Novel multi-component vaccine approaches for Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis. Historically believed to be a relatively rare human disease in tropical countries, a recent study estimated that, worldwide, there are approximately 165 000 human melioidosis cases per year, more than half of whom die. The bacterium is inherently resistant to many antibiotics and treatment of the disease is often protracted and ineffective. There is no licensed vaccine against melioidosis, but a vaccine is predicted to be of value if used in high-risk populations. There has been progress over the last decade in the pursuit of an effective vaccine against melioidosis. Animal models of disease including mouse and non-human primates have been developed, and these models show that antibody responses play a key role in protection against melioidosis. Surprisingly, although B. pseudomallei is an intracellular pathogen there is limited evidence that CD8+  T cells play a role in protection. It is evident that a multi-component vaccine, incorporating one or more protective antigens, will probably be essential for protection because of the pathogen's sophisticated virulence mechanisms as well as strain heterogeneity. Multi-component vaccines in development include glycoconjugates, multivalent subunit preparations, outer membrane vesicles and other nano/microparticle platforms and live-attenuated or inactivated bacteria. A consistent finding with vaccine candidates tested in mice is the ability to induce sterilizing immunity at low challenge doses and extended time to death at higher challenge doses. Further research to identify ways of eliciting more potent immune responses might provide a path for licensing an effective vaccine.

Analysis of radiation effects on some properties of GaAs:Cr and Si sensors exposed to a 22 MeV electron beam / Análisis de los efectos de la radiación en algunas propiedades de sensores de GaAs:Cr y Si expuestos a un haz de electrones de 22 MeV

Abstract Nowadays, the experiments related to High Energy Physics and others fields demand the use of detectors with greater radiation resistance, and the novel material GaAs:Cr has demonstrated excellent radiation hardness compared with other semiconductors. On the basis of evidence obtained in the JINR experiment with the use of 22 MeV electrons beam generated by the LINAC-800 accelerator, an analysis of electron radiation effects on GaAs:Cr and Si detectors is presented. The measured I-V characteristics showed a dark current increase with dose, and an asymmetry between the two branches of behaviors for all detectors. Analyzing the MIP spectra and CCE dose dependence measurements a deterioration process of detectors collection capacity with dose increase was found, although behaviors are somewhat different according to the detector type. The detailed explanation of these effects from the microscopic point of view appears in the text, and are generally linked to the generation of atomic displacement, vacancies and other radiation defects, modifying the energy levels structure of the target material. These changes affect the lifetime and concentration of the charge carriers, and other characteristics of the target material.

Resumen Actualmente, los experimentos relacionados con la física de altas energías y otros campos, demandan el uso de detectores con mayor resistencia a las radiaciones y el novedoso material GaAs:Cr ha demostrado poseer una excelente fortaleza comparado con otros semiconductores. En base a las evidencias obtenidas en el experimento del IUIN con el uso de un haz de electrones de 22 MeV generado por el acelerador LINAC-800, se presenta un análisis de los efectos de la radiación en detectores de Si y GaAs:Cr. Las características I-V medidas mostraron un incremento de la corriente de fuga con la dosis y una asimetría entre las dos ramas de estos comportamientos para todos los detectores. Analizando las mediciones de los espectros MIP y la dependencia de la CCE con la dosis, fue encontrado un proceso de deterioro de la capacidad de detección de los detectores con el aumento de la dosis, sin embargo, los comportamientos son diferentes de acuerdo al tipo de detector. La explicación detallada de estos efectos desde el punto de vista microscópico aparece en el texto, los cuales están relacionados generalmente con la generación de desplazamientos atómicos, vacancias y otros defectos producto de la radiación, modificando la estructura de los niveles energéticos en el material sensor. Estos cambios afectan el tiempo de vida y la concentración de los portadores de carga, así como otras características del material.

Kefir Grains Change Fatty Acid Profile of Milk during Fermentation and Storage.

Several studies have reported that lactic acid bacteria may increase the production of free fatty acids by lipolysis of milk fat, though no studies have been found in the literature showing the effect of kefir grains on the composition of fatty acids in milk. In this study the influence of kefir grains from different origins [Rio de Janeiro (AR), Viçosa (AV) e Lavras (AD)], different time of storage, and different fat content on the fatty acid content of cow milk after fermentation was investigated. Fatty acid composition was determined by gas chromatography. Values were considered significantly different when p<0.05. The highest palmitic acid content, which is antimutagenic compost, was seen in AV grain (36.6g/100g fatty acids), which may have contributed to increasing the antimutagenic potential in fermented milk. Higher monounsaturated fatty acid (25.8 g/100g fatty acids) and lower saturated fatty acid (72.7 g/100g fatty acids) contents were observed in AV, when compared to other grains, due to higher Δ9-desaturase activity (0.31) that improves the nutritional quality of lipids. Higher oleic acid (25.0 g/100g fatty acids) and monounsaturated fatty acid (28.2g/100g fatty acids) and lower saturated fatty acid (67.2g/100g fatty acids) contents were found in stored kefir relatively to fermented kefir leading to possible increase of antimutagenic and anticarcinogenic potential and improvement of nutritional quality of lipids in storage milk. Only high-lipidic matrix displayed increase polyunsaturated fatty acids after fermentation. These findings open up new areas of study related to optimizing desaturase activity during fermentation in order to obtaining a fermented product with higher nutritional lipid quality.

