An important factor influencing the transmission dynamics of vector-borne diseases is the contribution of hosts with different parasitemia (no. of parasites per ml of blood) to the infected vector population. Today, estimation of this contribution is often impractical since it relies exclusively on limited-scale xenodiagnostic or artificial feeding experiments (i.e., measuring the proportion of vectors that become infected after feeding on infected blood/host).
We developed a novel mechanistic model that facilitates the quantification of the contribution of hosts with different parasitemias to the infection of the vectors from data on the distribution of these parasitemias within the host population. We applied the model to an ample data set of Leishmania donovani carriers, the causative agent of visceral leishmaniasis in Ethiopia.
Calculations facilitated by the model quantified the host parasitemias that are mostly responsible for the infection of vector, the sand fly Phlebotomus orientalis. Our findings indicate that a 3.2% of the most infected people were responsible for the infection of between 53% and 79% (mean – 62%) of the infected sand fly vector population.
Our modeling framework can easily be extended to facilitate the calculation of the contribution of other host groups (such as different host species, hosts with different ages) to the infected vector population. Identifying the hosts that contribute most towards infection of the vectors is crucial for understanding the transmission dynamics, and planning targeted intervention policy of visceral leishmaniasis as well as other vector borne infectious diseases (e.g., West Nile Fever).
An important factor influencing the transmission dynamics of vector-borne diseases is the contribution of hosts with different parasitemia (no. of parasites per ml of blood) to the infected vector population. In this study we developed a novel mechanistic model that facilitates the quantification of this contribution and applied it to an ample data set of people infected with visceral leishmaniasis in Ethiopia. Among vector borne diseases, visceral leishmaniasis is the second most important killer after malaria. It is caused by infection with Leishmania parasites with most cases (∼90%) occurring in the Indian sub-continent, East Africa, and South America. The disease is transmitted between people and other mammalian hosts by blood-sucking sand flies. Our findings indicate that a 3.2% of the most infected people were responsible for the infection of about 65% of the infected sand fly vector population. Identifying the hosts that contribute most towards infection of the vectors is crucial for understanding the transmission dynamics, and planning targeted intervention policy of visceral leishmaniasis, as well as other vector borne infectious diseases (e.g., Dengue, West Nile Fever).
Sand flies remain the only proven vectors of Leishmania spp. but recent implementation of PCR techniques has led to increasing speculation about "alternative vectors", including biting midges. Here, we summarize that PCR has considerable limits for studing the role of bloodsucking arthropods in the epidemiology of leishmaniasis. The Leishmania life cycle in the sand fly includes a complex series of interactions which are in many cases species-specific, the early phase of the infection is, however, non-specific to sand flies. These facts should be considered in detection of Leishmania in ,"alternative" or "new" vectors to avoid mistaken speculation about their vector competence.
Culicoides; Phlebotomus; Leishmania
In East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands.
Two cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase.
This is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition.
Phlebotomus orientalis is the vector of visceral leishmaniasis (VL) caused by Leishmania donovani in Northeast Africa. Immunization with sand fly saliva or with individual salivary proteins has been shown to protect against leishmaniasis in different hosts, warranting the intensive study of salivary proteins of sand fly vectors. In our study, we characterize the salivary compounds of P. orientalis, thereby broadening the repertoire of salivary proteins of sand fly species belonging to the subgenus Larroussius. In order to find out whether there is any connection between the composition of P. orientalis saliva and the epidemiology of VL in two distinct Ethiopian foci, Addis Zemen and Melka Werer, we studied the transcriptomes, proteomes, enzymatic activities, and the main salivary antigens in two P. orientalis colonies originating from these areas. We did not detect any significant difference between the saliva of female sand flies originating in Addis Zemen (endemic area) and Melka Werer (non-endemic area). Therefore, the different epidemiology of VL in these Ethiopian foci cannot be related to the distant salivary gland protein composition. Identifying the sand fly salivary gland compounds will be useful for future research focused on characterizing suitable salivary proteins as potential anti-Leishmania vaccine candidates.
Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.
