Phlebotomus Papatasi Research Articles

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.

Effects of multiple feedings on sensitized rabbits on the fitness of Phlebotomus papatasi (Diptera: Psychodidae)

In the present study, we evaluated the effects of antibodies anti-sandfly saliva on the fecundity of Phlebotomus papatasi, vector of zoonotic cutaneous leishmaniasis in the Old World. Rabbits were repeatedly exposed to sandfly bites. Immune sera showed increased levels of anti-sandfly saliva antibody compared to the pre-exposition period. The analysis of biological parameters revealed no decline on the feeding success of females P. papatasi fed on rabbits repeatedly exposed to sandfly bites. Our results showed that anti-sandfly saliva antibodies of rabbits are not detrimental to the fitness of females P. papatasi. Thus, rabbits did not acquire resistance to sandflies following repeated exposures, and that contribute in maintaining a high density of P. papatasi. To control sandfly infestations and Leishmania transmission, more studies are needed for a better understanding of the mechanisms governing the resistance of hosts to bites of sandflies.

Phlebotomus papatasi

This datasheet on Phlebotomus papatasi covers Identity, Distribution, Hosts/Species Affected.

Antibody Responses to Phlebotomus papatasi Saliva in American Soldiers With Cutaneous Leishmaniasis Versus Controls

Leishmania major, transmitted in Iraq by the bite of a sand fly Phlebotomus papatasi, causes cutaneous leishmaniasis (CL). The sand fly saliva is immunogenic, with both systemic humoral and cellular human immune responses resulting from natural exposure. 248 Americans who developed L. major infection in Iraq were sex, race/ethnicity, year of Iraq deployment-matched to controls without CL. Using a case-control study design, we compared sand fly saliva-specific human IgG levels and recognized antigens between the two groups. Serologic responses to Ph. papatasi salivary gland homogenate were studied with ELISA and Western blot, using serial samples obtained from before travel, during CL treatment (CL) or at time of return to US (controls), as well as (for CL cases) six to 24 months after return to non-endemic US. The mean change in optical density (MCOD), reflecting the change in sand fly saliva-specific IgG before and after exposure in Iraq, was 0.296 (range -0.138 to 2.057) in cases and 0.151 (range -0.454 to1.085) in controls, p<0.001. Low levels of sand fly saliva specific antibody were noted in CL cases by 7-8 months after return to the US. The most frequently recognized Ph. papatasi salivary antigens were MW30 (PpSP32) and MW64, although other salivary proteins recognized were MW12/14, 15, 18, 28, 32, 36, 42, 44, 46, 52. Logistic regression suggested that MW15, 28 and 42 were associated with the largest effect on the MCOD. MW30 was the most frequently recognized antigen suggesting a role as biomarker for sand fly exposure and CL risk. Anti-Ph. papatasi saliva IgG waned within months of return to the US. We also discuss vector antigenic saliva proteins in the context of CL presentation and identify some salivary antigens that may correlate with less lesion area, ulcer versus papule/plaque, race among those with CL.

Establishment and mass breeding of Phlebotomus papatasi (Diptera:Pshychodidae) in Orzouieh county in Kerman Province, Southeast of Iran

Background & objectives: Sand flies are important carriers of Leishmania parasites. Leishmaniosis, spread by sand fly vectors, is a major public health problem worldwide Particular in the tropics and the subtropics. The parasite transmission capacity of vectors, life cycle of parasites in the body of vectors, disease transmission, and the physiology and behavior of carrier vectors can be extensively studied by establishing insect colonies in the insectarium. In addition, response of vectors to repellents and insecticides, mating behaviors, blood feeding habits, interaction between parasites, and vectors, and taxonomic studies can be investigated by establishing insect colonies. The goal of the present study was to establish a colony of Phlebotomus papatasi in the insectarium in a ZCL endemic area in the Kerman Province of Iran. Methods: The Killick -Kendrick and Killick -Kendrick method (1991) was used for individual rearing and the volf and volfa (2011) method was used for mass rearing of the collected sand flies. The larvaewere fed by a diet with liver powder, which is the recommended diet for Phlebotomus papatasi larvae. Adult sand flies were allowed to feed on Balb/c mice blood in the laboratory. Results: 80 sand flies specimens (75 female+5 male) were collected with aspirator and reared for f1 and f2 generations in the laboratory. Interpretation & conclusion: The Phlebotomus sand flies were colonized in laboratory condition. Then sand flies successfully maintained for the first time as a laboratory species in Kerman Province.

