Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage.

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.

Identification of Ochrobactrum as a bacteria with transstadial transmission and potential for application in paratransgenic control of leishmaniasis.

Leishmaniasis is a zoonotic vector-borne disease with worldwide distribution. All current approaches in leishmaniasis control or development of vaccines/cures showed only limited success. Recently, paratransgenesis has been marked as a promising strategy for leishmaniasis control. Thus, the investigations of the gut microbial content of sand flies have gained popularity. Gut microbial composition of the laboratory colony of Phlebotomus papatasi was investigated via microbial culturomics approach which refers to the combination of multiple culture conditions and different selective and/or enriched culture mediums, followed by 16S rDNA sequencing. Investigations were conducted on three offspring generations, with six samplings of immature stages (four larval samplings, one pre-pupa, one pupa) and samplings of adults before and after blood feeding. The aim was to determine if microbiome changes during the sand fly development and to identify bacteria with transstadial potential. The presence of 8 bacterial taxa (Bacillus sp., Terribacillus sp., Staphylococcus sp., Alcaligenes sp., Microbacterium sp., Leucobacter sp., Ochrobactrum sp. and Enterobacter sp.), 2 fungi (Fusarium sp. and Acremonium sp.) and 1 yeast (Candida sp.) were recorded. Gram-positive bacteria were more diverse, but gram-negative bacteria were more abundant. All taxa were recorded among immature stage samples, while only one bacterium was detected in adults. Microbial diversity among larval samples was stable, with a steady decrease in pre-pupa and pupa, resulting in the survival of only Ochrobactrum sp. in adults. Abundance of microbes was higher when larvae were actively feeding, with a gradual decrease after larvae stopped feeding and commenced pupation. Ochrobactrum sp. is the bacteria with transstadial potential, worthy of future in-depth analysis for the application in paratransgenic approach for the control of Leishmania sp.

Evaluation of the biodiversity and distribution of sand flies and tracking of Leishmania major in them in central Iran.

Many areas of Iran are endemic regarding Cutaneous leishmaniasis (CL) as a parasitic disease transmitted by a female sand fly vector's bite. The present study investigated the distribution of sand flies in Harand and Egieh in Isfahan province, Central Iran. Overall, 408 out of 1260 collected sand flies were identified morphologically, among which 353 and 55 were isolated from Harand and Egieh, respectively. Also, 66.4% and 33.6% of the sand flies were female and male, respectively. The most prevalent sand fly species were Phlebotomus papatasi (52%), followed by Ph. caucasicus (40.4%), Sergentomyia sintoni (4.9%), and Ph. kazeruni (0.7%). Among 180 molecularly-analyzed sand flies, 14 (7.77%) were found infected with L. major, with 9 out of 103 (8.73%) Ph. papatasi and 5 out of 75 (6.66%) Ph. caucasicus.

Leishmania donovani Transmission Cycle Associated with Human Infection, Phlebotomus alexandri Sand Flies, and Hare Blood Meals, Israel1.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

The role of sand flies as vectors of viruses other than phleboviruses.

Sand flies (Diptera: Phlebotominae) are proven vectors of various pathogens of medical and veterinary importance. Although mostly known for their pivotal role in the transmission of parasitic protists of the genus Leishmania that cause leishmaniases, they are also proven or suspected vectors of many arboviruses, some of which threaten human and animal health, causing disorders such as human encephalitis (Chandipura virus) or serious diseases of domestic animals (vesicular stomatitis viruses). We reviewed the literature to summarize the current published information on viruses detected in or isolated from phlebotomine sand flies, excluding the family Phenuiviridae with the genus Phlebovirus, as these have been well investigated and up-to-date reviews are available. Sand fly-borne viruses from four other families (Rhabdoviridae, Flaviviridae, Reoviridae and Peribunyaviridae) and one unclassified group (Negevirus) are reviewed for the first time regarding their distribution in nature, host and vector specificity, and potential natural transmission cycles.

Biodiversity and vector-borne diseases: Host dilution and vector amplification occur simultaneously for Amazonian leishmaniases.

