A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

[Lung and pleural lesions before and after implementation of a national eradication program against enzootic pneumonia and actinobacillosis as well as changes of slaughter carcass organs in slaughter pigs in Switzerland].

In a representative cross-sectional study during 12 months of the years 2008/2009 in four abattoirs in Switzerland, lung and pleura lesions as well as lesions of slaughter carcasses and organs of 34 706 pigs were studied for frequency and type of macroscopic lesions. Of the 24276 examined pigs, 91.2% of the lungs, 94.4% of the heart and 95.5% of the livers showed no macroscopically visible lesions. Pigs that were produced for a label program had significantly less bronchopneumonia and pneumonia residuals, pleuritis and liver lesions due to echinococcosis. Pigs supervised by the Swiss Pig Health Service (SGD), showed significantly less bronchopneumonia and pneumonia residuals, diffuse pleuritis, pleuritis/pericarditis and milkspots compared to the non-SGD supervised farms. Thanks to the national eradication program for enzootic pneumonia (EP) and actinobacillosis, the health-status of lungs has been considerably improved and the prevalence of pleurisy decreased considerably. The results of this study indicate a good herd health in Swiss pig production.

JTT-130, a Microsomal Triglyceride Transfer Protein (MTP) Inhibitor Lowers Plasma Triglycerides and LDL Cholesterol Concentrations without Increasing Hepatic Triglycerides in Guinea Pigs

BACKGROUND: Microsomal transfer protein inhibitors (MTPi) have the potential to be used as a drug to lower plasma lipids, mainly plasma triglycerides (TG). However, studies with animal models have indicated that MTPi treatment results in the accumulation of hepatic TG. The purpose of this study was to evaluate whether JTT-130, a unique MTPi, targeted to the intestine, would effectively reduce plasma lipids without inducing a fatty liver. METHODS: Male guinea pigs (n = 10 per group) were used for this experiment. Initially all guinea pigs were fed a hypercholesterolemic diet containing 0.08 g/100 g dietary cholesterol for 3 wk. After this period, animals were randomly assigned to diets containing 0 (control), 0.0005 or 0.0015 g/100 g of MTPi for 4 wk. A diet containing 0.05 g/100 g of atorvastatin, an HMG-CoA reductase inhibitor was used as the positive control. At the end of the 7th week, guinea pigs were sacrificed to assess drug effects on plasma and hepatic lipids, composition of LDL and VLDL, hepatic cholesterol and lipoprotein metabolism. RESULTS: Plasma LDL cholesterol and TG were 25 and 30% lower in guinea pigs treated with MTPi compared to controls (P < 0.05). Atorvastatin had the most pronounced hypolipidemic effects with a 35% reduction in LDL cholesterol and 40% reduction in TG. JTT-130 did not induce hepatic lipid accumulation compared to controls. Cholesteryl ester transfer protein (CETP) activity was reduced in a dose dependent manner by increasing doses of MTPi and guinea pigs treated with atorvastatin had the lowest CETP activity (P < 0.01). In addition the number of molecules of cholesteryl ester in LDL and LDL diameter were lower in guinea pigs treated with atorvastatin. In contrast, hepatic enzymes involved in maintaining cholesterol homeostasis were not affected by drug treatment. CONCLUSION: These results suggest that JTT-130 could have potential clinical applications due to its plasma lipid lowering effects with no alterations in hepatic lipid concentrations.

Guinea Pigs: A Suitable Animal Model to Study Lipoprotein Metabolism, Atherosclerosis and Inflammation

Numerous animal models have been used to study diet effects on cholesterol and lipoprotein metabolism. However, most of those models differ from humans in the plasma distribution of cholesterol and in the processing of lipoproteins in the plasma compartment. Although transgenic or knock-out mice have been used to study a specific pathway involved in cholesterol metabolism, these data are of limited use because other metabolic pathways and responses to interventions may differ from the human condition.Carbohydrate restricted diets have been shown to reduce plasma triglycerides, increase HDL cholesterol and promote the formation of larger, less atherogenic LDL. However, the mechanisms behind these responses and the relation to atherosclerotic events in the aorta have not been explored in detail due to the lack of an appropriate animal model. Guinea pigs carry the majority of the cholesterol in LDL and possess cholesterol ester transfer protein and lipoprotein lipase activities, which results in reverse cholesterol transport and delipidation cascades equivalent to the human situation. Further, carbohydrate restriction has been shown to alter the distribution of LDL subfractions, to decrease cholesterol accumulation in aortas and to decrease aortic cytokine expression. It is the purpose of this review to discuss the use of guinea pigs as useful models to evaluate diet effects on lipoprotein metabolism, atherosclerosis and inflammation with an emphasis on carbohydrate restricted diets.