Effect of Beauveria bassiana and Metarhizium anisopliae on different stages of Phlebotomus papatasi (Diptera: Psychodidae)

The sand fly, Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae), is the main vector of Leishmania major Yakimoff and Schokhor, 1914, the causative agent of zoonotic cutaneous leishmaniasis North Africa, the Middle East, and North Sinai. The purpose of this study was to determine the effect of fungi on P. papatasi larvae, pupae, and adults using light microscopic analysis, scanning electron microscope (SEM), and histopathological studies. Specifically, larvae, pupae, and adult P. papatasi were infected with the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. Scanning electron microscope and histopathological methods were used to investigate the destructive impact of the fungi on the external and internal structures of P. papatasi. The results revealed propagation of the conidia on the cuticles of all P. papatasi life stages, including on the compound eyes, leg setae, thorax, wings, and abdomen of the adults. Histological sections of the control and treated larvae, pupae, and adults showed many alterations and malformations in the body and tissues of all life stages after 72 h. These results demonstrated that B. bassiana was more effective than M. anisopliae as a biological control of phlebotomine sand flies. Further studies to determine the best methods for delivery and application in the diverse ecological settings of the various leishmaniasis vectors are recommended.

Detection of Leishmania donovani in Wild-Caught Phlebotomine Sand Flies in Endemic Focus of Leishmaniasis in Himachal Pradesh, India.

An endemic focus of cutaneous leishmaniasis (CL) is located in the Sutlej River Valley in Himachal Pradesh (India) in the north-western Himalaya where CL co-exists with visceral leishmaniasis (VL). In areas of the Indian subcontinent such as Rajasthan, cutaneous leishmaniasis is transmitted by Phlebotomus papatasi (Scopoli) and Phlebotomus salehi. In Himachal Pradesh, Phlebotomus longiductus (Parrot) is suspected to be the vector for CL. In the current study, sand flies were collected and tested for Leishmania infection and to confirm the vector species causing CL. Sand flies were collected during April and September of 2017–2019 from CL endemic villages of Rampur (Shimla), Nirmand (Kullu) and Nichar (Kinnaur) districts of Himachal Pradesh. The sand flies were identified as Phlebotomus (adlerius) longiductus (Parrot) and Phlebotomus (larrousisus) major (Annandale). The density of P. longiductus was found highest. The elevation of villages ranged from 947 m to 2,130 m and were far from the presence of subsoil water. Field collected sand flies tested positive (7.69%) for Leishmania donovani by PCR-RFLP. The L. donovani sequences detected from P. longiductus were 97% similar to L. donovani sequences reported from the cases of CL in Himachal Pradesh.The Leishmania positive sand flies were morphologically identified as Phlebotomus adlerious longiductus providing one step further evidence towards the vector status of CL in Himachal Pradesh. The findings of the study are of epidemiological significance for strategic planning of vector control for leishmaniasis in India.

A new cluster of rhabdovirus detected in field-caught sand flies (Diptera: Psychodidae: Phlebotominae) collected from southern Thailand

