Search

Leishmania infections in American veterans of Iraq and Afghanistan have raised concern that veterans could serve as reservoirs of Old World parasites for domestic vector populations. A survey of sand flies on three U.S. Army facilities in the southern United States was conducted to identify potential vectors. Five species, including two new state records, are reported for Fort Hood, TX. Very few flies were detected in Fort Bragg, NC. Large numbers of a man-biting species, Lutzomyia shannoni, were trapped on Fort Campbell, KY. Weekly activity patterns for dominant species are presented. In addition, an infection experiment was conducted to determine if a domestic sand fly is susceptible to infection with Old World Leishmania major. Lu. shannoni became infected and supported Le. major up to 6 days postprandial. Metacyclogenesis and actual transmission of Le. major to an uninfected mouse did not occur because infected flies did not take subsequent blood meals. [ABSTRACT FROM AUTHOR]

Laboratory colonies of phlebotomine sand flies are necessary for experimental study of their biology, behaviour and mutual relations with disease agents and for testing new methods of vector control. They are indispensable in genetic studies and controlled observations on the physiology and behaviour of sand flies, neglected subjects of high priority. Colonies are of particular value for screening insecticides. Colonized sand flies are used as live vector models in a diverse array of research projects, including xenodiagnosis, that are directed toward control of leishmaniasis and other sand fly-associated diseases. Historically, labour-intensive maintenance and low productivity have limited their usefulness for research, especially for species that do not adapt well to laboratory conditions. However, with growing interest in leishmaniasis research, rearing techniques have been developed and refined, and sand fly colonies have become more common, enabling many significant breakthroughs. Today, there are at least 90 colonies representing 21 distinct phlebotomine sand fly species in 35 laboratories in 18 countries worldwide. The materials and methods used by various sand fly workers differ, dictated by the availability of resources, cost or manpower constraints rather than choice. This paper is not intended as a comprehensive review but rather a discussion of methods and techniques most commonly used by researchers to initiate, establish and maintain sand fly colonies, with emphasis on the methods proven to be most effective for the species the authors have colonized. Topics discussed include collecting sand flies for colony stock, colony initiation, maintenance and mass-rearing procedures, and control of sand fly pathogens in colonies.

Small mammals have difficulty maintaining body temperature under anesthesia. This hypothermia is a potential detriment not only to the health and comfort of the animal but also to the integrity of any treatment given or data gathered during the anesthetic period. Using an external warming device to assist with temperature regulation can mitigate these effects. In this study, we investigated the ability of an advanced warming device that uses far-infrared (FIR) heating and responds to real-time core temperature monitoring to maintain a normothermic core temperature in guinea pigs. Body temperatures were measured during 30 min of ketamine-xylazine general anesthesia with and without application of the heating device. The loss of core body heat from anesthetized guinea pigs under typical (unwarmed) conditions was significant, and this loss was almost completely mitigated by application of the FIR heating pad. The significant difference between the temperatures of the actively warmed guinea pigs as compared with the control group began as early as 14 min after anesthetic administration, leading to a 2.6 °C difference at 30 min. Loss of core body temperature was not correlated with animals' body weight; however, weight influences the efficiency of FIR warming slightly. These study results show that the FIR heating device accurately controls core body temperature in guinea pigs, therefore potentially alleviating the effects of body heat loss on animal physiology.

Animal models are often used to study hematophagous insect feeding behavior and evaluate products such as topical repellents. However, when these models are used the study animals often experience significant drops in core body temperature because of the effects of anesthesia. This study used a guinea pig model to investigate whether maintaining a normothermic core body temperature during anesthesia influenced the rate of Anopheles stephensi and Phlebotomus papatasi blood feeding. Experiments were conducted with anesthetized animals that had their body temperatures either maintained with a warming device or were allowed to drop naturally. Results showed that when guinea pigs were actively warmed by a heating device, An. stephensi feeding behavior was similar at the beginning and end of anesthesia. However, when a warming device was not used, fewer An. stephensi took a blood meal after the animals' temperatures had dropped. Phlebotomus papatasi were not as sensitive to changes in temperature and feeding rates were similar whether a warming device was used or not. These results are discussed and it is recommended that warming devices are used when conducting feeding experiments with insects sensitive to changes in host body temperature, such as An. stephensi.

