Ultrasound and microwave techniques for assisting ageing on lees of red wines.

Ageing on lees is a slow process that carries microbiological and economic risks in the wineries. This study evaluates the possibility of enhancing the extraction of different compounds from the lees, using combined strategies, such as ultrasound (US) or microwaves (MW) and the addition of inactive dry yeasts (IDY), to reduce the lees ageing time. The complete chemical analysis of the wine was done, amino acids, polysaccharides, colour and volatile compounds, together with the sensory analysis. The combined treatments increased the release of total polysaccharides, mannoproteins and total monosaccharides in the wines, and some amino acids like proline. However, wines treated with US and MW, with and without lees, showed a decrease in tannins and colour intensity, and in some volatile compounds like fatty acid esters, acetates and terpenes. The wines treated with IDY and MW were the best valued for their floral and red berry flavours and less astringency.

Fining with purified grape pomace. Effect of dose, contact time and varietal origin on the final wine phenolic composition.

Fining, which involves the addition of adsorptive material in order to reduce or eliminate certain unwanted components, is a common winemaking practice. Fining agents affect the wine phenolic compounds, some of which may be reduced. When this reduction is experimented by the tannins, a positive effect may result by decreasing astringency in the wine, although a decrease in the wine color may also take place when the anthocyanins are involved, affecting its quality. Recently, grape cell wall material has been tested as a potential fining agent in wines, since it shows a high affinity for tannins so that its use could reduce wine astringency. In this work, the use of purified grape pomace as fining agent is proposed and the effect of different doses and contact times on wine chromatic characteristics was investigated as well as how differences in the composition of the purified pomace could alter the phenolic composition of a red wine. The results showed that a Monastrell purified grape pomace dose of 6 mg/ml and a contact time of 5 days could be suitable for decreasing the wine tannin content without producing great changes in the wine chromatic characteristics. When comparing the effect of purified pomaces from four grape varieties, some differences in their capacity to interact with the wine tannins and anthocyanins were found, however, the results confirm that the purified grape pomace, a byproduct of the enology industry could be a new interesting fining material.

Influence of methyl jasmonate and benzothiadiazole on the composition of grape skin cell walls and wines.

Phenolic compounds are very important in crop plants, particularly in grapes. The different strategies to increase their levels include the use of elicitors such as methyl jasmonate (MeJ) and benzothiadiazole (BTH). In an attempt to improve the quality of wines, our aim was to evaluate the effect of preharvest application of these elicitors on the composition and structure of the skin cell walls of Monastrell, Merlot and Cabernet Sauvignon grapes, and to ascertain any relationship with the extractability of phenolic compounds during winemaking. The results indicated that the exogenous application of MeJ and BTH during veraison caused significant changes in several components of the skin cell walls, such as phenolic compounds, proteins and structural sugars. However these changes manifested themselves in different proportions in each variety and year, pointing to the varietal and meteorological dependence of the response to the application of these elicitors. The treatments delayed the maturation process in all varieties when rainfall was low. This observation, together with the observed increase in proteins and phenols in the skin cell wall of Monastrell and Cabernet Sauvignon, could contribute to the strength necessary to maintain the integrity of berries and to increasing resistance to fungal pathogens as the phenolic compounds evolve, thus improving the phenolic profile. However, the structural integrity of Merlot variety tended to decrease in the same conditions.

Application of enzyme preparations for extraction of berry skin phenolics in withered winegrapes.

Postharvest withering of grapes strongly affects the content and extractability of phenolic compounds in the production of sfursat, fortified and passito wines. This work evaluated the effectiveness of enzymes applied individually and/or in multi-enzyme blends, on the extraction of anthocyanins, oligomeric flavanols and polymeric flavanols from withered grape skins during simulated maceration. The study was performed on Vitis vinifera L. cv. Nebbiolo and Barbera because of their different skin phenolic profile and cell wall composition. Our findings highlight that the relationship between skin mechanical properties (berry skin break force and energy) and extraction yield of phenolic compounds is variety dependent. Significant correlations were found between the skin softening associated with cell wall degradation and the extraction of anthocyanins and flavanols in Nebbiolo, for which polygalacturonase, individually or in multi-enzyme blends, plays an important role. In Barbera, the extractability of phenolic compounds was not affected by the presence of exogenous enzymes.

