Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

Vegetative propagation of Colutea istria Mill. from leafy stem cuttings

This study investigates the optimal conditions for the vegetative propagation of the multipurpose leguminous shrub Colutea istria from leafy stem cuttings. The conditions examined included 1) the origin of the cutting (apical, medial or basal), 2) indole-3-butyric acid (IBA) treatment (0, 50, 200 or 400 mg/l), 3) the relative humidity of the propagation environment (high or low), 4) wounding base of cutting, and 5) the season in which the cutting was taken. The rooting ability of the cuttings was significantly influenced by all these factors. Winter cuttings obtained from the middle and base of the branch showed a greater rooting capacity than those harvested from the apical area, while autumn cuttings harvested from the middle and apical areas of the branch showed a greater rooting capacity than those harvested from the base. Winter cuttings showed the best rooting capacity than autumn cuttings. IBA treatment (200 mg/l), high humidity (98-100%), and wounding increased the rooting capacity of the cuttings. The sprout of new leaves was higher in basal cuttings, while IBA and wounding also significantly enhanced the root number of rooted cuttings. A method for vegetatively propagating C. istria plants is given.     

Changes in Distribution and Numbers of CD4+ and CD8+ T‐lymphocytes in Lymphoid Tissues and Intestinal Mucosa in the Early Phase of Experimentally Induced Early Onset Mucosal Disease in Cattle

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune‐mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post‐inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post‐inoculation was paralleled by a decrease of B‐lymphocytes and an increase of CD4+ T‐lymphocytes. An increased number of CD8+ T‐lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T‐lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T‐lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T‐lymphocytes in sites with lesions. This might indicate a cell‐mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T‐lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.     

Overview on soil fertility management for cereals crops in semi-arid conditions of Spain

The management of the fertilization in integrated production intends to produce good yields and high product quality, while protecting the soil from different processes such as erosion, salinization, and acidification. Principally, it seeks to maintain and improve soil fertility by preventing loss of organic matter and nutrient imbalances, while also preventing surface and groundwater pollution, and the main objective being to produce food of maximum nutritional value. In this paper we assess, in prevailing soil types in central Spain (Cambisol and Regosol), several limiting factors of soil fertility, such as soil compaction due to conservative tillage systems and climatic conditions which influence the vegetation and soil conditions (mainly during the summer). The analysis of integrated fertilization allows us to highlight the importance of maximizing resources and natural production mechanisms. We evaluate the nutritional requirements of crops, aiming to ensure long-term sustainable compatible agriculture which satisfies both the demands of society (concerning environmental protection) and the agricultural productivity requirements. In this context, the official guidelines in Spain have been to meet the standards and requirements established in integrated management programs.     

Population structure,genetic diversity,and colonization history of the eastern pondmussel,Sagittunio nasutus,in the Great Lakes drainage

1. The eastern pondmussel, Sagittunio nasutus (Bivalvia: Unionida), has declined in abundance and distribution in eastern North America over the last few decades. The declines are predominantly the result of infestation by invasive dreissenid mussels and changes in habitat. The species is now considered imperilled across large portions of its distribution, especially in the Laurentian Great Lakes region.
2. The genetic diversity and structure of the remnant populations in the Great Lakes region were assessed using 10 newly developed microsatellite DNA loci for S. nasutus. Understanding the remaining populations can inform future management projects and determine whether the remnant populations have experienced a genetic bottleneck or a founder effect. We hope that this will inform the conservation of other species in regions founded by, and isolated from, a more genetically diverse source population or with disjointed geographical distributions.
3. Focusing on the Great Lakes region, samples (n = 428) from 62 collection sites in 28 water bodies were collected. Across the locations sampled for S. nasutus 11 genetic populations were identified, with significant genetic differentiation among them. The genetic structure of the species was assessed, with genetic similarities among populations compared and with geographical routes of colonization and gene flow evaluated.
4. Initial colonization from the Atlantic coast into Lake Erie and Lake Ontario is evident, followed by colonization events into nearby inland rivers and lakes. Analyses found evidence of inbreeding in all but one population and evidence of past genetic bottlenecks or strong founder effects in all but four populations. This study deepens our understanding of the genetic past and present of this imperilled species, providing conservation suggestions for the future management of the species.

