Epidemiological and virological findings during an outbreak of equine influenza in Uruguay in 2018.

Equine influenza is one of the major respiratory infectious diseases in horses. In 2018, equine influenza virus (EIV) was confirmed as the cause of outbreaks of respiratory disease in horses in Chile and Argentina. In the same year, for the first time in Uruguay, EIV infection was confirmed by isolation and molecular analysis to be the cause of respiratory disease among hundreds of clinically affected thoroughbred horses in training and racing facilities. The virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A realtime reverse transcription polymerase chain reaction (rRT-PCR). The partial nucleotide sequence of the haemagglutinin 1 (HA1 ) gene (994 base pairs) was determined and analysed phylogenetically using MEGA X software. Amino acid sequence alignments were constructed, and serum samples were tested by haemagglutination inhibition and single radial haemolysis. The diagnosis of EIV was confirmed by rRT-PCR, virus isolation and serological testing. The phylogenetic analysis of the partial HA1 gene sequence of the isolated virus indicated that it belongs to clade 1 of the Florida sub-lineage of the American lineage and is closely related to viruses isolated in the recent past. Study of the HA1 region (331 amino acids) of the virus identified in horses in racing facilities in Uruguay displayed the highest amino acid sequence identity with viruses detected in Argentina, Chile and the United Kingdom in 2018. The surveillance data reported illustrate the international spread of EIVs and support the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel to include viruses of the Florida sub-lineage in vaccines.

La grippe équine est l'une des principales maladies respiratoires infectieuses affectant les équidés. En 2018, il a été confirmé que des foyers de maladie respiratoire enregistrés chez des chevaux au Chili et en Argentine étaient dus au virus de la grippe équine. Cette même année en Uruguay, pour la première fois dans ce pays, il a été établi par isolement viral et par des méthodes moléculaires que le virus de la grippe équine était l'agent causal d'une maladie respiratoire affectant cliniquement des centaines de chevaux pur-sang dans des centres d'entraînement et des hippodromes. La détection du virus s'est faite à partir d'écouvillons prélevés par voie naso-pharyngée en appliquant une technique d'amplification en chaîne par polymérase couplée à une transcription inverse en temps réel (rRT­PCR) à large spectre pour les virus influenza de type A. Une séquence nucléotidique partielle correspondant au gène de l'hémagglutinine 1 (HA1) (994 paires de bases) a fait l'objet d'une analyse phylogénétique au moyen du programme informatique MEGA X. Il a été procédé à la construction d'une matrice d'alignements de ces séquences d'acides aminés. D'autre part, des prélèvements de sérum issus de chevaux atteints ont été soumis à l'épreuve d'inhibition de l'hémagglutination et à une hémolyse radiale unique. Aussi bien la rRT­PCR que l'isolement viral et l'analyse sérologique ont confirmé le diagnostic de l'infection par le virus de la grippe équine. Il ressort de l'analyse phylogénétique du fragment de séquence du gène HA1 du virus isolé que ce dernier appartient au clade 1 de la sous-lignée Florida de la lignée américaine et qu'il est étroitement apparenté à des virus isolés au cours des dernières années. L'étude de la région HA1 (331 acides aminés) du virus détecté chez des chevaux de course en Uruguay a montré que les virus qui présentaient la plus grande similitude avec cette séquence d'acides aminés étaient ceux détectés en Argentine, au Chili et au Royaume-Uni en 2018. Les données de surveillance rapportées illustrent la propagation à l'échelle internationale des virus de la grippe équine et renforcent la recommandation émise par le Groupe d'experts de l'Organisation mondiale de la santé animale (OIE) chargé de la surveillance de la composition des vaccins contre la grippe équine d'inclure les virus de la sous-lignée Florida dans la composition de ces vaccins.

