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The aim of the present study was to explore resistance markers and possible biochemical resistance mechanisms in the Phlebotomine sand fly Phlebotomus papatasi in Esfahan Province, central Iran. Homogenous resistant strains of sand flies were obtained by exposing P. papatasi collected from Esfahan to a single diagnostic dose of DDT. The adults from the colony were tested with papers impregnated with four pyrethroid insecticides: Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambdacyhalothrin 0.05% to determine levels of cross-resistance. To discover the presence of mutations, a 440 base pair fragment of the voltage gated sodium channel (VGSC) gene was amplified and sequenced in both directions for the susceptible and resistant colonies. We also assayed the amount of four enzymes that play a key role in insecticide detoxification in the resistant colonies. A resistance ratio (RR) of 2.52 folds was achieved during the selection of resistant strains. Sequence analysis revealed no knockdown resistance (kdr) mutations in the VGSC gene. Enzyme activity ratio of the resistant candidate and susceptible colonies were calculated for α-esterases (3.78), ß-esterases (3.72), mixed function oxidases (MFO) (3.21), and glutathione-S-transferases (GST) (1.59). No cross-resistance to the four pyrethroids insecticides was observed in the DDT resistant colony. The absence of kdr mutations in the VGSC gene suggests that alterations in esterase and MFO enzymes are responsible for the resistant of P. papatasi to DDT in central Iran. This information could have significant predictive utility in managing insecticide resistant in this Leishmania vector.

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RESUMEN

BACKGROUND: Esfahan Province is considered as one of the main focus of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Recently, ZCL distribution is expanding through this province leading to report of new cases in non-endemic areas. In the current study epidemiological aspects of ZCL has been investigated in Naein County in Esfahan Province. METHODS: Adult sand flies were collected from beginning to the end of their seasonal activity. Rodents were caught by Sherman live traps once a month for one year. To active case detection, a hundred households in each selected village were visited in November and December 2016. Nested-PCR was employed to detect Leishmania parasite in the vector, reservoir and human. RESULTS: Totally 1562 sand flies including Phlebotomus sergenti, Phlebotomus papatasi, Sergentomyia sintoni and Sergentomyia mervinae were collected and identified. No Leishmania infection was detected in the collected sand flies. All of the 30 collected rodents were identified as Rhombomys opimus, and of these 3.3% and 26.7% were infected by Leishmania major using microscopic and molecular technique respectively. Totally, 914 individuals were investigated and the ulcer and scar rates of ZCL calculated to be at 1.1 and 15.3 per 1000 population, respectively. Molecular results confirmed L. major infection in human and reservoir samples. CONCLUSION: It is concluded that ZCL is established in the area in low endemicity, and it is extrapolated the disease will not be a serious increasing health problem in the near future in this region.

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BACKGROUND: Sand flies of subgenus Adlerius has a wide geographical distribution in Iran and are mostly found in wild form in mountainous areas. They are always considered as probable vectors of visceral leishmaniasis. The objective of this study was to determine the Adlerius species and its composition in an endemic focus of zoonotic visceral leishmaniasis in northwest of the country. METHODS: Sand flies were collected from 6 different areas of Azarbaijan-e-Sharqi Province using sticky paper traps from August to September which is active season for sand flies in this area, in 2009. The flies were mounted and identified. The length of third antennal segments, ascoid, labrum, coxite, surstyle, style, aedeagus, genital filament, genital pump, width of style, and the end of aedeagus were measured and the number of costal hairs group was also counted as the morphological characters. RESULTS: A total of 30 adult sand flies, (26 males and 4 females) including Phlebotomus halepensis (46.8%), P. longiductus (13.3%), P. balcanicus (23.3%), P. comatus (3.3%), and Adlerius spp. (13.3%) belong to subgenus Adlerius were identified respectively in 6 counties. One P. comatus male was captured in front of a cave located in the hillside of a mountain covered with the vegetation in Varzeqan area. CONCLUSION: The presence of at least 5 species of the subgenus Adlerius in Azarbaijan-e-Sharqi Province, an endemic focus of zoonotic visceral leishmaniasis in Iran, shows that the risk of parasite transmission among man and reservoir animals is high during the active season of sand flies. P. comatus is a new record for Iran and needs to be added to the list of Iranian phlebotomines of subgenus Adlerius.

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Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations.

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