BackgroundThe distribution of phlebotomine sand flies is changing rapidly due to climate change. This issue has implications for the epidemiology of sand fly-borne diseases, especially sand fly-associated viruses. Few studies concerning sand fly-associated viruses have been conducted in Thailand. Therefore, this study aimed to perform a molecular survey of groups of pathogenic RNA viruses belonging to the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae in sand fly samples collected from southern Thailand.MethodsSand flies were collected at two locations in Trang and Songkhla provinces of southern Thailand, and individual sand fly samples were processed for species identification and virus detection. The Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae molecular determination was performed by RT-PCR, and positive samples were identified by cloning and sequencing, cell culture inoculation, and phylogenetic analysis.ResultsThe results presented in this study were based on the analysis of a total of 331 female sand flies. This molecular study revealed evidence of Rhabdoviridae family virus presence in Phlebotomus papatasi (3/331, 0.9%). The findings demonstrated a new cluster of rhabdovirus that was closely related to Bactrocera dorsalis sigmavirus strain BDSV.abc5 and the lineages of insect-specific Rhabdoviridae. In addition, the Bayesian tree suggested that the common ancestor of this group was the dimarhabdovirus clade. It was assumed that the virus may have switched hosts during its evolution. However, the detection of Orbivirus, Phlebovirus, and Flavivirus genera using specific primers for RT-PCR was negative in the collected sand flies.ConclusionsThere is limited knowledge on the genetic diversity and ecology of Rhabdoviridae in Thailand. This is the first data regarding the circulation of Rhabdoviridae in Ph. papatasi from Thailand. We found a new cluster of rhabdoviruses that was close to the new B. dorsalis sigmavirus. It is possible that there is a great deal of diversity in this family yet to be discovered, and a more extensive survey for new rhabdoviruses may uncover viruses from a wide diversity of host taxa and broaden our understanding of the relationships among the Rhabdoviridae.Graphical abstract

Phlebotomus papatasi Antimicrobial Peptides in Larvae and Females and a Gut-Specific Defensin Upregulated by Leishmania major Infection.

Phlebotomus papatasi is the vector of Leishmania major, causing cutaneous leishmaniasis in the Old World. We investigated whether P. papatasi immunity genes were expressed toward L. major, commensal gut microbes, or a combination of both. We focused on sand fly transcription factors dorsal and relish and antimicrobial peptides (AMPs) attacin and defensin and assessed their relative gene expression by qPCR. Sand fly larvae were fed food with different bacterial loads. Relish and AMPs gene expressions were higher in L3 and early L4 larval instars, while bacteria 16S rRNA increased in late L4 larval instar, all fed rich-microbe food compared to the control group fed autoclaved food. Sand fly females were treated with an antibiotic cocktail to deplete gut bacteria and were experimentally infected by Leishmania. Compared to non-infected females, dorsal and defensin were upregulated at early and late infection stages, respectively. An earlier increase of defensin was observed in infected females when bacteria recolonized the gut after the removal of antibiotics. Interestingly, this defensin gene expression occurred specifically in midguts but not in other tissues of females and larvae. A gut-specific defensin gene upregulated by L. major infection, in combination with gut-bacteria, is a promising molecular target for parasite control strategies.

The Effect of Geographical and Climatic Factors on the Distribution of Phlebotomus papatasi (Diptera: Psychodidae) in Golestan Province, an Endemic Focus of Zoonotic Cutaneous Leishmaniasis in Iran, 2014.

Background:Phlebotomus papatasi is known as the main vector of zoonotic cutaneous leishmaniasis. This study aimed to investigate the effect of geographical and bioclimatic factors on the Ph. papatasi distribution.Methods:A total of 34 villages were selected, and sampling was performed three times using 120 sticky traps in each selected village. All the collected species were mounted and identified their species. The densities of Ph. papatasi were measured in all the villages and entered into ArcMap as a point layer. The required bioclimatic and environmental variables were extracted from the global climate database and The normalized difference vegetation index was obtained from the MODIS satellite imagery, also, all variables entered into ArcMap as raster layers, so The numerical value of each independent variable in the cell where the selected village is located in this, was extracted using spatial analyst tools and the value to point submenu. All the data were finally entered into IBM SPSS, and the relationship was examined between the number of collected Ph. papatasi and the independent variables using Spearman’s correlation test.Results:A total of 1773 specimens of Ph. papatasi were collected. The findings of this study showed that max temperature of warmest month, temperature annual range, temperature seasonality, mean diurnal range, precipitation seasonality, mean temperature of driest and warmest quarter were positively associated with the density of Ph. papatasi.Conclusion:Air temperature and precipitation were shown as the most significant factors in the distribution of Ph. papatasi.

Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi.

