutaneous leishmaniasis using PCR method in central county, Qom province

Leishmaniasis is one of the most important health problems in many areas of Iran. This study aimed to identify leishmania species in patients with cutaneous leishmaniasis using PCR teqnique based on sequencing ITS1 primers in villages of the central county of Qom province. This descriptive cross-sectional study was done on 169 patients who with leishmaniosis, based on clinical and laboratory confirmation treated and were followed in health center of Qomrood in 2010. This data was recorded in epidemiologic data forms. The DNA was extracted by KIAGEN kits. The extracted DNA was exploited to identify the parasite by PCR technique. The data were analyzed by SPSS-17 software. Chi square and Fisher's exact test were used for the evaluation of the hypothesis. The PCR result confirmed the parasite positive slides and obtained bands from these slides were seen in the range of 350 bp which are expected band for the leishmania major parasite. The most frequent age group was above 15 years old [71.5%]. Hands and feet were the most common sites of ulcer [78.8%]. 19.5 of patients had 3 ulcers and more. Highest disease prevalence was observed in months October, December and November. The PCR results illustrated that the prevalent CL in the central county of Qom province is Zoonotic type [ZCL], and the agent of disease is leishmania major parasite. In conclusion, identification of the disease and parasite type can help the health officials to make appropriate strategies for its prevention and control

[Determination of reservoirs of cutaneous leishmaniasis by PCR molecular method in the villages of central county of Qom province]

Cutaneous leishmaniasis is a major health problem in many parts of Iran and its main foci are in various parts of the country. This study was carried out with the intention of examining the species composition of the possible disease reservoirs in the cutaneous leishmaniasis focus in the selected villages of central county of Qom province. Study was done as a descriptive, cross-sectional study on rodents [possible leishmaniasis reservoirs] in the selected rural areas of Qomrood and Ghanavat located in central parts of Qom province in 2010. In this research, a total of 46 small rodents were hunted by live traps from 5 selected villages. A Smear prepared from auricle or suspicious lesions on skin, and after Giemsa staining, the smears were examined for the presence of leishman's bodies. After extracting DNA from positive smears, PCR technique was exploited to determine the parasite species. A total 46 small mammals were hunted and identified by the authentic keys. They included 31 rodents [67.4%] were Meriones libycus, 8 [17.4%] Allactaga elater, 4 [8.7%] Mus musculus, 2 [4.3%] Nesokia indica and one [2.7%] Hemiechinus auritus. The results of PCR demonstrated that 3.7% of M. libycus rodents were infected by Leishmania major. The results of this study showed that epidemic cutaneous leishmaniasis in the central county of Qom province was zoonotic and its reservoir was species of rodents. Therefore, determining disease type and its reservoir can help health care authorities to adopt appropriate strategies for prevention and control of the disease in this area

Confirmation of two sibling species among anopheles fluviatilis mosquitoes in south and southeastern Iran by analysis of cytochrome oxidase I gene

Anopheles fluviatilis, one of the major malaria vectors in Iran, is assumed to be a complex of sibling species. The aim of this study was to evaluate Cytochrome oxidase I [COI] gene alongside 28S-D3 as a diagnostic tool for identification of An. Fluviatilis sibling species in Iran. DNA sample belonging to 24 An. Fluviatilis mosquitoes from different geographical areas in south and southeastern Iran were used for amplification of COI gene followed by sequencing. The 474-475 bp COI sequences obtained in this study were aligned with 59 similar sequences of An. Fluviatilis and a sequence of Anopheles minimus, as out group, from GenBank database. The distances between group and individual sequences were calculated and phylogenetic tree for obtained sequences was generated by using Kimura two parameter [K2P] model of neighbor-joining method. Phylogenetic analysis using COI gene grouped members of Fars Province [central Iran] in two distinct clades separate from other Iranian members representing Hormozgan, Kerman, and Sistan va Baluchestan Provinces. The mean distance between Iranian and Indian individuals was 1.66%, whereas the value between Fars Province individuals and the group comprising individuals from other areas of Iran was 2.06%. Presence of 2.06% mean distance between individuals from Fars Province and those from other areas of Iran is indicative of at least two sibling species in An. Fluviatilis mosquitoes of Iran. This finding confirms earlier results based on RAPD-PCR and 28S-D3 analysis

