The Diversity of Midgut Bacteria among Wild-Caught Phlebotomus argentipes (Psychodidae: Phlebotominae), the Vector of Leishmaniasis in Sri Lanka.

Phlebotomus argentipes is the main suspected vector for leishmaniasis in Sri Lanka. Investigations on the presence of aerobic bacteria in the gut of sand flies which evidence a potential approach to control leishmaniasis transmission through a paratransgenic strategy are still not available for the local sand fly populations. Field-caught unfed female sand flies collected from three selected Medical Officer of Health (MOH) areas (Polpithigama, Maho, and Galgamuwa) in Kurunegala District, Sri Lanka from August to December 2018 were used. Prokaryotic 16S ribosomal RNA partial gene was amplified and sequenced. Morphological identification revealed the presence of only one sand fly species, P. argentipes (n = 1,969). A total of 20 organisms belonging to two phyla (Proteobactericea and Furmicutes) were detected within the gut microbial community of the studied sand fly specimens. This study documents the first-ever observation of Rhizobium sp. in the midgut of P. argentipes. The presence of Bacillus megaterium, which is considered as a nonpathogenic bacterium with potential use for paratransgenic manipulation of P. argentipes suggest that it may be used as a delivery vehicle to block the vectorial transmission of Leishmania parasites. In addition, Serratia marcescens may be used as a potential candidate to block the parasite development in sand fly vectors since it has evidenced antileishmanial activities in previous investigations. Hence, further studies are required to gain full insight into the potential use of this bacterium in the control of Leishmania parasites through paratransgenesis.

Infectivity of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) to Phlebotomus papatasi (Diptera: Psychodidae) under laboratory conditions.

Susceptibility of Phlebotomus papatasi Scopoli (Diptera: Psychodidae) larvae to the entomopathogenic fungus Metarhizium anisopliae (Metschinkoff) Sorokin (Ma79) (Hypocreales: Clavicipitaceae) was evaluated at two different temperatures. The ability of the fungus to reinfect healthy sand flies was followed up for approximately 20 wk and the effect of in vivo repassage on the enhancement of its virulence was assessed. The fungus reduced the adult emergence at 26 +/- 1 degrees C when applied to larval diet. Six spore concentrations were used in the bioassays ranging from 1 x 10(6) to 5 x 10(8) spores/ml. Mortality decreased significantly when the temperature was raised to 31 +/- 1 degrees C at all tested concentrations. Fungus-treated vials were assayed against sand fly larvae at different time lapses without additional reapplication of the fungus in the media to determine whether the level of inocula persisting in the media was sufficient to reinfect healthy sand flies. Twenty weeks postapplication, there were still enough infectious propagules of Ma79 to infect 40% of P. papatasi larvae. A comparison between the infectivity of 10 subsequent in vitro cultures and the host-passed inocula of the fungus against sand fly larvae was conducted. Mortalities of P. papatasi larvae changed significantly when exposed to inocula passed through different insects. Presented data can provide vector control decision makers and end users with fundamental information for the introduction and application of M. anisopliae as an effective control agent against the main cutaneous leishmaniasis old-world vector P. papatasi.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Some snake venom proteins present enzymatic activities, such as L-amino acid oxidase (LAAO). The aim of this paper was to investigate the effect of Bothrops leucurus total venom (BleuTV) and its fraction LAAO (BleuLAAO) on bacteria, yeast, and promastigote forms of Leishmania amazonensis and Leishmania chagasi, and epimastigote forms of Trypanosoma cruzi. BleuTV was isolated with a Protein Pack 5PW® (Waters Corporation, USA), and several fractions were obtained. BleuLAAO was purified to high molecular homogeneity, and its N-terminal amino acid sequence shared a high degree of amino acid conservation with other LAAOs. BleuTV inhibited Staphylococcus aureus growth in a dose-dependent manner, with a minimum inhibitory concentration (MIC) of 25 ìg/mL, which corresponded to its minimum lethal concentration (MLC). BleuTV also inhibited the growth of promastigote forms of L. chagasi and L. amazonensis, with respective IC50 values of 1.94 ìg/mL and 5.49 ìg/mL. Furthermore, it repressed T. cruzi growth with an IC50 of 1.14 ìg/mL. However, BleuLAAO did not inhibit the growth of the microorganisms studied and was not toxic to macrophages. BleuTV had low toxicity against macrophages at the concentrations studied. In conclusion, whole venom from Bothrops leucurus inhibited the growth of some microorganisms, including S. aureus, Leishmania sp., and T. cruzi.

