Growth promotion in broilers by both oxytetracycline and Macleaya cordata extract is based on their anti-inflammatory properties.

The non-antibiotic anti-inflammatory theory of antimicrobial growth promoters (AGP) predicts that alternatives can be selected by simple in vitro tests. In vitro, the known AGP oxytetracycline (OTC) and a Macleaya cordata extract (MCE) had an anti-inflammatory effect with a half-maximal inhibitory concentration of 88 and 132 mg/l, respectively. In vivo, chickens received three different concentrations of MCE in drinking-water, OTC in feed and a control. Body weight (BW), feed intake (FI) and gain:feed (G:F) ratio were determined on days 14, 21 and 35. On day 35, body composition was determined. Plasma α1-acid glycoprotein (α1-AG) concentration was measured on days 21 and 35, and the expression of several jejunal inflammatory genes was determined on day 35. OTC-fed chickens showed a significantly higher BW, FI and G:F ratio compared with the control group at all time points. MCE had a significant linear effect on BW on days 21 and 35, and the G:F ratio was improved only over the whole period, whereas FI was not different. Only MCE but not OTC decreased the percentage of abdominal fat. Plasma α1-AG concentration increased from day 21 to 35, with the values being lower in the treatment groups. Both OTC and MCE significantly reduced the jejunal mucosal expression of inducible NO synthase. For most parameters measured, there was a clear linear dose-response to treatment with MCE. In conclusion, the results are consistent with the anti-inflammatory theory of growth promotion in production animals.

Effects of plant-derived isoquinoline alkaloids on growth performance and intestinal function of broiler chickens under heat stress.

Broiler chickens reared under heat stress (HS) conditions have decreased growth performance and show metabolic and immunologic alterations. This study aimed to evaluate the effect of supplementation with a standardized blend of plant-derived isoquinoline alkaloids (IQ) on the growth performance, protein catabolism, intestinal barrier function, and inflammatory status of HS-treated chickens. Three hundred sixty 0-day-old Ross 308 male broiler chickens were randomly distributed into 2 treatment groups: control diet (no additives) or diet supplemented with 100 ppm IQ. At day 14, the chicks in each diet group were further divided into 2 groups, each of which was reared under thermoneutral (TN) (22.4°C) or constant HS (33.0°C) conditions until day 42. Each group consisted of 6 replicates with 15 birds per replicate, and chickens were provided ad libitum access to water and feed. During days 15-21, the body weight gain (BWG) and feed intake (FI) were significantly lower in the HS treatment group than in the TN group, and feed conversion ratio was higher (P < 0.05); these factors were not alleviated by IQ supplementation. During days 22-42, the final BW, BWG, and FI of the HS birds were better among those administered IQ than those that were not (P < 0.05). HS treatment increased plasma lipid peroxide, corticosterone, and uric acid concentrations as well as serum fluorescein isothiocyanate-dextran, a marker of intestinal barrier function, and decreased plasma total protein content (P < 0.05). These changes were not observed in the IQ group, suggesting that IQ supplementation improved oxidative damage, protein catabolism, and intestinal barrier function of chickens under HS. Isoquinoline alkaloid supplementation inhibited the expression of intestinal inflammatory factors, IL-6, tumor necrosis factor-like factor 1A, and inducible nitric oxide synthase under HS treatment (P < 0.05). These results suggest that IQ supplementation can improve the growth performance of broiler chickens under HS conditions, which may be associated with amelioration of oxidative damage, protein catabolism, intestinal barrier function, and inflammation.

Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to Enterotoxigenic Escherichia coli, Escherichia coli, and E. coli Lipopolysaccharide.

IPEC-J2, a promising in vitro model system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenic Escherichia coli (ETEC), nonpathogenic E. coli, and E. coli endotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured with E. coli strains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction.

Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium.

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.

The early transcriptional response of pig small intestinal mucosa to invasion by Salmonella enterica serovar typhimurium DT104.

