Determination of the feeding behavior of Phlebotomus sergenti using multiplex PCR and tent-baited traps in a new focus of Anthroponotic Cutaneous Leishmaniasis in the southeast of Iran.

OBJECTIVES: This study aimed to determine feeding behaviors of Phlebotomus sergenti Parrot, in a new focus of Anthroponotic Cutaneous Leishmaniasis in Bam County, southeast Iran. METHODS: Two methods were used to determine the feeding behavior of Phlebotomus sergenti. In the first method, blood-fed sand flies were captured using a mouth aspirator in human and animal dwellings and consequently, blood meal identification was made using Multiplex PCR. The results were used for calculating Host Feeding Index (HFI) and Forage Ratio (FR) parameters. In the second method, human (Homo sapiens), goat (Capra aegagrus), cattle (Bos taurus), chicken (Gallus gallus) and dog (Canis lupus) were used as baits in tent-baited traps to determine the feeding behavior of Phlebotomus sergenti. RESULTS: Multiplex PCR analysis revealed that the most frequent blood in the stomack of sand flies' were from chicken, but the calculation of the FR revealed that this species prefers canine and poultary blood as meal. Human and animal tent-baited traps revealed that most Phlebotomus sergenti were attracted to chicken rather than the other hosts. CONCLUSIONS: Sand flies are attracted to animals for various reasons such as eating blood, mating on their bodies and laying eggs on their feces. Molecular methods are effective and accurate methods to determine the type of host that sandfly fed on, but they do not show host preferences. The results of the molecular analysis, along with the calculation of HFI and FR, can determine the preferred host of sand flies. The current study revealed that dogs, the secondary reservoir of ACL in Iran, is the first preferred host of Phlebotomus sergenti.

Correction to: An outbreak of cutaneous leishmaniasis due to Leishmania major in an endemic focus in central Iran.

[This corrects the article DOI: 10.1007/s12639-022-01474-6.].

An outbreak of cutaneous leishmaniasis due to Leishmania major in an endemic focus in central Iran.

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major is an important endemic disease and a major public health concern in Iran. Following an outbreak of leishmaniasis in 2013 in one of the important foci in Iran, the researchers were prompted to determine the underlying causes of the epidemic. Adult sand flies were collected using sticky traps and aspirating tubes and infection with Leishmania parasites was evaluated. Also, rodents were captured using Sherman live traps and stray dogs were hunted and were tested for the presence of leishmanial infection. Active case detection was also conducted and data related to each household were recorded using a researcher-designed form. Nested PCR and PCR-RLFP techniques were employed to determine Leishmania infection in the samples. Phlebotomus papatasi was the most dominant species among the 7 different species of sand flies collected in this study. Sergentomyia clydei, Sergentomyia theodori, and Sergentomyia grekovi were identified for the first time in the study area. 20% of collected Ph. papatasi species and one Meriones libycus were infected with Leishmania major. Stray dogs demonstrated no lesions on different parts of their body. The relative frequency of active lesions and scars on the dogs were respectively 5.49% and 1.23% in 2013 and 5.82% and 0.56% in 2014. The highest number of ZCL cases due to L. major was recorded in Ardestan city. Phlebotomus papatasi is the main vector and M. libycus seems to be the primary reservoir host of ZCL in the suburbs of this city. Furthermore, due to the low density of rodent reservoir hosts in the study area, we support the hypothesis that humans may have had a role in maintaining the parasite cycle in the population.

Molecular and Biochemical Detection of Insecticide Resistance in the Leishmania Vector, Phlebotomus papatasi (Diptera: Psychodidae) to Dichlorodiphenyltrichloroethane and Pyrethroids, in Central Iran.

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.

An Eco-Epidemiological Study on Zoonotic Cutaneous Leishmaniasis in Central Iran.

BACKGROUND: Leishmaniasis is an expanding neglected tropical disease in the world reporting from 98 countries including Iran. This study focused on eco-epidemiological determinants of the disease following a rapid and unexpected increase of leishmaniasis incidence in a strategic residential district in North-East of Isfahan County, Iran. METHODS: This study was accomplished from Apr 2012 to Jan 2014 in a strategic residential zone in North-East of Isfahan County, Esfahan, Iran. Monthly activity, parity, Leishmania infection and susceptibility tests, were determined on sand flies. Some portion of inhabitants and school children were surveyed to find active or passive cases of leishmaniasis and also wild rodents were collected to determine reservoir host. RESULTS: Totally 5223 sand flies belonging to Phlebotomus and Sergentomyia genus were collected and identified; Ph. papa-tasi was the dominant species and started to appear in May and disappeared in Oct. The majority of living dissected sand flies were unfed and parous. Ph. papatasi showed 4.6% Leishmania infection through direct examination and 39.54% by nested-PCR respectively. Phlebotomus papatasi was susceptible against deltametrin 0.05%. Totally 2149 people were surveyed and incidence and prevalence of zoonotic cutaneous leishmaniasis estimated as 45.39 and 314.40 per 1000 population. Rodents showed 73.91% and 80% Leishmania infection by direct examination and nested-PCR respectively. CONCLUSION: Cutaneous leishmaniasis due to L. major has been established in this area. Rodent control operation and personal protection are highly recommended to control the disease in this focus.