Infantile nosocomial Myiasis in Iran

Myiasis, the invasion of live human tissue by larva of Diptera, is reported in the nasal cavity of a 5.5-year-old Iranian girl. She was referred from Golestan Province to the Shaheed Rajaei Heart Center in Tehran. In the 41[th] day after admission, a live parasite was found in her nasal secretions suction identified presumably as a second instar larvae of a facultative myiasis, Woholfartia nuba [Diptera: Sarcophagidae], on the basis of mtDNA-COI and morphological characteristics. Since presence of the larva was recorded after hospitalization, by definition, this infestation is considered a nosocomial myiasis

Ecology of anopheles stephensi in a malarious area, southeast of Iran

District of Jiroft is situated in south-east of Iran which is one of the malarious regions. Anopheles stephensi is considered as one of the main malaria vector in this region. Ecology of this species was studied in the area to understand its vector behavior for implementation of effective vector control measures. Different methods like total catch, pit shelter, night bite collection on human and animal, larval dipping methods were used for species identification, seasonal activity, anthropophilic index and egg morphological characteristics. Anthropophilicity index was assessed by ELISA test. Activity of Anopheles species started at the beginning of April, and its peak occurs in late spring. The larvae were found in the river bed with pools, stagnant streams, slow foothill streams, temporary pools, and slowly moving water with and without vegetation, drainage containers of air conditioner and palm irrigation canals. From different methods of adult collection, it was found that spray sheet collection is the appropriate method. ELISA testing of 144 blood meals of females revealed the anthropophilicity of 11.8% indicating host preference on animal, mainly cow. Ridge length and their number on the egg floats confirmed Anopheles stephensi mysorensis form. This study showed that Anopheles stephensi is the main vector of malaria in the region, although some other species may play a role. Our findings could provide a valuable clue for epidemiology and control of malaria in the southeast of Iran

Phenology and population dynamics of sand flies in a new focus of visceral leishmaniasis in Eastern Azarbaijan Province, North western of Iran

OBJECTIVE@#To investigate species composition, density, accumulated degree-day and diversity of sand flies during April to October 2010 in Azarshahr district, a new focus of visceral leishmaniasis in north western Iran.@*METHODS@#Sand flies were collected using sticky traps biweekly and were stored in 96% ethanol. All specimens were mounted in Puri's medium for species identification using valid keys of sandflies. The density was calculated by the formula: number of specimens/m(2) of sticky traps and number of specimens/number of traps. Degree-day was calculated as follows: (Maximum temperature + Minimum temperature)/2-Minimum threshold. Diversity indices of the collected sand flies within different villages were estimated by the Shannon-weaver formula ( H'=∑i=1sPilog(e)Pi).@*RESULTS@#Totally 5 557 specimens comprising 16 Species (14 Phlebotomus, and 2 Sergentomyia) were indentified. The activity of the species extended from April to October. Common sand-flies in resting places were Phlebotomus papatasi, Phlebotomus sergenti and Phlebotomus mongolensis. The monthly average density was 37.6, 41.1, 40.23, 30.38 and 30.67 for Almalodash, Jaragil, Segaiesh, Amirdizaj and Germezgol villages, respectively. Accumulated degree-day from early January to late May was approximately 289 degree days. The minimum threshold temperature for calculating of accumulated degree-day was 17.32°. According on the Shannon-weaver (H'), diversity of sand flies within area study were estimated as 0.917, 1.867, 1.339, 1.673, and 1.562 in Almalodash, Jaragil, Segaiesh, Amirdizaj and Germezgol villages, respectively.@*CONCLUSIONS@#This study is the first detailed research in terms of species composition, density, accumulated degree-day and diversity of sand flies in an endemic focus of visceral leishamaniasis in Azarshahr district. The population dynamics of sand flies in Azarshahr district were greatly affected by climatic factors. According to this study the highest activity of the collected sand fly species occurs at the teritary week of August. It could help health authorities to predicate period of maximum risk of visceral leishamaniasis transmission and implement control program.

