Circle size and circle color indicates frequency and geographical location of haplotypes, respectively. Haplotype numbers are indicated next to the corresponding circle H_XX. Red numbers between haplotypes indicate number of mutations between haplotypes. (PPTX)

Circle size and circle color indicates frequency and geographical location of haplotypes, respectively. Haplotype numbers are indicated next to the corresponding circle H_XX. Red numbers between haplotypes indicate number of mutations between haplotypes. (PPTX)

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47615 generated sequences was cleaned and assembled into 17120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P.papatasi and will provide essential information for annotation of the full genome.

We characterized a cDNA from Phlebotomus papatasi, PpChit1, which encodes a midgut specific chitinase and show the presence of a functional, blood‐induced chitinolytic system in sand flies. PpChit1 is detected only in the midgut and is regulated by blood feeding. A recombinant protein (rPpChit1) produced in HEK 293‐F cells exhibited a similar activity profile to that found in the native protein against several specific substrates, including an oligomeric glycol chitin and synthetic 4‐methyl‐umbelliferone labelled substrates. Western blotting showed that the native protein is recognized by mouse polyclonal antibodies against rPpChit1. Additionally, the rPpChit1 and the native chitinase displayed similar retention times in a HPLC size fractionation column. When added to rPpChit1 or to midgut lysates, PpChit1 sera reduced chitinolytic activity by 65–70%.

Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector–parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1–Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40–60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.

Lcislunania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leislimania infection, while immunity to the saliva protects against infection as determined by coinoculation of parasites with vector salivary gland homogenates (SGHs) or by infected sand fly bites (Kamhawi, S., Y. Belkaid, G. Modi, E. Rowton, and D. Sacks. 2000. Science. 290:1351-1354). We have now characterized nine salivary proteins of Phlebotomus papatasi, the vector of Leishmania major. One of these salivary proteins, extracted from SDS gels and having an apparent mol wt of 15 kD, was able to protect vaccinated mice challenged with parasites plus SGH. A DNA vaccine containing the cDNA for the predominant 15-kD protein (named SP15) provided this same protection. Protection lasted at least 3 ino after immunization. The vaccine produced both intense humoral and delayed-type hypersensitivity (DTH) reactions. B cell-deficient mice immunized with the SP15 plasmid vaccine successfully controlled Leislimania infection when injected with Leislimania plus SGH. These results indicate that DTH response against saliva provides most or all of the protective effects of this vaccine and that salivary gland proteins or their cDNAs are viable vaccine targets against leishmaniasis.

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.

Some pathogens (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, Leishmania spp) have been shown to persist in their host after clinical cure, establishing the risk of disease reactivation. We analyzed the conditions necessary for the long term maintenance of Leishmania major in genetically resistant C57BL/6 mice after spontaneous healing of their dermal lesions. Interleukin (IL)-10 was found to play an essential role in parasite persistence as sterile cure was achieved in IL-10-deficient and IL-4/IL-10 double-deficient mice. The requirement for IL-10 in establishing latency associated with natural infection was confirmed in IL-10-deficient mice challenged by bite of infected sand flies. The host-parasite equilibrium was maintained by CD4+ and CD8+ T cells which were each able to release IL-10 or interferon (IFN)-gamma, and were found to accumulate in chronic sites of infection, including the skin and draining lymph node. A high frequency of the dermal CD4+ T cells released both IL-10 and IFN-gamma. Wild-type mice treated transiently during the chronic phase with anti-IL-10 receptor antibodies achieved sterile cure, suggesting a novel therapeutic approach to eliminate latency, infection reservoirs, and the risk of reactivation disease.

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.

The vectorial competence of Phlebotomus sergenti for 3 Old World species of Leishmania , L. tropica , L. major and L. donovani , was investigated in vivo and by in vitro midgut binding assays using living promastigotes and purified lipophosphoglycan (LPG). P. sergenti consistently showed a high specificity for L. tropica strains, which were able to develop mature, potentially transmissible infections. The loss of infection with L. major and L. donovani correlated with the excretion of the digested bloodmeal. These strains were able to produce sustained infections in the midguts of their appropriate vectors, P. papatasi and P. argentipes , respectively. In in vitro binding assays, a significantly higher number of L. tropica procyclic promastigotes attached to the midgut lining of P. sergenti , compared to those of L. major and L. donovani ( P < 0·05). The prediction that the species specificity of midgut attachment is controlled by polymorphic structures on the parasite LPG was supported by the finding that P. sergenti midguts were intensely stained following incubation with purified phosphoglycan (PG) from L. tropica compared with PGs from L. major or L. donovani . The results provide further evidence that LPG structural polymorphisms are driven by the species diversity of molecules present on the sandfly midgut that function as parasite attachment sites.

Sand fly saliva contains a rich array of pharmacologically active compounds whose primary function is to prevent the hemostatic mechanisms of the host. Several studies have ascribed immunosuppressive properties to sand fly saliva as well as an exacerbative effect on Leishmania infectivity for their mammalian hosts. This review provides a comprehensive account of sand fly salivary components, the immunomodulatory properties exhibited by some of its molecules, and describes the findings concerning the influence of saliva on Leishmania infections. The potential use of saliva as part of an anti-Leishmania vaccine for the mammalian host is also addressed.