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., (© 2014 The Society for Vector Ecology.)

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., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Cutaneous leishmaniasis is a neglected, vector-borne parasitic disease and is responsible for persistent, often disfiguring lesions and other associated complications. Leishmania, causing zoonotic cutaneous leishmaniasis (ZCL) in the Old World are mainly transmitted by the predominant sand fly vector, Phlebotomus papatasi. To date, there is no efficient control measure or vaccine available for this widespread insect-borne infectious disease., Methodology/principal Findings: A survey was carried out to study the abundance of different natural gut flora in P. papatasi, with the long-term goal of generating a paratransgenic sand fly that can potentially block the development of Leishmania in the sand fly gut, thereby preventing transmission of leishmania in endemic disease foci. Sand flies, in particular, P. papatasi were captured from different habitats of various parts of the world. Gut microbes were cultured and identified using 16S ribosomal DNA analysis and a phylogenetic tree was constructed. We found variation in the species and abundance of gut flora in flies collected from different habitats. However, a few Gram-positive, nonpathogenic bacteria including Bacillus flexus and B. pumilus were common in most of the sites examined., Conclusion/significance: Our results indicate that there is a wide range of variation of aerobic gut flora inhabiting sand fly guts, which possibly reflect the ecological condition of the habitat where the fly breeds. Also, some species of bacteria (B. pumilus, and B. flexus) were found from most of the habitats. Important from an applied perspective of dissemination, our results support a link between oviposition induction and adult gut flora.

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.

Our goal was to study the effectiveness of the insecticide imidacloprid as a systemic control agent. First, to evaluate the blood-feeding effect, we fed adult female Phlebotomus papatasi with imidacloprid-treated rabbit blood and monitored blood-feeding success and survival. Second, to evaluate the feed-through effectiveness of this insecticide, we fed laboratory rats and sand rats with insecticide-treated food and evaluated the survival of sand fly larvae feeding on rodents' feces. In the blood-feeding experiment, 89.8% mortality was observed with the higher dose (5 mg/ml) and 81.3% with the lower dose (1 mg/ml). In the larvicide experiments, both sand fly species demonstrated a typical dose-response curve with the strongest lethal effect for the 250 ppm samples. Lutzomyia longipalpis larvae, however, were less sensitive. In all experiments, 1(st) instar larvae were more sensitive than the older stages. First instar P. papatasi larvae feeding on sand rat feces passed the larvicidal threshold of 90% mortality at doses higher than 50 ppm. In comparison, in older stages 90% mortality was obtained with a dose of only 250 ppm. Overall, results support the feasibility of imidacloprid as a systemic control agent that takes advantage of the tight ecological association between the reservoir host and the sand fly vector., (© 2011 The Society for Vector Ecology.)

Oviposition behavior is a fairly neglected aspect in our understanding of the biology of sand flies. In this study, we used a comparative approach using both new- and old-world species (Lutzomyia longipalpis and Phlebotomus papatasi) in choice and no-choice oviposition chambers to evaluate the effect of old sand fly colony remains (frass), conspecific eggs, and their combination on oviposition rates of these sand flies. We also tested the effect of egg washing with de-ionized water on oviposition rates. In both choice and no-choice experiments, sand fly species laid more eggs on a substrate containing frass. The effect of eggs alone was not significant but showed a positive trend. Furthermore, for both sand fly species, the effect of the combined treatment was sub-additive suggesting a potential inhibitory effect of one factor on the other. Egg washing did not have a significant effect. The choice and no-choice experimental designs did not differ in their outcomes suggesting the choice-design could serve as an effective high throughput method for screening oviposition attractants/stimulants., (© 2011 The Society for Vector Ecology.)

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., (© 2011 The Society for Vector Ecology.)

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.