[Ecological characters of leishmaniasis vectors in Kalaleh district, Golestan province, Iran, [2006-07]]

Leishmaniasis is a Zoonotic disease that is transmited by sandfly to human. This study were carried out in order to demonstrate some ecological characters of leishmaniasis vectors in Kalaleh district, Golestan province Iran, during 2006-07. In present study 6 villages were selected. Sandfly were collected by sticky traps. 3 places were sampled in each village and in each places 20 traps were installed. After sampling collection, we used diagnostic criteria to identify the Sandflies, also confirmed human cases were recorded according to the months of identification. 4900 sandflies were detected in 6 villages and 12 species of sandflies were identified, which including P.papatasi, P.mongolensis, P.caucasieus, P.caucasicus group, P.sergenti, P.alexandri, P.kazeroni, P.brevis, P.[adlerhis] sp, S.sintoni, S.clydei, S.sogdiana. P.papatasi was predominant species in indoor places [46.1%] and S.sintoni was in outdoor places [36.7%]. Sandflies activities extended from early May through mid October with two peaks in mid June and September. Human infection had a important peak in January. During the collection of sandflies, the species of P.alexandri, P.kazeroni, P.brevis, P.[Adlerius sp.] S.clydei and S.sogdiana were collected for the first time from this area. In this study, P.papatasi was the predominant species in this area. Sandflies second activity peak occured in September that is crucial for transmission of disease. The incubation period for this disease was four months

Discrimination of Borrelia persica and B.microiti the causative agents of relapsing fever using PCR-RFLP and specific primers

Utility of PCR-RFLP and species-specific PCR as novel and fast methods for identification and discrimination of causative agents of relapsing fever, Borrelia persica and B. microtii in infected blood were investigated. Genomic DNA of B.persica and B.microtii species were extracted from the highly infected blood samples. Two fragments of GlpQ and 16SrDNA genes were amplified using specific primers and then the PCR products were sequenced. Based on sequence variation between the two species, species-specific primers as well as restriction enzymes were respectively designed and selected for discrimination of these species. The results showed that using PCR technique we could easily amplify and detect the Borrelia species within the infected blood samples. Two different profiles of RFLP were produced when GlpQ PCR products of B.persica and B.microtii treated by Sspl, Taql, Dral, Hinfl, and EcoRV restriction enzymes. Also when 16SrDNA was digested with Taql enzyme we could discriminate between these two species. Based on GlpQ sequence variation, a set of primer 795r-BMGLPF produced specific band of 451 bp for B.microtii and a set of primer 128f-BPGLPR produced specific band of 252 bp for B.persica which could discriminate the both species well. In this study the discrimination of the two species of B.persica and B.microtii was investigated by PCR-RFLP and species-specific PCR methods for the first time. Both methods could easily distinguish the species from each other. Due to accuracy and speed of the molecular methods, they could be replaced with the classic methods. These fast and accurate diagnostic methods could be recommended for diagnosis laboratories in Iran and middle-east countries where both B.persica and B.microtii are prevalent

Evaluation of PCR and Xenodiagnosis assays for detection of no Borrelia persica in soft ticks Ornithodoros tholozani

