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A review of organophosphate poisoning.

Many organophosphate compounds are pesticides widely used for the control of insect vectors. They are not ideal agents because they lack target vector selectivity, and have caused severe toxicity and even death in humans and domestic animals. Their toxicity has been recognised since the 1930s, when they were also developed for use as chemical warfare agents. The mechanism of action of organophosphates has been determined in some depth; the understanding of the toxic effects resulting from the inhibition of cholinesterase activity, causing accumulation of acetylcholine at nerve endings has played a major part in providing a rationale for specific antidote treatment using atropine and oximes. However, the most suitable oxime for reactivation of cholinesterases has still not been established with certainty, although pralidoxime is widely recommended. Chronic toxicity, particularly the neuropathic effects, merits further study because it contributes substantially to the long term morbidity in cases of severe acute, or chronic, exposure. Prevention of potentially toxic organophosphate exposure, particularly amongst employees in industries manufacturing or using the compounds and in the most susceptible groups of the population, such as the young and the elderly, should be sought wherever possible. Government authorities should be encouraged to control organophosphate product licensing, manufacture, storage, import, methods of use and delivery, food contamination and disposal.

Antidotes↗

Genetic mapping of maize stripe disease resistance from the Mascarene source.

Maize stripe virus (MStV) is a potentially threatening virus disease of maize in the tropics. We mapped quantitative trait loci (QTLs) controlling resistance to MStV in a maize population of 157 F(2:3) families derived from the cross between two maize lines, Rev81 (tropical resistant) and B73 (temperate susceptible). Resistance was evaluated under artificial inoculations in replicated screenhouse trials across different seasons in Réunion Island, France. Composite interval mapping was employed for QTL detection with a linkage map of 143 microsatellite markers. Heritability estimates across seasons were 0.96 and 0.90 for incidence and severity, respectively, demonstrating a high genotypic variability and a good control of the environment. Three regions on chromosomes 2L, 3 and 5, with major effects, and another region on chromosome 2S, with minor effects, provided resistance to MStV in Rev81. In individual seasons, the chr2L QTL explained 60-65% of the phenotypic variation for disease incidence and 21-42% for severity. The chr3 QTL, mainly associated with incidence and located near centromere, explained 42-57% of the phenotypic variation, whereas the chr5 QTL, mainly associated with severity, explained 26-53%. Overall, these QTLs explained 68-73% of the phenotypic variance for incidence and 50-59% for severity. The major QTLs on chr2 and 3 showed additive gene action and were found to be stable over time and across seasons. They also were found to be included in genomic regions with important clusters of resistance genes to diseases and pests. The major QTL on chr5 appeared to be partially dominant in favour of resistance. It was stable over time but showed highly significant QTL x season interactions. Possible implications of these QTLs in different mechanisms of resistance against the virus or the insect vector are discussed. The prospects for transferring these QTLs in susceptible maize cultivars and combining them with other resistances to virus diseases by conventional or marker-assisted breeding are promising.

Chromosome Mapping↗

Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR.

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0. 872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

Brazil↗

Lipophosphoglycan of the protozoan parasite Leishmania: stage- and species-specific importance for colonization of the sandfly vector, transmission and virulence to mammals.

Leishmaniasis is a major health problem to the human population of the tropics, subtropics and Mediterranean regions. This disease is caused by the parasitic protozoa Leishmania, which have adapted to survive in several hostile environments such as the vector insect midgut, blood and the mammalian macrophage phagolysosome. Several Leishmania glycoconjugates have been implicated as key molecules for these remarkable capabilities. This review summarizes the current knowledge on potential and proven functions of the most prominent of the Leishmania glycoconjugates, the lipophosphoglycan.

Animals↗

Induction of stress proteins in the plant trypanosome Phytomonas characias.

The present study of the synthesis of new proteins in plant trypanosomatids in the genus Phytomonas as a response to different types of stress demonstrates the production of a number of proteins that can be grouped into four families similar to those that appear in other organisms (heat-shock proteins). In the study of stress, Phytomonas cultures were subjected to changes in temperature from 22 degrees to 37 degrees C, deprived of glucose, grown in the presence of sodium arsenite, and treated with calcium ionophore. In addition, the culture medium was changed from Grace's medium (330 mosmol/1) to a plant-culture medium with an osmolarity of 286 mosmol/l, implying the exertion of stress during the parasite's normal biological cycle of passage from the insect vector to the plant host. The treatment with actinomycin D demonstrated that some of the mRNAs that codify these proteins are found in normal presynthesized conditions. To measure the effect of temperature on the macromolecule biosynthesis we compared the incorporation of labeled analogues ([3H]-thymidine, [3H]-uridine, and [3H]-leucine) by flagellates cultured at 22 degrees C with that by parasites cultivated at 37 degrees C.

Animals↗

Characterization and genetic diversity of potato yellow mosaic virus from the Caribbean.