Changes in biodiversity may impact infectious disease transmission through multiple mechanisms. We explored the impact of biodiversity changes on the transmission of Amazonian leishmaniases, a group of wild zoonoses transmitted by phlebotomine sand flies (Psychodidae), which represent an important health burden in a region where biodiversity is both rich and threatened. Using molecular analyses of sand fly pools and blood-fed dipterans, we characterized the disease system in forest sites in French Guiana undergoing different levels of human-induced disturbance. We show that the prevalence of Leishmania parasites in sand flies correlates positively with the relative abundance of mammal species known as Leishmania reservoirs. In addition, Leishmania reservoirs tend to dominate in less diverse mammal communities, in accordance with the dilution effect hypothesis. This results in a negative relationship between Leishmania prevalence and mammal diversity. On the other hand, higher mammal diversity is associated with higher sand fly density, possibly because more diverse mammal communities harbor higher biomass and more abundant feeding resources for sand flies, although more research is needed to identify the factors that shape sand fly communities. As a consequence of these antagonistic effects, decreased mammal diversity comes with an increase of parasite prevalence in sand flies, but has no detectable impact on the density of infected sand flies. These results represent additional evidence that biodiversity changes may simultaneously dilute and amplify vector-borne disease transmission through different mechanisms that need to be better understood before drawing generalities on the biodiversity-disease relationship.

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

Immune Response to LinB13, a Lutzomyia Intermedia Salivary Protein Correlates With Disease Severity in Tegumentary Leishmaniasis.

BACKGROUND: We have previously shown that seropositivity to rLinB-13, a salivary protein from Lutzomyia intermedia, predicted sand fly exposure and was associated with increased risk of developing cutaneous leishmaniasis (CL). METHODS: Here, we investigated the cellular immune response to saliva from Lu. intermedia, using rLinB-13 as a surrogate antigen in naturally exposed individuals presenting positive serology to LinB-13. We also investigated the response to rLinB-13 in leishmaniasis patients, displaying active ulcers and positive PCR for Leishmania braziliensis. RESULTS: Peripheral blood mononuclear cells (PBMCs) stimulated in vitro with rLinB-13 secreted elevated levels of interleukin-10 (IL-10), IL-4, IL-1ß, IL-1α, IL-6, and chemokines (CCL3, CCL4, CCL5, and CXCL5). CL and disseminated leishmaniasis (DL) patients displayed a significantly higher immunoglobulin G (IgG) response to rLinB-13 compared with healthy subjects, and anti-rLinB-13 IgG was positively correlated with the number of lesions in DL patients. Positive serology to rLinB-13 was also associated with chemotherapy failure. PBMCs from DL patients stimulated with rLINB-13 secreted significantly higher levels of IL-10 and IL-1ß compared with CL individuals. CONCLUSIONS: In this study, we observed an association between humoral and cellular immune response to the sand fly salivary protein rLinB-13 and disease severity in tegumentary leishmaniasis. This study brings evidence that immunity to rLinB-13 influences disease outcome in L. braziliensis infection and results indicate that positive serology to rLinB-13 IgG can be used as a marker of DL, an emerging and severe form of disease caused by L. braziliensis.

Revisiting the cave-dwelling sand flies (Diptera, Psychodidae, Phlebotominae) from Brazil: Diversity and potential role in the transmission of Leishmania Ross, 1903 (Kinetoplastida: Trypanosomatidae).

Brazilian caves, one of the many tourist attractions of the country, may act as a shelter for insects, such as sand flies (Diptera: Psychodidae), natural hosts of various microorganisms including parasites of the genus Leishmania Ross, 1903. In the last decades, with the increasing global need for sustainable development, ecotourism has emerged as one of the major activities in Brazil. However, the constant monitoring in environmentally protected areas is not often carried out, endangering visitors and professionals, especially due to the occurrence of zoonoses. Several sand fly species have already been recorded in Brazilian caves, drawing attention to the possibility of Leishmania transmission at this ecotope. Indeed, this current systematic review summarizes the fauna of cave-dwelling sand flies in Brazil, focusing on their biological behaviour and the occurrence of potential vectors of Leishmania parasites.

Field surveys in Croatia and North Macedonia reveal two novel phleboviruses circulating in sandflies.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae. Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi. BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Understanding the immune responses involved in mediating protection or immunopathology during leishmaniasis.

Leishmaniasis is a vector-borne Neglected Tropical Disease (NTD) transmitted by the sand fly and is a major public health problem worldwide. Infections caused by Leishmania clinically manifest as a wide range of diseases, such as cutaneous (CL), diffuse cutaneous (DCL), mucosal (MCL) and visceral leishmaniasis (VL). The host innate and adaptative immune responses play critical roles in the defense against leishmaniasis. However, Leishmania parasites also manipulate the host immune response for their survival and replication. In addition, other factors such as sand fly salivary proteins and microbiota also promote disease susceptibility and parasite spread by modulating local immune response. Thus, a complex interplay between parasite, sand fly and the host immunity governs disease severity and outcome. In this review, we discuss the host immune response during Leishmania infection and highlight the factors associated with resistance or susceptibility.

