Analysis of the sex pheromone extract of individual male Lutzomyia longipalpis sandflies from six regions in Brazil.

Although the phlebotomine sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) is generally accepted to be a species complex, it is unclear how many members there are, how they are related and which are the main vectors of leishmaniasis. The vectorial capacity of each sibling species is likely to differ, thus a means of identifying the most important vector species is of critical importance to the epidemiology and control of this debilitating disease in South and Central America. In Brazil four chemotypes have been distinguished by sex pheromone analysis. In this study the sex pheromone extracts of L. longipalpis from six regions of Brazil were analysed in detail. Samples included the sympatric 1-spot, 2-spot and intermediate spot morphotypes from Sobral, Ceará State. The results strongly suggest that members of the complex that produce different sex pheromones are reproductively isolated, thus strengthening the argument that the different chemotypes represent true sibling species. The study also found significant differences in morphology and the amounts of sex pheromone produced by members of each chemotype from different parts of Brazil, which suggests population substructuring that has not previously been recognized. Evidence of a fifth chemotype in Brazil is also presented.

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America.

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.

A fourth chemotype of Lutzomyia longipalpis (Diptera: Psychodidae) from Jaibas, Minas Gerais State, Brazil.

Lutzomyia longipalpis (Lutz and Neiva) is a species complex of Lutzomyia pseudolongipalpis (Arrivillaga and Feliciangeli) and at least three other as yet undefined siblings. Isozyme and mitochondrial studies of allopatric populations across Central and South America have suggested the presence of four "clades" that have been hypothesized to have arisen mainly because of geographical isolation mechanisms. Parallel studies of sexual behavior as well as cross-mating and genetic analysis, of both allopatric and sympatric populations, suggest at least four sibling species that do not seem to correspond to the defined four "clades." In an effort to understand this apparent discrepancy, sympatric populations of L. longipalpis from a single South American country, Brazil, are being studied. In Brazil, three putative species can be identified by their male-produced sex pheromones: (S)-9-methylgermacrene-B, 3-methyl-a-himachalene, and a cembrene. We report here that analysis by coupled gas chromatography-mass spectrometry shows that L. longipalpis from Jaibas, Minas Gerais State, Brazil, occurs as two sympatric sex pheromone chemotypes. One chemotype is the cembrene type previously recorded in a L. longipalpis population from Sobral, Ceará State, Brazil, and the other is a new cembrene isomer not previously observed in L. longipalpis. The finding of this new chemotype strongly suggests that the L. longipalpis species complex in Brazil consists of four members rather than the three previously recognized and confirms previous analysis of genetic variation that had suggested the presence of a complex in Brazil.

Genetic identification of two sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) that produce distinct male sex pheromones in Sobral, Ceará State, Brazil.

Lutzomyia longipalpis, the main sandfly vector for New World visceral leishmaniasis is a complex of an as yet undefined number of sibling species. At present, there is no consensus on the status (single species vs. species complex) of Brazilian populations. We applied five microsatellite loci to test the hypothesis that L. longipalpis occurs as two sympatric cryptic species in Sobral, Ceará State, Brazil as predicted by male sex pheromone chemotypes described previously for field specimens from this site [S-9-methyl-germacrene-B (9MGB) and a cembrene compound]. Abdominal spot morphology corresponds with pheromone type at this locality (9MGB in '1 spot' males and cembrene in '2 spot' males). Genotype data from 190 wild-caught L. longipalpis specimens collected in October 1999 and April 2001 were used to estimate genetic differentiation between the two sex pheromone populations and sampling dates. No significant (P > 0.05) genetic differences were found between the 1999 and 2001 9MGB samples (theta = 0.018; RST = -0.005), and genetic differentiation was low between the cembrene collections (theta = 0.037, P < 0.05; RST = -0.043, P > 0.05). By contrast, highly divergent allelic frequencies (largely at two microsatellite loci) corresponded to significant (P > 0.05) genetic differentiation (theta = 0.221; RST = 0.215) for all comparisons between samples with different pheromones. When pheromone samples were pooled across sample date, genetic differentiation was high (theta = 0.229; P < 0.001; Nem = 0.84). The allele frequency distribution at each of the five microsatellite loci was similar for males and females from the two collection years. Two of these loci showed highly divergent allele frequencies in the two sex pheromone populations. This was reflected in the highly significant genetic differentiation obtained from the male genotypes, between populations producing different pheromones (theta = 0.229-0.268; P < 0.0001 for the 2001 and theta = 0.254-0.558; P < 0.0001 for the 1999 collections, respectively). Similar results were obtained when the females, assigned to a pheromone type, were included in the analysis. Both a Bayesian analysis of the data set and a population assignment test provided strong evidence for two distinct populations corresponding to pheromone type. Given its genotype, the probability of assigning a 9MGB male to the original 9MGB population was 100% once the two years' collections were pooled. For cembrene-producing '2 spot' males this probability although still high, was lower than for 9MGB males, at 86%. This microsatellite data together with previously reported reproductive isolation between the two Sobral populations confirm that premating barriers are important in speciation of L. longipalpis.

