The vectorial competence of Phlebotomus sergenti is specific for Leishmania tropica and is controlled by species-specific, lipophosphoglycan-mediated midgut attachment.

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.

A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut.

The role of the peritrophic matrix (PM) in the development of Leishmania major infections in a natural vector, Phlebotomus papatasi, was investigated by addition of exogenous chitinase to the bloodmeal, which completely blocked PM formation. Surprisingly, the absence of the PM was associated with the loss of midgut infections. The chitinase was not directly toxic to the parasite, nor were midgut infections lost due to premature expulsion of the bloodmeal. Most parasites were killed in chitinase-treated flies within the first 4 h after feeding. Substantial early killing was also observed in control flies, suggesting that the lack of PM exacerbates lethal conditions which normally exist in the blood-fed midgut. Early parasite mortality was reversed by soybean trypsin inhibitor. Allosamadin, a specific inhibitor of chitinase, led to a thickening of the PM, and also prevented the early parasite mortality seen in infected flies. Susceptibility to gut proteases was extremely high in transitional-stage parasites, while amastigotes and fully transformed promastigotes were relatively resistant. A novel role for the PM in promoting parasite survival is suggested, in which the PM creates a barrier to the rapid diffusion of digestive enzymes, and limits the exposure of parasites to these enzymes during the time when they are especially vulnerable to proteolytic damage.

Experimental infection and transmission of Leishmania major by laboratory-reared Phlebotomus bergeroti parrot.

The ability of colony-reared Phlebotomus bergeroti Parrot to successfully acquire and transmit Leishmania major (strain IPAP/EG 89/SI-177) was demonstrated in the laboratory. Female P. bergeroti were fed naturally on infected mice and artificially on infected blood suspension using a chick-skin membrane apparatus. Groups of sand flies, either infected on mice or by membrane feeding, were dissected and examined using light microscopy at 2-6, 8, 10, and 11 days postfeeding. Heavy promastigote infection of the thoracic and abdominal midgut was observed in 10% (2 of 20) of the naturally infected flies. Promastigote maturation was observed in 87% (81 of 93) of the artificially infected sand flies, with promastigotes observed in the cibarium and mouthparts at five days postinfection, and infective metacyclic stage promastigotes observed at eight days postinfection. Ten days postinfection, 31% (10 of 32) of the remaining artificially infected sand flies refed on an uninfected BALB/c mouse. Twenty-eight days following exposure to the infective sand flies, leishmanial lesions were observed on the pads of the mouse's front feet. The development of lesions on mouse foot pads clearly suggests the potential of P. bergeroti to serve as a vector for L. major.

Changes in lipophosphoglycan and gene expression associated with the development of Leishmania major in Phlebotomus papatasi.

Stage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course of L. major infection in a natural vector, Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics in vitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promastigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesis in vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression in vitro, also showed selective expression by promastigotes in the fly, and was used in in situ hybridization studies to demonstrate the positioning of metacyclics in the anterior gut.

Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan.

Phlebotomine vectors can in some instances transmit only certain species of Leishmania. Comparison of a large number of vector/parasite pairs revealed that species-specific differences in vectorial competence were in every case directly correlated with the ability of promastigotes to attach to the sand-fly midgut, the variable outcomes of which were controlled by structural polymorphisms in the surface lipophosphoglycan (LPG) of the parasite. The ability of Phlebotomus papatasi to transmit only Leishmania major could be attributed to the unique, highly substituted nature of L. major LPG that provides for multiple terminally exposed beta-linked galactose residues for binding. While the relatively unsubstituted LPGs of other Leishmania species were unable to mediate promastigote attachment to P. papatasi, they could mediate binding to midguts of Phlebotomus argentipes, which was found to be a potentially competent vector for every Leishmania species examined. The data suggest that at least some phlebotomine vectors differ with respect to the parasite recognition sites which they express and that midgut adhesion is a sufficiently critical component of vectorial competence as to provide the evolutionary drive for LPG structural polymorphisms.

Epidemiology of cutaneous leishmaniasis at a focus monitored by the multinational force and observers in the northeastern Sinai Desert of Egypt.