La gripe equina es una de las principales infecciones respiratorias que afectan al caballo. En 2018 se confirmó que el virus de la gripe equina era la causa de diversos brotes de afección respiratoria que habían afectado a caballos de Chile y Argentina. Ese mismo año, por primera vez en el Uruguay, se confirmó por aislamiento y análisis molecular que el virus de la gripe equina era la causa de una infección respiratoria que, acompañada de manifestaciones clínicas, afectó a cientos de caballos purasangre de hipódromos y centros de adiestramiento. El virus fue detectado en muestras de frotis nasales mediante una técnica de reacción en cadena de la polimerasa acoplada a transcripción inversa en tiempo real (rRT­PCR, por sus siglas en inglés) que reacciona ante todos los virus gripales de tipo A. Tras secuenciar parcialmente el gen de la hemaglutinina 1 (HA1 ) (994 pares de bases), se procedió a su análisis filogenético empleando el programa informático MEGA X. Además de crear una matriz de alineamiento de secuencias de aminoácidos, se sometieron muestras de suero a pruebas de inhibición de la hemaglutinación y hemólisis radial simple. Así, el diagnóstico que apuntaba al virus de la gripe equina fue confirmado por rRT­PCR, aislamiento vírico y análisis serológico. El análisis filogenético de la secuencia parcial del gen HA1 del virus aislado puso de manifiesto que pertenece al clado 1 del sublinaje Florida del linaje americano y guarda estrecho parentesco con otros virus aislados en fechas recientes. El estudio de la región HA1 (331 aminoácidos) del virus detectado en caballos de hipódromos uruguayos reveló que el mayor nivel de concordancia de su secuencia de aminoácidos se daba con virus detectados en Argentina, Chile y el Reino Unido en 2018. Los datos de vigilancia comunicados dan fe de la propagación internacional de los virus de la gripe equina y avalan la recomendación formulada por el Panel de expertos en vigilancia de la composición de las vacunas contra la gripe equina de la Organización Mundial de Sanidad Animal (OIE), que aboga por incluir virus del sublinaje Florida en las vacunas.

Evaluation of VEGF gene polymorphisms and proliferative diabetic retinopathy in Mexican population.

AIM: To assess if the included vascular endothelial growth factor (VEGF) polymorphisms rs3025035, rs3025021 and rs2010963 are associated to proliferative retinopathy in a Mexican population with type 2 diabetes mellitus (T2DM). METHODS: A case-control study was conducted in adult individuals with T2DM associated to proliferative retinopathy or non-proliferative retinopathy from Oct. 2014 to Jun. 2015 from the Retina Department of the Asociation to Prevent Blindness in Mexico. The selected patients were adults with a diagnosis of T2DM ≥5y. All subjects had a comprehensive ocular examination and the classification of the retinopathy severity was made considering the Early Treatment Diabetic Retinopathy Study (ETDRS) standardization protocols. Genomic DNA was extracted from whole fresh blood. All samples were genotyped by qPCR for selected VEGF polymorphisms. Hardy-Weinberg equilibrium was calculated by comparing Chi-square values between the expected and the observed values for genotype counts. RESULTS: In total 142 individuals were enrolled, 71 individuals with T2DM and associated proliferative retinopathy and 71 individuals with non-proliferative retinopathy. One-sided Fisher's exact test was performed for rs3025021 [OR (95% CI)=0.44(0.08-2.2); P=0.25] and rs2010963 [OR (95% CI)=0.63(0.25-1.6); P=0.23]. The minor allelic frequencies obtained were 26% for rs3025021, 10% for rs3025035 and 61% for rs2010963. The pairwise linkage disequilibrium between the three SNP was assessed, and was as follows: rs3025021 vs rs3025035: D'=1.0, r2=0.1043, P≤0.0001; rs3025021 vs rs2010963: D'=0.442, r2=0.0446, P=0.149; rs3025035 vs rs2010963: D'=0.505, r2=0.0214, P=0.142. CONCLUSION: This is the first analysis involving VEGF polymorphisms and proliferative diabetic retinopathy in a Mexican population. A major finding of the present study is that none of the polymorphisms studied was significantly associated with proliferative retinopathy. Based on these results, we can infer that different populations have different associations for the same polymorphisms.