An in vitro feeding method using chicken-skin membranes and human blood was compared with an established in vivo method using anesthetized hamsters for blood-feeding mass-reared phlebotomine sand flies. Parameters measured were percentage of sand flies taking blood meals, number of eggs laid per female, and percentage of eggs that hatched. Females from a long established (>20 yr) colony of Phlebotomus papatasi (Scopoli) from Israel landed on and started feeding sooner on the hamster than on the membrane. However, when sand flies were allowed access to the membrane feeder for the same length of time as the anesthetized hamster, the feeding percentages were not significantly different and were usually better on the membrane feeder if flies were allowed access for a longer time. Egg production and percent hatch between the two feeding methods were not statistically different. Based on these results, we conclude that the chicken-skin membrane feeding method is a viable alternative to the use of live animals for feeding large numbers of P. papatasi.

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.

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.

One of the most significant modern day efforts to prevent and control an arthropod-borne disease during a military deployment occurred when a team of U.S. military entomologists led efforts to characterize, prevent, and control leishmaniasis at Tallil Air Base (TAB), Iraq, during Operation Iraqi Freedom. Soon after arriving at TAB on 22 March 2003, military entomologists determined that 1) high numbers of sand flies were present at TAB, 2) individual soldiers were receiving many sand fly bites in a single night, and 3) Leishmania parasites were present in 1.5% of the female sand flies as determined using a real-time (fluorogenic) Leishmania-generic polymerase chain reaction assay. The rapid determination that leishmaniasis was a specific threat in this area allowed for the establishment of a comprehensive Leishmaniasis Control Program (LCP) over 5 mo before the first case of leishmaniasis was confirmed in a U.S. soldier deployed to Iraq. The LCP had four components: 1) risk assessment, 2) enhancement of use of personal protective measures by all personnel at TAB, 3) vector and reservoir control, and 4) education of military personnel about sand flies and leishmaniasis. The establishment of the LCP at TAB before the onset of any human disease conclusively demonstrated that entomologists can play a critical role during military deployments.

Visceral leishmaniasis, caused by protozoa of the genus Leishmania donovani complex, is a vectorborne zoonotic infection that infects humans, dogs, and other mammals. In 2000, this infection was implicated as causing high rates of illness and death among foxhounds in a kennel in New York. A serosurvey of >12,000 foxhounds and other canids and 185 persons in 35 states and 4 Canadian provinces was performed to determine geographic extent, prevalence, host range, and modes of transmission within foxhounds, other dogs, and wild canids and to assess possible infections in humans. Foxhounds infected with Leishmania spp. were found in 18 states and 2 Canadian provinces. No evidence of infection was found in humans. The infection in North America appears to be widespread in foxhounds and limited to dog-to-dog mechanisms of transmission; however, if the organism becomes adapted for vector transmission by indigenous phlebotomines, the probability of human exposure will be greatly increased.

Using massive cDNA sequencing, proteomics and customized computational biology approaches, we have isolated and identified the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis. Out of 550 randomly isolated clones from a full-length salivary gland cDNA library, we found 143 clusters or families of related proteins. Out of these 143 families, 35 were predicted to be secreted proteins. We confirmed, by Edman degradation of Lu. longipalpis salivary proteins, the presence of 17 proteins from this group. Full-length sequence for 35 cDNA messages for secretory proteins is reported, including an RGD-containing peptide, three members of the yellow-related family of proteins, maxadilan, a PpSP15-related protein, six members of a family of putative anticoagulants, an antigen 5-related protein, a D7-related protein, a cDNA belonging to the Cimex apyrase family of proteins, a protein homologous to a silk protein with amino acid repeats resembling extracellular matrix proteins, a 5'-nucleotidase, a peptidase, a palmitoyl-hydrolase, an endonuclease, nine novel peptides and four different groups of proteins with no homologies to any protein deposited in accessible databases. Sixteen of these proteins appear to be unique to sand flies. With this approach, we have tripled the number of isolated secretory proteins from this sand fly. Because of the relationship between the vertebrate host immune response to salivary proteins and protection to parasite infection, these proteins are promising markers for vector exposure and attractive targets for vaccine development to control Leishmania chagasi infection.