Identificación de especies de Leishmania por la técnica de amplificación al azar del ADN polimórfico / Identification of Leishmania species by the random amplified polymorphic DNA technique

INTRODUCCIÓN: la leishmaniosis ha sido clasificada por la Organización Mundial de la Salud como una de las enfermedades tropicales más importantes. El control de esta parasitosis es muy difícil, porque no existen vacunas y el tratamiento es tóxico e insatisfactorio. Es de crucial importancia establecer un método de diagnóstico oportuno junto a la identificación del parsito, lo cual incide en la selección del tratamiento adecuado y en el diseño de las medidas de control apropiadas. Recientemente, los avances en biología molecular han permitido la caracterización de especies de Leishmania por diferentes métodos. La técnica de ADN polimórfico amplificado al azar es una técnica simple para detectar el polimorfismo genético del ADN. OBJETIVO: estandarizar la técnica de ADN polimórfico amplificado al azar para su utilización en la tipificación de especies de Leishmania del Nuevo Mundo. MÉTODOS: empleando una concentración de cebador de 5 pmol, 75 ng de ADN molde, 2 mM de cloruro de magnesio y 2 U de Taq ADN polimerasa en 25 mL de reacción, se obtuvieron patrones de amplificación reproducibles. La técnica optimizada de ADN polimórfico amplificado al azar se empleó para determinar las diferencias genéticas entre 10 cepas de referencia de Leishmania, con la utilización de 6 juegos de cebadores comerciales diseñados al azar. La relación filogenética entre las especies estudiadas se determinó utilizando la estrategia de agrupaciones mediante el método de UPGMA. RESULTADOS: los cebadores OPA 3, 4 y 8 permitieron diferenciar las 10 cepas de referencia de Leishmania estudiadas. Se obtuvieron 2 grupos genéticos bien definidos donde se agrupan las especies del subgénero Leishmania y Viannia, respectivamente; los 2 subgéneros mostraron diferencias genéticas. CONCLUSIONES: de esta forma se cuenta en el laboratorio con la técnica del ADN polimórfico amplificado al azar optimizada para la identificación de especies de Leishmania(AU)

INTRODUCTION: leishmaniosis has been regarded by the World Health Organization as one of the most important tropical diseases. It is very difficult to control such parasitosis because there are not vaccines, and therapy is generally toxic and unsatisfactory. It is of vital importance to set prompt diagnostic method along with identification of the parasite in order to select the suitable treatment and to design the most convenient control measures. Recently, the advances in molecular biology have made it possible to characterize Leishmania species by different methods. The random amplified polymorphic DNA technique is a simple method to detect the genetic polymorphic DNA. OBJECTIVE: to standardize the random amplified polymorphic DNA technique for its use in New World Leishmania species typing. METHODS: by using 5 pmol primer concentration, 75 ng of template DNA, 2 mM of magnesium chloride and 2 U of polymerase DNA Taq in 25µL reaction, two reproducible amplification patters were obtained. The optimized random amplified polymorphic DNA technique served to determine the genetic differences among ten reference strains of Leishmania, with 6 sets of randomly designed conventional primers. The UP GMA method-based grouping strategy determined the phylogenetic relation among the studied species. RESULTS: OPA primers -3, 4 and 8 allowed distinguishing the ten reference strains of Leishmania under study. Two well defined genetic groups including species of Leishmania and Viannia subgenres were obtained; these 2 subgenres showed genetic differences. CONCLUSIONS: in this way, our laboratory has the optimized random amplified polymorphic DNA for the identification of Leishmania species(AU)