Absceso del psoas como presentación de tuberculosis vertebral: reporte de un caso / Psoas abscess as spinal tuberculosis presentation: case report

Introducción: El absceso del psoas es un cuadro infrecuente cuyo principal agente etiológico es el Staphyloccocus aureus, y que con baja frecuencia puede ser de etiología tuberculosa como complicación de una afección vertebral por Mycobacterium tuberculosis. Presentación del caso: Mujer de 58 años, mapuche, con antecedentes de enfermedad pulmonar obstructiva crónica y alcoholismo crónico, consultó por cuadro de dos meses de evolución de dolor lumbar, baja de peso y paresia de extremidad inferior derecha que impedía la marcha. Tomografía computada (TC) de columna evidenció espondilodiscitis y absceso del psoas derecho, iniciándose tratamiento antibiótico cubriendo Staphyloccocus aureus. Los hemocultivos y los cultivos de la colección resultaron negativos por lo que se decidió mantener tratamiento ambulatorio. Paciente consultó cuatro meses después por exacerbación de su disnea basal de dos semanas de evolución, radiografía de tórax y TC de tórax de alta resolución compatibles con tuberculosis pulmonar con diseminación miliar bilateral, por lo cual, se inició tratamiento antituberculoso, realizándose baciloscopías que resultaron negativas. Además, se solicitó identificación del bacilo de Koch mediante reacción de polimerasa en cadena que resultó positiva, con lo que se confirmó el diagnóstico de tuberculosis miliar y mal de Pott. Se decidió reevaluar con TC de columna una vez finalizado el tratamiento antituberculoso para decidir conducta quirúrgica. Discusión: Es importante la sospecha activa de etiología tuberculosa ante una espondilodiscitis y un absceso del psoas, pese a su baja frecuencia. El inicio del tratamiento en forma precoz puede modificar la progresión de una infección que puede ser invalidante e incluso mortal.

Introduction: The Psoas abscess is an infrecuent condition which main ethiology is the Staphyloccocus aureus and with less frequency can be a vertebral tuberculosis complication caused by Mycobacterium tuberculosis. Case Report: a 58 years old female, Mapuche, with Chronic Obstructive Pulmonar Disease and chronic alcoholism antecedents, was admitted in the emergency room with a two month old lumbar pain, loss of weight and right inferior extremity paresia which didn't allowed her to walk. The column's Computed Tomography (CT) showed spondylodiscitis and a right psoas abscess, the antibiotherapy, covering Staphyloccocus aureus, was started. The hemoculives and abscess' cultives were negative, so it was decided to continue with ambulatory treatment. The patient was admitted four month later because of two weeks of exacerbation of her basal dysnea. The thorax Radiography and high resolution CT were compatibles with pulmonary tuberculosis with bilateral miliar disemination. Antituberculosis treatment was started, bacilloscopies were negative, also, a Polymerase Chain Reaction identification of Koch's bacillus was performed, resulting positive, confirming the diagnosis of miliar tuberculosis and Pott's disease. Once antituberculosis treatment was finished, a new column's CT was requested to decided surgical conduct. Discussion: Is important to actively suspect of tuberculosis ethiology in presence of spondylodiscitis and psoas abscess, despite it's low frequency. The early treatment start may modify the progresion of an infección that can be invalidating and even mortal.

Characterization and expression analysis of Toll-like receptor 3 cDNA from Atlantic salmon (Salmo salar).

Innate pathway activation is fundamental for early anti-viral defense in fish, but currently there is insufficient understanding of how salmonid fish identify viral molecules and activate these pathways. The Toll-like receptor (TLR) is believed to play a crucial role in host defense of pathogenic microbes in the innate immune system. In the present study, the full-length cDNA of Salmo salar TLR3 (ssTLR3) was cloned. The ssTLR3 cDNA sequence was 6071 bp long, containing an open reading frame of 2754 bp and encoding 971 amino acids. The TLR group motifs, such as leucine-rich repeat (LRR) domains and Toll-interleukin-1 receptor (TIR) domains, were maintained in ssTLR3, with sixteen LRR domains and one TIR domain. In contrast to descriptions of the TLR3 in rainbow trout and the murine (TATA-less), we found a putative TATA box in the proximal promoter region 29 bp upstream of the transcription start point of ssTLR3. Multiple-sequence alignment analysis of the ssTLR3 protein-coding sequence with other known TLR3 sequences showed the sequence to be conserved among all species analyzed, implying that the function of the TLR3 had been sustained throughout evolution. The ssTLR3 mRNA expression patterns were measured using real-time PCR. The results revealed that TLR3 is widely expressed in various healthy tissues. Individuals challenged with infectious pancreatic necrosis virus and immunostimulated with polyinosinic:polycytidylic acid exhibited increased expression of TLR3 at the mRNA level, indicating that ssTLR3 may be involved in pathogen recognition in the early innate immune system.