Evaluación de procesos de desgaste en turbinas aeronáuticas mediante espectrometría por disco rotativo(rde) y por contador de partículas láser(lnf)

En esta ponencia se realiza una revisión de diferentes metodologías para la evaluación de procesos de desgaste en turbinas aéreas derivadas. Mediante el análisis de las características del lubricante utilizado, a través de espectrometría de micropartículas y de la evaluación por contadores de partículas de tamaño superior a los 20 micrómetros, se pretende determinar procesos de desgaste y correlaciones entre diversos parámetros de funcionamiento de la turbina. Para ello se presentan las principales metodologías de análisis de aceite en uso, indicando la información que suministran, los ámbitos en los que su aplicación es pertinente, y se muestran los resultados de diferentes casos de aplicación correspondientes tanto a aeronaves de ala fija como de ala rotativa.

Bile Acids Induce Glucagon-Like Peptide 2 Secretion with Limited Effects on Intestinal Adaptation in Early Weaned Pigs

Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve intestinal adaptation in weanling pigs. During the first 6 d after weaning, piglets were intragastrically infused once daily with either deionized water -control-, chenodeoxycholic acid -CDC; 60mg/kg body weight-, or b-sitoesterol -BSE; 100 mg/kg body weight-. Infusing CDC increased plasma GLP-2 -P menor que 0.05- but did not affect plasma GLP-1 and feed intake. The intestinal expression of Glp2r -glucagon-like peptide 2 receptor-, Asbt -sodium-dependent bile acid transporter-, Fxr -farnesoid X receptor-, and Tgr5 -guanosine protein?coupled bile acid receptor- genes were not affected by CDC treatment. The intragastric administration of CDC did not alter the weight and length of the intestine, yet increased the activation of caspase-3 in ileal villi -P menor que 0.02- and the expression of Il6 -interleukin 6; P menor que 0.002- in the jejunum. In contrast, infusing BSE did not affect any of the variables that were measured. Our results show that the enteral administration of the bile acid CDC potentiates the nutrient-induced secretion of endogenous GLP-2 in early-weaned pigs. Bile acid?enhanced release of GLP-2, however, did not result in improved intestinal growth, morphology, or inflammation during the postweaning degenerative phase.

Influence of crude protein content, ingredient complexity, feed form, and duration of feeding of the phase I diets on productive performance and nutrient digestibility of Iberian pigs.

The influence of CP content and ingredient complexity, feed form, and duration of feeding of the Phase I diets on growth performance and total tract apparent digestibility -TTAD- of energy and nutrients was studied in Iberian pigs weaned at 28 d of age. There were 12 dietary treatments with 2 type of feeds -high-quality, HQ; and low-quality, LQ-, 2 feed forms -pellets vs. mash-, and 3 durations -7, 14, and 21 d- of supply of the Phase I diets.

Management factors affecting mortality, feed intake, and feed conversion ratio of grow-finishing pigs

The aim of this study was to determine the effect of animal management and farm facilities on total feed intake (TFI), feed conversion ratio (FCR) and mortality rate (MORT) of grower-finishing pigs. In total, 310 batches from 244 grower-finishing farms, consisting of 454 855 Pietrain sired pigs in six Spanish pig companies were used. Data collection consisted of a survey on management practices (season of placement, split-sex by pens, number of pig origins, water source in the farm, initial or final BW) and facilities (floor, feeder, ventilation or number of animals placed) during 2008 and 2009. Results indicated that batches of pigs placed between January and March had higher TFI (P=0.006), FCR (P=0.005) and MORT (P=0.03) than those placed between July and September. Moreover, batches of pigs placed between April and June had lower MORT (P=0.003) than those placed between January and March. Batches which had split-sex pens had lower TFI (P=0.001) and better FCR (P<0.001) than those with mixed-sex in pens; pigs fed with a single-space feeder with incorporated drinker also had the lowest TFI (P<0.001) and best FCR (P<0.001) in comparison to single and multi-space feeders without a drinker. Pigs placed in pens with <50% slatted floors presented an improvement in FCR (P<0.05) than pens with 50% or more slatted floors. Batches filled with pigs from multiple origins had higher MORT (P<0.001) than those from a single origin. Pigs housed in barns that performed manual ventilation control presented higher MORT (P<0.001) in comparison to automatic ventilation. The regression analysis also indicated that pigs which entered to grower-finisher facilities with higher initial BW had lower MORT (P<0.05) and finally pigs which were sent to slaughterhouse with a higher final BW presented higher TFI (P<0.001). The variables selected for each dependent variable explained 61.9%, 24.8% and 20.4% of the total variability for TFI, FCR and MORT, respectively. This study indicates that farms can increase growth performance and reduce mortality by improving farm facilities and/or modifying management practices.

