Rodents as vehicle for delivery of transgenic bacteria to make paratransgenic sand fly vectors of cutaneous leishmaniasis in field condition.

Vector-borne diseases, among them leishmaniasis, cause more than 700,000 deaths annually. The lack of an effective vaccination and the increasing resistance of sand flies to insecticides require the urgent development of innovative approaches to contain the disease. The use of engineered bacteria that express anti-parasite molecules (paratransgenesis) shows much promise. However, a challenge for implementation of this strategy is to devise means to introduce modified bacteria into sand flies in the field. In this study, we use rodent food bait as a delivery strategy to introduce two mCherry-fluorescent bacteria, Serratia AS1 and Enterobacter cloacae, into adult sand flies in field settings. Bacteria-infected food was provided to Rhombomys opimus rodents. These bacteria transiently pass through the rodent alimentary tract and are delivered to larval habitats with the rodent feces. The feces are ingested by sand fly larvae and, in the case of Serratia AS1, are trans-stadially transmitted to adults. This is the first report of targeting delivery of Serratia AS1 in a paratransgenic system to control transmission of leishmaniasis under field condition. This novel strategy shows promise for delivering transgenic bacteria to Leishmania vectors in the field.

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 Susceptibility Status of Phlebotomus papatasi, the Main Vector of Zoonotic Cutaneous Leishmaniasis, to Different WHO Recommended Insecticides in an Endemic Focus, Central Iran.

Background: Among neglected zoonotic diseases, leishmaniases caused by Leishmania parasite through infected female sand fly bite, are a group of diseases found in 98 countries and territories representing a critical burden of disease worldwide. Vector management plays a crucial role in reducing the burden of vector-borne diseases by WHO's global plan. The objective of the current study was to assess the susceptibility status of wild phlebotomine sand flies from Esfahan Province, central Iran, to the recommended insecticides by WHO. Methods: Sand flies were collected by mouth aspirator in Matin Abad desert Eco-resort and were tested using WHO adult mosquito test kit against Dichlorodiphenyltrichloroethane (DDT) 4%, Deltamethrin 0.05%, Malathion 5% and Propoxur 0.1%. The number of knockdown sand flies were recorded during exposure time in ten minutes interval for DDT and Deltamethrin and they were allowed to recover for 24 hours. Knockdown Time50 (KD50) and KD90 were generated for them using Probit software. They were mounted and identified by valid keys. Results: Among the tested insecticides against female Phlebotomus papatasi, DDT, Deltamethrin, and Malathion recorded the highest mortality rate of 100%, followed by Propoxur with 92.2% mortality for a one-hour exposure. For DDT, KD50 and KD90 were calculated 21.87 and 42.93 and for Deltamethrin, they were 23.74 and 56.50 minutes respectively. Total sand flies exposed with DDT and Deltamethrin shed their leg(s). Conclusion: It is concluded that Ph. papatasi from central Iran is susceptible to DDT, Deltamethrin, Malathion, and Propoxur.

Emerging of Cutaneous Leishmaniais Due to Leishmania major in a New Focus in Esfahan Province, Central Iran.

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.

Differential expression profiles of the salivary proteins SP15 and SP44 from Phlebotomus papatasi.