Cutaneous Leishmaniasis in Pakistan: a neglected disease needing one health strategy.

BACKGROUND: Cutaneous Leishmaniasis (CL) is a neglected tropical disease, which mainly affects poor communities. It is one of the major vector-borne disease and endemic in Pakistan. METHODS: A case-control study to evaluate potential risk factors of human-CL was conducted in Khewra region, District Jhelum, Pakistan from January-April 2014. Case data about 90 cases registered during October 2012 to November 2013 was retrieved from Municipal Hospital. Controls were matched (1,1 ratio) on the date of registration with cases from same hospital. Both cases and controls were invited to participate and data was collected in a face-to-face interview. A prospective study of canine leishmaniasis (canine-CL) was also conducted at Civil Veterinary Hospital in the same area. Suspected dogs with skin ulceration signs were included in the study and blood samples were collected. Statistical analyses were conducted to determine association between various parameters and outcome of interest. RESULTS: The ages of cases ranged from 1 to 76 years (median = 15 years) and proved to be protective factor i.e. increase in each year in age reduced the likelihood of being infected with human-CL [Odds Ratio (OR) = 0.4, 95% Confidence Interval (CI) = 0.25-0.76]. People sleeping outsides in an open area were more likely to become a case (OR = 8.7, 95% CI = 2.90-26.37) than a control. Poor sanitary condition inside the house (OR = 3.3, 95% CI 1.03-10.56) and presence of other animals in house (livestock, poultry) (OR = 3.6, 95% CI = 1.07-12.12) also identified as risk factors of high significance. The proportion of positive dogs with canine-CL was 21.05% and was significantly associated with human-CL cases in the same area (p < 0.05). CONCLUSIONS: We concluded that adopting self-protections measures against sand-fly, and maintaining good hygiene may lower the risk of human-CL. One-Health Strategy is suggested to control leishmaniasis in human and dog population.

Behavioural responses of Phlebotomus duboscqi to plant-derived volatile organic compounds.

Phlebotomine sand flies are vectors of Leishmania parasites that cause leishmaniases. Both sexes of sand flies feed on plants primarily for sugars, although the chemical cues that mediate attraction to host plants remain largely unknown. Previously, using coupled gas chromatography-mass spectrometry, the authors identified several volatile organic compounds (VOCs) common to preferred host plants for selected Afrotropical sand flies from the Fabaceae family. Of the identified volatiles, the significance of the monoterpenes linalool oxide, ocimene and p-cymene and the benzenoid m-cresol, p-cresol in sand fly behaviour is unknown. In olfactometer assays, the authors tested these compounds singly and in blends for their attractiveness to Phlebotomus duboscqi, cutaneous leishmaniasis vector in Kenya. In dose-response assays, single compounds increased the responses of males and females over controls, but their optimum attractive doses varied between the sexes. Two five-component blends, referred to as Blend-f and Blend-m for females and males respectively, were formulated and tested in dose-response assays against 1-octen-3-ol (positive control). The results of the present study showed that males and females were significantly attracted to varying levels of the two blends. In pairwise assays, the authors evaluated the most attractive of these blends to each sex (i.e., Blend Am for male against Blend Bf for female), revealing that males were attracted to both blends at varying levels, whereas females were indifferent. The study's results demonstrate that plant-derived VOCs can be exploited for sand fly management.

Determination of sand fly fauna and molecular detection of Leishmania in sand flies in Antalya Province, Southern Turkey.

Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are diseases transmitted by infected female sand flies. Since the eradication of malaria in Turkey, CL is the main vector-borne disease in the country, with more than 2000 cases per year, making it a significant public health problem. The aims of this study were to carry out an entomological survey in Antalya Province, an endemic area for CL in the Mediterranean Region of Turkey, to identify sand fly fauna and to screen female specimens for the presence of Leishmania parasites (Leishmania infantum, L. tropica, L. major, and L. donovani) using molecular analysis. Sand flies were collected in 42 localities of seven districts in Antalya Province using CDC miniature light traps in two different periods, June 2012 and September 2013. The specimens were kept in 96% ethanol until the dissection was done. The head and genitalia of the specimens were cut for preparing individual slides for species identification. The rest of the body of female specimens was kept separately. The specimens were identified at the species level, and 27 pools were generated according to the locations and species for screening the presence of Leishmania. A commercial kit was used for DNA extractions. Real-time and conventional polymerase chain reaction (PCR) targeting the internal transcribed spacer region (ITS1) were then performed. In total, 1306 specimens comprising nine species belonging to the Phlebotomus genus were collected in the study region, with Phlebotomus neglectus/syriacus (38.82%) the most abundant, followed by P. alexandri (21.67%) and P. tobbi (20.44%). In the 27 pools, Leishmania infantum DNA was detected in four pools containing P. neglectus/syriacus and one pool containing P. tobbi. In conclusion, the sand fly fauna in the Antalya Province is diverse. The probable vector sand fly species are P. neglectus/syriacus and P. tobbi with high dominance (59.26%), which indicates a high risk of CL transmission. The data presented here may help to shed more light on the transmission cycles of the Leishmania parasite in this CL endemic area.

Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

Leishmania infection induces a limited differential gene expression in the sand fly midgut.

BACKGROUND: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. RESULTS: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. CONCLUSION: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Emerging Sand Fly-Borne Phlebovirus in China.

We isolated 17 viral strains capable of causing cytopathic effects in mammalian cells and death in neonatal mice from sand flies in China. Phylogenetic analysis showed that these strains belonged to the genus Phlebovirus. These findings highlight the need to control this potentially emerging virus to help safeguard public health.

DNA extraction from individual Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) specimens: Which is the method with better results?

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are a group of small insects of great concern for Public Health. These dipterous are intensely studied worldwide due to their involvement in the transmission of several pathogens, mainly Leishmania spp. parasites. Nowadays, the molecular tools have been included in Phlebotomine sand flies studies and has shown to be powerful tools in bioecology studies of these dipterous. Thereby, when molecular approaches are employed, there is a great concern regarding the amount and quality of the DNA obtained for analysis. Here, seven methods of DNA extraction, between commercial kits and in house extraction protocols were evaluated. We considered measure of DNA concentration and purity ratios using a spectrophotometer to check the performance of each protocol. In addition, the quality evaluation of the DNA extracted was performed by endogenous gene PCR on samples. The results of the seven evaluated DNA extraction protocols and their implications are discussed.

Identification of sex determination genes and their evolution in Phlebotominae sand flies (Diptera, Nematocera).

BACKGROUND: Phlebotomine sand flies (Diptera, Nematocera) are important vectors of several pathogens, including Leishmania parasites, causing serious diseases of humans and dogs. Despite their importance as disease vectors, most aspects of sand fly biology remain unknown including the molecular basis of their reproduction and sex determination, aspects also relevant for the development of novel vector control strategies. RESULTS: Using comparative genomics/transcriptomics data mining and transcriptional profiling, we identified the sex determining genes in phlebotomine sand flies and proposed the first model for the sex determination cascade of these insects. For all the genes identified, we produced manually curated gene models, developmental gene expression profile and performed evolutionary molecular analysis. We identified and characterized, for the first time in a Nematocera species, the transformer (tra) homolog which exhibits both conserved and novel features. The analysis of the tra locus in sand flies and its expression pattern suggest that this gene is able to autoregulate its own splicing, as observed in the fruit fly Ceratitis capitata and several other insect species. CONCLUSIONS: Our results permit to fill the gap about sex determination in sand flies, contribute to a better understanding of this developmental pathway in Nematocera and open the way for the identification of sex determining orthologs in other species of this important Diptera sub-order. Furthermore, the sex determination genes identified in our work also provide the opportunity of future biotechnological applications to control natural population of sand flies, reducing their impact on public health.

Exploring sand fly salivary proteins to design multiepitope subunit vaccine to fight against visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by the parasites of Leishmania donovani complex, leads to the death of 20 000 to 40 000 people from 56 affected countries, worldwide. Till date, there is not a single available vaccine candidate to prevent the VL infection, and treatment only relies upon expensive and toxic chemotherapeutic options. Consequently, immunoinformatics approach was applied to design a multiepitope-based subunit vaccine to enhance the humoral as well as cell-mediated immunity. Constructed vaccine candidate was further subjected to evaluation on allergenicity and antigenicity and physiochemical parameters. Later on, disulfide engineering was performed to increase the stability of vaccine construct. Also, molecular docking and molecular dynamics simulation study were performed to check the binding affinity and stability of toll-like receptor-4 to vaccine construct complex. Finally, codon optimization and in silico cloning were performed to ensure the expression of proposed vaccine construct in a microbial expression system.