Jaagsiekte Sheep Retrovirus Can Reach Peyer's Patches and Mesenteric Lymph Nodes of Lambs Nursed by Infected Mothers.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). It is generally accepted that transmission by the respiratory route occurs under natural conditions. However recent studies strongly indicate that JSRV can also be transmitted to lambs perinatally via colostrum and milk (C/M). The aim of this work was to confirm that C/M can transmit JSRV infection to lambs under natural conditions and investigate the initial events associated with this transmission route. We have analyzed the presence of JSRV in C/M samples from 22 naturally infected, asymptomatic ewes throughout a lactation period, and in various tissues collected from a group of 36 of their lambs that were fed naturally. The lambs were euthanized at 12, 24, 48, and 72 hours and at 5 and 10 days after birth. We detected JSRV-provirus by PCR in the somatic C/M cells from 10/22 ewes (45.45%). The virus was also detected in 9/36 lambs (25%). JSRV-infected cells, with lymphoreticular-like morphology, were observed by immunohistochemistry (IHC) and in situ hybridization (ISH) in Peyer's patches (PP) from the small intestine of the youngest lambs and in mesenteric lymph nodes (MLN) from lambs older than 72 hours. The virus was also detected by PCR in white blood cells (WBC) in 2/36 lambs (5.5%). These results confirm colostral transmission of JSRV to lambs under natural conditions. Infected lymphoreticular cells contained in C/M appear to be involved. These cells can cross the intestinal barrier of newborn lambs, reach the MLN and enter into circulation.

Vaccination schedules in small ruminant farms.

Development and implementation of health management plans is the cornerstone of profitable farms; prevention of microbial diseases by means of vaccination is an integral part of such a plan. In every production type and management system in small ruminants, microbial diseases have a major significance, hence their proper control must be based in good health management practices, including use of effective and safe vaccines. Development of various types of vaccines is evolving very quickly in recent years and the improvement of new type of vaccines offers prospects. The article reviews and discusses vaccination programs and latest advances in development of vaccines against diseases that cause major economic losses in small ruminants. Specifically, vaccination schedules for the following diseases are reviewed: bacterial abortion (abortion associated with Brucella melitensis, Campylobacter spp., Chlamydophila abortus, Coxiella burnetii, Salmonella abortus ovis or Salmonella brandenburg), caseous lymphadenitis, clostridial diseases, colibacillosis, contagious echtyma, epididymitis caused by Brucella ovis, footrot, mammary diseases (contagious agalactia, mastitis), paratuberculosis and respiratory diseases (respiratory disease caused by Mannheimia haemolytica or other Pasteurellaceae).

Solitary tumours associated with Jaagsiekte retrovirus in sheep are heterogeneous and contain cells expressing markers identifying progenitor cells in lung repair.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). This study examines immunohistochemically solitary lung nodules considered as early OPA lesions from 11 sheep infected naturally by JSRV. All 11 neoplastic nodules exhibited features of adenocarcinoma and in four of them mesenchymal growth was also observed. Both types of lesion were labelled with antibody specific for JSRV-Env. In two cases infiltrating lymphoreticular cells also contained JSRV-Env. All tumours had a high Ki67 labelling index and variably contained cells expressing CC10 (a marker of Clara cells (CCs)), SPC (a marker of type II pneumocytes), p63 and keratin 14 (markers for stem/progenitor cells of the lung airway epithelia). Tumours with mesenchymal growth had intense expression of vimentin and desmin, weak expression of smooth muscle actin and did not express pancytokeratin and p63. Both epithelial and mesenchymal proliferations did not express the stem cell markers CD90 and CD117, but some tumour infiltrating cells expressed CD133. Solitary OPA tumours can therefore be adenocarcinomas or mixed tumours and have a heterogeneous cellular composition, containing groups of cells expressing markers that characterize local progenitor cells involved in lung repair.

Filter-based infrared detectors for high temperature size exclusion chromatography analysis of polyolefins: calibration with a small number of standards and error analysis.

Infrared detection has been shown to be very appropriate for high temperature analysis of polyolefins. After some early reports in which dispersive or single-band filter-based detectors were applied, Fourier transform detectors have been described for this application, in order to improve the method sensitivity. Modern simple filter-based detectors prove, however, comparable sensitivity while providing a number of practical advantages when coupled to chromatographic systems: reduced cell volume, simplified hardware, continuous generation of absorbance chromatograms, as well as simpler data collection and processing. A practical method for calibration, using multiple-band signals obtained with filter-based detectors and a small number of reference materials, is here discussed. Calibration data are used to compare the performance of detectors based on different opto-electronic technologies and filter designs. A procedure for estimation of errors in the slice-by-slice measured methyl frequency, based on signal-to-noise ratio considerations, is described. The good accuracy provided by the filter-based IR detectors was noticeable, considering that it was obtained using a small set of reference materials. A minimal concentration of 0.009 mg/mL was estimated to be required at the detector cell, in order to keep the errors below one unit of methyl per one thousand total carbons. This low minimal concentration requirement allows using standard SEC conditions, without compromising the molar mass distribution accuracy and resolution.

