Molecular epidemiology of bluetongue virus in Portugal during 2004-2006 outbreak

After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.     

Development of a candidate DNA vaccine against Maedi-Visna virus

DNA vaccine candidates against Maedi-Visna virus (MVV) infection in ovines were developed as an alternative to conventional vaccines. Candidates were constructed by cloning genes encoding the MVV gag polyprotein and gag proteins p16 and p25 fused to a beta-galactosidase reporter in a plasmid backbone. Transfection of different ovine cells showed a higher protein expression with plasmid lacZp16, which was hence further optimised by (i) removing a putative inhibitory sequence via reduction of the AU-content in the p16 gene or by (ii) introducing a secretory signal (Sc) to promote antigen secretion and increase its presentation to APCs. Unexpectedly, plasmids constructed on the basis of the first strategy by mutagenesis of lacZp16 (lacZp16mut(24)), led to a reduction in the expression of the antigen/reporter fusion in cultured ovine cells. This indicates that the high AU content in MVV does not inhibit protein expression. However, mice primed with lacZp16mut(24) and boosted with MVV protein displayed higher humoral response when compared with control lacZp16. The addition of the Sc signal (Sc-p16) led to lower amounts of intracellular antigen/reporter fusion in transfected ovine cells, thus confirming secretion. These findings correlate with in vivo experiments, which showed that mice primed with Sc-p16 and boosted with MVV exhibited stronger antibody responses when compared with control mice primed with lacZp16 and boosted with MVV. Stronger humoral responses were recorded by immunising mice with (i) Sc-p16 and lacZp16mut(24) plasmids together or with (ii) one plasmid containing both the mutations and the Sc signal.     

Identification and heterologous expression of a Sarcoptes scabiei cDNA encoding a structural antigen with immunodiagnostic potential

The mite Sarcoptes scabiei causes sarcoptic mange (or scabies), a disease of considerable human and veterinary significance. An S. scabiei cDNA clone of about 2 kb was isolated from a S. scabiei var. hominis expression library by immunological screening using blood serum from a naturally infected chamois (Rupicapra rupicapra). The nucleotide sequence of the identified cDNA contains an open reading frame of 1930 bp that encodes a 642 amino acid polypeptide. This polypeptide shows tandem repeats of a glycine-serine rich 20 residue sequence followed by a unique C-terminal glutamate rich 54 residue sequence. The cDNA or the deduced polypeptide did not show significant similarities to any of the sequences in the databases. A carboxyl-terminal fragment of this polypeptide (residues 380 to 642) was efficiently expressed in Escherichia coli as a fusion with Glutathione S-transferase and then was used to produce a specific antiserum. The antigen encoded by the cDNA was located at the integument of the mite's epidermis and the cavities surrounding its vital organs. Western blot analysis of mite extracts using the specific antiserum against the recombinant protein identified antigens larger that 60 kDa indicating that the isolated cDNA did not contain the full ORF. Moreover, we designed a diagnostic assay based on the carboxyl-terminal fragment of the antigen for the identification of infected animals.     

Genetic variability in natural populations of <Emphasis Type="Italic">Paspalum dilatatum</Emphasis> Poir. analyzed by means of morphological traits and molecular markers

Native species show adaptive traits that are difficult to find in introduced species. The Pampas region in Argentina is a valuable nature reserve of grasses and Paspalum dilatatum Poir. is one of the most important grasses found there. Based on ploidy level and on morphological traits, five biotypes of P. dilatatum have been described. Two of them were included in this study: a tetraploid biotype with sexual reproduction and a pentaploid biotype with apomictic reproduction. We analyzed the genetic diversity in eight native populations from the Salado basin, Argentina, using both quantitative traits and molecular data (RAPD) with these aims: to obtain information of the degree of phenotypic variation in that area, to know which the pattern of distribution of this variation is and to look for association between molecular markers with populational or biotypic differentiation. Cluster analysis based on morphological data grouped the individuals of the different populations by ploidy level. Molecular markers showed the inverse situation because individuals were grouped by geographic origin as opposed to biotype. Moreover, since RAPD did not discriminate between biotypes with sexual or apomictic reproduction, they are probably not associated with mating system. The results let us conclude that polygenic traits such as LP, LBSR, NRT and NSP can discriminate between biotypes and molecular markers such as bands 12, 40, 19 and 46 can be used to discriminate among populations, probably because they detect neutral variation. The article is part of a thesis presented by María Victoria García in partial fulfillment of the requirements for the Doctor’s degree, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Argentina.     

Identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets by polyclonal antibody-based enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the species identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. The assay was performed in two different formats, microtiter plates and immunostick tubes, and uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of grouper (anti-GSP), wreck fish (anti-WSP), and Nile perch (anti-PSP). The antibodies were made species-specific by blocking them with the heterologous soluble muscle proteins. Immunorecognition of polyclonal antibodies adsorbed to their specific fish samples was made with swine antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of the species studied.     

