Orientation of colonized sand flies Phlebotomus papatasi, P. duboscqi, and Lutzomyia longipalpis (Diptera: Psychodidae) to diverse honeys using a 3-chamber in-line olfactometer.

A 3-chamber in-line olfactometer designed for use with sand flies is described and tested as a high-throughput method to screen honeys for attractiveness to Phlebotomus papatasi (four geographic isolates), P. duboscqi (two geographic isolates), and Lutzomyia longipalpis maintained in colonies at the Walter Reed Army Institute of Research. A diversity of unifloral honey odors were evaluated as a proxy for the natural floral odors that sand flies may use in orientation to floral sugar sources in the field. In the 3-chamber in-line olfactometer, the choice modules come directly off both sides of the release area instead of angling away as in the Y-tube olfactometer. Of the 25 honeys tested, five had a significant attraction for one or more of the sand fly isolates tested. This olfactometer and high-throughput method has utility for evaluating a diversity of natural materials with unknown complex odor blends that can then be down-selected for further evaluation in wind tunnels and/or field scenarios.

The characterization of the Phlebotomus papatasi transcriptome.

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 47 615 generated sequences was cleaned and assembled into 17 120 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.

Oral treatment of rodents with insecticides for control of sand flies (Diptera: Psychodidae) and the fluorescent tracer technique (FTT) as a tool to evaluate potential sand fly control methods.

In laboratory studies, insecticides (diflubenzuron, novaluron, methoprene and, pyriproxyfen) that have been incorporated into rodent diets were effective as feed-throughs against sand fly larvae. Novaluron also was effective against sand fly larvae at low concentrations and under simulated field conditions. Ivermectin has been shown to be effective as a systemic insecticide, killing 100% of blood-feeding sand flies for up to seven d after rodents were treated. The fluorescent tracer technique (FTT) is the use of certain fluorescent dyes (rhodamine B or uranine O) as feed-through transtadial biomarkers for phlebotomine sand flies, systemic biomarkers for blood-feeding sand flies, and permanent markers for nectar-feeding sand flies. The results of these laboratory studies provide proof of concept for the FTT and indicate that the FTT could be used to delineate specific foci with rodent/sand fly associations that would be susceptible to control by using feed-through or systemic insecticides, or foci where insecticide-treated sugar baits could be used against sand flies.

Evaluation of juvenile hormone analogues as rodent feed-through insecticides for control of immature phlebotomine sandflies.

The juvenile hormone analogues methoprene and pyriproxyfen were evaluated as rodent feed-through insecticides for control of immature stages of the sandfly Phlebotomus papatasi Scopoli (Diptera: Psychodidae). The development and survival of P. papatasi second-instar larvae fed faeces from Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing methoprene (0, 9.788, 97.88 or 978.8 p.p.m.) or pyriproxyfen (0, 9.82, 98.2 or 982 p.p.m.) were evaluated. The faeces of methoprene-treated hamsters greatly reduced the percentage of larvae that pupated at all concentrations tested and prevented adult emergence at all but the lowest concentration (9.788 p.p.m.). Pyriproxyfen prevented both pupation and adult emergence at all concentrations tested. The results of this study suggest that a control strategy using rodent baits containing juvenile hormone analogues to control phlebotomine sandflies that live in rodent burrows and feed on rodent faeces may be possible. As rodent reservoirs and vectors of Leishmania major live in close association in many parts of the Middle East, control of the transmission of the agent of zoonotic cutaneous leishmaniasis may also be possible.

Ivermectin as a rodent feed-through insecticide for control of immature sand flies (Diptera: Psychodidae).

Ivermectin was evaluated as a potential rodent feed-through for the control of immature stages of Phlebotomus papatasi. The survival of sand fly larvae fed feces of Syrian hamsters (Mesocricetus auratus) that had been fed a diet containing 0, 2, 6, 10, 20, 60, or 100 ppm ivermectin was measured. Sand fly larvae fed the feces of ivermectin-treated hamsters had significantly reduced survival, with 100% mortality of larvae fed feces of hamsters fed a diet containing 20, 60, and 100 ppm ivermectin. The results of this study suggest that a control strategy using rodent baits containing ivermectin to control phlebotomine sand flies may be possible. Because rodent reservoirs and sand fly vectors of Leishmania major live in close association in many parts of the Middle East, the control of transmission of the agent of zoonotic cutaneous leishmaniasis also may be possible.