BACKGROUND: Sand fly saliva has been shown to help parasite establishment and to induce immune responses in vertebrate hosts. In the current study, we investigated the pattern of expression of two Phlebotomus papatasi salivary transcripts in specific physiological and seasonal conditions at a hyperendemic area of zoonotic cutaneous leishmaniasis (ZCL) in Iran. METHODS: Sand flies were collected during 2012-2013, and grouped according to physiological stages such as unfed, fed, semi-gravid, gravid, parous, nulliparous, infected or non-infected with Leishmania major and also based on the season in which they were collected. Quantitative Real-Time PCR was applied for assessment of the expression of two relevant salivary transcripts, PpSP15 and PpSP44, associated to protection from and exacerbation of ZCL, respectively. RESULTS: The expression of PpSP15 and PpSP44 transcripts was significantly up-regulated (1.74 and 1.4 folds, respectively) in blood fed compared to unfed flies. Among four groups of fed, unfed, semi-gravid and gravid flies, the lowest levels of PpSP15 and PpSP44 expression were observed in gravid flies. Additionally, the expression levels of both PpSP15 and PpSP44 transcripts in P. papatasi collected during summer were significantly up-regulated (3.7 and 4.4 folds, respectively) compared to spring collections. In addition, the PpSP15 transcript exhibited a significant up-regulation (P < 0.05) in non-infected flies compared to those infected with L. major. CONCLUSIONS: This study contributes to our knowledge of the differential expression of salivary genes among different groups within a P. papatasi population under natural field conditions. Cutaneous and visceral leishmaniasis are of public health importance in many parts of Iran and neighbouring countries where P. papatasi is the proven and dominant sand fly vector for ZCL, the most prevalent and endemic form of the disease in Iran. Therefore, the current study could be helpful in understanding the influence of salivary genes on Leishmania transmission by phlebotomine sand flies. Our findings demonstrate the differential expression of salivary transcripts under various physiological conditions potentially influencing the sand fly capacity for parasite transmission as well as the outcome of disease.

Seasonal and Physiological Variations of Phlebotomus papatasi Salivary Gland Antigens in Central Iran.

BACKGROUND: Sand fly saliva helps parasite establishment and induce immune responses in vertebrate hosts. In the current study, we investigated the modulation of Phlebotomus papatasi salivary gland antigen expression by seasonal and biological factors. METHODS: Sand flies were grouped according to physiological stages such as unfed, fed, semi-gravid, gravid, parous, nulliparous, infected or non-infected with Leishmania major and based on the season in which they were collected. Salivary gland antigens (SGAs) were analyzed using SDS-PAGE and the antibody response against SGAs in Rhombomys opimus was determined by ELISA and Western blot. RESULTS: The highest protein content was found in the salivary glands of unfed sand flies. The saliva content was higher in parous compared to nulliparous, in summer compared to spring, and in Leishmania-infected compared to non-infected flies. The salivary gland lysate (SGL) electrophoretic pattern variations were observed among sand flies with various physiological stages particularly from 4-9 protein bands of 14-70 kDa. The SGL of unfed and gravid flies had extra protein bands compared to fed and semi-gravid sand flies. There was missing protein bands in SGL of parous compared to nulliparous; and in summer compared to spring collected flies. Rhombomys opimus serum reacted strongly with an antigenic band of around 28 kDa in the SGL of all sand fly groups. CONCLUSION: Certain biological and environmental characteristics of wild populations of vector sand flies affect the protein content and antigenicity of saliva. This might have an important implication in the design of vector-based vaccines.

Rodenticide Comparative Effect of Klerat® and Zinc Phosphide for Controlling Zoonotic Cutaneous Leishmaniasis in Central Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected disease with public health importance that is common in many rural areas of Iran. In recent years, behavioral resistance and/or bait shyness against the common rodenticide among reservoir hosts of ZCL have been reported. The aim of this study was to evaluate the effectiveness of Klerat® and zinc phosphide against natural reservoir of ZCL. METHODS: This survey was carried out in four villages located 45 to 95 km far from Esfahan City Esfahan province, central Iran from April to November 2011. The rodent burrows were counted destroyed and reopened holes baited around all villages. Effect of rodent control operation on the main vector density and incidence of ZCL were evaluated. RESULTS: The reduction rate of rodent burrows after intervention calculated to be at 62.8% in Klerat® and 58.15% in zinc phosphide treated areas. Statistical analysis showed no difference between the densities of the vector in indoors and outdoors in intervention and control areas. The incidence of the disease between treated and control areas after intervention was statistically different (P< 0.05). CONCLUSION: Klerat® could be a suitable alternative for zinc phosphide in a specific condition such as behavior resistance or occurrence of bait shyness.

Epidemiological study on sand flies in an endemic focus of cutaneous leishmaniasis, bushehr city, southwestern iran.

