Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Evaluation of the Entomopathogenic Fungus Beauveria bassiana on Different Stages of Phlebotomus papatasi (Diptera: Psychodidae), Vector of Zoonotic Cutaneous Leishmaniasis in Iran.

Background: Zoonotic cutaneous leishmaniasis is a major public health problem in Iran with the main vector of Phlebotomus papatasi. The use of entomopathogenic fungi for biological control of the vector is a potential substitute for the current methods which are being used. The purpose of the current study was to assess the virulence of two local isolates of Beauveria bassiana (OZ2 and TV) against Ph. papatasi. Methods: To perform the bioassay test, fungal suspensions were applied for every stage of the sand fly life cycle. The mortality rate, longevity, and number of eggs laid were determined. Also, the probability of fungal survival on the surface of rodent's body was assessed. Results: The longevity of infected adult sand flies with both isolates of B. bassiana was significantly lower (P< 0.05) in comparison to the negative control. The estimated Lethal concentration 50 (LC50) values for adult female and male sand flies treated with OZ2 isolate were 1.4×106 and 2.2×107 conidia/ml, respectively, while they were 6.8×106 and 2.3×108 conidia/ml for TV isolate, respectively. Both isolates of B. bassiana exhibited nonsignificant mortality rates in sand fly larvae and pupae and fecundity rate (P> 0.05). According to our findings for both isolates, the fungus continued to spread throughout the surface of the rodent's body for 144 hours after spraying. Conclusion: The current study demonstrated that both isolates of B. bassiana have considerable biological control capacity against adult sand flies.

A Comparative Study on the Biodiversity and Species Richness of Phlebotomine Sand Flies (Diptera: Psychodidae) in Kermanshah and Khuzestan Provinces of Iran.

Background: Leishmaniasis is one of the most important vector-borne and neglected tropical diseases in many parts of the world. The main objective of this study was to determine the biodiversity of phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) in Khuzestan and Kermanshah Provinces of Iran. Methods: Sampling was conducted in Khuzestan and Kermanshah Provinces using sticky paper traps and CDC light traps. The samples were then stored in 96% alcohol-containing vials, mounted, and identified. Afterward, the alpha diversity (using Simpson's, Shannon-Weiner's diversity, evenness, Maghalef's, Menhinick's, Hill N 1 and N 2 indices) and beta diversity indices (using Sorensen's and Jaccard's coefficients) were calculated. Results: A total of 4302 sand flies were caught and identified which comprised mostly of Phlebotomus papatasi, Ph. alexandri and Ph. sergenti. It was found that among the four counties of Ahvaz, Shush, Shushtar and Dezful in Khuzestan Province, Shush had the lowest, and Shushtar, the highest amount of species diversity and evenness. In the four studied counties of Kermanshah Province, species diversity in Kermanshah County had the lowest amount and Sarpol-e-Zahab, the highest. The species richness in Kermanshah County was the lowest, while Qasr-e-Shirin County had the highest amount. Conclusion: An overview of the biodiversity of phlebotomine sand flies in Kermanshah County in Kermanshah Province, and Shush County in Khuzestan Province, showed less stability in community structure of these vectors, which can be counted as an alarm for emerging dominant vectors among the studied counties and have the potential to increase the prevalence of leishmaniasis.

Using Ecological Niche Modeling to Predict the Spatial Distribution of Anopheles maculipennis s.l. and Culex theileri (Diptera: Culicidae) in Central Iran.

BACKGROUND: Mosquitoes are very important vectors of diseases to human. We aimed to establish the first spatial database on the mosquitoes of Isfahan Province, central Iran, and to predict the geographical distribution of species with medical importance. METHODS: Mosquito larvae were collected from eight counties of Isfahan Province during 2014. Collected data were transferred to a database in ArcGIS and the distribution maps were created. MaxEnt model and jackknife analysis were used to predict the geographical distribution of two medical important species, and to find the effective variables for each species. RESULTS: Totally, 1143 larvae were collected including 6 species, Anopheles maculipennis s.l., An. superpictus s.l., An. marteri, Culex hortensis, Cx. theileri and Culiseta longiareolata. The area under curve in MaxEnt model was 0.951 and 0.873 rather 1 for An. maculipennis s.l. and Cx. theileri, respectively. Culex theileri had wider and more appropriate niches across the province, except for the eastern area. The environmental variable with highest gain was mean temperature of the wettest quarter for Cx. theileri and temperature seasonality for An. maculipennis. Culex theileri, An. maculipennis s.l. and An. superpictus, three important vectors of parasitic agents to humans, were collected in this study. CONCLUSION: The mosquito collected and mapped can be considered for transmission of malaria and filariasis in the region. Bearing in mind the results of niche modeling for vector species, more studies on vectorial capacity and resistance status to different insecticides of these species are recommended.