Visceral leishmaniasis is an endemic protozoal disease of humans and dogs in tropical and subtropical regions in Asia, Africa, southern Europe, Central America, and South America, where sand flies (genera Phlebotomus and Lutzomyia) act as vectors. An outbreak in a New York foxhound kennel and subsequent surveillance revealed widespread Leishmania infantum infection of dogs in the United States, outside the known range of the vector sand flies. For this study, we conducted surveillance for sand flies during the summers of 2001 and 2002 at two areas: on the grounds of the New York kennel and at the Institute of Ecosystem Studies (IES) 10 km away. CO2-baited light traps were used for surveillance. Populations of Lutzomyia vexator, not previously known in New York, were widespread and locally abundant (range, 0.26-1.16 flies/trap night) at the IES site. These populations showed a bimodal, midsummer activity peak and were most abundant on steep slopes within mature mixed hardwood forests. Further research will be necessary to determine whether the New York populations of L. vexator in the vicinity of the kennel could be involved in transmission of canine leishmaniasis.

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.

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.

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.

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., (Copyright 2000 Academic Press.)

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.

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.

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.

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 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.

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 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.

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.

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.

Genetic immunization is a promising gene therapy approach for the prevention and treatment of infectious disease. Plasmid DNA expressing genes of pathogens is directly introduced into host cells and specific cell-mediated and/or humoral immune responses are elicited against the encoded protein. Leishmaniasis is a significant world-wide health problem for which no vaccine exists. In susceptible animals, such as BALB/c mice, protection from leishmaniasis requires induction of a Thl immune response. In this study, cell-mediated immunity to Leishmania major (L. major) was induced by injecting BALB/c mice intradermally with plasmid DNA expressing the conserved L. major cell surface glycoprotein gp63 (gp63-pcDNA-3). CD4 T lymphocytes from gp63-pcDNA-3-immunized mice proliferated and produced IFN-gamma (but not IL-4) when stimulated in vitro with freeze-thawed parasites, consistent with a Th1 immune response. In contrast, lymphocyte proliferation in animals immunized with freeze-thawed parasites was associated with IL-4 (but not IFN-gamma) production, suggesting a nonprotective Th2 response. Challenge studies revealed that gp63-pcDNA-3 vaccination protected 30% of susceptible mice (21 of 70) from Leishmania infection while neither gp63 protein (0 of 20) nor freeze-thawed parasite vaccines (0 of 50) were efficacious. Dendritic cells derived from skin of gp63-pcDNA-3-injected mice also immunized naive recipients and protected them from leishmaniasis. We conclude that gp63-pcDNA-3 genetic vaccination results in a CD4-dependent Th1 immune response that correlates with protection from disease, and suggest that skin-derived dendritic cells are involved in priming this response.

An initial evaluation of insecticide barrier spraying directed against sand fly vectors of cutaneous leishmaniasis was done in a nonclimax forested area with heavy undergrowth in Peten, Guatemala. A 100 m-wide swath of vegetation was sprayed once with a 1:3 mixture of cyfluthrin insecticide and a palm oil carrier using back-pack sprayers to simulate a central cantonment area in one site while another site remained as an untreated control. Prior to spraying and throughout 87 days post-treatment, sand fly populations were monitored at both sites with light traps set at ground and canopy levels at 50-m intervals radiating out from the centers of the cantonments, 150-m in the four cardinal directions. A total of 2,876 female sand flies were captured, representing 16 species. Three species, Brumptomyia galindoi, Lutzomyia panamensis, and Lu, ovallesi, comprised 70% of the total collection. The single insecticide barrier significantly reduced sand flies from reaching the cantonment area for more than 80 days, while sand fly populations outside the treated cantonment and in the untreated (control) cantonment remained high (52 sand flies in the treated cantonment versus 235 sand flies in the untreated cantonment).

We have recently demonstrated that Lutzomyia longipalpis salivary gland homogenates (SGH) inhibited the multiplication of Leishmania promastigotes in vitro. The present work shows that Leishmania amazonensis sensitivity to SGH is correlated to the phase of promastigote in vitro growth and can be decreased by the addition of hemin to the culture medium. The possible relevance of these in vitro results is discussed in relation to the development of Leishmania parasites within their sand fly vectors.

Leishmania braziliensis is endemic in Guatemala and Belize in Central America. To help identify the vector(s) of this parasite in Guatemala, phlebotomine sand flies that were aspirated from the clothing of collectors at Tikal National Park in the Department of the Peten were examined for flagellates. Lutzomyia ovallesi was found infected with flagellates that were identified as L. braziliensis by isoenzyme electrophoresis. The isoenzyme profile of this isolate matched those from humans from the same area.