Detection of Salmonella enterica in Magellanic penguins (Spheniscus magellanicus) of Chilean Patagonia: evidences of inter-species transmission.

Patagonia in southern South America is among the few world regions where direct human impact is still limited but progressively increasing, mainly represented by tourism, farming, fishing and mining activities. The sanitary condition of Patagonian wildlife is unknown, in spite of being critical for the assessment of anthropogenic effects there. The aim of this study was the characterization of Salmonella enterica strains isolated from wild colonies of Magellanic penguins (Spheniscus magellanicus) located in Magdalena Island and Otway Sound, in Chilean Patagonia. Eight isolates of Salmonella were found, belonging to Agona and Enteritidis serotypes, with an infection rate of 0·38%. Resistance to ampicillin, cefotaxime, ceftiofur and tetracycline antimicrobials were detected, and some of these strains showed genotypic similarity with Salmonella strains isolated from humans and gulls, suggesting inter-species transmission cycles and strengthening the role of penguins as sanitary sentinels in the Patagonian ecosystem.

A comparison of neutral and immune genetic variation in Atlantic salmon, Salmo salar L. in Chilean aquaculture facilities.

Genetic diversity was assessed in samples of cultured Atlantic salmon, Salmo salar L., obtained from facilities in Chile between 2005 and 2010, a period of time during which the infectious pathogens Infectious Salmon Anemia (ISA) virus, Caligus rogercresseyi (sea lice), and Piscirickettsia salmonis (salmon rickettsial syndrome) were common. Two panels of microsatellite markers were utilized: one with microsatellites with no known gene associations (neutral) and one featuring microsatellites linked to putative immune-related genes (immune-related). Allelic richness and gene diversity across samples were significantly greater in neutral loci as compared to immune-related loci. Both diversity measures were homogeneous among samples for immune-related loci and heterogeneous among samples for neutral loci. Immune-related loci were identified as F(ST) outliers in pairwise comparisons of samples at a 10-fold higher frequency than neutral loci. These results indicate that neutral and immune-related portions of the Atlantic salmon genome may have differed in response to the gauntlet of pathogens and that monitoring of specific, well characterized immune-related loci as well as neutral loci in cultured species could be useful when disease control and prevention is a goal.

Phenotype gene expression differences between resistant and susceptible salmon families to IPNV.

Infectious pancreatic necrosis virus (IPNV) is an economically important pathogen of the salmonid aquaculture industry. Selective breeding has been employed to improve resistance to this infectious disease, and it is of importance to investigate the expression profile of immune genes of Atlantic salmon with different genetic background in response to this virus. This study examined the immune modulation response of eight candidate genes in head kidney tissue in two families of Atlantic salmon with high and low mortalities, after challenge with IPNV. The results showed that the expression pattern of target genes differed in the two families. Generally, higher expression of antiviral, pro-inflammatory genes and transcription factors such as tripartite motif, NF-κB, IFNI, STAT1, protein kinase R, and Vig-2 in the resistant family were observed at the same time point. One may speculate the functional importance of these putative candidate genes in the characterization of the IPNV-resistant (low mortalities) immune phenotype. Therefore, on our findings, we suggest that future salmonids studies aiming to identify candidate genes/pathway or vaccines evaluation should consider validating detected genes/pathway across different genetic backgrounds or immune phenotype.

Population structure in Chilean hake Merluccius gayi as revealed by mitochondrial DNA sequences.

Genetic variation and divergence among samples of Chilean hake Merluccius gayi, from three localities off the coast of Chile and one locality off the coast of northern Peru, were assessed using sequences from the control region of mitochondrial DNA. Homogeneity tests revealed occurrence of at least three distinct genetic stocks of M. gayi within the region sampled. Factors potentially contributing to genetic divergence among M. gayi probably include hydrodynamics and behaviour.

A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content.

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.

Converging pathways in the occurrence of endoplasmic reticulum (ER) stress in Huntington's disease.