Evaluation of novaluron as a feed-through insecticide for control of immature sand flies (Diptera: Psychodidae).

The development and survival of sand fly Phlebotomus papatasi Scopoli (Diptera: Psychodidae) larvae fed feces of Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing novaluron were evaluated. In total, six larval diets were used in sand fly larval bioassays. Four groups of larvae were fed feces of hamsters that had been maintained on a diet containing either 0, 9.88, 98.8, or 988 ppm novaluron. Two additional groups were fed a larval diet composed of equal parts composted rabbit feces and rabbit chow containing either 0 or 988 ppm novaluron. No pupation, hence no adult emergence, occurred when larvae were fed feces of hamsters that were fed diets containing novaluron. The mortality of sand flies fed feces of treated hamsters occurred during larval molts. The results of this study suggest that a control strategy using rodent baits containing novaluron to control phlebotomine sand flies and zoonotic cutaneous leishmaniasis may be possible.

Laboratory evaluation of diflubenzuron as a feed-through for control of immature sand flies (Diptera: Psychodidae).

The benzoylurea chitin synthesis inhibitor diflubenzuron was evaluated as a rodent feed-through for the control of immature stages of Phlebotomus papatasi Scopoli (Diptera: Psychodidae). The development and survival of second instars of P. papatasi larvae that were fed feces from Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing 0, 8.97, 89.7, or 897 ppm diflubenzuron was evaluated. No pupation or adult emergence occurred when larvae were fed feces from hamsters that were fed diets containing diflubenzuron. The mortality of sand flies fed feces from treated hamsters was coincident with pupation of the controls, suggesting a specific effect on the larval-to-pupal molt. The results of this study suggest that a control strategy using rodent baits containing diflubenzuron for phlebotomine sand flies and zoonotic cutaneous leishmaniasis may be possible.

Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi.

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.

Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein.

Leishmania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leishmania 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 mo 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 Leishmania infection when injected with Leishmania 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.

Rhesus monkey model for Leishmania major transmitted by Phlebotomus papatasi sandfly bites.

Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection.

Speciation and population structure in the morphospecies Lutzomyia longipalpis (Lutz & Neiva) as derived from the mitochondrial ND4 gene.

Recent studies have suggested that the phlebotomine sand fly Lutzomyia longipalpis (Diptera: Psychodidae), the principal vector of visceral leishmaniasis in the Neotropics, may consist of several allopatric sibling species. Phylogenetic and population genetic analyses of nucleotide variation in a 618-bp fragment of the mitochondrial ND4 gene were carried out on specimens of Lu. longipalpis from several locations in Central and South America. The analyses were concordant with previous findings, indicating that certain allopatric populations of Lu. longipalpis have become sufficiently differentiated as to represent sibling species. Phylogenetic analyses revealed deep genetic divisions between four clades represented by specimens from northern South America, Brazil, Central America, and an isolated Colombian population. Strong differentiation also was observed between certain populations within the first two clades. Partitioning of genetic diversity within and between Central American populations did not show the presence of more than one species in the region. However, distance, even within the 70-km range of the Honduran collection sites, was found to have a remarkably strong effect on gene flow. The highly subdivided population structure may be due to the patchiness of their distribution. F(ST) values comparing a Guatemalan population with several Honduran populations revealed a level of differentiation associated with a negligible rate of gene flow.

Simulium vittatum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psychodidae) salivary gland hyaluronidase activity.

Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum (Zetterstedt) is described, and its optimal pH determined. Salivary activity was reduced significantly after a blood meal, indicating that it was secreted after blood feeding. Phlebotomus papatasi (Scopoli) also exhibited salivary hyaluronidase activity. These results indicate that hematophagous pool-feeding insects may secrete this enzyme to help the spread of salivary antihemostatic agents in the vicinity of the feeding lesion, and perhaps to increase the size of the feeding lesion itself. Additionally, this enzyme may affect local host immune reactions and promote arboviral transmission.