Simple SummaryDiverse strains of Wolbachia bacteria, carried by many arthropods, as well as some nematodes, interact in many different ways with their hosts. These include male killing, reproductive incompatibility, nutritional supplementation and suppression or enhancement of the transmission of diseases such as dengue and malaria. Consequently, Wolbachia have an important role to play in novel strategies to control human and livestock diseases and their vectors. Similarly, cell lines derived from insect hosts of Wolbachia constitute valuable research tools in this field. During the generation of novel cell lines from mosquito and sand fly vectors, we isolated two strains of Wolbachia and demonstrated their infectivity for cells from a range of other insects and ticks. These new insect cell lines and Wolbachia strains will aid in the fight against mosquitoes, sand flies and, potentially, ticks and the diseases that these arthropods transmit to humans and their domestic animals.Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

Characterization of sand fly breeding sites in district Malakand, Khyber Pakhtunkhwa, Pakistan, and evaluation of risk factors for cutaneous leishmaniasis in the region.

Present study was carried to determine the sand fly species composition, breeding sites ecology, seasonal abundance, and spatial distribution in district Malakand, Khyber Pakhtunkhwa, Pakistan. In addition, risk factors associated with cutaneous leishmaniasis (CL) were also evaluated. Survey of indoor and outdoor habitats was carried out using sticky traps in 31 villages of Dargai and Batkhela tehsils of Malakand. Soil from habitats of adult and immature sand flies was analysed. Questionnaire-based household survey was also performed in these villages to assess risk factors associated with CL. Soil samples from selected CL positive households were analysed for its contents. Additionally, clinicoepidemiological data from local health centres was examined for the year 2019. Total of 3,140 sand flies belonging to 18 species were collected. Phlebotomus sergenti was the most abundant species (38.16%). Its abundance had a strong positive correlation with mean monthly relative humidity and negative correlation with average temperature. Phlebotomus sergenti and Phlebotomus papatasi were abundant at an elevation ranging from 320 to 1,120m above sea level and in agricultural lands near human settlements. Flight height preference apparatus collected maximum sand flies at 30cm (1ft) above the ground and all species associated negatively with height. Soil analysis from habitats of adult and immature flies showed that highest mean number of adults and immatures were recorded from silt loam which carried highest concentrations of K2 O, Mg, Ca, and Zn. Number of immature sand flies correlated moderately (r=.7, p<.05) with K2 O soil concentrations. There was significant similarity between organic matter contents in soil samples from positive breeding sites and CL households (Wilcoxon rank-sum test, p=.1976). In multivariate analysis model for CL risk factors, age (26-35 and >35years), knowledge of leishmaniasis, living in a middle and upper class, preachers visit to villages, and assumption that Afghan refugees are more prone to CL were significant. CL patient's archived data from health centres showed that majority of patients had lesions on face and hands. Patient's influx was highest in February and March.

Risk Assessment of the Role of the Ecotones in the Transmission of Zoonotic Cutaneous Leishmaniasis in Central Tunisia.

Zoonotic cutaneous leishmaniasis (ZCL), endemic in Central and Southern Tunisia, is caused by Leishmania major (Kinetoplastida: Trypanosomatidae), which is transmitted by the sand fly Phlebotomus papatasi. In Tunisia, the fat sand rat Psammomys obesus and the desert jird Meriones shawi are the principal reservoir hosts of L. major. The presence of the P. papatasi vector of the L. major etiologic agent of ZCL was assessed in the vicinity of villages in endemic areas of Central Tunisia. The study was performed from September through October 2019, a period corresponding to the main peak of activity of P. papatasi. Sand flies were collected from rodent burrows located at the ecotone level, which is the transition zone between the natural environment and human settlement. Sand flies were identified to species level and tested for the presence of L. major by PCR. Our entomological survey showed that P. papatasi is the most abundant sand fly species associated with rodent burrows, and this abundance is even higher in ecotones primarily occupied by P. obesus in comparison to ecotones occupied by M. shawi. Infections with Leishmania major were detected only in P. papatasi, with an overall minimum infection rate (MIR) of 2.64%. No significant difference was observed between the MIRs in ecotones of P. obesus and of M. shawi. Incidence of ZCL in the studied areas ranged from 200 to 700 cases per 100,000 inhabitants, with a mean incidence of 385.41 per 100,000. Higher ZCL incidence was identified in ecotones of M. shawi compared to ecotones of P. obesus. ZCL cases are positively correlated with the MIRs. Considering the short flight range of P. papatasi, increases in its densities associated with burrows of P. obesus or M. shawi at the ecotone level expand the overlap of infected vectors with communities and subsequently increase ZCL incidence. Therefore, control measures should target P. papatasi populations at the ecotones.