Recent technical and procedural advances in mass rearing of sand flies have resulted in larger, healthier, and less labor-intensive colonies. We now maintain closed colonies of Phlebotomus papatasi, P. duboscqi, P. argentipes, and Lutzomyia longipalpis which produce up to 1,000 females per week, in excess of colony-maintenance requirements, for use in research. Advances include larval food preparation in acrylic-plastic incubator cabinets, strict regulation of food quantity and moisture in 500-ml plaster-lined rearing jars, use of large plaster-lined adult holding/mating cages and vacuum-powered aspirators for trauma-free handling of adults.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Monoclonal antibodies specific to the infective-stage promastigotes of Leishmania major are needed for developing rapid diagnostic assays of infected sand flies. An in vitro immunization protocol was applied for the production of monoclonal antibodies using small amounts of L. major. Infective-stage promastigotes were isolated from sand flies (Phlebotomus papatasi) 7-10 days after infection and used as antigen for immunization. Two weeks after a primary immunization, murine splenocytes were removed and immunized in vitro with antigen in murine EL-4 thymoma cell conditioned medium. Three fusions were performed using X63-Ag.653 myeloma cells as fusion partners and two fusions were performed using FOX-NY cells. Antibodies specific to promastigotes were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Initially 56 monoclonal antibodies were selected, and their species and stage specificity were determined using both an ELISA and an indirect fluorescent antibody assay (IFA). Twelve monoclonal antibodies showed species specificity to L. major when tested against four sympatric species of Leishmania. Four other monoclonal antibodies showed species and infective-stage specificity to L. major promastigotes. When tested in immunoblots, all four species- and stage-specific monoclonal antibodies bound to five protein bands that were unique to the infective-stage promastigotes.

An agar plating technique was used to determine the number of amastigotes ingested by Lutzomyia longipalpis fed on papules on Mesocricetus auratus caused by Leishmania mexicana amazonensis and on lesions on Mystromys albicaudatus caused by Leishmania braziliensis panamensis. The technique involved homogenizing sand flies after bloodfeeding on the infected animals and spreading the homogenate over the surface of agar plates. A great variation in the number of amastigotes ingested by individual sand flies was demonstrated. Not all amastigotes ingested developed anterior stomodeal infections.

Leishmania parasites from animals, man or insect vectors were characterized by the gel electrophoresis of restriction endonuclease enzyme-produced mitochondrial (kinetoplast) DNA (kDNA) fragments and/or by DNA-DNA hybridization with 32P-labelled cloned, or uncloned, kDNA fragment probes from type isolates. The electrophoretic separation of kDNA fragments is a sensitive method for detecting genetic similarities and differences among Leishmania. Parasites with similar kDNA restriction fragment patterns belong to the same schizodeme and schizodeme analysis is useful for studying Leishmania populations. Cloned, species-specific kDNA probes detected Leishmania in sandflies and in liver, spleen or blood preparations from infected animals. Cloned DNA probes also hybridized to immobilized kDNA from in vitro cultivated promastigotes and detected as few as 100 parasites in a species-specific manner. Sensitive DNA hybridization probes should be useful in research on the immunology, chemotherapy or epidemiology of animal and human leishmaniasis.

Cross-infection experiments demonstrated that Ascogregarina barretti, from Aedes triseriatus, completes its life cycle in Aedes geniculatus. Parasite numbers were comparable to infection with Ascogregarina geniculati, making the separation of these parasites by host preference difficult. However, electrophoresis readily distinguished isoenzymes from the two morphologically similar gregarine species. Different migration rates were obtained for isocitrate dehydrogenase 1 and 2, lactate dehydrogenase, and malate dehydrogenase. The migration rates were also different for parasite and host isoenzymes. When a single, heavily infected gut was subjected to electrophoresis the isocitrate dehydrogenase bands of each were clearly distinguishable on the same electrophoretic track. Electrophoresis appears to be a reliable method for resolving taxonomic complications of mosquito gregarines, a group often with wide host specificities and variable taxonomic characters.