Sexual reproduction is predicted to be a rare event in Leishmania parasites, as evidenced by detection of rare parasite hybrids in natural populations using molecular methods. Recently, a sexual cycle has been detected experimentally in parasites within the sand fly vector (that transmits this pathogenic microorganism to mammalian species including man, causing human leishmaniasis). In this study, we have used whole genome sequencing to investigate genetic variation at the highest level of resolution in Leishmania parasites isolated from sand flies in a defined focus of leishmaniasis in southeast Turkey. Using a range of analytical tools, we show that variation in these parasites arose following a single cross between two diverse strains and subsequent recombination between the progeny, despite mainly clonal reproduction in the parasite population. We have thus been able to derive quantitative estimates of the relative rates of sexual and asexual reproduction during the Leishmania life cycle for the first time, information that will be critical to our understanding of the epidemiology and evolution of this genus.
Phlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches.
Six laboratory-reared colonies of phlebotomine sand flies belonging to five species and four subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Various storage conditions and treatments, including the homogenization in either distilled water or given concentrations of formic acid, were tested on samples of both sexes.
Specimens of all five analysed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their conclusive species identification. The method also distinguished between two P. sergenti colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens.
Species identification of sand flies by MALDI-TOF MS is feasible and represents a novel promising tool to improve biological and epidemiological studies on these medically important insects.
Species identification; Molecular taxonomy; Phlebotomus; MALDI-TOF MS
Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs.
The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate.
Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector exposure and for estimating the risk of L. infantum transmission in dogs.
The protozoan parasite Leishmania infantum is a causative agent of zoonotic visceral leishmaniasis, an important and potentially fatal human disease. The main reservoir hosts of this Leishmania species are dogs, and the only proven vectors are phlebotominae sand flies, Phlebotomus perniciosus being considered the major vector in the western Mediterranean area. During feeding on the host, sand flies spit saliva into the host skin; hosts develop a specific antibody response directed against sand fly salivary proteins and levels of these antibodies reflect the intensity of sand fly exposure. As the availability of salivary gland homogenate is limited, recombinant salivary proteins have been suggested as antigens suitable for measuring specific antibody levels. In the present work, we expressed six of the most-antigenic salivary proteins, and studied the mice and canine humoral immune responses to these recombinant proteins. We demonstrated that three proteins, a yellow-related protein and two apyrases, are suitable antigens for measuring anti-P. perniciosus antibody levels and estimating the host exposure to this sand fly species.
The development of pathogens transmitted by haematophagous invertebrate vectors is closely connected with the digestion of bloodmeals and is thus affected by midgut enzymatic activity. Some studies have demonstrated that avian blood inhibits Leishmania major infection in the Old World vector Phlebotomus papatasi; however, this effect has never been observed in the New World vectors of the genus Lutzomyia infected by other Leishmania species. Therefore, our study was focused on the effect of chicken blood on bloodmeal digestion and the development of Leishmania major in its natural vector Phlebotomus duboscqi, i.e. in a vector-parasite combination where the effect of blood is assumed. In addition, we tested the effect of avian blood on midgut trypsin activity and the influence of repeated feedings on the susceptibility of sand flies to Leishmania infection.
Phlebotomus duboscqi females were infected by rabbit blood containing L. major and either before or after the infection fed on chickens or mice. The individual guts were checked microscopically for presence and localization of Leishmania, parasite numbers were detected by Q-PCR. In addition, midgut trypsin activity was studied.
Sand fly females fed on chicken blood had significantly lower midgut trypsin activity and delayed egg development compared to those fed on rabbits. On the other hand, there was no effect detected of avian blood on parasite development within the sand fly gut: similar infection rates and parasite loads were observed in P. duboscqi females infected by L. major and fed on chickens or mouse one or six days later. Similarly, previous blood feeding of sand flies on chickens or mice did not show any differences in subsequent Leishmania infections, and there was equal susceptibility of P. duboscqi to L. major infection during the first and second bloodmeals.
In spite of the fact that avian blood affects trypsin activity and the oocyte development of sand flies, no effect of chicken blood was observed on the development of L. major in P. duboscqi. Our study unambiguously shows that sand fly feeding on avian hosts is not harmful to Leishmania parasites within the sand fly midgut.
Leishmaniases; Vectors; Bloodmeal digestion; Trypsin; Chicken blood
Sand fly species of the genus Sergentomyia are proven vectors of reptilian Leishmania that are non-pathogenic to humans. However, a consideration of the role of Sergentomyia spp. in the circulation of mammalian leishmaniasis appears repeatedly in the literature and the possibility of Leishmania transmission to humans remains unclear. Here we studied the susceptibility of colonized Sergentomyia schwetzi to Leishmania donovani and two other Leishmania species pathogenic to humans: L. infantum and L. major.