A variety of neurological diseases including Huntington's disease (HD), Alzheimer's disease and Parkinson's disease share common neuropathology, primarily featuring the presence of abnormal protein inclusions containing specific misfolded proteins. Mutations leading to expansion of a poly-glutamine track in Huntingtin cause HD, and trigger its misfolding and aggregation. Recent evidence indicates that alterations in the secretory pathway, in particular the endoplasmic reticulum (ER), are emerging features of HD. Although it is not clear how cytoplasmic/nuclear located mutant Huntingtin alters the function of the ER, several reports indicate that mutant Huntingtin affects many essential processes related to the secretory pathway, including inhibition of ER-associated degradation, altered ER/Golgi vesicular trafficking and axonal transport, disrupted autophagy and abnormal ER calcium homeostasis. All these alterations are predicted to have a common pathological outcome associated to disturbance of protein folding and maturation pathways at the ER, generating chronic ER stress and neuronal dysfunction. Here, we review recent evidence involving ER stress in HD pathogenesis and discuss possible therapeutic strategies to target organelle function in the context of disease.

Avaliação preliminar da atividade antimicrobiana do extrato de erva-mate (Ilex paraguariensis A. St. - Hil.) obtido por extração com CO2 supercrítico / Preliminary evaluation of the antimicrobial activity of "erva mate" (Ilex paraguariensis A. St. - Hil.) extract obtained by using supercritical CO2

Ilex paraguariensis A. St. - Hil. (erva-mate) é uma árvore pertencente à família Aquifoleaceae, nativa da região sul da América do Sul e de grande importância cultural e econômica para os três estados da região sul do Brasil. O presente trabalho determinou o potencial antimicrobiano do extrato de erva-mate obtido por fluido supercrítico, frente a alguns microrganismos. As folhas de Ilex paraguariensis foram coletadas na região norte do RS. O extrato bruto da planta desidratada foi obtido pelo método de extração com fluido supercrítico, utilizando dióxido de carbono (CO2) como solvente. Foi obtida a solução mãe desse extrato (100 mg mL-1) que a seguir foi diluído (50; 25; 12,5; 6,25 mg mL-1) para avaliação da atividade antimicrobiana por meio da técnica de difusão do disco em ágar Müeller Hinton, sobre cepas de Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) e Enterococcus faecalis (ATCC 29212). As análises foram efetuadas em triplicata, utilizando como controle negativo disco estéril sem impregnação e como controle positivo discos de antibióticos comerciais variáveis de acordo com a espécie bacteriana. Após 16 a 24h de incubação a 35ºC os halos com significância foram medidos. Observou-se atividade antimicrobiana frente a S. aureus (halos de inibição variaram entre 7 e 18 mm) e para P. aeruginosa (halos entre 6 e 11 mm), ambos nas concentrações de 100 até 25 mg mL-1. O extrato obtido por extração em fluido supercrítico de Ilex paraguariensis apresentou atividade antimicrobiana frente a S. aureus e P. aeruginosa.

Ilex paraguariensis A. St. - Hil. ("erva mate") is a tree of the Aquifoliaceae family, native to the south region of South America and of great cultural and economic importance for the three states of the south region of Brazil. The present study aimed to determine the antimicrobial potential of "erva mate" extract obtained by supercritical fluid against some microorganism strains. The leaves of Ilex paraguariensis were collected in the north region of Rio Grande do Sul State. The crude extract of dehydrated plant was obtained by supercritical fluid extraction, using carbon dioxide (CO2) as solvent. The extract mother solution (100 mg mL-1) was diluted (50; 25; 12, 5; 6.25 mg mL-1) for antimicrobial activity evaluation, by means of disk diffusion method on Müeller-Hinton agar, against strains of Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212). The analyses were done in triplicate, using as negative control sterile disk without impregnation and as positive control, commercial antibiotic disks according to the bacterial species. After 16-24h incubation at 35ºC, the diameters of inhibition zones were measured. Antimicrobial activity was observed against S. aureus (inhibition zones between 7 and 18 mm) and P. aeruginosa (inhibition zones between 6 and 11 mm), both at the concentrations of 100 mg mL-1 to 25 mg mL-1. The extract of Ilex paraguariensis obtained by supercritical fluid presented antimicrobial activity against S. aureus and P. aeruginosa.

Economic effect of bovine abortion syndrome in commercial dairy herds in Southern Chile.