The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis.

In the process of sequencing a subtracted cDNA library from the salivary glands of the sand fly Lutzomyia longipalpis, we identified a cDNA with similarities to gene products of the adenosine deaminase family. Prompted by this cDNA finding, we detected adenosine deaminase activity at levels of 1 U/mg protein in salivary gland homogenates. The activity was significantly reduced following a blood meal indicating its apparent secretory fate. The native enzyme has a K(m) of approximately 10 microM, an isoelectric pH between 4.5 and 5.5, and an apparent molecular weight of 52 kDa by size exclusion chromatography. The possible role of this enzyme, which converts adenosine to inosine, in the feeding physiology of L. longipalpis is discussed.

Salivary amylase activity of the phlebotomine sand fly, Lutzomyia longipalpis.

Both male and female adult stages of the sand fly Lutzomyia longipalpis have detectable amylase activity in their salivary glands, as indicated by formation of p-nitrophenyl-alpha-D-maltoside from p-nitrophenyl-alpha-D-octoside and by hydrolysis of 4-nitrophenyl-alpha-D-maltoheptaoside-4,6,-O-ethylidene. No salivary alpha-glucosidase was detected. Amylase activity was also found in the crop and midgut of female flies, although in a smaller amount. Salivary amylase is significantly reduced from the salivary glands immediately after a blood meal, as is the case with salivary alpha-glucosidases in mosquitoes. Presence of salivary gland amylase in these sand flies, and absence of salivary alpha-glucosidase, indicates that in nature these insects may have a significant intake of carbohydrates in the form of starch, as suggested by their plant-feeding behavior, previously demonstrated by Schlein and Warburg (Schlein, Y., Warburg, A., 1986. Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera: Psychodidae) under experimental conditions. Journal of Medical Entomology 23, 11-15), and Alexander and Usma (Alexander, B., Usma, M.C., 1994. Potential sources of sugar for the phlebotomine sandfly Lutzomyia youngi (Diptera: Psychodidae) in a Columbia coffee plantation. Ann. Trop. Med. Parasitol. 88, 543-549).

The salivary 5'-nucleotidase/phosphodiesterase of the hematophagus sand fly, Lutzomyia longipalpis [corrected].

Salivary gland homogenates from adult female Lutzomyia longipalpis sand flies contain large amounts of 5'-nucleotidase and phosphodiesterase activities. Phosphodiesterase activity was found to be associated with 5'-nucleotidase in several independent experiments: (i) it coelutes with 5'-nucleotidase on a molecular sieving column, (ii) it coelutes with 5'-nucleotidase on a chromatofocusing column, and (iii) it has the same thermal inactivation kinetics as the 5'-nucleotidase activity. Additionally, both activities are independent of divalent cations, and both are decreased following a blood meal, suggesting that they reside in the same molecule. The role of salivary nucleotidases and purine nucleotides in blood-feeding by sand flies is discussed.

Laboratory transmission of Rift Valley fever virus by Phlebotomus duboscqi, Phlebotomus papatasi, Phlebotomus sergenti, and Sergentomyia schwetzi (Diptera: Psychodidae).

We examined the potential for Phlebotomus papatasi (Scopoli), Phlebotomus duboscqi (Neveu-Lemarie), Phlebotomus sergenti (Parrot), and Sergentomyia schwetzi (Adler, Theodor, & Parrot) to transmit Rift Valley fever (RVF) virus. After feeding on hamsters that had been inoculated with RVF virus, P. papatasi, P. sergenti, and S. schwetzi became infected and developed disseminated infections. All P. papatasi and P. duboscqi inoculated with RVF virus developed high-titer infections. In contrast, only 41% of the inoculated S. schwetzi contained detectable virus, and infected individuals contained significantly less virus than the two Phlebotomus species. Although 50% of the inoculated P. duboscqi transmitted RVF virus to hamsters, only 14% of P. papatasi and none of the S. schwetzi transmitted this virus. Additional studies are needed to determine the role of sand flies as vectors of RVF virus.