Effects of malaria infection on vitellogenesis in Anopheles gambiae during two gonotrophic cycles.

We report changes in the abundance of vitellogenin (Vg) mRNA, and concentration of haemolymph Vg and ovarian vitellin (Vn) in Anopheles gambiae following infection with Plasmodium yoelii nigeriensis. A parasite-induced reduction in Vg mRNA abundance was first detected 24 h after feeding on an infective blood meal, when ookinetes were invading the midgut. During a second gonotrophic cycle post-infection, developing oocysts reduced Vg mRNA abundance by up to 33% and the effect was detected from 2 h post blood meal. Concentrations of Vg were initially reduced by infection during the second cycle, as predicted from Vg mRNA measurements. However, after 24 h, excess Vg had accumulated in the haemolymph. This accumulation may be due to impaired uptake, since ovarian vitellin accumulation was significantly decreased by infection during both gonotrophic cycles.

The Egyptian mongoose, Herpestes ichneumon, is a possible reservoir host of visceral leishmaniasis in eastern Sudan.

Investigations were made on possible reservoir hosts of Leishmania donovani in 2 zoonotic foci of visceral leishmaniasis (VL) in Dinder National Park (DNP) and the peri-domestic habitats of adjacent villages of eastern Sudan. Animals were captured, in November 1997-1998 and April-May 1999 and examined for L. donovani infection using light microscopy and 2 sensitive Polymerase Chain Reaction (PCR) systems. Microscopy and PCR investigations were also used to determine the infection rates of L. donovani in Phlebotomus orientalis captured from the uninhabited site of DNP. Infections of L. donovani were detected in 2 out of 14 Egyptian mongooses (Herpestes ichneumon), 1 out of 168 Arviconthus niloticus and 1 out of 8 Mastomys natalensis. Samples from 68 other animals captured from the study area were all negative for the infection. Active zoonotic transmission of L. donovani at the time of animal sampling in the uninhabited site of DNP was demonstrated by finding the parasite in 3.4% (7 out of 184) and 3.2% (5 out of 157) of flies collected in March 1998 and May 1999, respectively. We suggest that the Egyptian mongoose is a possible reservoir host of L. donovani. The importance of other animals in maintaining the infection is also discussed.

The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) complex and the lizard Leishmania: their classification and evidence for a neotropical origin of the Leishmania-Endotrypanum clade.

The relationships of the Leishmania hertigi complex and the lizard Leishmania species to the main groups of mammalian Leishmania and Endotrypanum parasites were examined. Restriction fragment length polymorphisms and sequences of small subunit ribosomal RNA genes and hybridization studies of kinetoplast DNA indicated that the L. hertigi complex was more closely related to the genus Endotrypanum than to the genus Leishmania. The lizard Leishmania species were found to be at the crown of the Leishmania tree. The data provides strong evidence for a Neotropical origin of the Endotrypanum/Leishmania clade since the parasites closest to the root of the tree are all found exclusively in the Neotropics. The evolution of the Leishmania/Endotrypanum clade in relation to the evolution of the known hosts of these parasites is discussed.

Leishmania chagasi: genotypically similar parasites from Honduras cause both visceral and cutaneous leishmaniasis in humans.

In the Mediterranean region Leishmania infantum causes both visceral and cutaneous leishmaniasis. These two pathologies tend to be caused by distinct parasite zymodemes. We have studied 33 isolates of Leishmania, 2 from sandflies, 5 from visceral cases, and 26 from cutaneous cases in Honduras, to determine if there is a correlation between pathology and parasite type in the New World similar to that in the Mediterranean region. Nine of the 26 cutaneous cases were caused by L. mexicana parasites, which have not been previously reported from Honduras; the remaining 17 cutaneous cases were due to L. chagasi. Only minor differences were found between the Honduran L. chagasi parasites by random amplified polymorphic DNA, differential display, pulsed-field gel electrophoresis, and schizodemes. This suggests that in Honduras the parasite type may not be the only factor determining the clinical outcome of L. chagasi infections.