Cutaneous leishmaniasis is the most important health problem in the city of Bushehr, southwestern Iran. The objective of the study was to determine some ecological aspects of sand flies in the city during 2010-2011. Sand flies were collected monthly from outdoors and indoors by sticky traps at four selected districts of the city. They were also dissected and examined by nested-PCR for identification of the parasite during August-September of 2011. A total of 1234 adult sand flies were collected and 6 species including 3 of Genus Phlebotomus and 3 of Genus Sergentomyia were identified. Four species including P. papatasi (3.98%), P. sergenti (1.14%), S. tiberiadis (87.18%), and S. baghdadis (7.7%) were found indoors. Six species including P. papatasi (3.47%), P. sergenti (3.17%), P. alexandri (0.1%), S. tiberiadis (77.74%), S. baghdadis (15.41%), and one female of S. clydei (0.11%) were collected from outdoors. Sand flies started to appear from March and disappear at the end of January. There was only one peak in the density curve in July. The study revealed that S. tiberiadis and S. baghdadis could enter indoors which 89 and 81.8% of them were found blood-fed, respectively. Moreover, P. papatasi, S. tiberiadis, and S. baghdadis were active indoors and outdoors in most months of the year. Nested-PCR of P. papatasi females was positive against kinetoplast DNA of L. major and L. turanica and also mixed natural infections were found by L. gerbilli and L. turanica. Moreover, mixed infections by L. major and L. turanica were observed in this species. Sergentomyia clydei and S. tiberiadis were found to be negative to any DNA of Leishmania species. Phlebotomus sergenti females were found infected with DNA of L. turanica and this is the first report of natural infection and detection of the parasite from this sand fly species in worldwide.

Aerobic bacterial flora of biotic and abiotic compartments of a hyperendemic Zoonotic Cutaneous Leishmaniasis (ZCL) focus.

BACKGROUND: Identification of the microflora of the sand fly gut and the environmental distribution of these bacteria are important components for paratransgenic control of Leishmania transmission by sand flies. METHODS: Biotic and abiotic bacterial communities of four compartments of a hyper-endemic focus of Zoonotic Cutaneous Leishmaniasis (ZCL) were investigated using 16S ribosomal DNA sequencing and phylogenetic tree construction. These compartments include Phlebotomus papatasi's gut, skin and intestinal tract of great gerbil Rhombomys opimus, the gerbil nest supplies, and plant food sources of the vectors and reservoirs. RESULTS: Sequence homology analysis using nine available 16S rDNA data bases revealed 40, 24, 15 and 14 aerobic bacterial species from the vector guts, the gerbil bodies, the gerbil nests, and the plants, respectively. The isolated bacteria belong to wide ranges including aerobic to facultative anaerobic, pathogen to commensals, sand fly oviposition inducers, land to air and ocean habitats, animal and human probiotics, and plant growth-promoting rhizobacteria. Matching data analysis suggested that the adult P. papatasi gut bacteria could be acquired from three routes, adult sugar feeding on the plant saps, adult blood feeding on the animal host, and larval feeding from nest supplies. However, our laboratory experiment showed that none of the bacteria of the reservoir skin was transmitted to female sand fly guts via blood feeding. The microflora of sand fly guts were associated with the sand fly environment in which the predominant bacteria were Microbacterium, Pseudomonas, and Staphylococcus in human dwellings, cattle farms, and rodent colonies, respectively. Staphylococcus aureus was the most common bacterium in sand fly guts. Presence of some sand fly ovipoisition inducers such Bacillus spp. and Staphylococcus saprophyticus support association between gut flora and oviposition induction. CONCLUSIONS: Results of this study showed that Bacillus subtilis and Enterobacter cloacae particularly subsp. dissolvens are circulated among the sand fly guts, the plants, and the sand fly larval breeding places and hence are possible candidates for a paratransgenic approach to reduce Leishmania transmission.

Biodiversity of Aquatic Insects of Zayandeh Roud River and Its Branches, Isfahan Province, Iran.