The Potential Role of Humans in the Transmission Cycle of Leishmania major (Kinetoplastida: Trypanosomatidae), the Causative Agent of the Old World Zoonotic Cutaneous Leishmaniasis.

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major (Yakimoff and Schokhor, 1914) is known as a neglected tropical disease, transmitted by Phlebotomus papatasi (Scopoli, 1786) in North Africa and the Middle East. The main reservoirs of ZCL are desert gerbils and the role of humans as a reservoir host of the disease is not clearly defined and is therefore investigated in this study. In order to achieve this objective, Ph. papatasi sand flies were allowed to take blood through indirect blood sources (human, Rhombomys opimus (Lichtenstein, 1823) (great gerbil), and BALB/c mice) using artificial feeding or direct xenodiagnoses from ZCL patients. To detect Leishmania promastigotes inside digestive canal, blood-fed sand flies were dissected and examined under light microscope and then confirmed by nested-PCR. In indirect xenodiagnoses, promastigote forms of Leishmania parasite were observed in 7.1%, 12%, and 13.6% of sand flies which fed on Rh. opimus, BALB/c mice, or human blood sources, respectively. In direct xenodiagnoses, among 93 female lab-reared sand flies which fed on active L. major lesion(s), 2.1% Leishmania infection was subsequently observed in the sand flies. No infection was detected in those sand flies that fed on nonlesioned skin of CL patients. Humans can serve as a reservoir of ZCL since the data indicate that Ph. papatasi is able to acquire L. major parasites from an active lesion of ZCL patients and the parasites can complete metacyclogenesis in the sand fly.

Designing and Introducing a New Artificial Feeding Apparatus for Sand Fly Rearing.

BACKGROUND: Due to strict ethical rules, the risk of accidental disease transmission and the most importantly, inconvenience regarding using of live animals, artificial feeding apparatus has been developed for colonization of haematophagous insects. Rearing of sandfly is more difficult than other haematophagous insects. METHODS: In the current study, a new apparatus for membrane feeding of Phlebotomus papatasi was designed, made and compared with available apparatus in Sand Fly Insectary, Tehran University of Medical Sciences, Tehran, Iran, in 2014. RESULTS: In comparison to other apparatus designed for artificial feeding of other arthropods, our designed apparatus had the highest performance which after up to 1h, the majority of sand flies landed and took blood and among tested membranes, chicken skin was proved the most efficient membrane. CONCLUSION: Sand fly artificial feeding apparatus can be used at least for rearing of Ph. papatasi.

Assessing the Ovarian Accessory Glands to Determine the Parity of Phlebotomus papatasi, Vector of Zoonotic Cutaneous Leishmaniasis, under Laboratory Condition.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease prevailed in many rural areas of 17 out of 31 provinces in Iran. The main vector of the disease is Phlebotomus papatasi and the causative agent is Leishmania major in ZCL foci of Iran. In the current study we investigated the validity of accessory glands secretions as an indicator to recognize parous from nulliparous Ph. papatasi females under laboratory conditions. METHODS: Over 235 laboratory-reared females of Ph. papatasi were dissected in 6 groups including: newly emerged, one hour, one day, two days, and three days after blood feeding and also after oviposition under stereo microscope for their parity in 2014-2015. RESULTS: Transparent glands were compatible with nulliparous only in newly emerged sand flies. In sand flies dissected after oviposition, accessory glands were rather large as a result of oviposition though they were transparent. CONCLUSION: The accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of Ph. papatasi females.

Rearing and Biology of Phlebotomus sergenti, the Main Vector of Anthroponotic Cutaneous Leishmaniasis in Iran.

BACKGROUND: Establishment of sand flies laboratory colonies is essential to understand various biological aspects of Phlebotominae sand flies. The aims of the current study were to establish the colony of Phlebotomus sergenti Parrot (1917), the main vector of anthroponotic cutaneous leishmaniasis in old world, and to study biological parameters of this species. METHODS: The sand flies were reared at 26-28 °C temperature, 14:10 (light: dark) photoperiod and 70-80% relative humidity. Larval diet was a composted mixture of rabbit faces and rabbit pellets which is prepared through a special process. First to fifth generations of P. sergenti were used to define biological parameters. RESULTS: Results showed that, P. sergenti blood feeding percentage were 42% on chicken, 21% on BALB/c and 37% on golden hamster. Average time of blood digestion, egg incubation, 1st instar larva, pupa and adult emerging was recorded at 3.4, 8.7, 15, 33.3 and 41.2 days after blood feeding, respectively. Mean number of laid eggs was 55.1 and retained eggs were 35 per a female. Fecundity and production rate were 61.6%, and 42.2% respectively. Average longevity recorded at 15.2 days for females and 14.8 days for males. CONCLUSION: Colony of P. sergenti has been established for the first time in Iran. Average interval time from egg to adult of this species was 32.5 days. Chicken and golden hamster were recommended as a blood source for colony initiation and routine blood feeding, respectively.