Isolation and characterization of immortalized porcine aortic endothelial cell lines

Primary porcine endothelial cells have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (AOCs) to facilitate in vitro studies of different pathological process involving the endothelium. Primary porcine aortic endothelial cells (PAECs) were transfected with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. Flow cytometry analysis demonstrated uptake of acetylated low density lipoproteins (Ac-LDL) and constitutive expression of SLA class I, CD29, CD31, CD41/61, CD80/86, CD46, SWC3, and LAMP-1 antigens by all analyzed lines and showed little differences to primary cells. The functional similarity between primary and immortalized endothelial cells was demonstrated in a cytotoxicity assay using a human natural killer cell line (NKL) as effector. The AOCs cell lines should be valuable tools for in vitro study of the human immune response against pig endothelial cells. In addition, they would be very useful to gain insight in the pathogenesis of some viral haemorrhagic diseases of pig such as African swine fever (ASF) or classical swine fever (CSF).     

Osteomalacia as a result of phosphorus deficiency in beef cattle grazing subtropical native pastures in Uruguay

We investigated 2 outbreaks of osteomalacia as a result of phosphorus (P) deficiency in herds of lactating beef cows grazing subtropical native pastures in Uruguay. Cows exhibited pica, difficulty to stand and walk, rib fractures, and body weight loss even with adequate forage availability. Osteopenia and severe osteomalacia were observed on gross and histologic examination. The concentrations of bicarbonate-extractable P in soil (4.0, 4.1 mg P/kg), total P in pasture (0.9, 1.1 g P/kg), inorganic P in serum (1.0, 0.71 mmol P/L), and P in bone (73 mg P/mL) were all low. Although injectable and mineral salt supplements provided additional P in both outbreaks, these supplementary amounts were insufficient to prevent P deficiency. The P ingested by the cows from the pasture and supplements would have provided 20–55% of their daily P requirements of ~21 g P/d. Osteomalacia occurred in cattle at the 2 ranches as a result of severe P deficiency in the soil and forage, and inadequate P supplementation. Following diagnosis, control of P deficiency in beef cattle requires estimation of the amount of pasture P ingested and provision of sufficient additional supplementary P to meet the animals’ requirements.     

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8 kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs.     

The importance of dietary HUFA for meagre larvae (Argyrosomus regius; Asso, 1801) and its relation with antioxidant vitamins E and C

Despite the interest of meagre (Argyrosomus regius) as a fast‐growing candidate for Mediterranean aquaculture diversification, there is a lack of information on nutrition along larval development. Importance of highly unsaturated fatty acids (HUFA) and the antioxidant vitamins E and vitamin C has not been investigated yet in this species. Six diets with two levels of HUFA (0.4% and 3% dw), two of vitamin E (1500 and 3000 mg kg^?1) and two of vitamin C (1800 and 3600 mg kg^?1) were fed to 15 dah meagre larvae. Larval growth in total length and dry body weight was significantly lowest in larvae fed diet 0.4/150/180 and showed few lipid droplets in enterocytes and hepatocytes and lower HUFA contents than the initial larvae. Increase in dietary HUFA up to 3%, significantly improved larval growth and lipid absorption and deposition. Besides, among fish fed 3% HUFA, increase in vitamin E and vitamin C significantly improved body weight, as well as total lipid, 22:6n‐3 and n‐3 fatty acids contents in the larvae. Thus, the results showed that 0.4% dietary HUFA is not enough to cover the essential fatty acid requirements of larval meagre and a high HUFA requirement in weaning diets is foreseen for this species. Besides, the results also pointed out the importance of dietary vitamin E and C to protect these essential fatty acids from oxidation, increase their contents in the larvae and promote growth, suggesting high vitamin E and C requirements in meagre larvae (higher than 1500 and 1800 mg kg^?1 for vitamin E and vitamin C respectively).     

Variability of the quality and quantity of organic matter in soil affected by multiple wildfires

Purpose

Fire in mountainous areas can lead to increased variability of their soil organic matter (SOM) due to spatial inhomogeneity and pre-fire fuel distribution. Here, we elucidated if this was the case in our study area and how this affected the reliability of solid-state ¹³C NMR spectroscopy applied for the study of the medium-term impact of fire on SOM

Materials and methods

The study occurred in the Sierra de Aznalcóllar, Southern Spain, which experienced their last intense fire 7 years before sampling. In a first approach (method 1), the corners and the center of a randomly chosen square with a side length of 15 m were sampled and analyzed separately. For comparison, composite samples (method 2) were obtained from three soils. We characterized material from unburnt, burnt, and double burnt regions. Data describing the physical and chemical properties of the soils together with the NMR spectroscopic characterization were analyzed using ANOVA.

Results and discussion

Both sampling methods yielded comparable results with comparable standard errors. No major differences between the fire-affected and unburnt soils were observed with respect to physical and chemical properties and C and N contents, but solid-state ¹³C NMR spectroscopy indicated a small but significant elevation of aromaticity in the soils with fire history.

Conclusions

The analysis showed that sampling with reduced replicates (method 1) can still lead to representative NMR data. The more complex sampling of comparing three composite samples (method 2) did not decrease the standard error. Our results also indicate that in the study area typical properties of the soil and its SOM induced by former burnings will not persist beyond a few decades.