BACKGROUND: Aquatic insects are the major groups of arthropods that spend some parts of their life cycle in the water. These insects play an important role for transmission of some human and animal diseases. There is few information about the aquatic insects fauna of Iran. METHODS: To study the aquatic insects fauna, adult, nymphal and larval collections were carried out from different habitats using the standard technique in Zayandeh Roud River, Isfahan Province,central Iran, during summer 2011. RESULTS: In total, 741 speimens of aquatic insects were collected and morphologically identified. They include 7 families and 12 genera representing 2 Orders. The order of Diptera (92.31%) and Coleoptera (7.69%). The families Culicidae, Syrphidae and Chironomidae from Diptera order, Gyrinidae, Dytiscidae, Haliplidae, Hydrophilidae from Coleoptera order were identified. CONCLUSION: Some aquatic insects play an important role for transmission of human and animal diseases. These insects also are important for biological control. Therefore ecological study on aquatic insects can provide information about ecology of insects in an area for any decision making.

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.

Molecular epidemiological study of cutaneous leishmaniasis in the focus of bushehr city, southwestern iran.

BACKGROUND: Cutaneous leishmaniasis (CL) represents the most frequent vector borne parasitoses in Iran. The objective of this study was to determine the epidemiological features of CL including human infection and the reservoir host in the city of Bushehr, Bushehr Province, Iran during 2010-2011. METHODS: Studies on human infection was carried out on 2962 school children aged 7-14 years old from 60 primary schools and among 400 households with a total population of 1568 in four infected districts of the city in December 2010. Serosity materials from patients on glass slides were collected for molecular identification of causative agent. Rodents were caught by Sherman traps and examined for identification of the parasite. RESULTS: Prevalence of scars and ulcers among the inhabitants were 5.86% and 0.12% respectively. Molecular study indicated the presence of two coexisting species: Leishmania major and L. tropica among patients. The scar rate was 1.24% but no ulcers were seen among the students. Nineteen rodents were caught and identified as Tatera indica (47.4%) and Rattus norvegicus (52.6%). Specimens from 7 T. indica and 9 R. norvegicus were examined by two techniques, microscopic examination and nested-PCR. Out of 7 T. indica, 14.3% were infected with L. major and 42.9% with L. turanica by nested-PCR. Out of 9 R. norvegicus 22.2% were infected with L. turanica and 11.1% with L. gerbilli. CONCLUSION: Based on this survey L. major and L. tropica are the causative agents of the disease among patients and T. indica plays a predominant role in the dissemination of L. major in the city.

Laboratory Evaluation of a Rodenticide-insecticide, Coumavec®, against Rhombomys opimus, the Main Reservoir Host of Zoonotic Cutaneouse Leishmaniasis in Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis is a growing health problem in many rural areas of Iran. Rhombomys opimus, the great gerbil, is the main animal reservoir of ZCL in the northeast and central part of Iran. The aim of the current study was to evaluate the rodenticidal effect of Coumavec® (a mixture of Coumatetralyl 0.5% and Etofenprox 0.5%) on R. opimus under laboratory condition. METHODS: Great gerbils were collected from Sejzi rural district, Esfahan Province, Iran. Four groups of 19 great gerbils were treated with the poisoned baits of different concentrations and one group was considered as control. The bating procedure was conducted in three stages: first, second (a week after first) and third (a month after first stage), in each stage baits were offered in 1 day, based on national protocol for rodent control operation in purpose of ZCL control. RESULTS: The mortality rate for 0.03, 0.0625, 0.125 and 0.25% concentrations in the first stage of baiting were obtained 36.8%, 31.5%, 52.6% and 36.8%, in the second stage 47.3%, 52.6%, 68.4% and 52.6%, and in the third stage 52.6%, 63.1%, 68.4% and 57.8% respectively. The maximum and minimum mortality has occurred in 5-6 days and 31-40 days intervals consequently. CONCLUSION: The results of this study showed that, Coumavec® has some rodenticidal effects on R. opimus in laboratory condition. For the appropriate rodenticide-insecticide contamination of the rodent body and also considering to the economic issues, we suggest the use of 0.125% concentration for rodent control operation in the field condition.

Leishmania species: detection and identification by nested PCR assay from skin samples of rodent reservoirs.

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.