Bovine abortion is a limiting factor for dairy business, as it decreases milk production and the potential, number of herd replacements, increases feeding and medical treatment costs, increases the number of artificial inseminations to obtain a calf as well as culling rates of cows. An estimation of the economic impact of abortion in dairy farms in Chile is not available yet. The aim of this study was to estimate the economic consequences of bovine abortion syndrome (BAS) in dairy cows from Chile. A stochastic model was proposed to evaluate the cost of an abortion on a yearly basis to include variability in cost and income by dairy and by year. The marginal total net revenue (ΔTNR) for a typical, lactation was obtained by the calculating the difference between total revenues (retail milk and calf sales) and total expenses (production cost (cows, feeding, labor, health) plus administrative and, general costs) for lactation with and without abortion. Production data were obtained from a retrospective study of 127 dairy herds located in southern Chile between 2000 and 2006. Milk production from cows with and without abortion was estimated by a mixed model using milk test day data. Production cost and prices paid to farmers were obtained from service company records (TODOAGRO S.A.). Cost and income value was corrected for inflation and expressed in the values from 2006. In addition, a separate analysis for different parities (1, 2, 3 or more) was performed. Distributions for the stochastic variables were obtained by fitting distributions from our database using @Risk. The stochastic variables included in the analysis were all related to income, feeding, depreciation, health, Artificial Insemination and general costs like fuel, salaries, taxes, etc. There was a high probability (89.20%) of a negative ΔTNR in lactations with abortion for overall, parities, with a mean loss of $ -143.32. Stratifying by parity, the predicted mean of the distribution for ΔTNR in each parity (1, 2, 3 or more) was also negative and the probability of a negative ΔTNR was 89.40%, 95.30% and 97.00%, respectively, but differs between them (p<0.05). For parity 1, mean ΔTNR was $ -120.92, parity 2 $ -116.35 and for parities ≥3 it was $ -132.26 and the mean was statistically different from the others (p<0.05). The age of culled cows was the input variable most correlated with TNR and dairy production was the second. However, the sale price of milk resulted in a low correlation with abortion cost.

Simple DNA extraction protocol for a 16S rDNA study of bacterial diversity in tropical landfarm soil used for bioremediation of oil waste.

Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oil-contaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 micro g DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria - alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments.

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus.

A set of expressed sequence tag-simple sequence repeat (EST-SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di-, tri- and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross-species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.

Application of Origanum majorana L. essential oil as an antimicrobial agent in sausage.

This work reports on the antimicrobial activity in fresh sausage of marjoram (Origanum majorana L.) essential oil against several species of bacteria. The in vitro minimum inhibitory concentration (MIC) was determined for 10 selected aerobic heterotrophic bacterial species. The antimicrobial activity of distinct concentrations of the essential oil based on the highest MIC value was tested in a food system comprising fresh sausage. Batch food samples were also inoculated with a fixed concentration of Escherichia coli and the time course of the product was evaluated with respect to the action of the different concentrations of essential oil. Results showed that addition of marjoram essential oil to fresh sausage exerted a bacteriostatic effect at oil concentrations lower than the MIC, while a bactericidal effect was observed at higher oil concentrations which also caused alterations in the taste of the product.

Distribution of putative adhesins in Shiga toxin-producing Escherichia coli (STEC) strains isolated from different sources in Chile.

The distribution of three putative adhesin genes in 123 Shiga toxin-producing (STEC) strains was determined by PCR. The STEC strains were isolated from human patients (n=90) and food (n=33) and were characterized by serogroup, virulence markers (eae, stx(1), stx(2)) and adherence factors (efa1, lpfA(O157), saa) genes. Serogroups O157 (64.4%) and O26 (28.8%) were the most frequent among human strains and the majority (60.6%) of food strains were serologically non-typable. The adhesin genes efa1 (90%) and lpfA(O157) (73.3%) were the most common in humans strains and saa (45.5%) in food strains. The presence of these genes in addition to eae in STEC from different sources may suggest a relevant role in their pathogenesis.

Sorptive behavior of sorgoleone in ultisol in two solvent systems and determination of its lipophilicity.

