Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103

Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes.     

肠道微生物区系中与肉鸡生产性能相关菌属的鉴定

正肉鸡的胃肠系统在提高饲料转化率方面发挥着极其重要的作用,它参与分解日粮组分、吸收营养物质及将机体代谢物排出体外。肉鸡肠道具有独特的微生物生态系统,可与肉鸡形成共生关系。肠道微生物可使宿主完全消化日粮成分,是细菌密度最高的生态系统,含有有益菌、有害菌和良性菌等,因而可对动物健康与营养造成潜在的影响。日粮营养物质摄入的速度、宿主的免疫应答状态及肠道结构等均可影响肠道微生物;同时日粮组分、宿主免疫或肠道结构     

Treatment of intracranial neoplasia in dogs using higher doses: A randomized controlled trial comparing a boosted to a conventional radiation protocol

BackgroundLocal progression of intracranial tumors can be the consequence of insufficient radiation dose delivered. Dose increases in the brain must be made carefully so as not to risk debilitating adverse effects such as radiation necrosis.HypothesisA new protocol with 10 × 4 Gy + 11% physical dose increase limited to the macroscopic tumor volume results in a clinically better outcome compared to a 10 × 4 Gy protocol.AnimalsFifty‐seven client‐owned dogs with primary intracranial neoplasia.MethodsRandomized controlled trial. Twenty‐eight dogs were assigned to the control protocol (10 × 4 Gy) and 29 to the simultaneous integrated boost (SIB) protocol with 4.45 Gy dose increase. Treatment groups were compared for outcome and signs of toxicity.ResultsMild, transient acute or early‐delayed adverse radiation effects were observed in 5 dogs. Severe late adverse effects were not seen. Between the protocols, no significant differences were found for outcome (intention‐to‐treat analysis): overall time to progression (TTP) was 708 days (95% confidence interval (95% CI) [545,872]), in the control group it was 828 days (95% CI [401,1256]), and in the SIB group 627 days (95% CI [282,973]; P = .07). Median overall survival (OS) was 684 days (95% CI [516,853]), in the control group it was 724 days (95% CI [623,826]), and in the SIB group 557 days (95% CI [95,1020]; P = .47). None of the tested variables was prognostic in terms of outcome.Conclusion and Clinical ImportanceThe dose escalation used with an 11% physical dose increase did not result in better outcome.     

Biotechnological production of vitamin B2-enriched bread and pasta

Lactic acid bacteria (LAB) were obtained from durum wheat flour samples and screened for roseoflavin-resistant variants to isolate natural riboflavin-overproducing strains. Two riboflavin-overproducing strains of Lactobacillus plantarum isolated as described above were used for the preparation of bread (by means of sourdough fermentation) and pasta (using a prefermentation step) to enhance their vitamin B2 content. Pasta was produced from a monovarietal semolina obtained from the durum wheat cultivar PR22D89 and, for experimental purposes, from a commercial remilled semolina. Several samples were collected during the pasta-making process (dough, extruded, dried, and cooked pasta) and tested for their riboflavin content by a high-performance liquid chromatography method. The applied approaches resulted in a considerable increase of vitamin B2 content (about 2- and 3-fold increases in pasta and bread, respectively), thus representing a convenient and efficient food-grade biotechnological application for the production of vitamin B2-enriched bread and pasta. This methodology may be extended to a wide range of cereal-based foods, feed, and beverages. Additionally, this work exemplifies the production of a functional food by a novel biotechnological exploitation of LAB in pasta-making.     

Effects of Iron, Ammonium and Temperature on Microcystin Content by a Natural Concentrated Microcystis Aeruginosa Population

We report results of a study conducted to evaluate effects of ammonium, iron and temperature on microcystins (MC) and proteins content in cultures of a natural Microcystis aeruginosa population, concentrated from a field sample of San Roque reservoir (Córdoba – Argentina). Based on a previous field study, we tested two temperatures (20 and 28 ^°C), two iron concentrations (1 μM and 10 μM) and two ammonium-nitrogen conditions (absence and 54 μM) in semi-continuous cultures. Total MC (TMC = MC-LR + MC-RR) and protein content per cyanobacteria cell increased when Fe concentration was enlarged. However, the ratio TMC: protein was almost the same for both iron concentrations. Thus, a high level of iron enhances both protein and MC content in the same proportion. TMC and protein content are significantly lowered in presence of 54 μM-N-ammonium. Additionally, the ratio TMC: protein is lowered by almost two folds in cultures having ammonium. Increasing the temperature does not affect the protein content or TMC per cell. However, cultures carried out at 28 ^°C maintain the same MC-LR: MC-RR ratio during all the studied period, while cultures developed at 20 ^°C show that MC-RR content is increased by 82-fold after four growth cycles (20 days total), while MC-LR remain almost constant in the same time. As a result, in our case, different temperatures produce a significant change in the pattern of MC content but not in the content of TMC per cell. The two-fold drop observed in MC content in presence of ammonium are within the range expected for physiological responses of cyanobacteria, raising questions on the probable inhibitory role of ammonium in MC production. On the contrary, the 82-fold increase of MC-RR observed at 20 ^°C exceed the effects attributable to a cell physiological response, and could be better explained by an ecological shift from the starting genotype composition. Metagenomics, or similar molecular techniques, would provide the necessary tools to elucidate the ecological effect of temperature on cyanobacteria populations.     