Multiple cranial neuropathy and intracranial hypertension associated with all-trans retinoic acid treatment in a young adult patient with acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) induces complete remission in 64-100 % of patients with acute promyelocytic leukemia (APL), and is considered to be a safe agent. Pseudotumor cerebri is a neurological side effect of ATRA reported in pediatric patients, and which is characterized by raised cerebrospinal fluid pressure in the absence of any intracranial pathology or secondary causes of intracranial hypertension. Involvement of cranial nerves other than II and VI is very uncommon in idiopathic intracranial hypertension (IIH); peripheral facial nerve palsy is exceptional and has rarely been described in the context of treatment with ATRA. We describe the case of a 15-year-old female patient with APL who developed an IIH and involvement of cranial nerves (bilateral papilledema, left facial and right sixth nerves) after receiving induction therapy including ATRA. Viral infections and other causes of secondary cranial nerve lesions were excluded. Symptoms completely subsided with the temporary withdrawal of ATRA and did not recur after reintroducing the drug. To date, the patient has managed to receive the treatment as per protocol. In conclusion, we report an atypical presentation of IIH that merits consideration, especially with respect to young patients with APL receiving ATRA; our most important observation is that the drug could be safely reintroduced once the symptoms had resolved.

The distribution of immune cells in the lungs of classical and atypical ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA) is a contagious tumour caused by infection of sheep with Jaagsiekte sheep retrovirus. Two forms of OPA have been identified, classical and atypical, which can be distinguished clinically and pathologically. Most notably classical OPA is progressive until death, while atypical OPA remains subclinical. In the present study the local immune responses in the lungs of cases of atypical OPA were compared with those from classical cases by immunohistochemistry using a panel of mouse anti-sheep mAbs. Distinct differences in the distribution of immune cell subsets in the two forms of OPA were observed. In particular there was an intratumoural influx of T cell subsets and MHC Class II expression on the tumour cells in atypical OPA, neither of which was seen in classical OPA. It is possible that these differences may contribute, at least in part, to determining the progressive course of classical OPA compared with the subclinical nature of atypical OPA.

A complementary mobile phase approach based on the peak count concept oriented to the full resolution of complex mixtures.

Situations of minimal resolution are often found in liquid chromatography, when samples that contain a large number of compounds, or highly similar in terms of structure and/or polarity, are analysed. This makes full resolution with a single separation condition (e.g., mobile phase, gradient or column) unfeasible. In this work, the optimisation of the resolution of such samples in reversed-phase liquid chromatography is approached using two or more isocratic mobile phases with a complementary resolution behaviour (complementary mobile phases, CMPs). Each mobile phase is dedicated to the separation of a group of compounds. The CMPs are selected in such a way that, when the separation is considered globally, all the compounds in the sample are satisfactorily resolved. The search of optimal CMPs can be carried out through a comprehensive examination of the mobile phases in a selected domain. The computation time of this search has been reported to be substantially reduced by application of a genetic algorithm with local search (LOGA). A much simpler approach is here described, which is accessible to non-experts in programming, and offers solutions of the same quality as LOGA, with a similar computation time. The approach makes a sequential search of CMPs based on the peak count concept, which is the number of peaks exceeding a pre-established resolution threshold. The new approach is described using as test sample a mixture of 30 probe compounds, 23 of them with an ionisable character, and the pH and organic solvent contents as experimental factors.

Finding the best separation in situations of extremely low chromatographic resolution.

Samples with a large number of compounds or similarities in their structure and polarity may yield insufficient chromatographic resolution. In such cases, however, finding conditions where the largest number of compounds appears sufficiently resolved can be still worthwhile. A strategy is here reported that optimises the resolution level of chromatograms in cases where conventional global criteria, such as the worst resolved peak pair or the product of elementary resolutions, are not able to detect any separation, even when most peaks are baseline resolved. The strategy applies a function based on the number of "well resolved" peaks, which are those that exceed a given threshold of peak purity. It is, therefore, oriented to quantify the success in the separation, and not the failure, as the conventional criteria do. The conditions that resolve the same amount of peaks are discriminated by either quantifying the partial resolution of those peaks that exceed the established threshold, or by improving the separation of peaks below it. The proposed approach is illustrated by the reversed-phase liquid chromatographic separation of a mixture of 30 ionisable and neutral compounds, using the acetonitrile content and pH as factors.