Females of laboratory-reared S. schwetzi were infected by cultured Leishmania spp. by feeding through a chicken membrane, dissected at different time intervals post bloodmeal and examined by light microscopy for the abundance and location of infections.
All three Leishmania species produced heavy late stage infections in Lutzomyia longipalpis or Phlebotomus duboscqi sand flies used as positive controls. In contrast, none of them completed their developmental cycle in Sergentomyia females; Leishmania promastigotes developed within the bloodmeal enclosed by the peritrophic matrix (PM) but were defecated together with the blood remnants, failing to establish a midgut infection. In S. schwetzi, the PM persisted significantly longer than in L. longipalpis and it was degraded almost simultaneously with defecation. Therefore, Leishmania transformation from procyclic to long nectomonad forms was delayed and parasites did not attach to the midgut epithelium.
Sergentomyia schwetzi is refractory to human Leishmania species and the data indicate that the crucial aspect of the refractoriness is the relative timing of defecation versus PM degradation.
Visceral leishmaniasis; Phlebotomine sand flies; Phlebotomus; Sergentomyia; Peritrophic matrix
Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ).
Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (2×103 per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding.
Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 µl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.
Phlebotomus orientalis is the main vector of Leishmania donovani in East Africa and is, therefore, a sand fly species of high importance. We studied various properties of P. orientalis populations from both endemic (Addis Zemen) and non-endemic (Melka Werer) areas in Ethiopia. We successfully demonstrated the ability of laboratory colonies arising from these populations to crossbreed by obtaining 1st and 2nd generation hybrid progeny. Hybrids had similar or even higher fecundity than parental colonies. Comparison of the populations by sequencing of two genes (cytB and COI) and by RAPD (a multilocus method) revealed no genetic differences. We demonstrated that both populations are highly susceptible to experimental infection with L. donovani and even small numbers of parasites are able to initiate heavy infections in P. orientalis females. As the development pattern of L. donovani was similar for females from both colonies, we deduce that the absence of visceral leishmaniasis in the non-endemic area of Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.
Leishmaniases are vector-borne parasitic diseases with 0.9 – 1.4 million new human cases each year worldwide. In the vectorial part of the life-cycle, Leishmania development is confined to the digestive tract. During the first few days after blood feeding, natural barriers to Leishmania development include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood meal and sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted with the blood remnant. This binding is strictly stage-dependent as it is a property of nectomonad and leptomonad forms only. While the attachment in specific vectors (P. papatasi, P. duboscqi and P. sergenti) involves lipophosphoglycan (LPG), this Leishmania molecule is not required for parasite attachment in other sand fly species experimentally permissive for various Leishmania. During late-stage infections, large numbers of parasites accumulate in the anterior midgut and produce filamentous proteophosphoglycan creating a gel-like plug physically obstructing the gut. The parasites attached to the stomodeal valve cause damage to the chitin lining and epithelial cells of the valve, interfering with its function and facilitating reflux of parasites from the midgut. Transformation to metacyclic stages highly infective for the vertebrate host is the other prerequisite for effective transmission. Here, we review the current state of knowledge of molecular interactions occurring in all these distinct phases of parasite colonization of the sand fly gut, highlighting recent discoveries in the field.
Phlebotomus; Lutzomyia; Kinetoplastida; Proteolytic enzymes; Peritrophic matrix; Chitinase; Innate immunity
In Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear.
In this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy.
Late-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi.
Our data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L. turanica to establish late-stage infections in these specific vectors of L. major suggests that the lipophosphoglycan of this species must be identical or similar to that of L. major.
Leishmania turanica; L. major; Mixed infections; Competition; Genetic exchange; Vector competence; Phlebotomus
Phlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins.
Sera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera.
Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi–mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission.
Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis and Phlebotomus papatasi serve as the major vector. In endemic foci, rodents are the natural reservoirs of this disease. Thus, we studied anti-P. papatasi saliva antibody response in BALB/c and C57BL/6 mice that are commonly used as model organisms sensitive and resistant to cutaneous leishmaniasis, respectively. We followed the kinetics and persistence of specific antibody response in both mice strains and we characterized the main P. papatasi salivary antigens. We demonstrated that sand fly bites elicit production of specific IgG that reflect the intensity of sand fly exposure. In endemic areas, this could provide useful information about the effectiveness of anti-vector control programs. We also examined the reaction of mice sera with four P. papatasi recombinant proteins. Our data indicate that a combination of these proteins could be used instead of crude salivary gland homogenate for the monitoring of anti-sand fly saliva antibodies in natural hosts in endemic foci.
Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. Le. infantum and Le. tropica typically cause visceral or cutaneous leishmaniasis, respectively, but Le. infantum strains transmitted by P. tobbi can cause cutaneous disease. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared.
cDNA libraries of P. tobbi and P. sergenti female SGs were constructed, sequenced, and analyzed. Clones (1,152) were randomly picked from each library, producing 1,142 high-quality sequences from P. tobbi and 1,090 from P. sergenti. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41.9-kDa superfamily of proteins. Phylogenetic analyses and multiple sequence alignments of putative proteins were used to elucidate molecular evolution and describe conserved domains, active sites, and catalytic residues. Proteomic analyses of P. tobbi and P. sergenti SGs were used to confirm the identification of 35 full-length sequences (18 in P. tobbi and 17 in P. sergenti). To bridge transcriptomics with biology P. tobbi antigens, glycoproteins, and hyaluronidase activity was characterized.
This analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research.
Phlebotomine female sand flies require a blood meal for egg development, and it is during the blood feeding that pathogens can be transmitted to a host. Leishmania parasites are among these pathogens and can cause disfiguring cutaneous or even possibly fatal visceral disease. The Leishmania parasites are deposited into the bite wound along with the sand fly saliva. The components of the saliva have many pharmacologic and immune functions important in blood feeding and disease establishment. In this article, the authors identify and investigate the protein components of saliva of two important vectors of leishmaniasis, Phlebotomus tobbi and P. sergenti, by sequencing the transcriptomes of the salivary glands. We then compared the predicted protein sequences of these salivary proteins to those of other bloodsucking insects to elucidate the similarity in composition, structure, and enzymatic activity. Finally, this descriptive analysis of P. tobbi and P. sergenti transcriptomes can aid future research in identifying molecules for epidemiologic assays and in investigating sand fly-host interactions.
New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.
The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.
The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.
In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought.
Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.
Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.
Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.
Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in the Mediterranean Basin and Phlebotomus perniciosus serve as the major vector. In the endemic foci, Leishmania parasites are transmitted mostly to dogs, the main reservoir host, and to humans. We studied the canine humoral immune response to Phlebotomus perniciosus saliva and its potential use as a marker of sand fly exposure and consequently as a risk marker for Leishmania transmission. We also characterized major salivary antigens of P. perniciosus. We demonstrated that under laboratory conditions, the levels of anti-P. perniciosus saliva antibodies positively correlated with the number of blood-fed sand flies and therefore, may be used to evaluate the need for, and the effectiveness of, anti-vector campaigns. In parallel, we studied sera of dogs naturally exposed to P. perniciosus in highly active focus of canine leishmaniasis in Southern Italy. Specific antibodies against P. perniciosus saliva were significantly increased according to the ongoing sand fly season. Moreover, the levels of anti-P. perniciosus antibodies in naturally bitten dogs negatively correlated with anti-Leishmania seropositivity. Thus, for dogs living in endemic areas, specific antibody response against saliva of the vector is an important marker for estimating the risk of Leishmania transmission.
Visceral leishmaniasis is the world' second largest vector-borne parasitic killer and a neglected tropical disease, prevalent in poor communities. Long-lasting insecticidal nets (LNs) are a low cost proven vector intervention method for malaria control; however, their effectiveness against visceral leishmaniasis (VL) is unknown. This study quantified the effect of LNs on exposure to the sand fly vector of VL in India and Nepal during a two year community intervention trial.
As part of a paired-cluster randomized controlled clinical trial in VL-endemic regions of India and Nepal we tested the effect of LNs on sand fly biting by measuring the antibody response of subjects to the saliva of Leishmania donovani vector Phlebotomus argentipes and the sympatric (non-vector) Phlebotomus papatasi. Fifteen to 20 individuals above 15 years of age from 26 VL endemic clusters were asked to provide a blood sample at baseline, 12 and 24 months post-intervention.
A total of 305 individuals were included in the study, 68 participants provided two blood samples and 237 gave three samples. A random effect linear regression model showed that cluster-wide distribution of LNs reduced exposure to P. argentipes by 12% at 12 months (effect 0.88; 95% CI 0.83–0.94) and 9% at 24 months (effect 0.91; 95% CI 0.80–1.02) in the intervention group compared to control adjusting for baseline values and pair. Similar results were obtained for P. papatasi.