A longitudinal epidemiologic study of cutaneous leishmaniasis (CL) transmission was conducted between July 1989 and June 1991 in a 1,200-km2 sector of the northeastern Sinai Desert monitored by the Multinational Force and Observers (MFO), an international peace keeping mission between Egypt and Israel. The occurrence of human cases, sand fly density, rodent collection, and isolations of Leishmania confirmed only one of four surveyed locations as a significant focus of CL transmission. Phlebotomus papatasi, the only anthropophilic sand fly species encountered at this focus, comprised more than 96% of the sand fly population and attained human landing densities exceeding 100 sand flies/person/hr during 1990. Seasonal activity of this species ranged from April to November, with highest densities occurring during the period May-September. A peak promastigote infection rate of 2.4% (13 of 534) was observed in P. papatasi during July 1990. Twelve of the 60 (20%) persons at risk during the six months of intense sand fly activity at this site developed lesions consistent with CL; L. major was isolated from nine (75%) of these cases. Leishmania major infection was acquired by two of 22 (9%) sentinel hamsters used during the same period. More than 97% of the 897 wild rodents trapped at this site were desert gerbil species. Leishmania major was the only Leishmania isolated from human, sand fly, wild rodent (Gerbillus pyramidum), and sentinel hamster infections that originated at site Check point 1-Delta, the focus of CL transmission within jurisdiction of the MFO. The altered ecology of this area, created by construction of a dam, may contribute significantly to the transmission dynamics of CL at this focus.

Refractory barriers in the sand fly Phlebotomus papatasi (Diptera: Psychodidae) to infection with Leishmania panamensis.

The life cycle of Leishmania panamensis in Phlebotomus papatasi was studied to characterize barriers limiting parasite colonization, differentiation, migration, and attachment in an unnatural sand fly host. The insects were fed a suspension of L. panamensis-infected macrophages and human erythrocytes, and were examined up to 16 days post-infection by light and electron microscopy. Histologic examination of 401 flies showed the peritrophic membrane to be the first important barrier to parasite establishment in the gut lumen. In most flies, parasites were unable to escape from the closed peritrophic sac, which was either excreted or retained intact in the midgut. After five days, only 31% of the flies were infected; attached parasites colonized the pylorus-ileum and/or colon regions of the hindgut. Anterior migration into the cardia region of the midgut occurred in less than 1% of infected flies; no parasites colonized the foregut. In the bloodmeal and residual bloodmeal, five morphologic forms developed from ingested amastigotes: stumpy, spatulate, elongate, short nectomonad promastigotes, and paramastigotes. Abnormal retention of amastigotes in macrophages and delayed development of promastigote stages was observed. The primary form attached in the hindgut was a pear-shaped haptomonad promastigote. Differentiation of L. panamensis in Ph. papatasi appeared to be similar to that described in natural hosts, except that metacyclic infective forms were not observed, and some forms developed in unusual locations. Phlebotomus papatasi was a partly refractory biological host for L. panamensis. The peritrophic membrane adversely affected the infection rate; rare anterior migration and a lack of metacyclic promastigotes may preclude transmission by bite.

Vesicular stomatitis virus, New Jersey serotype: replication in and transmission by Lutzomyia shannoni (Diptera: Psychodidae).

Laboratory-reared female sand flies (Lutzomyia shannoni) were experimentally infected, orally and by intrathoracic inoculation, with the New Jersey serotype of vesicular stomatitis (VSNJ) virus. Virus replication occurred in the insects following infection by both routes. Virus titers greater than 10(4) plaque forming units of VSNJ virus were present in heads of orally infected sand flies 12 days after virus ingestion, confirming that a persistent disseminated infection had occurred. Both orally and parenterally infected Lu. shannoni transmitted VSNJ virus by bite to susceptible rodents and by transovarial transmission to a small percentage of their F1 progeny. The significance of these findings in the epizootiology of VSNJ virus on Ossabaw Island, Georgia, an enzootic focus of this virus, is discussed.

Ultrastructural development of Leishmania chagasi in its vector, Lutzomyia longipalpis (Diptera: Psychodidae).