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge.

The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E. coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M.luteus infection since anti-E.coli activity was similar in haemolymph from both E.coli or M.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4 kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.

Appraisal of various random amplified polymorphic DNA-polymerase chain reaction primers for Leishmania identification.

Pathogenic Leishmania can cause a variety of symptoms ranging from asymptomatic or mild infections to severe mucocutaneous disease, partly according to the species of Leishmania involved. Genomic and kinetoplast-derived DNA probes as well as species complex-specific kinetoplast-derived polymerase chain reaction (PCR) primers have been successful for parasite identification in epidemiologic and taxonomic studies. However, the lack of a species DNA probe or PCR primer set, their relatively poor availability, and unknown comparative sensitivity and specificity have precluded their routine and widespread use as identification tools in many laboratories. This study addresses this problem for 28 different random amplified polymorphic DNA (RAPD)-PCR primers that have been assessed regarding their sensitivity, specificity, and reliability for distinguishing each of four closely related New World Leishmania species. The degree of relatedness between species was quantified and estimates were made of the accuracy and precision of the determinations. The results compared well with standard methods for Leishmania classification. The application of RAPD-PCR for screening isolates for possible interspecific hybrids is also demonstrated.

Leishmania and Sauroleishmania: the use of random amplified polymorphic DNA for the identification of parasites from vertebrates and invertebrates.

We used the RAPD-PCR method for distinguishing the main Old World Leishmania parasites Leishmania tropica, L. major, and L. infantum and applied it to Sauroleishmania species from Iran. Twelve out of 21 tested primers were suitable for identification of these parasites. The Jaccard similarity index was 0.30 for L. tropica and L. infantum as well as for L. major and L. infantum. The similarity coefficient for L. major and L. infantum was 0.22 and for L. tropica and L. major it was 0.26. These data agree well with established phylogenetic/ taxonomic classification. The index between different isolates derived from various hosts and sandfly vectors for the same Leishmania species was 1, indicating that this method is suitable for epidemiological analysis.

Synthetic peptide-based enzyme-linked immunosorbent assay for serodiagnosis of visceral leishmaniasis.

Synthetic peptides, derived from the amino acid sequence of a Leishmania donovani clone, were used to develop an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against L. donovani. For this purpose, five peptides were conjugated to a protein carrier, human serum albumin (HSA), by using a heterobifunctional reagent, epsilon-maleimidocaproic acid N-hydroxysuccinimide ester, to obtain a well-defined product. The sensitivity and the specificity of the peptide-specific ELISA were determined with a panel of 106 serum samples from individuals living in areas where visceral leishmaniasis is endemic; sera from post-kala azar dermal leishmaniasis-infected patients and from individuals suffering from other infectious diseases were also included. ELISAs were performed with either a single peptide-HSA conjugate or a mixture of two peptide-HSA conjugates. Ninety-seven percent of the serum samples from patients with visceral leishmaniasis had detectable antibodies to one or more of the single synthetic peptides. ELISA with a single peptide-HSA conjugate proved to be less sensitive (less than 71%) but more specific (up to 93%) than ELISA with crude promastigote antigens (80% sensitivity and 79% specificity); when a combination of two different peptide-HSA conjugates was used, the test increased both in sensitivity and in specificity. Chemically defined peptide-protein conjugates improve the reproducibility and reliability of ELISA for the serodiagnosis of L. donovani infection.

The EUROLEISH network: an exercise in partnership.

The EUROLEISH network arose in 1989 as a spontaneous initiative from contract holders funded by the health component of the Science and Technology for Development programme of the European Commission (EC). Leishmaniasis projects from other programmes (International Scientific Co-operation and Avicenna) joined the network later on. The network has three central objectives: to improve interactions between laboratory and field scientists; to rank the queries raised by health services in order of importance for research; and to develop partnerships between European countries and between these and developing countries. The EUROLEISH network is run consensually by the contract-holders and meets yearly, the subject of each meeting alternating between biology (in Europe) and epidemiology (in an endemic country). Presently, the network comprises 66 contract-holders from 18 projects in 28 countries, with a research budget of 4.6 million ecus during the Third EC Framework Programme (1990-1994). The next main issue to be addressed by the network will probably be the strengthening of thematic research, by promoting 'bridge building' between projects at regional level.

Molecular techniques in the characterization of Leishmania isolates from Central America.

