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PubMed1990

Detection of bluetongue virus serotype 17 in Culicoides variipennis by nucleic acid blot and sandwich hybridization techniques.

Molecular hybridization techniques were developed for the detection and surveillance of bluetongue virus (BTV) serotype 17 in the insect vector Culicoides variipennis, a biting midge. Radiolabeled RNA and cDNA probes were generated from sequences of the L3 segment of BTV serotype 17. These probes were used to detect BTV RNA in pools of infected C. variipennis by hybridizing the probes directly to analyte immobilized on nylon membranes or by using a nucleic acid sandwich hybridization test. Hybridization procedures were able to detect 1 infected C. variipennis in a pool of 50 and as little as 3.55 log10 50% tissue culture infective doses per ml of virus. These hybridization techniques provide an alternative to virus isolation for the surveillance of BTV in vector populations.

Animals↗
PubMed1998

Evaluation of three commercial enzyme-linked immunosorbent assays for diagnosis of Chagas' disease.

Chagas' disease is a common cause of morbidity in Latin American countries. In Brazil, naturally occurring transmission of its etiologic agent, Trypanosoma cruzi, has been almost completely abolished through effective control programs aimed at the triatomid insect vector. Thus, transfusion of blood from infected donors has become the major route for contracting Chagas' disease due to the socioeconomically motivated migration of residents from areas where the disease is endemic to the larger urban centers. Therefore, the employment of screening tests is mandatory for all blood banks throughout the country. We compared the diagnostic performances of three commercially available screening assays used in routine testing in Brazilian blood banks: the Abbott Chagas antibody enzyme immunoassay (Abbott Laboratórios do Brasil, São Paulo), the BIOELISACRUZI kit (Biolab-Mérieux, Rio de Janeiro, Brazil), and the BIOZIMA Chagas kit (Polychaco S.A.I.C., Buenos Aires, Argentina). The evaluation was performed with sera obtained from chagasic patients and healthy residents of four different areas in Brazil where Chagas' disease is either endemic or emergent and where clinical manifestations of the disease and circulating parasite strains vary. The results obtained with each kit were compared to matched in-house enzyme-linked immunosorbent assay and immunofluorescence assay data obtained for each sample. Depending on the area under investigation, the three commercial kits produced specificity values between 93.3 and 100.0%, sensitivity values between 97.7 and 100%, and accuracies ranging from 93.6 to 100.0%.

Animals↗
PubMed2006

Recovery of infectious bluetongue virus from RNA.

Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.

Animals↗
PubMed2025

Degradation of ribosomal RNA during Plasmodium falciparum gametocytogenesis.

The life cycle of Plasmodium falciparum is characterized by complex regulatory changes that allow adaptation of the parasites to different environmental conditions, which are especially pronounced during transmission between the mammalian host and the insect vector. Previous studies have shown that P. falciparum uses three types of ribosomal RNAs (rRNA A, S1, and S2 types) at different stages of its life cycle. We used Oxford Nanopore Technologies direct RNA sequencing to investigate the dynamics of rRNA usage throughout the parasite's intraerythrocytic development, as well as in salivary gland sporozoites. Our study revealed a preponderance of A-type rRNAs during the intraerythrocytic cycle and gametocytogenesis, while S-type rRNAs slowly increase in abundance in mosquito stages starting 3 days post-infection. Salivary gland sporozoites showed an even proportion of all rRNA types. By examining the length distributions of rRNA molecules, we detected an extensive and specific degradation of rRNAs during gametocytogenesis, starting in stage II gametocytes and continuing until the final stages of gametocyte development. We hypothesize that rRNA degradation may be linked to the global translational repression and metabolic quiescence described in stage V gametocytes, similar to mechanisms observed in bacterial and eukaryotic stress responses.IMPORTANCEOur study uses Oxford Nanopore direct RNA sequencing of tightly synchronized blood-stage Plasmodium falciparum parasites to investigate the expression of ribosomal RNAs during asexual and sexual development. P. falciparum utilizes distinct types of rRNA during its development. However, due to the challenges of differentiating these highly similar molecules, their regulation and the mechanism underlying the switch between rRNA types remain unclear. We observe significant rRNA degradation in mature gametocytes, leading us to propose that this potentially leads to a reduced number of functional ribosomes when parasites become quiescent and translation is repressed.