Salmonella enterica serovar typhimurium (S. typhimurium) species are a leading cause of human invasive gastroenteritis. There is increasing in vitro evidence about Salmonella interaction with isolated cells or cell lines (macrophages, and enterocytes) on the molecular level, however, very little is known about in vivo interactions during actual invasion. We investigated the early interaction of S. typhimurium with intact small intestinal mucosa, in a pig model. Intestinal segments were infected with or without S. typhimurium DT104, and perfused. Whole mucosal gene expression was analyzed by cDNA array on 0, 2, 4, and 8h post-infection. Invasion resulted in the upregulation of only eight transcripts in jejunal mucosa, among those the proinflammatory IL-8 (at 4h only), and the antiinflammatory STAT3 (at 4 and 8h). The limited number of differentially expressed genes found here in vivo compared to in vitro is most likely due to the presence of multiple, heterogenous cell interactions in intact mucosa. Furthermore, it is concluded that S. typhimurium evades strong host responses by downregulating the local inflammatory response.

Expression of beta-defensins pBD-1 and pBD-2 along the small intestinal tract of the pig: lack of upregulation in vivo upon Salmonella typhimurium infection.

Defensins are antimicrobial peptides that play an important role in the innate immune response in the intestine. Up to date, only one beta-defensin (pBD-1), has been described in pig, which was found to be expressed at low levels in the intestine. We set-up a quantitative PCR method to detect the gene expression of pBD-1 and a newly discovered porcine beta-defensin, pBD-2. Expression of pBD-1 mRNA increased from the proximal to the distal part of the intestine whereas pBD-2 expression decreased. The main gene expression sites for pBD-2 were kidney and liver, whereas pBD-1 was mainly expressed in tongue. The porcine small intestinal segment perfusion (SISP) technique was used to investigate effects of Salmonella typhimurium DT104 on intestinal morphology and pBD-1 and pBD-2 mRNA levels in vivo. The early responses were studied 2, 4 and 8 h post-infection in four separate jejunal and ileal segments. Immunohistochemistry showed invasion of the mucosa by Salmonella and changes in intestinal morphology. However, no concomitant changes in expression of either pBD-1 or pBD-2 were observed. We conclude that at least two defensins are differentially expressed in the intestine of pigs, and that expression of both defensins is not altered by S. typhimurium under these conditions.

Dietary L-carnitine supplementation enhances the lipopolysaccharide-induced acute phase protein response in broiler chickens.

The objective of the present study was to evaluate the potential effects of dietary L-carnitine supplementation on acute phase protein response upon a lipopolysaccharide (LPS) challenge of male broiler chickens receiving a commercial broiler diet supplemented with 15 or 100 mg L-carnitine/kg or an unsupplemented (control) diet from 14 days of age onwards. At 28 days of age, eight chickens per dietary treatment were weighed and subcutaneously injected with 300 microg LPS from E. coli (100 microg LPS/ml saline) or 3 ml saline (unsupplemented group only). During the next 10 days, blood samples were taken repeatedly and analysed for their hemopexin (HX) and alpha-1 acid glycoprotein (AGP) levels. Extra dietary L-carnitine did not affect broiler performance. At day 1 postinjection, plasma HX and AGP levels were significantly increased in all treatment groups. However, the elevations in circulating HX and AGP levels were more pronounced in the L-carnitine supplemented chickens, especially in the 100mg L-carnitine group. It is concluded that extra L-carnitine in the diet of broiler chickens enhances or advances the acute phase protein response. The exact mode of action needs to be elucidated but seems to be consistent with a glucocorticoid mimicking effect.

Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis.