This trial provides evidence that LNs have a limited effect on sand fly exposure in VL endemic communities in India and Nepal and supports the use of sand fly saliva antibodies as a marker to evaluate vector control interventions.
Visceral leishmaniasis (VL), also known as kala azar, is one of the major public health concerns of the Indian subcontinent, caused by Leishmania donovani transmitted by the bite of the sand fly Phlebotomus argentipes. To date, Indoor Residual Spraying (IRS) campaigns have been unable to control the disease. This makes Long-lasting insecticidal nets (LNs) an attractive alternative or complement to IRS. Therefore, it is important to assess the extent that LNs reduce bites from P. argentipes. When female sand flies bite they require their saliva to efficiently bloodfeed. For humans and animals alike, the host' immune response against components of sand fly saliva can be used as a marker of exposure to the vector. Here we describe how comprehensive coverage of LNs in trial communities over two years reduced antibody levels to the saliva of P. argentipes and P. papatasi (a man-biting sand fly that co-exists with P. argentipes but does not transmit VL) sand flies by 9–12% compared to communities without LNs. Our results demonstrate that the large-scale distribution of LNs did not confer significant additional protection against sand fly bites in VL-endemic regions of India and Nepal and questions the indoor transmission of L. donovani in these regions.
We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5–1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19×104 promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.
Leishmaniasis is a disease caused by protozoan parasites which are transmitted through the bites of infected insects called sand flies. The World Health Organization has estimated that leishmaniases cause 1.6 million new cases annually, of which an estimated 1.1 million are cutaneous or mucocutaneous, and 500,000 are visceral, the most severe form of the disease and fatal if left untreated. The development of a more natural model is crucial for the evaluation of new drugs or vaccine candidates against leishmaniases. The main aim of this study was to quantify the number of Leishmania infantum parasites transmitted by a single sand fly female into the skin of a vertebrate host (mouse). Two L. infantum strains, viscerotropic IMT373 and dermotropic CUK3, were compared in two natural sand fly vectors: Phlebotomus perniciosus and Lutzomyia longipalpis. We found that the parasite number transmitted by individual sand flies ranged from 4 up to 4.19×104. The maximal natural infective dose found in our experiments was about 250 times lower than the experimental challenge dose used in most previous studies.
The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described.
Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro.
For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits.
Parasite-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.
A 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.
This 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.
The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood.
To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.
The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.
Many infectious diseases such as leishmaniasis are transmitted to people by biting insects, in this case by female sand flies. To control this and similar diseases we need to understand why particular species of sand fly transmit particular species of Leishmania. One important feature of the Leishmania parasite-sand fly interaction is the ability of the parasite to bind to the midgut wall of the fly, as it is within the gut that the parasite lives. Here we have studied the specificity of this interaction and report two main findings. The first is that only specific stages in the parasite life-cycle are capable of binding to the gut. The second is that, providing these life-cycle stages are analysed, parasite species that can be transmitted by particular sand flies are always capable of binding to their guts, but in some cases they are also capable of binding to non-transmitting sand fly species. This shows that gut binding by parasites is necessary but not sufficient to explain transmission. This research advances our understanding of Leishmania biology, but also shows us that there are further aspects that need to be investigated before we can fully understand the Leishmania-sand fly relationship.
Antibody (IgG) responses to the saliva of Phlebotomus argentipes were investigated using serum samples from regions of India endemic and non-endemic for visceral leishmaniasis (VL). By pre-adsorbing the sera against the saliva of the competing human-biting but non-VL vector P. papatasi, we significantly improved the specificity of a P. argentipes saliva enzyme-linked immunosorbent assay. Using this method, we observed a statistically significant correlation between antibodies to P. argenitpes saliva and the average indoor density of female sand flies. Additionally, the method was able to detect recent changes in vector exposure when sera from VL patients were assayed before, during, and after hospitalization and protected from sand fly bites under untreated bed nets. Collectively, these results highlight the utility of antibodies to P. argentipes saliva as an important tool to evaluate VL vector control programs.
Sand flies deliver Leishmania parasites to a host alongside salivary molecules that affect infection outcomes. Though some proteins are immunogenic and have potential as markers of vector exposure, their identity and vector specificity remain elusive.