Enteropathogenic Escherichia coli: foe or innocent bystander?

Enteropathogenic Escherichia coli (EPEC) remain one the most important pathogens infecting children and they are one of the main causes of persistent diarrhoea worldwide. Historically, typical EPEC (tEPEC), defined as those isolates with the attaching and effacement (A/E) genotype (eae(+)), which possess bfpA(+) and lack the stx(-) genes are found strongly associated with diarrhoeal cases. However, occurrence of atypical EPEC (aEPEC; eae(+)bfpA(-)stx(-)) in diarrhoeal and asymptomatic hosts has made investigators question the role of these pathogens in human disease. Current epidemiological data are helping to answer the question of whether EPEC is mainly a foe or an innocent bystander during infection.

Effective stabilization of CLA by microencapsulation in pea protein.

CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications.

Development of a pododermatitis score in breeding does using clustering methods.

Pododermatitis in rabbit production is an important welfare problem and there is less information on this type of lesion in rabbits than in many other species. The aim of this work was to develop a scoring system to assess the presence and severity of pododermatitis through observation of 1367 photos of rabbit feet by two observers. Different groups of lesions were established according to color, size, presence of chaps, presence of ulcers, shape, appearance and presence of blood in each observed foot. A two-step cluster methodology was used to gather the results in homogenous and objective units. The inter-rater agreement was moderate, and after the cluster analysis four main clusters were obtained. These clusters were later comprehensively described in terms of pododermatitis severity. Finally, attending to cluster description, a five-level score was defined and this scale resulted in a practical and objective way to assess pododermatitis in rabbit does. Cluster analysis provided a detailed characterization of this type of lesions and helped to obtain uniform scores.

The influence of broiler activity, growth rate, and litter on carbon dioxide balances for the determination of ventilation flow rates in broiler production.

Carbon dioxide balances are useful in determining ventilation rates in livestock buildings. These balances need an accurate estimation of the CO(2) produced by animals and their litter to determine the ventilation flows. To estimate the daily variation in ventilation flow, it is necessary to precisely know the daily variation pattern of CO(2) production, which mainly depends on animal activity. The objective of this study was to explore the applicability of CO(2) balances for determining ventilation flows in broiler buildings. More specifically, this work aimed to quantify the amount of CO(2) produced by the litter, as well as the amount of CO(2) produced by the broilers, as a function of productive parameters, and to analyze the influence of broiler activity on CO(2) emissions. Gas concentrations and ventilation flows were simultaneously measured in 3 trials, with 1 under experimental conditions and the other 2 in a commercial broiler farm. In the experimental assay, broiler activity was also determined. At the end of the experimental trial, on the day after the removal of the broilers, the litter accounted for 20% of the total CO(2) produced, and the broilers produced 3.71 L/h of CO(2) per kg of metabolic weight. On the commercial farm, CO(2) production was the same for the 2 cycles (2.60 L/h per kg of metabolic weight, P > 0.05). However, substantial differences were found between CO(2) and broiler activity patterns after changes in light status. A regression model was used to explain these differences (R(2) = 0.52). Carbon dioxide increased with bird activity, being on average 3.02 L/h per kg of metabolic weight for inactive birds and 4.73 L/h per kg of metabolic weight when bird activity was highest. Overall, CO(2) balances are robust tools for determining the daily average ventilation flows in broiler farms. These balances could also be applied at more frequent intervals, but in this case, particular care is necessary after light status changes because of discrepancy between animal activity and CO(2) production.

Profiles of fatty acids and triacylglycerols and their influence on the anaerobic biodegradability of effluents from poultry slaughterhouse.