Evaluation of Different Attractive Traps for Capturing Sand Flies (Diptera: Psychodidae) in an Endemic Area of Leishmaniasis, Southeast of Iran.

BACKGROUND: The attraction of phlebotomine sand flies to plant and animal hosts is due to the produced chemical compounds, affecting the olfactory receptors of the insects. Therefore, novel and effective methods, such as Attractive Toxic Sugar Baits (ATSB) and Attractive Toxic Baits (ATB), are based on the effective materials that attract sand flies toward the host. The present study was designed to identify the attractive materials in plants and animals for using in ATSB and ATB. METHODS: This cross-sectional study was carried out in July 2018 on endemic areas of leishmaniasis in Iran. Different baits, including mango, nectarine, grape, banana, melon and watermelon, defibrinated blood of cattle, sheep, goat and chicken, urine of cattle, sheep, goat and ultimately, simple and complex chemicals, such as CO2, 1-octanol, lactic acid and human sweat were placed inside the traps, and the rate of the sand flies attraction to these materials was studied. Furthermore, data were analyzed using the Kruskal-Wallis test and Mann Whitney U test. RESULTS: There was a significant difference in the sand flies attraction between the traps containing watermelon, urine of cattle, and sheep, and chemicals such as CO2 and human sweat and the control trap (p< 0.05). CONCLUSION: This study showed that watermelon and CO2 are the potential candidates for using in ATSB and ATB, respectively.

Monitoring of Laboratory Reared of Phlebotomus papatasi (Diptera: Psychodidae), Main Vector of Zoonotic Cutaneous Leishmaniasis to Different Imagicides in Hyper endemic Areas, Esfahan Province, Iran.

BACKGROUND: In domestic and per domestic area, insecticides such as DDT, malathion, fenitrothion, propoxur and, more recently, synthetic pyrethroids such as deltamethrin and lambda-cyhalothrin, have been successfully used to control sand flies in many countries. The present study reports the results of time-mortality bioassay to DDT 4%, lambda-cyhalothrin 0.05%, permethrin 0.75%, cyfluthrin 0.15% and deltamethrin 0.05% in recently colonized Phlebotomus papatasi populations in Iran. METHODS: The insecticide susceptibility status of P. papatasi laboratory population was assessed during 2016-2017, following the standard WHO technique for mosquito (WHO, 2013) based on diagnostic dose. Sand flies collected from rural area of Badrood (Matin Abad), Natanz County, Esfahan Province, using aspirator. RESULTS: Susceptibility test to DDT and pyrethroids was assessed on 3534 laboratory-reared P. papatasi (1746 females and 1788 males). The LT 50 and LT 90 values were measured using probit analysis and regression lines. The test results against males of P. papatasi revealed that LT 50 values to DDT 4%, Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 439.28, 108.90, 97.75, 5.00 and 57.84 seconds. The figures for females were 641.62, 136.15, 146.44, 8.71 and 72.69 seconds, respectively. CONCLUSION: According to presented results, the reared population of sand flies collected from a hyper-endemic region of Esfahan Province is still susceptible to prethroids and Resistance candidate to DDT 4%.

Control of American Cockroach (Periplaneta americana) in Municipal Sewage Disposal System, Central Iran.

BACKGROUND: Cockroaches consists of 4000 species, of which 40 species are serious domestic pests. They have involved the mechanical transmission of various pathogenic viruses, bacteria, and protozoans to humans. This study aimed to determine different control methods of P. americana in sewage system of Esfahan City, central Iran using different insecticides recommended by WHO. METHODS: Totally, 164 manholes with at least more than three cockroaches were selected until 2017 for this study. The species of cockroaches were identified; the adults and nymphs were counted and recorded. Each sewer shaft was assigned to one treatment method in an ascending order of number of cockroaches counted, coding the treatment method with paint on the lower part of the wall near the manhole cover. The manhole shafts were then inspected at 1 and 5 months' post-treatment. Data analyzed by statistical methods. RESULTS: Almost all of the products (Excluding boric acid with bait formulation) resulted in appropriate control within one month of application. The appropriate products for chemical control of cockroaches were the chlorpyrifos 5% Emulsifiable Concentrate (EC), diazinon 5% (EC), diazinon 0.05% (EC) and cypermethrin 5% Fog. These pesticides achieved an optimal reduction of population providing more than 90% control of cockroaches for five consecutive months. CONCLUSION: The emulsifiable concentrates and fog formulations in the control were more successful compared to other methods and this was penetration deep into the hiding places of cockroaches.