Identificación de Lutzomyia spp. (Diptera: Psychodidae) grupo verrucarum por medio de microscopía electrónica de sus huevos / Identification of lutzomyia spp. (Diptera: Psychodidae) verrucarum group through electron microscopy of eggs

The value of Colombian phlebotomine eggs for species determination was studied with a scanning electron microscope. The species diversity and medical importance of the verrucarum group were the bases to select Lutzomyia youngi, Lutzomyia evansi, Lutzomyia coumbiana and Lutzomyia longiflocosa. The egg surface was poligonal. Lutzomyia yungi, and Lutzomyia columbiana had pentagonal or hexagonal patterns; Lutzomyia evansi elongated polygons and Lutzomyia longiflocosa irregular polygonal sculpturing, frequently rectangular. Egg scannin electron microscopy is reliable to identify species of the verrucarum group. Key words: Electronic microscopy, Lutzomya, verrucarum group, sculpturing models, poligonal model

Producciòn local de Leishmanina / Local Leishmanin production / Annual reports 1996

La prueba cutànea de la leishmanina (reacciòn de Montenegro) es ùtil como mètodo de tamizaje para el diagnòstico epidemiològico de las leishmaniasis, permitiendo definir las zonas endèmicas para la enfermedad. Sòlo con una buena confiabilidad en los antìgenos se asegura que la prueba cutànea responda a las expectativas en estudios epidemiològicos. En el IICS se han producido hasta el momento seis lotes de dicho antìgeno a partir de promastigotes de L. amazonensis, que han aprobado los diversos ensayos de control, segùn dictados internacionales para productos de administraciòn parenteral. De todos los ensayos realizados, sòlo el ensayo de acividad, desarrollado a nivel experimental en animales, no arroja aùn resultados concluyentes. La disponibilidad de esta producciòn en el IICS permitirà desarrollar estudios epidemiològicos acerca de la prevalenciaq de la leishmaniasis en el Paraguay.

Producciòn local de Leishmanina / Local Leishmanin production / Annual reports 1996

La prueba cutànea de la leishmanina (reacciòn de Montenegro) es ùtil como mètodo de tamizaje para el diagnòstico epidemiològico de las leishmaniasis, permitiendo definir las zonas endèmicas para la enfermedad. Sòlo con una buena confiabilidad en los antìgenos se asegura que la prueba cutànea responda a las expectativas en estudios epidemiològicos. En el IICS se han producido hasta el momento seis lotes de dicho antìgeno a partir de promastigotes de L. amazonensis, que han aprobado los diversos ensayos de control, segùn dictados internacionales para productos de administraciòn parenteral. De todos los ensayos realizados, sòlo el ensayo de acividad, desarrollado a nivel experimental en animales, no arroja aùn resultados concluyentes. La disponibilidad de esta producciòn en el IICS permitirà desarrollar estudios epidemiològicos acerca de la prevalenciaq de la leishmaniasis en el Paraguay

Autonomous replication of bacterial DNA plasmid oligomers in Leishmania.

Extrachromosomal amplicons are frequently observed in drug-resistant Leishmania. A dominant selectable marker, the neomycin phosphotransferase gene, was introduced by gene targeting in a circular amplicon derived from the H locus of Leishmania in a mutant cell. This recombinant amplicon was isolated and transfected in a wild-type cell. The amplicon was kept in the wild-type cells, provided the selective pressure was maintained, suggesting that it was capable of autonomous replication. Novel Leishmania expression vectors suited for stable transfections were made to isolate, by a high transformation assay, the putative origin of replication in the amplicons. However, these plasmids, which did not contain a single Leishmania nucleotide, were found as extrachromosomal circular oligomers in Leishmania transfectants. Their relative stability, in addition to changes in their methylation pattern, indicated that these plasmids were most likely replicating. No specific sequences seem to be required for replication (and expression) in Leishmania, therefore precluding the isolation of origins of replication by genetic transformation.