Human immune response to sand fly salivary gland antigens: a useful epidemiological marker?

Antibody (IgG) responses to salivary gland homogenate and to a recombinant salivary protein from the sand fly Lutzomyia longipalpis were investigated using sera from children living in an endemic area of visceral leishmaniasis in Brazil. We classified children into four groups according to their responses to Leishmania antigen: (Group I) positive serology and positive delayed type hypersensitivity (DTH), (Group II) positive serology and negative DTH, (Group III) negative serology and positive DTH, and (Group IV) negative serology and negative DTH. A highly significant correlation was found between anti-salivary gland IgG levels and DTH responses. An L. longipalpis salivary recombinant protein used as an antigen in an enzyme-linked immuno sorbent assay (ELISA) gave a significant but different result. A positive correlation was found between anti-Leishmania IgG and anti-recombinant protein IgG titers. The results indicate that sand fly salivary proteins may be of relevance to the study the epidemiology of leishmaniasis.

Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius apyrase, (ii) a 5'-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the BLAST databases. Following these findings, we observed that the salivary apyrase activity of L. longipalpis is indeed similar to that of Cimex apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for Lutzomyia salivary apyrase. A 5'-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to alpha-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.

Comparison of adjuvants with Leishmania antigens in a guinea pig model to induce delayed-type hypersensitivity responses.

BACKGROUND AND PURPOSE: Guinea pigs have been a traditional model for studies of delayed-type hypersensitivity. They are the natural host of Leishmania enriettii and have been experimentally infected with other species of Leishmania. They have been used as a skin-test model to screen potential antigens for use in diagnostic tests for Leishmania. Use of complete Freund's adjuvant (CFA), along with whole promastigote Leishmania antigen, was necessary to sensitize guinea pigs to invoke a sufficient cell-mediated immune response. However, use of CFA has come under scrutiny by Animal Care and Use Committees due to the pathologic changes associated with its use. METHODS: Thirty-two specific-pathogen-free male Hartley guinea pigs were inoculated with Leishmania antigens alone or mixed with one of three adjuvants (CFA, TiterMax, and liposomes), and were skin tested 2 weeks later. RESULTS: For the Leishmania antigens tested, guinea pigs that received liposomes as an adjuvant had skin-test responses comparable to those of guinea pigs that received CFA. TiterMax was also tested, but cellular responses at antigen test sites were poor. CONCLUSIONS: Liposomes can be used in this model as a safe, effective adjuvant.

Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms.

Resistance to murine leishmaniasis correlates with development of a CD4(+) T helper 1 (Th1)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN), known to promote a Th1 immune response, could provide protection from Leishmania infection, CpG-ODN and freeze-thawed (F/T) Leishmania major were coinjected intradermally into susceptible BALB/c mice. A Leishmania-specific Th1-predominant immune response was induced, and 40% of animals were protected from subsequent challenge with infectious organisms, with 0% protection of animals injected with F/T Leishmania organisms and PBS, F/T organisms and control ODN, or F/T organisms alone. More striking protection (65-95%) was seen in mice first infected with intact Leishmania organisms and then injected with CpG-ODN, either at the site of infection or at a remote site. To determine whether the therapeutic protection provided by CpG-ODN depended on IL-12 and IFN-gamma production, both IFN-gamma-deficient BALB/c mice and BALB/c mice treated with neutralizing anti-IL-12 mAb were first inoculated with Leishmania and then treated with either CpG-ODN, ODN, or PBS. None of these IFN-gamma-deficient mice survived (0/20, 0/20, and 0/20 respectively). Furthermore, neutralization of IL-12 completely abolished the therapeutic protection provided by CpG-ODN (0/20 mice surviving). We conclude that immunostimulatory DNA sequences likely exert systemic effects via IL-12 and IFN-gamma-dependent mechanisms and hold considerable promise as both vaccine adjuvants and potential therapeutic agents in the prevention and treatment of leishmaniasis.