Effect of addition of soybean trypsin inhibitor to colostrum on immunological status in goat kids.

The aim of the study was to evaluate the influence of soybean trypsin inhibitor (TI) on immunoglobulin G (IgG) serum levels and growth in neonatal goat kids. Twenty-four newborn kids were fed with natural colostrum (group A), and 24 kids received the same colostrum with 1 g of TI per litre (group B). Blood samples were obtained at birth and on days 1, 2 and 4 of life to analyze serum proteins, IgG and haematological parameters. There were no clinical signs of disease and no significant differences in body weight between the groups. Haematological parameters were not affected by treatment. The peak of serum IgG was reached at 24 h of life, but no effects of soybean TI was observed on serum IgG levels. The apparent efficiency of absorption of IgG was similar in both groups (group A 24.5% vs. group B 25.2%, p > 0.05). The addition of TI to colostrum did not change the concentration of serum proteins and their fractions in goat kids. The correlation between serum IgG and gamma-globulin was positive and significant (p < 0.01, r = 0.64) in group A, but not in group B (p > 0.05, r = 0.08), suggesting a negative influence of soybean TI on gamma-globulin absorption. These results show that addition of soybean TI to colostrum did not improve the performance or immunological status in goat kids.

Unilateral scrotal pyocele in ram caused by Staphylococcus capitis.

A massive unilateral scrotal pyocele caused by Staphylococcus capitis in a 6-year-old ram is reported. Ultrasound examination of the right hemiscrotum showed an irregular hyperechoic mass in an anechoic fluid. A dense exudate was collected from the scrotum for microbiological analysis. Grossly, there was an extensive greenish purulent exudate and a completely atrophied right testis. Coagulase-negative S. capitis was isolated in pure culture. To the authors' knowledge, this is the first report of genital infection by S. capitis in rams. This microorganism should be included in the differential diagnosis of ovine genital infections.

Bronchioloalveolar carcinoma not related to jaagsiekte sheep retrovirus in a goat.

A spontaneous lung tumor in a 5-year-old goat of the Murciano-Granadina breed is described in this paper. Clinical signs of cachexia and tachypnoea were evident, and a considerable amount of white mucous foamy fluid was discharged from the nostrils when the animal's head was lowered. A lung tumor with the characteristics of bronchioloalveolar carcinoma was detected during histopathologic examination. The tumor cells were positive for surfactant proteins C and B, confirming that alveolar type II cells were the origin of the neoplasia. Tumor samples were tested by polymerase chain reaction, immunoblotting, and immunohistochemistry for the presence of Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), another retrovirus very closely related to JSRV, but all tests were negative. Therefore, this is the first reported case of spontaneous bronchioloalveolar carcinoma not related to JSRV or ENTV infection in a goat.

In-situ demonstration of mitogen-activated protein kinase Erk 1/2 signalling pathway in contagious respiratory tumours of sheep and goats.

Ovine pulmonary adenocarcinoma (OPA) and enzootic nasal adenocarcinoma (ENA) are two contagious neoplastic diseases of secretory epithelial cells in the respiratory system of sheep and goats. Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of OPA, and enzootic nasal tumour virus (ENTV) is associated with ENA. The genomes of these retroviruses do not contain known oncogenes but products of the env gene are important in the generation of transforming stimuli. However, the cell signalling pathways activated in vivo are not completely understood. This study was based on the use of activation stage antibodies specifically detecting proteins of the extracellular signal regulated kinase Erk 1/2 cell signalling pathway and transcription factors. Tissue sections were collected from four natural cases of OPA, four experimentally induced OPA tumours, four ENA tumours in sheep, four ENA tumours in goats, two normal sheep lungs and two lungs with chronic inflammation. Routine immunohistochemical procedures with phosphorylation stage-specific antibodies were carried out. Representative proteins of the Erk1/2 pathway (Raf-1, Mek1/2 and p44/42MAPK) were activated in natural cases of OPA and ENA in sheep and goats and also in experimentally induced OPA. Transcription factors 90Rsk and Elk-1 were activated in OPA and ENA tumours. However, c-Myc was activated only in OPA tumours. In contagious respiratory neoplasms of sheep and goats the Erk1/2 pathway appears to be important for the in-vivo generation of the transforming stimuli.