The public-health problems caused by leishmaniasis in most countries in Central America are becoming more severe. This is partly because of the increasing size of the human populations that are at risk and their migratory patterns. Annual incidence of the disease in Costa Rica, Honduras, Guatemala, Panama and Nicaragua is estimated to be as high as 20,000 cases. Regional changes in the epidemiology of the various Leishmania spp. present have emphasized the need for innovative, sensitive and accurate diagnostic tools. PCR and isoenzyme, monoclonal antibody, schizodeme, DNA-probe and random-amplified, polymorphic DNA analyses have been tested. Preliminary indications that Leishmania chagasi was present in Costa Rica and Honduras and that interspecific hybrids occurred in Nicaragua have been confirmed using these methods. The distribution of the mexicana complex was also found to be broader and more heterogeneous than initially expected. Overall, there was 87% concordance between the results produced using the different techniques.

Cutaneous leishmaniasis in Tachira State, Venezuela.

Cutaneous and mucocutaneous leishmaniases are widely spread in the mountainous Andean regions of South America. In Venezuela, these regions consist of the coffee-growing states of Trujillo, Merida and Tachira. Entomological and parasitological investigations in three geographically different climatic zones (Lomas Bajas, Delicias and La Grita) in Tachira state have shown a predominance of the sandfly species Lutzomyia spinicrassa (verrucarum group) and two Leishmania species, Leishmania mexicana and Leishmania braziliensis. Two transmission cycles appear to occur: a peridomestic cycle in Lomas Bajas and a sylvatic one in Delicias and La Grita.

Antigenic analysis of Leishmania isolates from Tachira state, Venezuela.

Studies of cutaneous leishmaniasis in 3 endemic foci in Tachira state, western Venezuela have revealed sympatric populations of parasites causing both cutaneous and mucocutaneous disease. Immunological techniques and measurement of protease/acid phosphatase activities have been used to detect species-specific parasite antigens from 3 isolates from Tachira. Identified antigens of particular interest had molecular masses of 100, 82, 66, 50 and 27 kDa, but there was a high degree of heterogeneity between the antigens of the Tachira isolates and other Venezuelan strains of Leishmania braziliensis and L. mexicana. This heterogeneity has implications concerning the selection of antigens for use in serodiagnosis of leishmaniasis.

The application of random amplified polymorphic DNA for sandfly species identification.

We have applied the recently developed Random Amplified Polymorphic DNA (RAPD) method to produce species-specific, DNA profiles for two sympatric, Venezuelan sandfly species, thought to be the vectors responsible for recent outbreaks of cutaneous and mucocutaneous leishmaniasis in the Andean State of Tachira. Moreover, within the profile, it was possible to identify a diagnostic DNA band for Lu. youngi of 0.32 kb. Results showed that the size of this diagnostic DNA band remained constant and did not vary with sex or geographical distribution.

Molecular approaches applied to the epidemiology of leishmaniasis in Venezuela.

Leishmaniasis is on the increase in Venezuela (ca 30,000 new cases per year) due to deterioration in health management, increased risk groups among inmunosuppressed individuals and increased human penetration into the ecological habitats of sandfly vectors. An STD2-funded project (1989-1992) focused on the Andean state of Táchira, which showed the highest annual index of new cases (ca 200-250). The project aimed at contributing to vector/parasite identification through a combination of molecular and well established field techniques: Newly developed molecular methods distinguished among Lu. spinicrassa, Lu. youngi and Lu. townsendi. These three species of the Verrucarum group are sympatric in the Northeast of the state and could be successfully identified by CHA, DNA probes and RAPD. A Le. braziliensis specific KDNA probe used with squash blots indicated that Lu. spinicrassa is the main vector and that Le. braziliensis is the main parasite species in Táchira state, Venezuela. PCR and the Le. brasiliensis specific DNA probe, schizodemes, isoenzymes and polyclonal antibodies agreed as taxonomic criteria for classification of Leishmania isolated from parasitologically confirmed cases in Tachira. Considerable degree of antigen heterogeneity in Venezuelan Le. braziliensis complex and Le. mexicana complex isolates from Tachira suggests multiple candidate antigens for improving the specificity of immunological diagnosis. The methods developed and tested in Táchira state should be valuable in order to help solving other outstanding epidemiological problems such as following of the epidemiological impact of intervention and vector control measures in highly endemic areas. Future work (STD3 funded, 1993-1996) aims to apply these molecular techniques to a vector control pilot study in Lara state, an area showing the highest incidence of new cases in the country.