A checklist of the cultivated plants of Cuba

Summary Checklists of cultivated plant species of a geographical area have proven to be a very useful tool for the further exploration and collection in this area and for the compilation of local floras of cultivated plants. They contain valuable information for ethnobotanists, plant breeders as well as for other researchers engaged in cultivated plants. A checklist of the cultivated plant species of Cuba compiled from Cuban literature sources and own field studies is presented. It contains 755 taxa belonging to 740 different species, 414 genera and 103 families. These findings are in striking contrast to the opinions of previous foreign investigators about the poverty of plant genetic resources in Cuba. The alphabetically ordered articles for the taxa contain nomenclatural information (accepted name, synonymy and their literature references), the plant family, Cuban folk names, details of plant uses, additional remarks and references to relevant Cuban literature sources, including reports from own explorations. Three appendices provide indexes by families and genera, by synonyms, and by folk names respectively, which allow to locate the information by different ways. The information given in the checklist is maintained in the Database of the Cultivated Plants of Cuba (DBCPC), a microcomputer-based system. Its main purpose is to produce the list and the indexes mentioned from the same set of data, but it allows also to retrieve the information and to check the data for consistency.

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Eine Liste der kubanischen Kulturpflanzen

Zusammenfassung Listen kultivierter Pflanzenarten eines geografischen Gebietes, sog. Checklists, haben sich als nützliches Werkzeug für die weitere Erkundung und Sammlung pflanzlicher genetischer Ressourcen und für die Erarbeitung lokaler Kulturpflanzenfloren erwiesen. Sie enthalten wertvolle Informationen für Ethnobotaniker, Pflanzenzüchter und andere Wissenschaftler, die sich mit Kulturpflanzen befassen. Es wird eine Liste der kultivierten Pflanzenarten Kubas vorgelegt, die auf der Grundlage kubanischer Literaturquellen und eigener Feldforschungen zusammengestellt wurde. Sie enthält 755 Taxa, die 740 verschiedenen Arten, 414 Gattungen und 103 Familien angehören. Diese Ergebnisse stehen in auffälligem Gegensatz zu den Ansichten früherer ausländischer Forscher über die angebliche Armut Kubas an pflanzlichen genetischen Ressourcen. Die alphabetisch angeordneten Artikel über die einzelnen Taxa enthalten nomenklatorische Angaben (akzeptierter Name, Synonymie und zugehörige Literaturzitate), die Pflanzenfamilie, kubanische Volksnamen, Angaben über die Nutzung der Pflanzen, weitere Bemerkungen und Verweise auf kubanische Literaturquellen, einschließlich Berichte über eigene Sammelreisen. Drei Register gestatten das Aufsuchen relevanter Informationen in der Liste nach Pflanzenfamilien und Gattungen, nach Synonymen bzw. nach Volksnamen. Die in der Liste enthaltene Information wird mit Hilfe der Datenbank der Kulturpflanzen Kubas (DBCPC) verwaltet. Dies ist ein mikrocomputer-gestütztes System für die Erzeugung der Liste und der Register auf der Grundlage ein und desselben Datenbestandes; darüberhinaus gestattet es eine Informationsrecherche und unterstützt die Vereinheitlichung der Daten.

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Metal Remobilisation during Resuspension of Anoxic Contaminated Sediment: Short-Term Laboratory Study

The sediments of the Tagus estuary North Channel arecharacterised by high concentrations of trace metals andmonosulphides. During dredging operations Cd, Cu and Pb wereanalysed in water and suspended sediments collected 50 to 100 m around the dredging point. Concentrations in bothfractions fluctuated randomly: 2–3 fold for Cd and Cu and 10 for Pb. Since sampling in the dredging point reflectsintegration of rapid chemical reactions, a short-termlaboratory experiment was conducted to follow the geochemicalalterations occurring in the highest turbidity sites. Theexperiment was monitored as a function of time over a period of4 hr in short time intervals. Dissolved oxygen, pH, E_H,AVS, SO₄ ^2-, Cl^- and metals were monitored in theslurry samples. Iron, Mn, Cd, Pb and Cu were determined in thedissolved fraction (<0.45 μm), in the reactive solid phaseand in the total fraction. Resuspension resulted in asignificant release of Fe, Mn, Cd, Cu and Pb from the solids.Following the release Pb and Cu were almost totally scavengedin the 4 hr by the newly precipitated Fe oxyhydroxides, while more than 50% of the mobilised Cd remained in the dissolved fraction. The less efficient removal of Cd from solution implies a prolonged availability of this metal in the environment.