The aim of the study was to determine the intraarticular serum amyloid A (SAA) response pattern in horses with inflammatory arthritis. Inflammatory arthritis was induced by injection of lipopolysaccharide (LPS) into the radiocarpal joint of four horses. Serum and synovial fluid (SF) samples were collected before and at 4, 8, 12, 24, 48, 72, 96, and 144 h after injection. Concentrations of SAA were measured by immunoturbidometry, and expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. The LPS injection caused systemic and local clinical signs of inflammation. Serum amyloid A appeared in serum and SF within 8h after LPS injection. Isoelectric focusing showed three major SAA bands with apparent isoelectric points (pI) of 7.9, 8.6, and >9.3 in serum and SF. Synovial fluid contained two additional isoforms with highly alkaline apparent pI values (apparent pI value extrapolated from standard curve=10.0 and 10.2), which were not present in any of the serum samples. In conclusion, intraarticular injection of LPS induced systemic and local inflammatory responses in the horses. By demonstrating SF-specific SAA isoforms the results of the present study suggest that SAA is synthesized locally in the equine inflamed joint, similar to what has been demonstrated in humans previously. The marked local SAA synthesis suggests an important pathophysiological role in inflammatory arthritis.

A protocol for sustained reduction of Total Parenteral Nutrition and cost savings by improvement of nutritional care in hospitals.

BACKGROUND AND AIMS: Malnutrition and the use of Total Parenteral Nutrition (TPN) contribute considerably to hospital costs. Recently, we reported on the introduction of malnutrition screening and monitoring of TPN use in our hospital, which resulted in a large (40%) reduction in TPN and improved quality of nutritional care in two years (2011/12). Here, we aimed to assure continuation of improved care by developing a detailed malnutrition screening and TPN use protocol involving instruction tools for hospital staff, while monitoring the results in the following two years (2013/14). METHODS: A TPN decision tree for follow up of TPN in patients and a TP-EN instruction card for caregivers was introduced, showing TPN/EN introduction schedules based on the energy needs of patients according to EB guidelines, also addressing the risk of refeeding syndrome. TPN patients were monitored by dietitians and TPN usage and costs were presented to the (medical) staff. Screening and treatment of malnourished patients by dietitians is simultaneously ongoing. RESULTS: In 2014 48% of patients, hospitalized for at least 48 h, were screened on malnutrition, 17% of them were diagnosed at risk, 7.9% malnourished and treated by dietitians. TPN usage dropped by 53% and cost savings of 51% were obtained due to 50% decrease of TPN users in 2014 versus 2010. TPN over EN ratio dropped from 2.4 in 2010 to 1.2 in 2014. CONCLUSION: Sustained improvement of nutritional care and reduction of TPN usage and costs is possible by introduction of procedures embedded in the existing structures.

Chemical typing of porcine systemic amyloid as AA-amyloid.

Systemic AA amyloidosis is frequently reported in a wide variety of domestic and wild animal species. Porcine amyloidosis is rare and the amyloid has not been typed chemically thus far. In the present study, we have extracted porcine amyloid from formalin-fixed tissue sections. By subsequent amino acid sequencing, an N-terminal fragment was obtained identifying porcine systemic amyloid as AA amyloid. The N-terminal sequence had a great homology to bovine and ovine SAA1, suggesting that pig AA amyloid is derived from the systemic isoform of SAA. It is argued that the low incidence of amyloidosis in pigs is not likely to be attributed to unique features of porcine amyloid precursor protein. Elucidation of the basis for the high apparent resistance of pigs against amyloidosis may yield important clues for treatment and prevention of amyloidosis in other species. This is the first report on chemical identification of porcine amyloid.

Intestinal translocation of Streptococcus suis type 2 EF+ in pigs.

Sepsis with subsequent multisystem organ failure after translocation of bacteria from the gut is a serious risk associated with stress situations. We showed that intestinal bacterial translocation could be one of the pathways for pathogenic Streptococcus suis infections in the pig. In 24 piglets weighing 10-14 kg, free of the extracellular factor (EF+) producing phenotype of S. suis serotype 2, a silicon canula was placed in the proximal jejunum to enable intestinal inoculation and bypassing the upper alimentary tract. The pigs were individually housed. After stress induction in 18 pigs by means of a truck drive in individual cages for 1h, pigs were inoculated through the intestinal canula either with S. suis type 2 EF+ or with growth medium only, and put back in their original housing. The six not transported pigs were also inoculated with the same strain. To prevent oral self-infection, faeces were collected in a bag that was glued around the anus. Clinical and behavioral symptoms were recorded for 72 h post inoculation, and then the animals were sacrificed for pathological and bacteriological examination. In three animals, the inoculation strain was re-isolated from mesenterial lymph nodes and typically affected organs. No S. suis type 2 EF+ was detected by specific polymerase chain reaction (PCR) in any of the tonsil-swabs and -homogenates. We concluded that infection of the organs had taken place after bacterial translocation out of the gut and that the intestinal tract can be a porte d'entree for S. suis type 2 EF+.