Sorgoleone (SGL) exuded by sorghum roots inhibits the development of some weeds. Due to its high hydrophobicity, it is expected that SGL presents low soil mobility and limited allelopathic activity in the field. This work aims to evaluate the sorptivity of sorgoleone in octanol-water and in soil under two solvent systems. The two solvent systems were methanol:water (60:40) (MeOH:H2O) and pure methanol (MeOH). These two solvent systems promote different conditions for SGL solubility. Treatments were arranged in a 2 x 6 factorial (solvent systems x equilibrium concentrations in the solution (EC)). For each solvent, the sorption was achieved by shaking 500 mg of soil with 10 ml of 0, 5, 10, 15, 25, 40, and 60 mg L-1 of SGL solution, during 24 h. After centrifugation, the supernatant was filtered and the SGL concentration was determined by high performance liquid chromatography (HPLC). Data of sorbed amount of SGL were submitted to variance analysis, using a hierarchic factorial model. The data of sorbed amount (x/m) and equilibrium concentration (C) were fitted to the linear (x/m = a + KdC) and to the Freundlich (x/m = KfC1/n) models. The isotherm obtained for the MeOH:H2O system presented linear shape, whereas for the MeOH system a two subsequent linear isotherm was fitted. Sorgoleone is a highly hydrophobic compound, presenting a log Kow of 6.1. The sorption of sorgoleone to the soil was very high. The organic environment stimulated the sorgoleone sorption to the soil.

Multiple resistance of acetolactate synthase and protoporphyrinogen oxidase inhibitors in Euphorbia heterophylla biotypes.

Resistance to acetolactate synthase (ALS)-inhibiting herbicides in Brazil has been documented for six species. The probability to select biotypes of Euphorbia heterophylla (EPPHL) with multiple resistance increases in the same order of magnitude as the use of other herbicides belonging to only one mechanism of action. The objectives of this work were to evaluate the distribution of resistant populations (R) in the states of the Parana and Santa Catarina; to determine the existence of populations of EPHHL with multiple resistance to ALS and PROTOX inhibitors, and to confirm the occurrence of cross resistance to compounds of these mechanisms of action. Seeds of EPHHL of areas with suspected resistance had been sampled in 97 places during 2003. In the greenhouse experiment samples of each population were sprayed with imazethapyr or fomesafen, at only one rate. To identify the resistant ones they were sprayed with different levels of the herbicides imazethapyr and fomesafen. Later they were sprayed with diverse herbicides of the same mechanisms of action to confirm the multiple/cross resistance. There is widespread distribution in the region of populations with resistance to ALS inhibitors. Some biotypes demonstrated resistance to herbicides from the two mechanisms of action. The resistance factor (FR), or the relation of resistance between R and susceptible biotypes, confirms the existence of two biotypes of EPHHL with cross resistance to several herbicides inhibitors of ALS and PROTOX.

Hippocampal infection with HSV-1-derived vectors expressing an NMDAR1 antisense modifies behavior.

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Gene transfer of NMDAR1 subunit sequences to the rat CNS using herpes simplex virus vectors interfered with habituation.

1. The aim is to study some roles of the hippocampal NMDA receptor, by modifying the expression of the essential NR1 subunit, with temporal and spatial restrictions in the central nervous system (CNS) of the rat. 2. Due to their neurotropism and the size of inserts they can accomodate, herpes simplex virus type-1 (HSV-1) derived amplicon vectors were used to transfer sequences, either in sense (+) or antisense (-) orientations, of the NR1 subunit gene, or of the green fluorescent protein (GFP) gene, into the CNS. 3. Vector expression in cell lines was followed by GFP autofluorescence, immunofluorescence and western blot. 4. The vectors were inoculated into the dorsal hippocampus of adult male Wistar rats, which were evaluated for habituation to an open field, and then, for expression of the transgenes, by autofluorescence and western blot; the expression mainly happened in pyramidal cells of CA1. 5. The animals injected with vectors carrying the NR1(+) transgene showed habituation to the new environment, as also happened with rats injected with vectors carrying only the GFP transgene. 6. In contrast, animals injected with vectors carrying NR1(-) sequence, did not show habituation. This might be retrograde amnesia or disability to record the trace, suggesting that the NR1 subunit in the dorsal hippocampus, is involved in habituation to a new environment. 7. HSV-1 derived amplicon vectors appear to be useful tools to modify endogenous gene expression, at a defined period, in restricted regions of the CNS.