Complete sequence of Leishmania RNA virus 1-4 and identification of conserved sequences.

In order to understand the coding strategies and identify potential cis-acting sequences in Leishmania RNA virus 1 (LRV1), a complete cDNA sequence was obtained for LRV1-4 and compared to the sequence reported for LRV1-1. The results show that the 5' end of LRV1 is conserved at the nucleotide level while open reading frames (ORFs) 2 and 3 are conserved at the amino acid level. A simple translation initiation consensus sequence is conserved at the 5' end of ORF2 but absent from ORF3, consistent with a possibility that ORF3 is expressed as a gag-pol fusion protein. Comparison of secondary structure predictions obtained for both isolates identified nucleotide sequences capable of forming conserved stem-loops at the virus termini and in the putative frameshift region between ORF2 and ORF3. Although direct evidence is lacking, the appearance of compensatory nucleotide substitutions suggests that the structures may form in vivo. Possible functions for the conserved structures are discussed.

Virus-like particles in Eimeria nieschulzi are associated with multiple RNA segments.

RNA preparations from sporulated oocysts of Eimeria nieschulzi were found to contain 2 double-stranded RNA segments of 5.0 kb and 5.7 kb that were not present in other species of Eimeria. Treatment of crude lysates with RNase A revealed that in addition to these two segments, 3 other segments of 0.57 kb, 0.72 kb and 11.5 kb were protected from digestion, suggesting that they were enclosed within particles. Virus-like particles with a diameter of approximately 39 nm were purified by caesium chloride buoyant density centrifugation. Four of the five RNA segments copurified with these particles. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this new isolate ENV 1. The largest RNA segment does not cosediment with ENV 1 particles and may be derived from another RNA-protein complex that is unstable under the conditions used. The particle size and genome structure of ENV 1 both differ from that of the Eimeria stiedae virus (ESV), which is the only other virus to have been isolated from Eimeria to date. Short cDNA clones derived from ENV 1 show significant homology to a region of the Leishmania virus (LRV 1) genome that encodes an RNA-dependent RNA polymerase. The polymerase sequences from ENV 1 and LRV 1 are more closely related to each other than to any other protein sequences in the GenEMBL Database. This raises intriguing questions about the origins of the two viruses, since Eimeria and Leishmania normally infect different hosts and also show different cell tropisms within these hosts.

Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system.

The putative capsid open reading frame (ORF2) of the Leishmania RNA virus LRV1-4 was expressed in a baculovirus expression system. The expressed protein was identified by Western immunoblot analysis with polyclonal antiserum raised to purified LRV1-4 virus. Electron microscopy and sedimentation analysis indicated that the expressed protein self-assembles into empty viruslike particles of similar size and shape to authentic virus particles, thus confirming that ORF2 encodes the viral capsid. The expressed particles are present exclusively in the cytoplasm of infected SF9 cells and are able to assemble in the absence of LRV1-4 RNA, viral polymerase, or any Leishmania host factors.

Detection of Leishmania RNA virus 1 proteins.

Polyclonal antiserum was raised against the peak viral fraction of a sucrose gradient from LRV1-4-infected cells and used in Western immunoblot analysis to identify viral proteins from various isolates. Consistent with this result, in vitro-translated protein from cloned RNA was immunoprecipitated with the same antiserum. The putative capsid at times appeared as a doublet; relative amounts of the two species varied, depending on the method of purification.

Successful transient introduction of Leishmania RNA virus into a virally infected and an uninfected strain of Leishmania.

Viruses of Leishmania have recently been identified and characterized. These viruses are consistently double-stranded RNA viruses of approximately 5 kb. To date, they have not been reported to exist outside their protozoan host, nor have they been shown to be infectious. We report here the ability to transiently transfer these viruses to two strains of Leishmania, one previously infected and one that did not previously carry a virus. A PCR-based assay was used to detect viral negative-stranded RNA. Input RNA was ruled out as the source of template because a replication-incompetent (UV inactivated) virus was not detectable after transfer into Leishmania.

Molecular organization of Leishmania RNA virus 1.

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.