Eco-physiological and Antioxidant Responses of Holm Oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.) Leaves to Cd and Pb

Plants of the urban environment are exposed to a wide range of pollutants, including heavy metals. This research studies in situ the eco-physiological and antioxidant responses of holm oak (Q. ilex) leaves to Pb and Cd to assess the mechanisms of metal tolerance in this species, widely used as biomonitor. Leaves of plants grown at parks and roadsides were analyzed for photosynthetic activity, Pb and Cd concentration in tissues and cell-free extracts, thiol groups, D1 and Rubisco protein content, ascorbic acid (AsA) amount, and catalase (CAT) activity. The main results evidenced that Cd concentration was higher in leaves collected at the park out from the downtown; whereas Pb was most abundant in leaves sampled at the roadside nearby the highway. Pb in cell-free extracts was higher in park than in roadside leaves. Although Cd in the leaf tissues was twofold lower than Pb, it was more abundant than Pb in cellular extracts deprived of all particulate matter. Leaves responded to different concentration of Cd and Pb modulating some eco-physiological and biochemical traits, roadside leaves showed reduced leaf lamina, higher content of photosynthetic pigments, hydrogen peroxide, and AsA, as well as higher CAT activity compared to park leaves. In the roadside leaves, a stress condition for photosynthetic apparatus can be hypothesized on the basis of the decline of photochemical activity, the increase of NPQ, and the reduction of Rubisco and D1 protein content. The elevated presence of thiol groups in these leaves suggests a possible role of Pb and Cd in activation of antioxidant responses.     

Mechanical and water barrier properties of glutenin films influenced by storage time

The goal of this work was to study the effect of storage time on the functional properties of glutenin films plasticized with selected hydrophilic low molecular weight compounds: glycerol (GL), triethanolamine (TEA), and sorbitol (S). Glutenins were extracted from wheat gluten, and films were cast from film-forming solutions. The glutenin-based films were homogeneous, flexible, translucent, and easy to handle. Films were stored in an environmental chamber at 50 +/- 5% realtive humidity and 23 +/- 2 degrees C. Optical, mechanical, and water vapor permeability properties were monitored at regular intervals for 16 weeks. Films plasticized with GL and TEA had similar mechanical and water vapor barrier properties during the first few days of fabrication. Films plasticized with S were stronger, with better water vapor barrier properties. Mechanical and water vapor permeability properties of films plasticized with GL changed dramatically over time, whereas the properties of films plasticized with TEA and S remained stable during storage. Color properties of films plasticized with GL, TEA, and S did not change within the time period studied.     

Molecular markers for promoting agro-biodiversity conservation: a case study from Italy. How cowpea landraces were saved from extinction

Landraces (LRs) are crop populations that are locally grown by farmers; they have a strategic role to play in rebuilding healthy and complex agro-ecosystems, but are at risk of extinction. This paper outlines how some LRs were rescued with the support of modern techniques such as molecular markers. Cowpea LRs from Umbria, Italy, were initially characterized for morphological, organoleptic and genetic traits and reintroduced among farmers. These activities led to an expansion of the area under LR based cowpea cultivation and increased farmers’ income. To encourage further LR cultivation a request for the Protected Designation of Origin (PDO) status has been promoted. Investigations were carried out to determine if it is possible to certify that the product actually belongs to the traditional agricultural and cultural context of the cultivation area. The genetic diversity and phylogenetic relationships among local LRs, other LRs, commercial varieties, and other taxa were investigated with five AFLP markers. Although genetic diversity is limited, genetic analysis showed that Umbrian cowpea LRs can be distinguished from commercial varieties, LRs from abroad and other taxa. There is a tight phylogenetic connection among Umbrian LRs. These data confirmed the available historical and sociological data and were included in the PDO specification. AFLP markers can be used to monitor the origin of seed lots that are on the market; this protects the rights of both consumers and farmers. This case study could be a resource for planning activities for the future on-farm conservation of other LRs in different countries.     

Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection

The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8⁺ IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8^?IFN-γ⁺ phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8⁺IFN-γ⁺ as well as CD8^?IFN-γ⁺ cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.