Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253 +/- 0.01 mM and a maximum velocity of 0.246 +/- 0.04 nmol/min per 107 cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host. (C) 2008 Elsevier B.V. All rights reserved.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

Resposta imune à saliva de flebotomíneos (diptera: psychodidae) em indivíduos residentes em áreas urbana e periurbana no nordeste do brasil

Visceral leishmaniasis (VL) has a wide geographical distribution in tropical and subtropical areas of the planet, which is a protozoan parasite of the genus Leishmania. This pathogen is transmitted to the host through the sandflies bite, with its saliva, the immune response that leads to both. In the state of Rio Grande do Norte, 85% of the sand flies captured is Lutzomyia longipalpis, but the second most abundant, Lutzomyia evandroi, it deserves emphasis because its wide distribution and eclectic behavior. The exposure of people living in endemic areas for the insect vector VL greatly increases the chances of infection. This study aimed to evaluate aspects of the epidemiological profile of VL in endemic areas of human and nonendemic in the metropolitan area of Natal, as well as verify the abundance and seasonal fluctuations of sandflies species in two counties endemic for VL. Were collected in the municipalities of Nísia Floresta, Parnamirim, São Gonçalo do Amarante and Macaíba, of which groups of females were separated for further dissection of the salivary glands and identification of species. The blood samples used were from individuals of two Natal s districts where it has never been reported cases of VL and neighborhoods of Parnamirim applicants who present cases of VL. In the municipality of Nísia Floresta, the most abundant species was L. evandroi with 38.39%, followed by L. longipalpis with 36.22%, L. walkeri 19.67% L. lenti 3.81%, L. wellcomei 1.39% and L. whitmani 0.52%. Already in Parnamirim the proportions were L. walkeri with 73.15%, L. evandroi with 10.55%, L. wellcomei 7.63%, L. longipalpis 6.37%, L. whitmani 1.46%, L. sordellii 0.52%, L. intermedia 0.21 and L. shanonni 0.1%. In both municipalities was observed higher abundance of species distributed in the initial months of the year, as February and March. The study showed that no difference in exposure to the vector of VL among individuals from endemic and non endemic area for this disease. But there are differences in exposure between individuals of L. longipalpis and L. evandroi, confirming the great powers of the first vector. It was also characterized as predominant phenotype in the population of endemic areas who had negative serologic responses to antigens of Leishmania and result in negative Montenegro skin test (DTH), indicating that much of the population hasn t been bitten by infected insects

Estudo teórico-bioclimático da potencialidade de desenvolvimento do Aedes aegypti no estado do Rio Grande do Norte

In the current climate of global public health there is the emergence of urban dengue, a disease regarded as acute infectious fever. The disease annually, has affected millions of people worldwide, mostly in the range of the intertropical globe. The disease's main vector in urban areas, the Aedes aegypti mosquito. Recent studies indicate that the distribution of the insect vector of dengue in the geographical area is directly tied to the behavior of environmental restrictions that area, especially among those, the air temperature and relative humidity. From that context, the work aims to estimate and spatializing, monthly, for each municipality in the state of Rio Grande do Norte, the potential of biophysical conditions conducive to the development of Aedes aegypti. Yet, made use of the following methodology: collection of epidemiological data and climatological, Normal climatological, descriptive statistics (measures of central tendency and scatter), uniform distribution, estimation geostatistics and sufer program, version 8.0. The results flagged for a behavior very heterogeneous, both in space and in time, in the case of the potential of biophysical conditions conducive to the development of Aedes aegypti in the state of Rio Grande do Norte. Still, he noted that there is a tendency for lifting the potential of development for the entire state, from the month of January, ending in the month of April mainly in central and western portions of the state. By contrast, there is the permanence of increased potential for development in the eastern portion of the state. The latter record maximum potential in the month of July, resulting probability of greater than 70% have been favorable conditions for the development of Aedes aegypti in that area. In the period between the months of August to December, it is small potential for development of Aedes aegypti in all parts of the state