Effects of nutrition on digestión efficicency and gaseous emissions from slurry in growing-finishing pigs. I. Influence of the inclusión of two levels of orange pulp and carb meal in isofibrous diets

An experiment was conducted to investigate the effects of increasing the level of two sources of fibrous by-products, orange pulp (OP) and carob meal (CM), in iso-NDF growing-finishing pig diets on nutrient balance, slurry composition and potential ammonia (NH3) and methane (CH4) emissions. Thirty pigs (85.4 ± 12.3 kg) were fed five iso-nutritive diets: a commercial control wheat/barley (C) and four experimental diets including two sources of fibrous by-products (OP and CM) and two dietary levels (75 and 150 g/kg) in a 2 × 2 factorial arrangement. After a 14-day adaptation period, faeces and urine were collected separately for 7 days to measure nutrient digestibility and the excretory patterns of N from pigs (6 replicates per diet) housed individually in metabolic pens. For each animal, the derived NH3 and CH4 emissions were measured in samples of slurry over an 11- and 100-day storage periods, respectively. Source and level of the fibrous by-products affected digestion efficiency in a different way as the coefficients of total tract apparent digestibility (CTTAD) for dry matter (DM), organic matter (OM), fibre fractions and gross energy increased with OP but decreased with CM (P < 0.05). Crude protein CTTAD decreased with the inclusion of both sources of fibre, being lower at the highest dietary level. Faecal concentration of fibre fractions increased (P < 0.05) with the level of inclusion of CM but decreased with that of OP (P < 0.01). High dietary level for both sources of fibre increased (P < 0.02) CP faecal content but urine N content decreased (from 205 to 168 g/kg DM, P < 0.05) in all the fibre-supplemented compared to C diet. Additionally, the proportions of undigested dietary, water soluble, and bacterial and endogenous debris of faecal N excretion were not affected by treatments. The initial slurry characteristics did not differ among different fibre sources and dietary levels, except pH, which decreased at the highest by-product inclusion levels. Ammonia emission per kg of slurry was lower in all the fibre-supplemented diets than in C diet (from 2.44 to 1.81 g, P < 0.05). Additionally, slurries from the highest dietary level of by-products tended (P < 0.06) to emit less NH3 per kg of initial total Kjeldahl N and showed a lower B0, independently of the fibre source. Thus, the fibre sources and their dietary levels affected pig nutrient digestion and composition of urine and faeces, showing potential to decrease NH3 and CH4 emissions at high levels of inclusion, independently of type of fibre.

Effects of nutrition on digestión efficiency and gaseous emissions from slurry in growing pigs: III. Influence of varying the dietary level of calcium soap of palm fatty acids distillate with or without orange pulp supplemention

The aim of this study was to establish the relationships between faecal fat concentration and gaseous emissions from pig slurry. Five diets were designed to meet essential nutrient requirements: a control and four experimental feeds including two levels (35 or 70 g/kg) of calcium soap fatty acids distillate (CSP) and 0 or 200 g/kg of orange pulp (OP) combined in a 2 × 2 factorial structure. Thirty growing pigs (six per treatment) were used to measure dry matter (DM) and N balance, coefficients of total tract apparent digestibility (CTTAD) of nutrients, faecal and urine composition and potential emissions of ammonia (NH3) and methane (CH4). Increasing dietary CSP level decreased DM, ether extract (EE) and crude protein (CP) CTTAD (by 4.0, 11.1 and 3.5%, respectively, P < 0.05), but did not influence those of fibrous constituents. It also led to a decrease (from 475 to 412 g/kg DM, P < 0.001) of faecal concentration of neutral detergent fibre (aNDFom) and to an increment (from 138 to 204 g/kg, P < 0.001) of EE in faecal DM that was related to greater CH4 emissions, both per gram of organic matter (P = 0.021) or on a daily basis (P < 0.001). Level of CSP did not affect N content in faeces or urine, but increased daily DM (P < 0.001), and N (P = 0.031) faecal excretion with no effect on urine N excretion. This resulted in lesser (P = 0.036) NH3 potential emission per kg of slurry. Addition of OP decreased CTTAD of EE (by 7.9%, P = 0.044), but increased (P < 0.05) that of all the fibrous fractions. As a consequence, faecal EE content increased (from 165 to 177 g/kg DM; P = 0.012), and aNDFom decreased greatly (from 483 to 404 g/kg DM, P < 0.001), which in all resulted in a lack of effect of OP on CH4 potential emission. Inclusion of OP in the diet also led to a significant decrease of CP CTTAD (by 6.85%, P < 0.001), and to an increase of faecal CP concentration (from 174 to 226 g/kg DM, P < 0.001), with no significant influence on urine N content. These effects resulted in higher N faecal losses, especially those of the undigested dietary origin, without significant effects on potential NH3 emission. No significant interactions between CSP and OP supplementation were observed for the gaseous emissions measured.

Effects of nutrition on digestión efficicency and gaseous emissions from slurry in growing-finishing pigs. II. Effect of protein source in practical diets.

Uso de diferentes fuentes de proteína en la alimentación de cerdos en fase de cebo y su relación con las emisiones de gases amoniaco y metano.