Edgar D. Rowton

Canine Visceral Leishmaniasis, United States and Canada, 2000-2003

Foxhounds infected with Leishmania spp. were found in 18 states and 2 Canadian provinces.

Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi

SUMMARY 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.

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.

The role of the lipophosphoglycan of Leishmania in vector competence

The surface lipophosphoglycans (LPG) of Leishmania promastigotes express stage- and species-specific polymorphisms that are defined by variations in the type and number of phosphorylated oligosaccharide repeats. We have studied how these polymorphic structures control the development of transmissible infections in the sandfly vector as well as the species-specificity of vectorial competence. Procyclic promastigotes displayed an inherent capacity to bind to midgut epithelial cells of a competent vector. This capacity was lost during their transformation of metacyclic promastigotes, permitting the selective release and anterior migration of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release were found to be controlled by developmental modifications in terminally exposed saccharides on LPG, which, depending on the species of Leishmania, involved either substitution or capping of terminal side-chain sugars, loss of terminal side-chain sugars, substitution or loss of neutral capping sugars. The stage-specific terminal sugars involved in midgut adhesion are, in some cases, also species-specific, and the extent to which these differences affect midgut attachment, forcefully predicted vectorial competence.

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.

Impact of phlebotomine sand flies on U.S. Military operations at Tallil Air Base, Iraq: 1. background, military situation, and development of a "Leishmaniasis Control Program".

Abstract 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.

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.

A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in nonhuman primates

Immunity to sand fly salivary protein correlates to protection against cutaneous leishmaniasis in rhesus macaques. Leishmania vaccine targets the messenger The old adage “Don’t kill the messenger” may not hold true when vaccinating against leishmaniasis. Oliveira et al. demonstrate that a vaccine against sand fly salivary protein can protect nonhuman primate from leishmania infection. Leishmaniasis is transmitted by the bite of infected phlebotomine sand flies, which also transfer some of their saliva with the bite. Most macaques vaccinated against PdSP15, a sand fly salivary protein, had decreased parasite burden after induction of cutaneous leishmaniasis initiated by infected bites. Moreover, people exposed to sand fly bites developed an immune response to PdSP15 as well, suggesting that this approach may be translatable to humans. Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous leishmaniasis initiated by infected bites. Uninfected sand fly–exposed and 7 of 10 PdSP15-immunized rhesus macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4+IFN-γ+ lymphocytes, suggesting that immunity to saliva or PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably, the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-specific immunity. Sera and peripheral blood mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

Simulium vittatum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psychodidae) Salivary Gland Hyaluronidase Activity

Abstract 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.

Comparative Resistance of Anopheles albimanus and Aedes aegypti to N,N-Diethyl-3-methylbenzamide (Deet) and 2-Methylpiperidinyl-3-cyclohexen-1-carboxamide (AI3-37220) in Laboratory Human-Volunteer Repellent Assays

Abstract The insect repellents N,N-diethyl-3-methylbenzamide (Deet) and the racemate and 1S,2′S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (AI3-37220) were tested against Anopheles albimanus Wiedemann and Aedes aegypti (L.) in laboratory human-volunteer assays. Estimated skin doses of Deet or racemic AI3–37220 required to reduce biting by 95% in Ae. aegypti were 2.3 and 3.5 × 10–2 μmol/cm2 skin, respectively, whereas estimated doses for 95% bite reduction of An. albimanus in an ≈40-yr-old laboratory colony established from El Salvador were 5 times higher at 12 × 10–2 μmol Deet/cm2 skin and >20 × 10–2 μmol/cm2 skin for AI3-37220. In tests with the 1S,2′S stereoisomer of AI3-37220, a newly established colony of An. albimanus from Belize bit less aggressively than El Salvador An. albimanus. However, the Belize-derived mosquitoes were as resistant as the old El Salvador colony to repellent effects of 1S,2′S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide. Earlier workers surmised that usual skin doses of Deet would offer only limited protection against An. albimanus in the field. Our findings support this speculation, but they also indicate that doses of Deet higher than those needed for protection against Ae. aegypti might offer reasonable protection against An. albimanus. Results indicate that neither racemate nor 1S,2′S stereoisomer of 2-methylpiperidinyl-3-cyclohexene-1-carboxamide offer as much protection as Deet against An. Albimanus, despite being highly effective against Ae. aegypti.