The development of Leishmania chagasi, etiologic agent of American visceral leishmaniasis, was studied by light and electron microscopy in the gut of the sand fly, Lutzomyia longipalpis, a natural vector. New aspects of suprapylarian Leishmania behavior were elucidated. In the sand fly midgut, amastigotes transformed into promastigotes (division promastigote I) during a first division sequence within the bloodmeal. Secondary division of these promastigotes resulted in a second form (division promastigote II), and these subsequently elongated into nectomonad promastigotes. Nectomonads existed in long and short populations which divided in the bloodmeal and throughout the midgut lumen after escape from the peritrophic membrane. Nectomonads adhered to the midgut cells in a highly organized manner, with their flagella embedded deep into microvilli and cytoplasm. Migration of parasites from the posterior midgut into the cardia/stomodeal valve region at 36 hr was associated with breakdown of the peritrophic membrane anteriorly. Posterior breakdown at 48 hr resulted in a peritrophic tube open at both ends containing some parasites within the digesting bloodmeal for up to 6 days postinfection. At the stomodeal valve, a myriad of slender and rounded promastigotes attached to the intima by flagellar hemidesmosomes; these may represent a transformation sequence from slender nectomonads to pear-shaped haptomonads. Pear-shaped forms appear to be precursors of paramastigotes, which also attached to the valve intima. Both rounded haptomonads and paramastigotes were found in the esophagus, dividing in a complex sequence initiated by posterior cleavage of the cytoplasm producing unique heart-shaped forms. Dividing paramastigotes also colonized the pharynx up to the cibarial valve. The ultrastructure of paramastigotes suggested that they may be infective forms, capable of some motility in the foregut. Free-swimming "infective" promastigotes were observed throughout the midgut and foregut, were attached in the pharynx (armature region), and were associated with the labrum-epipharynx of the proboscis in 3.6% of flies (16 days). The fine structure of hemidesmosomes in the foregut showed regional specializations, including the presence of plasmalemmar bridges in the gap space.

Characterization of five new phleboviruses recently isolated from sand flies in tropical America.

Five new phlebotomus fever virus serotypes (Bunyaviridae: Phlebovirus) are described. These viruses, designated Ambe, Ixcanal, Mariquita, Armero, and Durania, were isolated from sand flies (Diptera: Psychodidae) collected in Brazil, Colombia, and Guatemala. Two of the agents were recovered from pools of male sand flies. The new viruses are antigenically related to other members of the phlebotomus fever serogroup by immunofluorescence, but are distinct from the other 39 members of this serogroup by plaque reduction neutralization test.

A novel vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis.

Salivary gland lysates of the sand fly Lutzomyia longipalpis contain a potent vasodilator that aids the fly to feed on the blood of its vertebrate hosts. Chromatographic analysis, antibody reactivity, and data obtained from bioassays of the salivary erythema-inducing factor indicate striking similarity with human calcitonin gene-related peptide. The erythema-inducing factor is, however, at least one order of magnitude more potent than calcitonin gene-related peptide.

Ultrastructural biology of Leishmania (Viannia) panamensis (=Leishmania braziliensis panamensis) in Lutzomyia gomezi (Diptera: Psychodidae): a natural host-parasite association.

The development of Leishmania (Viannia) panamensis in a natural sand fly host, Lutzomyia gomezi, was studied by light and transmission electron microscopy. New aspects of peripylarian parasite behavior and morphology in the sand fly gut, early bloodmeal stages, and ultrastructural development in the anterior gut were documented. Eight distinct morphological forms were observed in the life cycle of the parasite within the insect. In the bloodmeal, amastigotes (1) transformed into stumpy promastigotes (2) which rapidly multiplied, resulting in spatulate-shaped nectomonad promastigotes (3) and elongate nectomonad promastigotes (4). These latter forms migrated primarily into the hindgut, where both were observed attached (=haptomonad phase) to the cuticular intima by hemidesmosomes within extremely shortened flagella. Spatulate haptomonad promastigotes predominated, colonizing the entire length of the hindgut, with the greatest density at 2 disjunct sites: the pylorus/ileum and the anterior rectum/rectal sac. Paramastigotes and dividing flagellates were rare. Some parasites migrated directly to the cardia/stomodeal valve region without a hindgut phase; however, major movement anteriorly was from the hindgut beginning at 6 days postinfection. In the cardia lumen, dividing short Type A promastigotes (5) predominated, intermixed with short Type B promastigotes with longer flagella (6). Paramastigotes (7) were free-swimming in the lumen as well as attached to the stomodeal valve. The primary colonizers of the valve were pear-shaped haptomonad promastigotes (8), with flagella of variable lengths and multi-segmented hemidesmosomal attachment points to the intima. Promastigotes and paramastigotes colonized the esophagus-pharynx region and attached to the foregut lining by flagellar hemidesmosomes. Both forms may represent infective stages of L. (V.) panamensis; however, no parasites were detected in the cibarium or proboscis. L. (V.) panamensis appeared well-adapted to the gut of Lu. gomezi, multiplying extensively at 2 sites, changing morphological form, and adhering to host surfaces by variously modified flagellar hemidesmosomes.

Natural infection of humans, animals, and phlebotomine sand flies with the Alagoas serotype of vesicular stomatitis virus in Colombia.