Plasmodium falciparum↗
PubMed1991

Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).

Amino Acid Sequence↗
PubMed1984

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Animals↗
PubMed1986

Metacyclic variant surface glycoprotein genes of Trypanosoma brucei subsp. rhodesiense are activated in situ, and their expression is transcriptionally regulated.

During the metacyclic stage in the life cycle of Trypanosoma brucei subsp. rhodesiense, the expression of variant surface glycoproteins (VSGs) is restricted to a small subset of antigenic types. Previously we identified cDNAs for the VSGs expressed in metacyclic variant antigen types (MVATs) 4 and 7 and found that these VSG genes do not rearrange when expressed at the metacyclic stage (M. J. Lenardo, A. C. Rice-Ficht, G. Kelly, K. Esser, and J. E. Donelson, Proc. Nathl. Acad Sci. USA 81:6642-6646, 1984). We now provide further evidence that these genes do not rearrange and demonstrate that their 5' upstream regions lack the 72 to 76-base-pair repeats which are considered the substrate for duplication and transposition events. Pulsed field gradient electrophoresis showed that the MVAT VSG genes were located on the largest chromosome-sized DNA molecules, and the lack of the MVAT 4 gene in one of two different serodemes suggested that one mechanism for the evolution of MVAT repertoires is gene deletion. When MVATs were inoculated into the bloodstream of a mammalian host by a bite from the insect vector, they rapidly switched into nonmetacyclic VSG types. We found that this switch was accomplished by a loss of MVAT RNA concomitant with the loss of metacyclic VSGs. Transcription studies with isolated metacyclic nuclei showed that the MVAT genes were expressed in situ from a single locus and were regulated at the level of transcription.

Animals↗
PubMed1989

Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site.

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.

Animals↗
PubMed2002

Secretory pathway of trypanosomatid parasites.

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs.

Animals↗
PubMed2002

Whole-genome analysis of transporters in the plant pathogen Xylella fastidiosa.

The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing-among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits-we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with > or =4 alpha-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%).

Bacterial Outer Membrane Proteins↗
PubMed1998

Should we be frightened of bracken? A review of the evidence.

OBJECTIVE: To assess the risk to human health of the plant bracken (Pteridium sp). DESIGN: An evaluation of studies of human and animal populations exposed to bracken, together with a review of expert reports and advice to the public. MAIN RESULTS: Bracken induced disease has been demonstrated in animals in both laboratory and field studies. Depending on the species, diseases in animals associated with the plant have included; cancers of the alimentary and urogenital tract, lung and breast; haematuria; retinal degeneration; and, thiamine deficiency. Potential exposure of human populations is through: food either directly (people in some parts of the world eat bracken as a traditional dish) or indirectly by consuming animals fed on bracken; milk; water; inhalation and ingestion of spores; and insect vectors. Four studies of human populations (two analytical and two observational) failed to assess adequately confounding factors and other sources of bias, so that conclusions about a risk to human health from bracken cannot firmly be drawn. Establishing exposure is also extremely difficult in populations (such as the United Kingdom) where direct consumption of bracken is rare. CONCLUSION: Bracken is a common plant worldwide. It is toxic to many animal species and to several organ systems. There is no tumour (or other disease) that is pathognomic of exposure in animals, though cancers of the alimentary and urogenital tract seem to be the most commonly associated. It is not possible to extrapolate from animal models to humans. Studies of human populations, do not establish a clear risk of bracken to human health, largely because of methodological problems. Testing the evidence against traditional criteria of causality only fulfils the criterion of biological plausibility. Despite this, current public information implies a serious risk to human health from bracken, and increasing media coverage of the subject is likely to lead to greater public concern. Further epidemiological studies are required.

Animals↗
PubMed1989

Molecular biology of Leishmania.

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.

Amino Acid Sequence↗
PubMed1994

The role of pH and temperature in the development of Leishmania parasites.