Control of zoonotic cutaneous leishmaniasis vector, Phlebotomus papatasi, using attractive toxic sugar baits (ATSB).

INTRODUCTION: Attractive Toxic Sugar Baits (ATSB) is a new vector control method that meets Integrated Vector Management (IVM) goals. In an experimental design, this study aimed to determine effects of ATSB on control of Phlebotomus papatasi, as a main vector of Zoonotic Cutaneous Leishmaniasis (ZCL), in Qom Province, center of Iran. METHODS: In a cross-sectional design, boric acid was mixed with brown sugar solution and tested as toxic baits for P. papatasi. Two methods were utilized to use the baits: (a) spraying ATSB on vegetation, bushes, and shrubs; and (b) setting ATSB-treated barrier fences in front of colonies at 500 m distance from the houses in outskirts of villages. In order to examine the residual efficacy rate of ATSB-treated barrier fences, the bioassay test was used. Density of P. papatasi sandflies was measured using sticky and light traps biweekly. For data analysis, Mann-Whitney U Test and Kruskal-Wallis were used. Results ATSB-treated barrier fences led to 3 times reduction in P. papatasi population. Besides that, ATSB spraying on plants led to more than 5 times reduction in P. papatasi population. CONCLUSIONS: Comparing the incidence of leishmaniasis in treated villages before and after the study showed that the incidence was statistically reduced. Therefore, ATSB is an effective method to control vectors and prevent leishmaniasis.

Spatial Distribution of Phlebotomine Sand Fly Species (Diptera: Psychodidae) in Qom Province, Central Iran.

Zoonotic cutaneous leishmaniasis (ZCL) is transmitted to humans by phlebotomine sand fly bites. ZCL is a major health problem in Iran, where basic knowledge gaps about sand fly species diversity persist in some ZCL-endemic areas. This paper describes the richness and spatial distribution of sand fly species, collected with sticky traps, in Qom province, a ZCL-endemic area in central Iran, where sand fly fauna has been poorly studied. Collected species were mapped on urban and rural digital maps based on a scale of 1/50,000. All analyses were undertaken with rural- and urban-level precision, i.e., rural and urban levels were our basic units of analysis. After identifying the sand flies, high-risk foci were determined. For spatial analysis of vector species population, the entomological sampling sites were geo-referenced using GPS. Arc GIS 9.3 software was used to determine the foci with leishmaniasis vector species. Following the analyses, two genera (Phlebotomus and Sergentomyia) and 14 species were identified. Based on the mapping and sand fly dispersion analysis, the rural districts were categorized into three groups-infection reported, without infection, and no report. Based on Geographical Information System analyses, Kahak and Markazi districts were identified as high-risk foci with leishmaniasis vector species. These findings can act as a help guide to direct active control measures to the identified high-risk foci and, eventually, lead to reduction in incidence of the disease.

Epidemiological Study on Cutaneous Leishmaniasis in an Endemic Area, of Qom Province, Central Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) is one of the most important health problems in many areas of Iran. There are two forms of the disease in Iran, anthroponotic and zoonotic CL. This study conducted to assess the epidemiological situation of CL in an endemic area of Qom Province, central Iran from Apr to Nov 2015. METHODS: The sticky paper traps and aspirating tubes were used for collecting adult sand flies. Sherman traps and small insect nets were used to capture rodents and small mammals. Giemsa staining was used for preparing the expanded smear and followed by PCR for identifying the causative agent in human, vectors, and reservoirs. In this study, relative frequency of CL was also calculated. RESULTS: Fourteen species of Phlebotomine sand flies were collected. Phlebotomus papatasi (61.74%) was the predominant species through the period of activity. Overall, 62 Meriones libycus, 8 Nesokia indica, 4 Mus musculus, 16 Allactaga elater and 2 Hemiechinus auritis were caught. PCR technique showed 6 out of 150 P. papatasi (2%), two out of 62 M. libycus (3.23%) and all of suspected human's skin tissue samples (100%) were infected with Leishmania major. The relative frequency of CL was 0.30%. CONCLUSION: This is the first detection of L. major within P. papatasi, M. libycus and human in Kahak District in Qom Province of Iran. Zoonotic cycle of CL exists in this area, L. major is the causative agent, P. papatasi is the main vector and M. libycus is the main reservoir of the disease.

Bioassay evaluation of residual activity of attractive toxic sugar-treated barrier fence in the control of Phlebotomus papatasi (Diptera: Psychodidae).