The saliva of bloodsucking arthropods contains a large array of pharmacologically active compounds that assist hematophagy, Arthropod saliva is also responsible for causing uncomfortable allergic responses in its vertebrate hosts. In this article, we investigate whether the sand fly Phlebotomus papatasi, known to produce a strong delayed-type hypersensitivity (DTH) in humans. could benefit from, and possibly adaptively induce, this response in their vertebrate hosts. In this study, we show that flies fed on humans to completion nearly twice as fast in DTH sites as compared with normal skin sites. DTH sites had significantly larger blood flow as measured by the laser Doppler method. Sand flies feeding at sites in mouse ears that had a DTH response also fed faster than at normal sites. We conclude that in the case of P, papatasi, and possibly other arthropods such as fleas and bed bugs, the strong saliva-induced DTH response may reflect an adaptation of the fly to manipulate host immunity for the insect's own advantage.

Neurocysticercosis (NCC), which is caused by infection with the larval stage of the pork tapeworm (Taenia solium), is now recognized as a major cause of neurologic diseases in countries where the infection is endemic. Migration of persons from these countries is resulting in diagnosis and local transmission in nonendemic countries at increasing rates. In the present study, immunoblotting and an ELISA were carried out using antigens of T. solium cysticerci fractionated by isoelectric focusing and serum samples from patients with NCC, alveolar (AE) or cystic echinococcosis (CE), and other diseases. Immunoblot analysis revealed antigens fractionated by isoelectric focusing (pH 9.2-9.6) either from cyst fluid of T. solium cysticerci or from intact cysts had unique components (glycoproteins) highly specific and sensitive for detection of NCC exclusively. All confirmed NCC serum samples (53 of 53) recognized at least three major bands of 10-26-kD of fractions with pH 9.2-9.6 from either intact cysts or cyst fluid. These bands were not recognized by sera from patients with other parasitic diseases including AE (0 of 34), CE (0 of 36), or other heterologous parasitoses (0 of 77), patients with hepatoma (0 of 19) or sarcoidosis (0 of 11), or sera from healthy controls (0 of 29). The ELISA using the antigens showed the same sensitivity and specificity for differentiation of NCC (53 of 53) from other diseases (0 of 107) or healthy individuals (0 of 29). Both immunoblotting and the ELISA using the fractionated antigens readily differentiated all NCC from AE or CE in a blind test of 29 serum samples of persons with NCC, CE, and AE. Antigens fractionated from cyst fluid of T. solium cysticerci by a simple, single-step isoelectric focusing (pH 9.2-9.6) are highly specific and sensitive for differential serodiagnosis of NCC in immunoblotting and/or an ELISA.

We have developed a model of cutaneous leishmaniasis due to Leishmania major that seeks to mimic the natural conditions of infection. 1,000 metacyclic promastigotes were coinoculated with a salivary gland sonicate (SGS) obtained from a natural vector, Phlebotomus papatasii, into the ear dermis of naive mice or of mice preexposed to SGS. The studies reveal a dramatic exacerbating effect of SGS on lesion development in the dermal site, and a complete abrogation of this effect in mice preexposed to salivary components. In both BALB/c and C57Bl/6 (B/6) mice, the dermal lesions appeared earlier, were more destructive, and contained greater numbers of parasites after infection in the presence of SGS. Furthermore, coinoculation of SGS converted B/6 mice into a nonhealing phenotype. No effect of SGS was seen in either IL-4- deficient or in SCID mice. Disease exacerbation in both BALB/c and B/6 mice was associated with an early (6 h) increase in the frequency of epidermal cells producing type 2 cytokines. SGS did not elicit type 2 cytokines in the epidermis of mice previously injected with SGS. These mice made antisaliva antibodies that were able to neutralize the ability of SGS to enhance infection and to elicit IL-4 and IL-5 responses in the epidermis. These results are the first to suggest that for individuals at risk of vector-borne infections, history of exposure to vector saliva might influence the outcome of exposure to transmitted parasites.

An endemic focus of cutaneous leishmaniasis caused by Leishmania tropica was identified in the district of Bani Kinana, northern Jordan. Fifty-five autochthonous cases were detected from February to September 1994 in a population of about 50,000. Most cases (74.5%) were from the villages of Malka and Um-Quais. The lesions were typically small, dry and mostly located on the face and extremities. Multiple lesions were common. Incidence was higher in females than in males and familial clustering of cases was observed in houses on the outskirts of villages. A leishmanin survey showed a 23.3% positivity rate in the populations of Malka and Um-Quais. The rate of positivity was low in the younger age groups and increased with age. There was some evidence that the disease is a zoonosis in this focus.

The status of sandflies as vectors of cutaneous leishmaniasis in the southern Jordan Valley was investigated during 1992. Sandflies were collected from domestic habitats and from burrows of Psammomys obesus, Of 686 Phlebotomus papatasi females collected from burrows, fourteen harboured promastigotes in their guts. On the other hand, none of 1446 P.papatasi females collected from domestic habitats were found infected. The highest infection rate (5.5%) was recorded in November at the end of the sandfly season. Six leishmanial stocks isolated from P.papatasi females were typed by cellulose acetate electrophoresis using the six enzymes G6PDH, 6PGDH, PGI, PGM, FK and ME. Five of the leishmanial stocks were identical to a Leishmania major reference strain (MHOM/SU/73/5-ASKH), The sixth isolate was a 6PGDH variant of L.major. These findings present the first direct evidence of the role of P.papatasi as a vector of L.major in Jordan.