The begomovirus Potato yellow mosaic virus (PYMV) is responsible of significant yield losses in tomato in Guadeloupe. Four field isolates from Guadeloupe were analyzed in term of their host range using three inoculation methods (mechanical, grafting and insect vector), sequences analysis of PCR fragments and phylogenetic analysis of an infectious clone, PYMV-[GP]. Capsicum annuum, Datura stramonium, Nicotiana benthamiana, N. tabacum 'Xanthi NC', Petunia hybrida, and Solanum tuberosum were found to be hosts. All isolates from Guadeloupe, Martinique, Puerto Rico and the Dominican Republic were closely related to PYMV-[GP]. Sequence identity between PYMV-[GP] and PYMV-Ve from Venezuela and PYMTV from Trinidad and Tobago clearly confirmed that it is a new strain of PYMV.

Caribbean Region↗

Pns4 of rice dwarf virus is a phosphoprotein, is localized around the viroplasm matrix, and forms minitubules.

Rice dwarf virus (RDV), a member of the family Reoviridae, has a 12-segmented dsRNA genome. Seven segments, designated S1, S2, S3, S5, S7, S8, and S9, encode structural proteins, while the remainder encode nonstructural proteins. One of the nonstructural proteins, Pns4, which is encoded by S4, was characterized. Pns4 was a phosphorylatable substrate in a phosphorylation assay in vivo; it associated with large cytoplasmic fibrils and formed novel minitubules in infected cultured cells of its leafhopper insect vector, as revealed by immunofluorescence and immunoelectron microscopy. Early in infection, Pns4 was detected at the periphery of the viroplasm, and it was then observed on amorphous or fibrillar inclusions, which were identified as bundles of minitubules, at later stages of infection. Since viroplasms are believed to be the site of RDV replication, the intracellular location of Pns4 suggests that this protein might be involved in the process of assembly of the RDV virion.

Animals↗

Expression of rice yellow mottle virus coat protein enhances virus infection in transgenic plants.

The disease caused by rice yellow mottle virus (RYMV) is a major, economically important constraint to rice production in Africa. RYMV is mechanically transmitted by a variety of agents, including insect vectors. The production of resistant rice varieties would be an important advance in the control of the disease and increase rice production in Africa. We produced transgenic plants of the Oryza sativa japonica variety, TP309, to express a RYMV coat protein gene (CP) and mutants of the CP under the control of a ubiquitin promoter. Transgenic plants expressing genes that encode wild-type CP (wt.CP), deleted CP (DeltaNLS.CP), mRNA of the CP, or antisense CP sequences of the CP gene were characterised. Eighty per cent (80%) of independent transgenic lines analysed contained CP gene sequences. Transgenic plants were challenged with RYMV and produced two types of reactions. Most of the plants expressing antisense sequences of the CP and untranslatable CP mRNA exhibited a delay in virus accumulation of up to a week, and the level of virus accumulation was reduced compared with non-transgenic TP309 plants. Transgenic plants expressing RYMV wild-type CP (wt.CP) and deleted CP (DeltaNLS.CP) accumulated the highest levels of virus particles. These results suggest that antisense CP and untranslatable CP mRNA induced moderate resistance, whereas transgenic CP enhanced virus infection.

Capsid Proteins↗

An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach.

Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication. By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure. Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences. A model of potential Rep-iteron contacts is proposed. The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a beta-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae. The evolutionary implications of these findings are discussed.

Amino Acid Motifs↗

Detection and localization of Rice stripe virus gene products in vivo.

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2-4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N(.)and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.

Animals↗

Molecular variation in Leishmania.

The surface coat of the protozoan parasite Leishmania affords remarkable protection in the harsh environments encountered within the insect vectors and vertebrate hosts. It also provides specificity for the interaction of these parasites with the cells in the sandfly gut and with the human macrophage. Surprisingly few molecules have been identified on the Leishmania surface. The major surface molecules of both promastigotes and amastigotes are the glycoconjugates lipophosphoglycan and a glycoprotein of approximately 63 kDa. These major surface molecules vary structurally between Leishmania species and throughout the life-cycle of the parasite. In addition to these major glycoconjugates, Leishmania produce a number of less abundant surface molecules, including a family of glycosyl-inositol phospholipids, the Promastigote Surface Antigen-2 complex of glycoproteins and a glycoprotein of M(r) 46,000. These molecules share the common feature of attachment to the plasma membrane via glycosylphosphatidylinositol lipid anchors. Leishmania also release molecules from their surface in a species specific manner. In this review we will examine the molecular variation of these molecules and their biological importance. We will also discuss the potential of these molecules as targets for chemotherapy and as candidate vaccines.

Amino Acid Sequence↗

Comparison of chagasic and non-chagasic myocardiopathies by ELISA and immunoblotting with antigens of Trypanosoma cruzi and Trypanosoma rangeli.