BACKGROUND & OBJECTIVES: Phlebotomus papatasi is the main vector of the zoonotic cutaneous leishmaniasis (ZCL) in Qom Province and many other provinces of Iran. Attractive toxic sugar baits (ATSB) treated barrier fence is one of the new methods for controlling the vectors such as sandflies. The present study was designed to evaluate the residual activity of ATSB-treated barrier fence that was used in control of P. papatasi. METHODS: Following the selection of villages in Markazi district of Qom Province, central Iran during 2015 for ATSB and ASB (bait containing no active ingredient) methods; barrier fences on the ground in front of the rodent's colony were installed. A total of four conical tubes were installed and fixed on surfaces of treated barrier net of dimension 25 Χ 25 cm at biweekly interval. In each conical tube, 10 sand flies were released and after 3 min of exposure they were transferred to sterile cups. After 24 h, the obtained results were recorded according to the survival and mortality rate of sandflies. These tests were carried out five days after the installation of barrier fences, and repeated every 15 days until the mortality rate decreased to 60-65%. RESULTS: The bioassay tests results showed that the mortality rate of P. papatasi on ATSB-treated barrier fence for 5, 15, 30 and 45 days after spraying was 100, 95.83, 88.18 and 66.67% respectively, which decreased to 50.83% after 60 days. INTERPRETATION & CONCLUSION: Persistence and residual activity of the active ingredient of the bait in the hot and dry climatic conditions of Qom Province remained significantly effective for at most 45 days, which subsequently decreased at a high rate. Hence, every 45 days barrier fences need to be impregnated with ATSB bait. The method also appeared cost-effective and could be practical in implementation of vector control programmes against ZCL.

Sand Flies of the Subgenus Adlerius (Diptera: Psychodidae) in an Endemic Focus of Visceral Leishmaniasis and Introduction of Phlebotomus (Adlerius) comatus as a New Record for Iran.

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.

Deltamethrin-impregnated bed nets and curtains in an anthroponotic cutaneous leishmaniasis control program in northeastern Iran.

BACKGROUND: Anthroponotic cutaneous leishmaniasis (ACL) has long been a significant public health problem in northeastern Iran. The objective of this study was to determine the efficacy of deltamethrin-impregnated vs. nonimpregnated bed nets (NIBs) and curtains (NICs) in ACL control. PATIENTS: Deltamethrin-impregnated bed nets (IBs) and curtains (ICs) with 25 mg ai/m2 were distributed among 160 households in one district and NIBs and NICs were distributed among the same number of households in another district. A third district with a similar numbers of households served as a control. Health education mes- sages were disseminated to ensure the populationâs complicance with the proper use of bed nets and curtains. Sticky paper traps were used to assess the effect of insecticide-impregnated bed nets and curtains on the density of Phlebotomus sergenti. Deltamethrin susceptibility and also bioassay tests were carried out on the species by WHO standard method. Case findings were done by house-to-house visits once a season and all the inhabitants of the selected households in each district were examined. RESULTS: IBs and ICs provided good protection against sandfly bites and reduced the transmission of ACL in the intervention district, while NIBs and NICs provided no protection. There was no significant difference in monthly density of P. sergenti indoors and outdoors among the districts (P>0.05). This species was susceptible to delta- methrin in the field population in the area. Bioassays confirmed that the nets treated with deltamethrin remained effective for more than 3 months. CONCLUSION: Personal protection is an effective and sustainable means of preventing and controlling ACL and can reduce dependence on insecticides. We encourage the use of IBs and ICs to control ACL in other high-risk areas of Iran and Afghanistan during the active season of sandflies.

Rodent control operations against zoonotic cutaneous leishmaniasis in rural Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is a serious and increasing public health problem in many rural areas of Iran. Previous studies showed that rodent control operations are effective in destroying rodents and reducing the incidence of ZCL on a small scale and in special circumstances. The objective of this study was to determine suitable timing for rodent control operations in reducing the incidence of ZCL in an endemic area in Badrood, Iran. METHODS: We conducted rodent control operations by baiting rodent holes using zinc phosphide once a month in May, June, July and September in 1997 within a 500-meter circle of houses in the intervention area. From 1999 to 2002, the numbers of active rodent holes were counted in May and October in the intervention and control areas. When the numbers of rodent holes increased to 30% or more of the number before the first baiting in May 1997, holes in the intervention area were baited again with zinc phosphide in the intervention area; no baiting was done in the control village. Case findings were done by house-to-house visits once every season during 2000 to 2002. RESULTS: Changes in the number of rodent holes over time in the intervention and control villages were statistically significant (P<0.000001). There were also significant differences in the incidence of ZCL between the intervention and control villages (P<0.005) during 2000 to 2002. The incidence of ZCLwas unchanged in the years 2000 and 2001, but increased in one village in 2002 after an increase in the number of rodent holes. CONCLUSION: We suggest that rodent control operations using zinc phosphide be done within a 500-meter circle of houses once every two years before the beginning of the active season of sandflies, which begins in late April.