We screened human, dog, and fox sera from endemic areas of visceral leishmaniasis to identify potential markers of specific exposure to saliva of Lutzomyia longipalpis. Human and dog sera were further tested against additional sand fly species. Recombinant proteins of nine transcripts encoding secreted salivary molecules of Lu. longipalpis were produced, purified, and tested for antigenicity and specificity. Use of recombinant proteins corresponding to immunogenic molecules in Lu. longipalpis saliva identified LJM17 and LJM11 as potential markers of exposure. LJM17 was recognized by human, dog, and fox sera; LJM11 by humans and dogs. Notably, LJM17 and LJM11 were specifically recognized by humans exposed to Lu. longipalpis but not by individuals exposed to Lu. intermedia.
Salivary recombinant proteins are of value as markers of vector exposure. In humans, LJM17 and LJM11 emerged as potential markers of specific exposure to Lu. longipalpis, the vector of Leishmania infantum chagasi in Latin America. In dogs, LJM17, LJM11, LJL13, LJL23, and LJL143 emerged as potential markers of sand fly exposure. Testing these recombinant proteins in large scale studies will validate their usefulness as specific markers of Lu. longipalpis exposure in humans and of sand fly exposure in dogs.
Leishmania parasites are transmitted by the bite of an infected vector sand fly that injects salivary molecules into the host skin during feeding. Certain salivary molecules can produce antibodies and can be used as an indicator of exposure to a vector sand fly and potentially the disease it transmits. Here we identified potential markers of specific exposure to the sand fly Lutzomyia longipalpis, the vector of visceral leishmaniasis in Latin America. Initially, we determined which of the salivary proteins produce antibodies in humans, dogs, and foxes from areas endemic for the disease. To identify potential specific markers of vector exposure, we produced nine different recombinant salivary proteins from Lu. longipalpis and tested for their recognition by individuals exposed to another human-biting sand fly, Lu. intermedia, that transmits cutaneous leishmaniasis and commonly occurs in the same endemic areas as Lu. longipalpis. Two of the nine salivary proteins were recognized only by humans exposed to Lu. longipalpis, suggesting they are immunogenic proteins and may be useful in epidemiological studies. The identification of specific salivary proteins as potential markers of exposure to vector sand flies will increase our understanding of vector–human interaction, bring new insights to vector control, and in some instances act as an indicator for risk of acquiring disease.
Leishmaniases are serious parasitic diseases the etiological organisms of which are transmitted by insect vectors, phlebotominae sand flies. Two sand fly species, Phlebotomus papatasi and P. sergenti, display remarkable specificity for Leishmania parasites they transmit in nature, but many others are broadly permissive to the development of different Leishmania species. Previous studies have suggested that in ‘specific’ vectors the successful parasite development is mediated by parasite surface glycoconjugates and sand fly lectins, however we show here that interactions involving ‘permissive’ sand flies utilize another molecules. We did find that the abundant surface glycoconjugate lipophosphoglycan, essential for attachment of Leishmania major in the specific vector P. papatasi, was not required for parasite adherence or survival in the permissive vectors P. arabicus and Lutzomyia longipalpis. Attachment in several permissive sand fly species instead correlated with the presence of midgut glycoproteins bearing terminal N-Acetyl-galactosamine and with the occurrence of a lectin-like activity on Leishmania surface. This new binding modality has important implications to parasite transmission and evolution. It may contribute to the successful spreading of Leishmania due to their adaptation into new vectors, namely transmission of L. infantum by Lutzomyia longipalpis; this event led to the establishment of L.infantum/chagasi in Latin America.
Parasite transmission; Emerging infectious disease; Trypanosomatid protozoan
Development of Leishmania infantum/Leishmania major hybrids was studied in two sand fly species. In Phlebotomus papatasi, which supported development of L. major but not L. infantum, the hybrids produced heavy late-stage infections with high numbers of metacyclic promastigotes. In the permissive vector Lutzomyia longipalpis, all Leishmania strains included in this study developed well. Hybrids were found to express L. major lipophosphoglycan, apparently enabling them to survive in P. papatasi midgut. The genetic exchange of the hybrids thus appeared to have enhanced their transmission potential and fitness. A potentially serious consequence is the future spread of the hybrids using this peridomestic and antropophilic vector.
Leishmania transmission; Parasite-vector interaction; Emerging diseases