Five isolations of the Alagoas serotype of vesicular stomatitis virus (Rhabdoviridae: Vesiculovirus) were made from naturally infected phlebotomine sand flies (Lutzomyia spp.) collected in Colombia. These are the first isolations of Alagoas virus from an arthropod. Replication of the virus occurred in laboratory-reared sand flies (Lutzomyia longipalpis) after inoculation. Bite and transovarial transmission of the virus was also demonstrated in experimentally infected sand flies. Alagoas virus neutralizing antibodies were found in sera of humans and animals living near the insect collection site; antibody rates among human residents of two nearby towns were 63% and 83%, respectively. Results of comparative serologic studies demonstrated that Alagoas virus is closely related antigenically to Indiana, Cocal, and Maraba viruses and that these four agents form a complex within the vesicular stomatitis virus serogroup. The antigenic similarity among these four viruses makes their differentiation difficult; it also raises doubts about the accuracy of current laboratory methods used for identifying isolates in this serogroup. A discussion follows on the significance of human antibodies to these agents and on the role of sand flies in their ecology.

Host-parasite relationship of Leishmania mexicana mexicana and Lutzomyia abonnenci (Diptera: Psychodidae).

The life cycle of Leishmania mexicana mexicana in the gut of the sand fly, Lutzomyia abonnenci, was studied by light and electron microscopy. Development was suprapylarian with initial establishment of parasites in the bloodmeal (posterior midgut), and anterior migration of parasites to the cardia/stomodeal valve region beginning at 2.5 days post-infection. Flagellates were first observed in the esophagus at 3.5 days, in the posterior armature region of the pharynx at 5 days, and in the anterior pharynx at 7 days; but they were not detected in the cibarium or proboscis. Infection of the pylorus region of the hindgut and of the Malpighian tubules was also commonly observed. Three different morphological forms of L. m. mexicana developed in the gut: nectomonad promastigotes, short promastigotes, and paramastigotes. Nectomonads occurred primarily in the abdominal midgut after bloodmeal digestion, where they were oriented in longitudinal masses in the lumen, or interdigitated with epithelial microvilli via the flagellum. Short promastigotes found in the cardia/stomodeal valve region are described for the first time. These forms were smaller than nectomonads, showed an amplification of the kinetoplast, apposition of kinetoplast and nucleus, and were embedded in a gel-like matrix. To maintain position in the cardia, parasites commonly inserted the flagellum deep into microvilli or cytoplasm of the epithelium; adherence to the cuticular intima of the stomodeal valve was by flagellar modification and formation of hemidesmosome plaques. Paramastigotes occurred in the esophagus, were sometimes degenerated in appearance, and were attached via flagellar hemidesmosomes. Paramastigotes observed in the lumen of the pharynx were commonly degenerated and were not attached to the intima. L. m. mexicana was able to colonize the various gut habitats of Lu. abonnenci by a number of adaptations; this sand fly appears to be a suitable biological host for the parasite.

Maintenance of Toscana virus in Phlebotomus perniciosus by vertical transmission.

Toscana virus was maintained in a laboratory colony of Phlebotomus perniciosus by vertical (transovarial) transmission for 13 consecutive generations over a 23-month period. No significant biological changes were noted in the virus after prolonged vertical passage in the sand flies, and transovarially infected females were able to transmit the agent by bite to susceptible animals. Chronic infection of Ph. perniciosus with Toscana virus had no apparent effect on the insects' rate of eclosion. In the absence of selection and with random matings, the virus infection rates in each subsequent generation of the colony decreased, suggesting that Toscana virus cannot be maintained in Ph. perniciosus by transovarial transmission alone. Alternative mechanisms for virus maintenance are discussed.

Biology of Arboledas virus, a new phlebotomus fever serogroup virus (Bunyaviridae: Phlebovirus) isolated from sand flies in Colombia.

Six isolates of a new phlebotomus fever serogroup virus, designated Arboledas virus, were obtained from sand flies (Lutzomyia spp.) collected in northeastern Colombia. One of the isolates was made from a pool of male sand flies. By immunofluorescence, Arboledas virus is related to Caimito and Pacui viruses; by neutralization test, it is distinct. Arboledas virus neutralizing antibodies were found in the sera of opossums (Didelphis marsupialis) and humans living in the study area. D. marsupialis inoculated with the virus developed a viremia of four days' duration, and sand flies (Lutzomyia gomezi) feeding on a viremic opossum were readily infected. Transovarial transmission of Arboledas virus was also demonstrated in experimentally infected Lu. gomezi. Results of the above laboratory studies suggest that Arboledas virus is maintained in nature by two mechanisms: vertical (transovarial) transmission in the insect vector, and an alternating marsupial-sand fly cycle. The implications of this complex maintenance cycle for other phleboviruses are discussed.