The hydrolysis of effluent from a poultry slaughterhouse containing 800 mg oil and grease (O&G)/L was conducted with 1% (w/v) of an enzymatic pool obtained by solid-state fermentation with the fungus Penicillium restrictum. The chromatographic evaluation of the lipid profile during hydrolysis indicated a higher concentration of acids after 4h of reaction (2954 mg/L), with a predominance of oleic, palmitic, and linoleic acids. Effluent aliquots were collected after 4, 8, and 24h of hydrolysis and tested for anaerobic biodegradation in sequential batches. An adaptation of the biomass was observed, both in the control experiment (with non-hydrolyzed raw effluent) and in the experiments with enzymatically pre-treated effluent. The specific methane production in the control experiment was 0.248 L CH(4)/g COD(consumed), and in the experiment with effluent pre-treated for 4h, this production was 0.393 L CH(4)/g COD(consumed), indicating a higher methane production after enzymatic hydrolysis.

Characterization of gas emissions from a Mediterranean broiler farm.

Gas emissions from broiler production have been the subject of intensive research. However, little experimental information exists for farms under the particular management and environmental conditions of the European Mediterranean area. In this study, ammonia, carbon dioxide, methane, and nitrous oxide concentrations and emissions were measured in a commercial broiler farm located in Spain. Gas concentrations were measured using a photoacoustic gas monitor, whereas the ventilation flow was evaluated by controlling the operation status of each fan. Two rearing periods were studied, one in summer and one in winter. All gas emissions increased with bird age. Ammonia emission rates averaged 19.7 and 18.1 mg/h per bird in the summer and winter, respectively, and increased with indoor temperature (r(2) = 0.51 in summer; r(2) = 0.42 in winter). Average CO(2) emission rates were 3.84 and 4.06 g/h per bird, CH(4) emission was 0.44 and 1.87 mg/h per bird, and N(2)O emission was 1.74 and 2.13 mg/h per bird in summer and winter, respectively. A sinusoidal daily variation pattern was observed in all emissions except for CH(4). These patterns were characterized in terms of time of maximum emission and amplitude of the daily variation.

Mitochondrial 16S DNA variation in populations of Triatoma infestans from Argentina.

Variation in the mtDNA 16S ribosomal RNA gene in populations of Triatoma infestans (Klug) was surveyed. DNA sequence comparisons yielded 18 haplotypes among 130 individuals from 16 localities that represent a large proportion of the range of T. infestans in Argentina. The most common genotype in all populations was found in 76.9% of individuals and two other haplotypes were shared among different populations. The remaining 15 haplotypes were present exclusively in one of the populations, suggesting currently low levels of genetic exchange. Analysis of mtDNA 16S sequences uncovered substantial genetic variation among T. infestans populations. Haplotype and nucleotide diversities varied among populations, from 0% to 0.84% and 0% to 0.29%, respectively. It appears that this locus has a low mutation rate. Uncorrected pairwise differences of T. infestans haplotypes ranged from 0% to 1.2%. The molecular phylogeny supported the monophyly of T. infestans haplotypes and clustered two different pairs of haplotypes with a moderate degree of bootstrap support (approximately 60%). Mitochondrial DNA phylogeographic differentiation was not evident, suggesting a recent rapid spread of the species. Analysis of molecular variance showed hierarchical structure in the data. Considerably less variation was found among T. infestans populations from the northwest and northeast regions than among those belonging to the central area. Such a lack of variation may be indicative of one or more past population bottlenecks.

n-6 and n-3 Long-chain polyunsaturated fatty acids in the erythrocyte membrane of Brazilian preterm and term neonates and their mothers at delivery.

Placental transfer of the long-chain polyunsaturated fatty acids (LCPUFA) arachidonic (AA) and docosahexaenoic (DHA) acids is selectively high to maintain accretion to fetal tissues, especially the brain. The objectives of the present study were to investigate the essential fatty acid (EFA) and LCPUFA status at birth of preterm and term Brazilian infants and their mothers, from a population of characteristically low intake of n-3 LCPUFA, and to evaluate the association between fetal and maternal status, by the determination of the fatty acid composition of the erythrocyte membrane. Blood samples from umbilical cord of preterm (26-36 weeks of gestation; n = 30) and term (37-42 weeks of gestation; n = 30) infants and the corresponding maternal venous blood were collected at delivery. The LCPUFA composition of the erythrocyte membrane and DHA status were similar for mothers of preterm and term infants. Neonatal AA was higher (P < 0.01) whereas its precursor 18:2n-6 was lower (P < 0.01) than maternal levels, as expected. There was no difference in LCPUFA erythrocyte composition between preterm and term infants, except for DHA. Term infants presented a worse DHA status than preterm infants (P < 0.01) and than their mothers (P < 0.01) at delivery. There was a negative correlation of neonatal DHA with maternal AA and a positive correlation between neonatal AA and maternal AA and 18:2n-6 only at term. These results suggest that the persistent low DHA maternal status, together with the comparatively better AA and 18:2n-6 status, might have affected maternal-fetal transfer of DHA when gestation was completed up to term, and possibly contributed to the worse DHA status of term neonates compared with the preterm neonates.

