Uninfected Sand Flies Research Articles

Assessing public perception of a sand fly biting study on the pathway to a controlled human infection model for cutaneous leishmaniasis

BackgroundA controlled human infection model (CHIM) involves deliberate exposure of volunteers to pathogens to assess their response to new therapies at an early stage of development. We show here how we used public involvement to help shape the design of a CHIM to support future testing of candidate vaccines for the neglected tropical disease cutaneous leishmaniasis, a disease transmitted by the bite of infected sand flies in tropical regions.MethodsWe undertook a public involvement (PI) consultation exercise to inform development of a study to test the safety and effectiveness of a sand fly biting protocol using uninfected sand flies (FLYBITE: ClinicalTrials.gov ID NCT03999970) and a CHIM using Leishmania major-infected sand flies (LEISH_Challenge: ClinicalTrials.gov ID NCT04512742), both taking place in York, UK. We involved 10 members of the public including a patient research ambassador and a previous CHIM volunteer. The session took place at The University of York, UK and examined draft study volunteer-facing material and included the CHIM study design, potential adverse events and therapeutic interventions at study endpoints. A discussion of the scientific, ethical, humanitarian and economic basis for the project was presented to the participants to provoke discourse. An inductive, thematic analysis was used to identify the participants’ key concerns.ResultsThemes were identified relating to i) quality of volunteer-facing written information, ii) improving study design, and iii) factors to motivate involvement in the research. Group participants responded positively to the overall study aims. Initial concerns were expressed about potential risks of study involvement, but further explanation of the science and mitigations of risk secured participant support. Participants provided advice and identified improved terminology to inform the volunteer-facing material. Lastly, treatment options were discussed, and excision of any cutaneous lesion was favoured over alternatives as a treatment.ConclusionThe consultation exercise provided invaluable information which led to improved study design and enhanced clarity in the volunteer-facing material. The session also reinforced the need to maintain public trust in scientific rigour prior to initiation of any study. The investigators hope that this description strengthens understanding of PI in clinical research, and encourages its use within other studies.

Exploring the relationship between susceptibility to canine leishmaniosis and anti-Phlebotomus perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in Mallorca, Spain

BackgroundCanine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response.MethodsThis study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods.ResultsResults indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT.ConclusionsTo our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds.

Correction: DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica

[This corrects the article DOI: 10.1371/journal.pntd.0007067.].

DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

BackgroundThe vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection.Methods and resultsIn this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls.ConclusionThis study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens.

Use of In Vivo Imaging System Technology in Leishmania major BALB/c Mouse Ear Infection Studies.

Novel treatments for cutaneous leishmaniasis (CL) are needed, due to current lack of effective universal treatments, increasing resistance among the parasite, and toxic effects or impracticality of the current therapeutics. Models of direct infection with high number of Leishmania parasites in the current research of CL involving the BALB/c mouse or Golden Syrian Hamster are considered not suitable for the assessment of antileishmanial drug efficacy because of the lack of disease similarities with humans. The saliva of the sand fly vector is known to affect the host response to infection by the Leishmania parasite. Here, we build upon a previous BALB/c model infected with luciferase-expressing Leishmania major parasites. In the present study, we infect the ear dermis instead of the foot pad or base of the tail, and compare multiple methods of infection, using parasites alone or mixed with either bites from uninfected sand flies (Phlebotomus duboscqi Diptera Psychodidae:Neveu-Lemaire) or salivary gland sonicate from sand flies. Our data show a dose-response of bioluminescent signal (which represents the parasite load at the infection site), dermal lesion development, and Leishmania Donovan Units in liver and spleen. This in vivo L. major ear infection model, once optimized, can be used for assessing the efficacy of drug compounds that have been determined as very effective in the other, high inoculum CL models.

Increased Transmissibility of Leishmania donovani From the Mammalian Host to Vector Sand Flies After Multiple Exposures to Sand Fly Bites.

Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. The role of the blood or skin as a source of infection to sand flies remains unclear, and the possible effect of multiple exposures to fly bites on transmissibility has not been addressed. L. donovani-infected hamsters underwent xenodiagnoses with Lutzomyia longipalpis on the same or different sites on the abdomen on 2 consecutive days or by artificial feeding on the skin or blood. The transmission of L. donovani from sick hamsters to flies was surprisingly low (mean, 24% of fed flies). New flies fed on the same site acquired significantly more infections (mean, 61%; P < .0001). By artificial feeding, flies could acquire infection from blood and skin. However, only artificial feeding on blood produced infections that correlated with the natural feeding (R = 0.792; P < .0001). Infections acquired from blood increased dramatically for blood obtained after exposure to bites, as did the parasitemia level and the number of monocytes in the circulation. The bites of uninfected sand flies favor the transmissibility of L. donovani by infected hosts, owing to a systemic effect that exposure to bites has on the parasitemia. Patients with active visceral leishmaniasis are important reservoirs in the anthroponotic transmission cycle of Leishmania donovani. Using the hamster model of visceral disease, we demonstrate that prior exposure to bites of uninfected sand flies potentiates their ability to transmit infection to the vector.

Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

Identifying salivary antigens of Phlebotomus argentipes by a 2DE approach

In the Indian subcontinent visceral leishmaniasis, also known as kala-azar, is caused by the protozoa Leishmania donovani and is transmitted to humans by the bite of infected female sand flies Phlebotomus argentipes in an anthroponotic cycle. Sand fly saliva is known to play an important role in host infection outcome after an infective bite. Immunogenicity of P. argentipes saliva has already been described. However, specific antigens that can contribute to these immunogenic properties are unknown. This work focuses on the identification of antigens present in P. argentipes saliva through the combination of two-dimensional electrophoresis (2DE) and Western blot (WB). Analysis of the salivary protein profile showed a gradual increase of the protein content in relation to the age of sand flies, reaching the complete salivary protein pattern at day five, which marked the minimum age for dissections. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). The resulting salivary proteomic map consisted of at least 30 spots located between 10 and 60kDa. According to their isoelectric points, spots were mostly distributed around pH ranges: 5–6 and 9–10. In the proteomic maps, the presence of isoforms or posttranslational modifications was also highlighted since several spots were identified as the same protein. Analysis by in silico prediction programs located several potential glycosylation and phosphorylation sites in the aminoacidic sequences. On the other hand, pooled sera of immunized hamsters through the bite of uninfected sand flies showed elevated anti-saliva IgG levels. These sera permitted the detection of 4 protein bands and at least 20 protein spots in 1DE and 2DE respectively, followed by WB. The antigens were identified by MALDI-TOF, MALDI-TOF/TOF and de novo sequencing as D7-related proteins, PpSP15-like proteins, antigen 5-related proteins, apyrases, and several proteins without assigned protein family. Absence of cross-reactivity between P. argentipes and Phlebotomus perniciosus saliva antibodies determined by ELISA and WB was highlighted in this study, confirming that specific salivary antigens from different sand fly vectors need to be sought when designing vector-borne vaccines and markers for vector exposure assays.

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies

BackgroundParasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles.ResultsA total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal.ConclusionThis midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

Effects of Ivermectin on Blood-Feeding Phlebotomus papatasi, and the Promastigote Stage of Leishmania major

Ivermectin (IVM) is a chemically modified macrocyclic lactone of Streptomyces avermitilis that acts as a potent neurotoxin against many nematodes and arthropods. Little is known of IVM's effect against either blood-feeding Phlebotomus sand flies, or the infective promastigote stage of Leishmania transmitted by these flies. We injected hamsters subcutaneously with two standard IVM treatments (200 and 400 μg/kg body weight) and allowed cohorts of Leishmania major-infected Phlebotomus papatasi to blood-feed on these animals at various posttreatment time points (4 h, 1, 2, 6, and 10 days). Infected and uninfected sand flies that bit treated and untreated hamsters served as controls. Serum levels of IVM in low- and high-dose-treated hamsters were determined at the five time points. Sand fly mortality following blood feeding was recorded at 24-h intervals and, in relation to IVM treatment, was time and dose dependent. Mortality was most rapid and greatest among infected flies that fed nearest to time of dosing. Mean survival of infected sand flies after feeding on untreated hamsters was 11.5 days, whereas that of infected sand flies that fed 4 h, 1 day, or 2 days posttreatment on high-dose-treated hamsters (400 μg/kg) was 1.6, 2.1, and 2.7 days, respectively. Infected and uninfected sand flies that blood fed 6 days following low-dose IVM treatment (200 μg/kg) still experienced significantly greater mortality (p < 0.02) than controls. Promastigotes dissected out of surviving flies that fed on IVM-treated hamsters showed typical motility and survival. Moreover, 21.7% of IVM-treated hamsters developed lesions after being fed upon by infected sand flies. L. major promastigotes appeared to be tolerant to ng/mL blood levels of IVM that caused significant mortality for up to 10 days posttreatment in blood-feeding P. papatasi.