Experimental Study of the Mechanical Transmission of Rabbit Hemorrhagic Disease Virus (RHDV2/b) by Aedes albopictus (Diptera: Culicidae) and Phlebotomus papatasi (Diptera: Psychodidae).

Rabbit hemorrhagic disease (RHD) is caused by a lagovirus mainly affecting European rabbits (Oryctolagus cuniculus), although other European and North American lagomorph species are also susceptible to fatal infection by the new viral variant RHDV2/b. In the present work, direct mechanical transmission of the rabbit hemorrhagic disease virus (RHDV2/b variant) by the hematophagous Diptera Aedes albopictus (Skuse) (Diptera: Culicidae) and the sand fly Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) was tested. For each species, six and three laboratory rabbits were exposed to bites of dipterous females partially fed on RHDV2/b viral suspension 2 h and 24 h prior to exposure, respectively. The rabbits were then monitored for clinical changes and mortality for 35 d, and seroconversion was assessed by indirect ELISA. No rabbit died or showed clinical signs of disease, and seroconversion was recorded in two rabbits challenged with P. papatasi females fed the viral suspension 2 h prior to exposure. The number of RHDV2/b RNA copies/female was higher in Ae. albopictus than in P. papatasi but the decrease over time of RNA load in Ae. albopictus was greater than that in P. papatasi. The results of this study suggest the inability of Ae. albopictus to serve as a direct mechanical vector of RHDV2/b, but sand flies could play a role in the local transmission of RHD.

A clinical study to optimise a sand fly biting protocol for use in a controlled human infection model of cutaneous leishmaniasis (the FLYBITE study)

Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) . In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019)

Phlebotomine sand fly survey in the Republic of Moldova: species composition, distribution and host preferences

BackgroundPhlebotomine sand flies (Diptera: Psychodiae) in the Republic of Moldova have been understudied for decades. Our study provides a first update on their occurrence, species composition and bloodmeal sources after 50 years.MethodsDuring 5 seasons (2013–2017), 58 localities from 20 regions were surveyed for presence of sand flies using CDC light traps and manual aspirators. Species identification was done by a combination of morphological and molecular approaches (DNA barcoding, MALDI-TOF MS protein profiling). In engorged females, host blood was identified by three molecular techniques (RFLP, cytb sequencing and MALDI-TOF peptide mass mapping). Population structure of most abundant species was studied by cox1 haplotyping; phylogenetic analyses of ITS2 and cox1 genetic markers were used to resolve relationships of other detected species.ResultsIn total, 793 sand flies were collected at 30 (51.7%) localities from 12 regions of Moldova. Three species were identified by an integrative morphological and molecular approach: Phlebotomus papatasi, P. perfiliewi and Phlebotomus sp. (Adlerius), the first being the most abundant and widespread, markedly anthropophilic based on bloodmeal analyses, occurring also indoors and showing low population structure with only five haplotypes of cox1 detected. Distinct morphological and molecular characters of Phlebotomus sp. (Adlerius) specimens suggest the presence of a yet undescribed species.ConclusionsOur study revealed the presence of stable sand fly populations of three species in Moldova that represent a biting nuisance as well as a potential threat of pathogen transmission and shall be further studied.Graphical abstract

Amplification and Characterization of DDT Metabolizing Delta Class GST in Sand Fly, Phlebotomus argentipes (Diptera: Psychodidae) From Bihar, India.