TonB-dependent systems of uropathogenic Escherichia coli: aerobactin and heme transport and TonB are required for virulence in the mouse.

The uropathogenic Escherichia coli strain CFT073 has multiple iron acquisition systems, including heme and siderophore transporters. A tonB mutant derivative of CFT073 failed to use heme as an iron source or to utilize the siderophores enterobactin and aerobactin, indicating that transport of these compounds in CFT073 is TonB dependent. The TonB(-) derivative showed reduced virulence in a mouse model of urinary tract infection. Virulence was restored when the tonB gene was introduced on a plasmid. To determine the importance of the individual TonB-dependent iron transport systems during urinary tract infections, mutants defective in each of the CFT073 high-affinity iron transport systems were constructed and tested in the mouse model. Mouse virulence assays indicated that mutants defective in a single iron transport system were able to infect the kidney when inoculated as a pure culture but were unable to efficiently compete with the wild-type strain in mixed infections. These results indicate a role for TonB-dependent systems in the virulence of uropathogenic E. coli strains.

Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

TonB is required for intracellular growth and virulence of Shigella dysenteriae.

To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.

The aerobactin iron transport system genes in Shigella flexneri are present within a pathogenicity island.

Genes encoding the synthesis and transport of aerobactin, a hydroxamate siderophore associated with increased virulence of enteric bacteria, were mapped within a pathogenicity island in Shigella flexneri. The island, designated SHI-2 for Shigella pathogenicity island 2, was located downstream of selC, the site of insertion of pathogenicity islands in several other enteric pathogens. DNA sequence analysis revealed the presence of multiple insertion sequences upstream and downstream of the aerobactin genes and an integrase gene that was nearly identical to an int gene found in Escherichia coli O157:H7. SHI-2 sequences adjacent to selC were similar to sequences at the junction between selC and pathogenicity islands found in E. coli O157:H7 and in enteropathogenic E. coli, but the junctions between the island and downstream yic genes were variable. SHI-2 also encoded immunity to the normally plasmid-encoded colicins I and V, suggesting a common origin for the aerobactin genes in both S. flexneri and E. coli pColV. Polymerase chain reaction and Southern hybridization data indicate that SHI-2 is present in the same location in Shigella sonnei, but the aerobactin genes are not located within SHI-2 in Shigella boydii or enteroinvasive E. coli. Shigella dysenteriae type 1 strains do not produce aerobactin but do contain sequences downstream of selC that are homologous to SHI-2. The presence of the aerobactin genes on plasmids in E. coli pColV and Salmonella, on a pathogenicity island in S. flexneri and S. sonnei and in a different chromosomal location in S. boydii and some E. coli suggests that these virulence-enhancing genes are mobile, and they may constitute an island within an island in S. flexneri.

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria.

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli. We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA. These shuA-positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA, so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae. The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA. The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA-positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae, indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae, and it is located in the same relative position on the chromosome.

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7.

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene (chuA) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependent, Fur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis. A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.

Determination of trace heavy metals in biological samples by inductively-coupled plasma atomic emission spectrometry after extraction with 1,5-bis-(di-2-pyridylmethylene)thiocarbonohydrazide.

A sensitive inductively-coupled plasma atomic emission spectrometric sequential method for the determination of trace heavy metals (cadmium, cobalt, copper and nickel) in biological samples after extraction of the metals into isobutyl methyl ketone (IBMK) containing 1,5-bis-(di-2-pyridylmethylene)thiocarbonohydrazide (DPTH) is described. A systematic study was made to determine the optimum conditions for extraction of the metals into IBMK. The complexes formed are quite soluble in IBMK, so much so that this allows the use of aqueous-to-organic phase volume ratios of up to 40 and hence the determination of concentrations down to 40 times lower than those afforded by the direct non-extractive method. The method has been used for the determination of these elements in various biological materials with good results.

Simultaneous spectrophotometric determination of cadmium, copper and zinc.

A method for the simultaneous spectrophotometric determination of cadmium, copper and zinc based on the formation of their complexes with 1,5-bis(di-2-pyridylmethylene)thiocarbonohydrazide is proposed. The absorption curves of these complexes overlap severely in the scanning range 380-480 nm. The analyte concentrations are calculated by a least squares fit of the pure spectra to the mixture spectra. A linear determination range of 0.1-1.7 mug/ml for cadmium, 0.1-1.3 mug/ml for copper and 0.2-1.2 mug/ml for zinc were obtained. The effect of interference was studied. The method has been applied to the determination of these metal ions in various type of materials.