     

Secreted proteases from Actinobacillus pleuropneumoniae serotype 1 degrade porcine gelatin, hemoglobin and immunoglobulin A.

It was found that 48 hour cultures of Actinobacillus pleuropneumoniae secreted proteases into the medium. Electrophoresis in polyacrylamide gels (10%) copolymerized with porcine gelatin (0.1%), of the 70% (NH4)2SO4 precipitate from the culture supernatants, displayed protease activities of different molecular weights: > 200, 200, 90, 80, 70 and 50 kDa. They had activity over a broad range of pHs (4-8), with an optimal pH of 6-7. All were inhibited by 10 mM EDTA, and reactivated by 10 mM calcium. They were stable at -20 degrees C for more than a month. The proteases also degraded porcine IgA and porcine, human, and bovine hemoglobin, although they appeared to be less active against the hemoglobins. The IgA was totally cleaved in 48 h, using supernatants concentrated with polyvinyl pyrrolidone or the 70% (NH4)2SO4. Extracellular proteases could play a role in virulence.     

Reaction of potato varieties toPotato mop-top virus infection in the andes

The unexpected detection ofPotato mop-top virus (PMTV) in potatoes growing at several locations in the U.S.A. and Canada in 2002 has led to the realization that this soil-borne virus may be widespread throughout the potato-producing regions of both countries. A lack of information concerning the response of U.S. cultivars to PMTV infection caused us to examine the effects of PMTV on field-grown plants at three locations in the Peruvian Andes where the virus and its fungal vector,Spongospora subterranea, are endemic. All 21 U.S. cultivars tested proved susceptible to PMTV infection, especially at La Victoria where the overall incidence of both PMTV and powdery scab was high. Symptom expression in the foliage proved to be an unreliable indicator of virus infection, and thus, more reliable serological (NCM-ELISA) and nucleic acid-based techniques (NASH and RT-PCR) were used to monitor tuber tissue for the presence of PMTV. As much as 25% of field-grown tubers of cvs Kennebec, Monona, and Norland were infected with PMTV, but no correlation between virus infection and either the incidence or severity of powdery scab for any variety was apparent. Although PMTV infection did not result in the appearance of spraing or other internal necrotic reactions resembling those reported from many North American and European locations, a disproportionate proportion of infected tubers did exhibit a reticulate surface cracking. In hydroponic culture, the proportion of cracked tubers increased dramatically when the nutrient solution was seeded with virus-infestedS. subterranea. Comparatively few tubers of cvs Monona and Russet Burbank exhibited this surface cracking, suggesting that these two varieties may be somewhat tolerant to PMTV infection.     

Effects of tillage on winter wheat production in Mediterranean dryland fields

The benefits of conservation agriculture (CA) and associated technologies are not equal for all agro ecosystems. This study used a field experiment to examine winter-wheat yield and weeds under conservational and conventional systems in the central region of Spain. The three tillage treatments were conventional tillage (CT), minimum tillage (MT) and no-tillage (NT). The climatic conditions influenced wheat yield, yield components, soil water content and weeds. When the autumn-winter rainfall was abundant and constant (69.7% of annual rainfall), wheat grain yield (4465?kg?ha^?1) and yield components (3897?kg?ha^?1 of straw biomass and 584.5 ear m^?2) were highest. Wheat grain yield was highest with NT: 3549.9?kg?ha^?1 (compared to MT: 2955.1?kg?ha^?1 and CT: 2950.3?kg?ha^?1) and ear number per m² was significantly lower with MT (332 no ear m^?2, compared to 426 and 411.6?ear?m^?2 in CT and NT-systems respectively). Soil water content, at earing stage, was the highest in NT (27.36% of soil moisture) while MT showed the lowest content (11.83% of soil moisture). The higher weed measurements (means of 2.557 plants m^?2; 1.443 species m^?2 and 2.536 g m^?2) was with higher annual rainfall (488?mm). Throughout the experiment it was the dominant presence, in MT-wheat plots, of Lolium rigidum Gaudin (with means from 4.87 to 7.71 plants m^?2), which reduced the ear number per m². Our study revealed that in the short term, under semi-arid conditions, only the adoption of NT system (rather than MT) showed economic benefits.