Detection and partial characterization of a carlavirus causing stem necrosis of soybean in Brazil

No ano agrícola de 2000/2001, ocorreu um surto de nanismo e necrose da haste em soja (Glycine max) plantada em duas áreas do Brasil Central e na safra seguinte, esta anomalia foi constatada em outras regiões produtoras, mesmo distantes mais de 2.000 km de onde fora inicialmente constatada. Estudos envolvendo ensaios de transmissão (enxertia, mecânica e insetos vetores), microscopia eletrônica, purificação, sorologia e ensaios moleculares indicaram que a enfermidade foi causada por um carlavirus transmitido por mosca branca, possivelmente relacionado ao Cowpea mild mottle virus (CpMMV).

Nucleolar changes in malpighian tubules cells of Rhodnius prolixus during starvation

This study focused on nuclear and nucleolar area changes in Malpighian tubule cells of Rhodnius prolixus, an hematophagous insect, vector of Chagas' disease. Male and female adult insects were dissected after a 28-day starvation period, as well as insects which had been fed again after a 30-day starvation period. Malpighian tubules were fixed and silver stained. In both, males and females, nucleolar fusions and regions of nucleolar corpuscles were observed to be differentially impregnated by silver during feeding stress. In the males and females that were fed again, nucleolar corpuscles were partially fusioned, indicating a slight recovery upon the 30-day starvation period. The changes observed in the nucleolar phenotype and in both nuclear and nucleolar areas indicated that, as a result of stress, a more intense, compensatory activity occurred to supply the metabolic rate. The mechanism of this phenomenon may be associated to the decondensation and activation of chromatin that carries rDNA in order to increase rRNA transcription, and,consequently, protein synthesis.

Dissipation of DDT in a heavily contaminated soil in Mato Grosso, Brazil

After the prohibition of organochlorine-pesticide use in Brazil for controlling insect vector diseases, Mato Grosso State gathered the exceeding DDT and stored it irregularly in an open air area that belongs to the National Health Foundation, causing soil contamination. This study aimed to evaluate the contamination level and dissipation of p,p′-DDT and p,p′-DDE in this area. For that, surface soil samples were collected on 19 September 2000, 15 December 2000, 4 April 2001 and soil samples 30-40 cm; 60-70 cm and 90-100 cm deep were taken from five points in the studied area on 17 July 2001. The contaminants were determined by a small scale method which consists on extraction and clean-up steps combined into one step by transferring soil samples mixed with neutral alumina to a chromatographic column prepacked with neutral alumina and elution with hexane:dichloromethane (7:3 v:v). The eluate was concentrated and the analytes were quantified by gas chromatography with an electron-capture detector. p,p′-DDT at surface soil ranged from 3800 to 7300 mg kg -1. 30-40 cm deep soil sample concentrations varied from 0.036 to 440 mg kg -1 while 90-100 cm deep samples varied from 0.069 to 180 mg kg -1. Volatilization is probably the main dissipation process. The p,p′-DDT is moving slowly downward in the soil profile, however, the levels of this contaminant are high enough to present risk to underground waters. © 2005 Elsevier Ltd. All rights reserved.

Bacillus thuringiensis var. israelensis SPS1: caracterização da região promotora de genes cry e efeito em larvas de Aedes aegypti (L.) (Diptera: Culicidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sistemas de produção de mudas cítricas em viveiros protegidos no Estado de São Paulo

Pós-graduação em Agronomia (Horticultura) - FCA

Multiplicação de Diaphorina citri e de Candidatus Liberibacter asiaticus em híbridos de citros indicados como porta-enxerto

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite

Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ecto-phosphatase of living T. ranged epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and beta-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyr-residues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Characterization of Bean Necrotic Mosaic Virus: A Member of a Novel Evolutionary Lineage within the Genus Tospovirus