Trypanosoma cruzi associated myocardiopathy, or Chagas disease, continues to be a serious problem in Venezuela, for which there is neither a vaccine nor a cure. In order to learn more about the humoral immune response to trypanosomal antigens, and to try to identify dominant antigens, we used ELISA and immunoblotting to study the reactivity of sera from patients with chagasic and non-chagasic myocardiopathies, against surface and secreted proteins from T. cruzi and T. rangeli. Both species are found in the same insect vector, but only T. cruzi is thought to be pathogenic in vertebrates. The ELISA results fell into three patterns: (1) high reactivity values with both T. cruzi and T. rangeli surface and secreted proteins; (2) high values to T. cruzi but low values with T. rangeli; and (3) high values to T. rangeli and low values with T. cruzi. This finding that some chagasic sera react more strongly against T. rangeli than against T. cruzi is intriguing, and warrants further investigation. When chagasic sera were tested on Western blots of total extracts of T. cruzi and T. rangeli, the pattern of reactive bands was similar against both parasites, but no two sera showed an identical pattern. Furthermore, there was no correlation between a particular immunoblotting pattern and either the antibody titer, or the severity of the disease. Several T. cruzi and T. rangeli antigens were recognized by sera from healthy controls as well as from patients with other tropical diseases endemic in Venezuela. Overall, our results suggest that the humoral immune response to trypanosomal antigens is complex, and no single antigen may be the determining factor in the pathogenesis of chagasic myocardiopathy.

Adult↗

Hybridization on squashed flies: a method to detect gene sequences in individual Drosophila.

Specific gene sequences can be detected by DNA hybridization to individual Drosophila squashed on cellulose or nylon filters. This "squash-blot" method permits the rapid survey of DNA polymorphism in large Drosophila population samples. It could also be useful for studying chromosome aberrations, departure from diploidy, and detection of pathogenic agents in vector insects.

Animals↗

Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase.

The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.

Animals↗

Lumpy skin disease, an African capripox virus disease of cattle.

Lumpy skin disease is an infectious viral disease of cattle, which often occurs in epizootic form. The disease is characterized by the eruption of nodules in the skin, which may cover the whole of the animal's body. Systemic effects include pyrexia, anorexia, dysgalactia and pneumonia; lesions are often found in the mouth and upper respiratory tract. The severity of the disease varies considerably between breeds and strains of cattle. Many cattle suffer severe emaciation and loss of production for several months. The skin lesions cause permanent damage to the hides. The mode of transmission of the disease has not been clearly established. Contact infections do not readily occur and the evidence from the epizootiology strongly suggests that insect vectors are involved. The disease has been confined to sub-Saharan Africa, until it recently appeared in epizootic form in Egypt and in Israel. Transmission occurs in a wide variety of biotypes, from semi-desert to temperate grasslands and irrigated land. It has the potential to extend its range further.

Africa↗

Trypanosoma cruzi: differentiation to metacyclic trypomastigotes in the presence of ADP-ribosyltransferase inhibitors.

The participation of ADP-ribosyltransferase in Trypanosoma cruzi differentiation to the metacyclic stage was evaluated by analyzing morphogenesis blockage by specific enzyme inhibitors: benzamide, 3-aminobenzamide, theophylline, and nicotinamide. In vitro assays showed a statistically significant reduction in the number of metacyclic forms only when any one of the four inhibitors was added during the period of interaction between epimastigote and Triatoma infestans intestinal homogenate or when present throughout the subsequent culture period in Grace's medium. When nicotinamide or benzamide was present during both interaction and culture period, morphogenesis was virtually abolished (less than or equal to 2%). In the in vivo assays, mice inoculated with parasites obtained from the insect vectors fed with trypomastigote-infected blood containing one of the four enzyme inhibitors developed lower parasitemias and showed longer survival in every case, compared with the respective controls. These findings suggest ADP-ribosyltransferase participation in T. cruzi differentiation both in vitro and in vivo.

Animals↗

Separation of viable and non-viable Onchocerca microfilariae using an ion exchanger.

A technique has been developed for separating viable and non-viable Onchocerca lienalis microfilariae (mff) by passing a suspension of the parasites through a column of DEAE cellulose. A proportion (between 42% and 87%) of normally motile parasites, whether unfrozen or cryopreserved, passed through the cellulose columns and retained their viability. Non-motile and sluggish mff were retained by the cellulose. The proportion of viable cryopreserved mff was greater after passage through the column as assessed by motility, migration in mice and development in the insect vector. Numbers of cryopreserved mff eluted through the columns peak quickly at a relatively high level after about 20 ml of eluate had been passed through, whereas normal unfrozen parasites rose and fell in numbers more slowly. By using sodium chloride gradients and buffers of differing conductivity it was concluded that the separation of the mff is probably due to net charge changes as well as motility. It is believed that this technique offers considerable promise as a tool to provide populations of mff of more uniform viability following freezing and thawing with existing cryopreservation techniques.

Animals↗