Setting the stage for Leishmania

When a host is infected with a parasite, a symbiosis ensues that functions as a new complex organism; the infected host. The co-existence of these organisms requires integration of both physiological complexes. It has been shown that the immune system plays an important role in the fate of Leishmania in the host. White blood cells produce a cytokine context that either allows survival of the parasite or its elimination. This is dependent on the type of T-cell response activated upon infection. A T-helper 1 (Th1) response (characterized by the production of IFN-γ and IL-12) that leads to parasite killing owing to the activation of infected macrophages by IFN-γ. By contrast, a Th2 response (characterized by the production of IL- 4) allows survival of the parasite, probably because of downregulation of IFN-γ production by IL-10. Results from a mouse model indicated that salivary gland lysates of Plebotomus papatasi, the mosquito vector that transmits Leishmania major, provokes a Th2 response, which allows parasite survival (M.L. Mbow. et al., J. Immunol. 161, 5571–5577, 1998). Recently, it was shown that prior exposure of mice to bites of uninfected sandflies induced strong Th1 responsiveness with IFN-γ production, and associated resistance to Leishmania infection. These results suggest an alternative means of immunization against Leishmania infection (S. Kamhawi et al., Science 290, 1351–1354, 2000) and might have implications to the epidemiology of the disease. TS

Sensitization to sand fly bite prevents cutaneous leishmaniasis

Until recently, from both epidemiological and clinical perspectives, arthropods have been considered as little more than delivery systems for the pathogens that they carry. However, insofar as components of their saliva have been shown to modify the outcome of infection, an active modulation by arthropod vectors of disease transmission is starting to gain recognition. The disease most studied in this context is cutaneous leishmaniasis, which is transmitted exclusively by the bite of infectious sand flies. Kamhawi et al.1xProtection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. Kamhawi, S et al. Science. 2000; 290: 1351–1354Crossref | PubMed | Scopus (190)See all References1 have now described a reproducible murine model of Leishmania major infection transmitted by its natural vector, Phlebotomus papatasi, in which the ear is the site of parasite inoculation. The respective lesion-healing and lesion-non-healing phenotypes of C57BL/6 and BALB/c mice that have proved a powerful tool for dissection of the T helper type 1 (Th1)/Th2 immunological paradigm, established using high-dose needle inocula, were maintained in fly-transmitted infections. demonstrated that prior exposure of bites of uninfected sand flies alone provides a significant degree of protection against L. major, sufficient to confer resistance subsequent biting by infected sand flies. For both BALB/c and C57BL/6 mice, prior exposure to P. papatasi bites resulted striking reduction in both the severity dermal lesions and the parasite load.The protective mechanism involves a strong delayed-type hypersensitivity reaction and production of interleukin (IL)-12 and interferon (IFN)-. at the bite site. The rapidly raised secretion of these cytokines might activate infected macrophages for killing while the infection is initially establishing and might also promote an early polarized Leishmania-specific Th1-type response that is the immunological hallmark of a healing infection. The molecular identity of the stimulus that triggers this protective response remains to be elucidated but is thought to be a component of Phlebotomus saliva. These findings are very relevant to leishmaniasis of humans, for which many individuals in endemic regions are exposed to the bites of Leishmania-free sand flies before receiving an infective bite. Thus, a person's exposure history to uninfected phlebotomines could influence their susceptibility to, and the severity of, infection. Moreover, salivary antigens might be effective components of vaccines against this and other vector-borne infectious diseases.

Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies.

Despite the fact that Leishmania are transmitted exclusively by sand flies, none of the experimental models of leishmaniasis have established infection via sand fly bites. Here we describe a reproducible murine model of Leishmania major infection transmitted by Phlebotomus papatasi. Prior exposure of mice to bites of uninfected sand flies conferred powerful protection against Leishmania major that was associated with a strong delayed-type hypersensitivity response and with interferon-gamma production at the site of parasite delivery. These results have important implications for the epidemiology of cutaneous leishmaniasis and suggest a vaccination strategy against this and possibly other vector-borne diseases.