Protozoans of the genus Leishmania are obligate intracellular parasites that cycle between the midgut of sandflies and the phagolysosomes of mammalian macrophages and therefore are exposed to extreme environmental changes. Recent evidence obtained from in vitro experiments indicate that such environmental changes trigger a developmental program in the parasites. Thus, following heat shock, promastigotes from certain Leishmania species differentiate to amastigotes. Promastigotes also respond to acidification of their environment by changing the expression of a number of genes. However, the combination of both low pH and high temperature induces the transformation of the promastigote to the amastigote in all Leishmania species examined to date. This review discusses the role of pH and heat shock in gene regulation and its contribution to the differentiation processes in Leishmania spp. Cycling between cold-blooded insect vectors and the warm-blooded mammalian host is not unique to Leishmania spp., but typical to most parasitic protozoa. It is therefore likely that the mechanism of stress-induced differentiation is shared by other mammalian parasites.

Animals↗
PubMed2001

Antigenic variation at the infected red cell surface in malaria.

Many pathogens that either rely on an insect vector to complete their life cycle (e.g., Trypanosoma spp. and Borrelia spp.) or exist in a unique ecological niche where transmission from host to host is sporadic (e.g., Neisseria spp.) have evolved strategies to maintain infection of their mammalian hosts for long periods of time in order to ensure their survival. Because they have to survive in the face of a fully functional immune system, a common feature of many of these organisms is their development of sophisticated strategies for immune evasion. For the above organisms and for malaria parasites of the genus Plasmodium, a common theme is the ability to undergo clonal antigenic variation. In all cases, surface molecules that are important targets of the humoral immune response are encoded in the genome as multicopy, nonallelic gene families. Antigenic variation is accomplished by the successive expression of members of these gene families that show little or no immunological cross-reactivity. In the case of malaria parasites, however, some of the molecules that undergo antigenic variation are also major virulence factors, adding an additional level of complication to the host-parasite interaction. In this review, we cover the history of antigenic variation in malaria and then summarize the more recent data with particular emphasis on Plasmodium falciparum, the etiological agent of the most severe form of human malaria.

Animals↗
PubMed2000

Genetic Diversity and Evolution of Closteroviruses.

The family Closteroviridae comprises more than 30 plant viruses with flexuous, filamentous virions and includes representatives with either mono- or bipartite positive-strand ssRNA genomes. Closteroviruses are transmitted semipersistently by insects from three families of Homoptera, in infected plants are associated with phloem tissue, and demonstrate an astonishing genetic diversity that suggests extensive, on-going evolution. Phylogenetic analyses of their replicative genes as well as the conserved HSP70 demonstrate that closteroviruses co-evolved with their insect vectors, resulting in three major lineages, i.e. aphid-, mealybug-, and whitefly-transmitted viruses. Closteroviruses apparently represent an ancient and diverse virus family that may pose threats to agriculture and needs serious attention.

Journal Article↗
PubMed2002

Use of multiline cultivars and cultivar mixtures for disease management.

The usefulness of mixtures (multiline cultivars and cultivar mixtures) for disease management has been well demonstrated for rusts and powdery mildews of small grain crops. Such mixtures are more useful under some epidemiological conditions than under others, and experimental methodology, especially problems of scale, may be crucial in evaluating the potential efficacy of mixtures on disease. There are now examples of mixtures providing both low and high degrees of disease control for a wide range of pathosystems, including crops with large plants, and pathogens that demonstrate low host specificity, or are splash dispersed, soilborne, or insect vectored. Though most analyses of pathogen evolution in mixtures consider static costs of virulence to be the main mechanism countering selection for pathogen complexity, many other potential mechanisms need to be investigated. Agronomic and marketing considerations must be carefully evaluated when implementing mixture approaches to crop management. Practical difficulties associated with mixtures have often been overestimated, however, and mixtures will likely play an increasingly important role as we develop more sustainable agricultural systems.

Agriculture↗
PubMed2007

Regulation of carbon metabolism in the mollicutes and its relation to virulence.

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes.

Bacterial Proteins↗