Background: Tospoviruses (Genus Tospovirus, Family Bunyaviridae) are phytopathogens responsible for significant worldwide crop losses. They have a tripartite negative and ambisense RNA genome segments, termed S (Small), M (Medium) and L (Large) RNA. The vector-transmission is mediated by thrips in a circulative-propagative manner. For new tospovirus species acceptance, several analyses are needed, e. g., the determination of the viral protein sequences for enlightenment of their evolutionary history. Methodology/Principal Findings: Biological (host range and symptomatology), serological, and molecular (S and M RNA sequencing and evolutionary studies) experiments were performed to characterize and differentiate a new tospovirus species, Bean necrotic mosaic virus (BeNMV), which naturally infects common beans in Brazil. Based upon the results, BeNMV can be classified as a novel species and, together with Soybean vein necrosis-associated virus (SVNaV), they represent members of a new evolutionary lineage within the genus Tospovirus. Conclusion/Significances: Taken together, these evidences suggest that two divergent lineages of tospoviruses are circulating in the American continent and, based on the main clades diversity (American and Eurasian lineages), new tospovirus species related to the BeNMV-SVNaV clade remain to be discovered. This possible greater diversity of tospoviruses may be reflected in a higher number of crops as natural hosts, increasing the economic impact on agriculture. This idea also is supported since BeNMV and SVNaV were discovered naturally infecting atypical hosts (common bean and soybean, respectively), indicating, in this case, a preference for leguminous species. Further studies, for instance a survey focusing on crops, specifically of leguminous plants, may reveal a greater tospovirus diversity not only in the Americas (where both viruses were reported), but throughout the world.

Interactions between trypanosomes and tsetse flies

African trypanosomes are insect-borne parasites that cause sleeping sickness in humans and nagana in domesticated animals. Successful transmission is the outcome of crosstalk between the trypanosome and its insect vector, the tsetse fly. This enables the parasite to undergo successive rounds of differentiation, proliferation and migration, culminating in the infection of a new mammalian host. Several stage- and species-specific parasite surface molecules have been identified and there are new insights into their regulation in the fly. Tsetse flies are often refractory to infection with trypanosomes. While many environmental and physiological factors are known to influence infection, our detailed understanding of tsetse-trypanosome relationships is still in its infancy. Recent studies have identified a number of tsetse genes that show altered expression patterns in response to microbial infections, some of which have also been implicated in modulating trypanosome transmission.

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei.

The procyclic form of Trypanosoma brucei colonises the gut of its insect vector, the tsetse fly. GPEET and EP procyclins constitute the parasite's surface coat at this stage of the life cycle, and the presence or absence of GPEET distinguishes between early and late procyclic forms, respectively. Differentiation from early to late procyclic forms in vivo occurs in the fly midgut and can be mimicked in culture. Our analysis of this transition in vitro delivered new insights into the process of GPEET repression. First, we could show that parasites followed a concrete sequence of events upon triggering differentiation: after undergoing an initial growth arrest, cells lost GPEET protein, and finally late procyclic forms resumed proliferation. Second, we determined the stability of both GPEET and EP mRNA during differentiation. GPEET mRNA is exceptionally stable in early procyclic forms, with a half-life >6h. The GPEET mRNA detected in late procyclic form cultures is a mixture of transcripts from both bona fide late procyclic forms and GPEET-positive 'laggard' parasites present in these cultures. However, its stability was clearly reduced during differentiation and in late procyclic form cultures. Alternatively processed GPEET transcripts were enriched in samples from late procyclic forms, suggesting that altered mRNA processing might contribute to repression of GPEET in this developmental stage. In addition, we detected GPEET transcripts with non-templated oligo(U) tails that were enriched in late procyclic forms. To the best of our knowledge, this is the first study reporting a uridylyl-tailed, nuclear-encoded mRNA species in trypanosomatids or any other protozoa.