Relapsing fever caused by Borrelia persica is an acute tick-borne disease which is transmitted by soft ticks of Ornithodoros tholozani to human. The disease is reported from Middle East and many regions of Iran. Detection of infection is problematic since the suspected infected ticks should be fed on animal hosts such as guinea pigs and subsequently after 7-14 days, the animal blood should be microscopically investigated for Borellia spirochetes on a Giemsa stainined thick smear. This classic method named xenodiagnosis is hard, time consuming, and less reliable. In this study, the application of PCR technique has been examined for detection of Borellia persica in soft ticks of O. tholozani. Tick specimens were collected from northwestern Iran and were fed on Borellia persica infected guinea pigs. DNA of the animal blood were extracted and used as target for PCR amplification of 16rDNA gene. Subsequently the products were subjected to sequencing. The effect of tick sex and post digestion as well as the minimum nyjcrilper of spirochetes on the efficiency of PCR were also tested. The xenodiagnosis assay was able to detect infection in only 13.3% of the tick-bitten animal bloods whereas all of these blood specimens were PCR positive against the 16rDNA gene. There was no difference in results of PCR for male and female of the ticks. Post digestion of infected blood meal in ticks did not affect the efficacy of PCR and the recently-fed samples showed similar results to those of completely gravid ones. A test on the threshold sensitivity of PCR assay indicated that only one spirochete is enough for the primers to anneal and to amplify the target gene. This study describes the first molecular assay for diagnosis of B. persica infected ticks in Iran and due to its high speed, accuracy, and applicability is a substitution method for diagnostic purposes inTBRF foci

Detection and identification of Leishmania parasites sandflies using kDNA, rDNa and CPB loci

Molecular epidemiology of Visceral Leishmaniasis [VL] is currently used widely for different objectives such as vector incrimination studies. In this study three different loci including kinetoplast DNA [kDNA], ribosomal DNA [rDNA], cystein protease B [CPB[ of Leishmania parasite genome were used for detection and identification of natural infection of sand flies of Germi district of Ardebil province, the most important VL or Kala-azar foci in Iran. The results showed that the three loci of kDNA, rDNA and CPBs are respectively more appropriate for leptomonad infection/initial screening, identification of the L.donovani complex, and discrimination of the species complex. It was also verified that both members of the complex, L.donovani and L.infantum, are present in the study area and are transmitted to the hosts by Phlebotomus perfiliewi transcaucasicus sandflies. This is the first report on natural infection of sand flies to L.donovani in the country and since the ecology and biology of L.donovani differs extensively from L.infantum, it is necessary to perform further studies to highlight the role of L.donovani in epidemiology of VL in the region and country

Natural infection of sand files Sergentomyia dentata in Ardebil to Lizard Leishmania

An entomological survey was carried out for Leishmania vector incrimination of sand flies in northwestern Iran. Among other specimens, 358 sand flies belong to the Sergentomyia Genus were tested for leptomonad infection using semi-nested PCR method as well as sequence analysis of ITS-rDNA fragment. Results of semi-nested PCR against kietoplast DNA showed reptile leptomonad infection in two specimens of S.dentata. The ITS2 sequence analysis of the specimens revealed 76% identity with those of Leishmania [sauroleishmania] adleri of Genbank. However, further studies need to clarify the species identity of the leptomonads. Interestingly, blood meal analysis of the sand flies determined an S.sintoni specimen with mammalian hemoglobin. This reptile related sauroleishmania parasites lacks the Lipophosphoglican [LQG] necessary for entrance to human phagocytes cells, and hence are not human pathogen. However, the GlycoInositoPhosphLipid[GIPL] molecules of this parasite reacts with sera of kala-azar patients and may cause false positive scores in sero-epidemiological surveys for kala-azar. Sauroleishmania can be transmitted to human infected bite of some Sergentomyia subgenera that show intermediate characteristics of Phlebtomus Genus. They are able to feed on human blood. This is the first report on presence of L. [sauroleishmania] adleri as well as ingestion of mammalian hemoglobin Sergentomyia sand flies in Iran