Phlebotomus argentipes is an established vector for Visceral leishmaniasis prevalent in the Indian subcontinent. Insect Glutathione S-transferases (GST) enzyme plays a pivotal role in the metabolism of xenobiotics and chemical insecticides. We report herein the identification and characterization of a delta class GST from the sandfly, P. argentipes. The resulting clone (rParg-GSTδ) is successfully sequenced, which revealed 76.43% and 66.32% gene identity with GST from Phlebotomus papatasi (Scopoli; Diptera: Psychodidae) and Lutzomiya longipalpis (Lutz and Neiva; Diptera: Psychodidae), respectively. The identified rParg-GST amino acid Blast results revealed 82.6% homology to delta class GST of Phlebotomus papatasi and more than 50% homology to Lepidoptera which comprises butterflies and moths. The Phylogenetic analysis of Parg-GST with different classes of Insect GSTs further supported its classification as delta class. A functional recombinant Parg-GSTδ protein (rParg-GSTδ) was expressed in Escherichia coli (Migula; Enterobacterales: Enterobacteriaceae) cells in a soluble form, purified to homogeneity and found to be active against a substrate 1-chloro-2,4-dintrobenzene (CDNB) and lipid peroxidation by-product 4-Hydrxynonenal (4-HNE). Interestingly, rParg-GSTδ demonstrates high dehydrochlorination activity against dichlorodiphenyltrichloroethane (DDT) i.e., 16.27 nM/µg in high performance liquid chromatography (HPLC) assay. These results provide evidence of direct DDT metabolism property exhibited by P. argentipes GST and set the foundation to decipher the metabolic resistance mechanism in P. argentipes against insecticides.

A clinical study to optimise a sand fly biting protocol for use in a controlled human infection model of cutaneous leishmaniasis (the FLYBITE study).

Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) . In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019).

Insect vectors' saliva and gut microbiota as a blessing in disguise: probability versus possibility.

Drawing of host blood is a natural phenomenon during the bite of blood-probing insect vectors. Along with the blood meal, the vectors introduce salivary components and a trail of microbiota. In the case of infected vectors, the related pathogen accompanies the aforementioned biological components. In addition to Anopheles gambiae or Anopheles stephensi, the bites of other nonmalarialvectors cannot be ignored in malaria-endemic regions. Similarly, the bite incidence of Phlebotomus papatasi cannot be ignored in visceral leishmaniasis-endemic regions. Even the chances of getting bitten by uninfectedvectors are higher than the infectedvectors. We have discussed the probability or possibility of uninfected, infected, and/or nonvector's saliva and gut microbiota as a therapeutic option leading to the initial deterrent to pathogen establishment.

Species diversity and spatial distribution of CL/VL vectors: assessing bioclimatic effect on expression plasticity of genes possessing vaccine properties isolated from wild-collected sand flies in endemic areas of Iran

BackgroundLeishmaniasis is one of the ten most important neglected tropical diseases worldwide. Understanding the distribution of vectors of visceral and cutaneous leishmaniasis (VL/CL) is one of the significant strategic frameworks to control leishmaniasis. In this study, the extent of the bioclimatic variability was investigated to recognize a rigorous cartographic of the spatial distribution of VL/CL vectors as risk-maps using ArcGIS modeling system. Moreover, the effect of bioclimatic diversity on the fold change expression of genes possessing vaccine traits (SP15 and LeIF) was evaluated in each bioclimatic region using real-time PCR analysis.MethodsThe Inverse Distance Weighting interpolation method was used to obtain accurate geography map in closely-related distances. Bioclimatic indices were computed and vectors spatial distribution was analyzed in ArcGIS10.3.1 system. Species biodiversity was calculated based on Shannon diversity index using Rv.3.5.3. Expression fold change of SP15 and LeIF genes was evaluated using cDNA synthesis and RT-qPCR analysis.ResultsFrequency of Phlebotomus papatasi was predominant in plains areas of Mountainous bioclimate covering the CL hot spots. Mediterranean region was recognized as an important bioclimate harboring prevalent patterns of VL vectors. Semi-arid bioclimate was identified as a major contributing factor to up-regulate salivary-SP15 gene expression (P = 0.0050, P < 0.05). Also, Mediterranean bioclimate had considerable effect on up-regulation of Leishmania-LeIF gene in gravid and semi-gravid P. papatasi population (P = 0.0109, P < 0.05).ConclusionsThe diversity and spatial distribution of CL/VL vectors associated with bioclimatic regionalization obtained in our research provide epidemiological risk maps and establish more effectively control measures against leishmaniasis. Oscillations in gene expression indicate that each gene has its own features, which are profoundly affected by bioclimatic characteristics and physiological status of sand flies. Given the efficacy of species-specific antigens for vaccine production, it is essential to consider bioclimatic factors that have a fundamental role in affecting the regulatory regions of environmentally responsive loci for genes used in vaccine design.