Alpha4-integrin (CD49d) expression on bovine peripheral blood neutrophils is related to inflammation of the respiratory system.

Neutrophil emigration from the pulmonary vasculature, is mediated by cellular adhesion molecules (CAM) expressed on the outer membranes of endothelial cells and neutrophils. Although beta(2)-integrin-dependent migration is a major mechanism of neutrophil migration, which was demonstrated by extensive invasion of neutrophils in pulmonary tissue of calves suffering from a genetic deficit in expression of beta(2)-integrins, termed bovine leukocyte adhesion deficiency (LAD), the role of alternative CAM is still unclear. We investigated whether an alternate CAM for beta(2)-integrin function, i.e. the alpha(4)-integrin, was expressed on peripheral blood neutrophils of calves. As we detected basal but significant expression, the effect of naturally acquired pulmonary infection on the expression of either integrin was determined, as an indication for its function in the migration process. In our experiments, basal expression of alpha(4)-integrins on peripheral blood neutrophils from clinically healthy calves was detected. On neutrophils of calves, experiencing field outbreaks of enzootic bronchopneumonia, higher expression of the alpha(4)-integrin was detected, which returned to normal after successful treatment of the disease. In addition, its level of expression was linearly related to plasma acute phase protein (haptoglobin) concentrations, which is a sensitive parameter for severity of respiratory inflammation. Increased expression of the alpha(4)-integrin on peripheral blood neutrophils during pulmonary inflammation indicates a role for this CAM in neutrophil migration in the lung.

Proteomic approaches to study the pig intestinal system.

One of the major challenges in pig production is managing digestive health to maximize feed conversion and growth rates, but also to minimize treatment costs and to warrant public health. There is a great interest in the development of useful tools for intestinal health monitoring and the investigation of possible prophylactic/ therapeutic intervention pathways. A great variety of in vivo and in vitro intestinal models of study have been developed in the recent years. The understanding of such a complex system as the intestinal system (IS), and the study of its physiology and pathology is not an easy task. Analysis of such a complex system requires the use of systems biology techniques, like proteomics. However, for a correct interpretation of results and to maximize analysis performance, a careful selection of the IS model of study and proteomic platform is required. The study of the IS system is especially important in the pig, a species whose farming requires a very careful management of husbandry procedures regarding feeding and nutrition. The incorrect management of the pig digestive system leads directly to economic losses related suboptimal growth and feed utilization and/or the appearance of intestinal infections, in particular diarrhea. Furthermore, this species is the most suitable experimental model for human IS studies. Proteomics has risen as one of the most promising approaches to study the pig IS. In this review, we describe the most useful models of IS research in porcine and the different proteomic platforms available. An overview of the recent findings in pig IS proteomics is also provided.

Optimizing culture conditions of a porcine epithelial cell line IPEC-J2 through a histological and physiological characterization.

The high similarity between pigs and humans makes pigs a good gastrointestinal (GI) model for humans. Recently an epithelial cell line originating from the jejunum of pig (IPEC-J2) became available. Once validated, this model can be used to investigate the complex interactions occurring in the intestine. The advantages of using IPEC-J2 as in vitro model of the GI tract are the high resemblance between humans and pigs, and the ease of extrapolating in vitro to in vivo characteristics. In this study, the IPEC-J2 cells were functionally characterized by measuring the trans-epithelial electrical resistance (TEER), and by histological and ultrastructural studies. IPEC-J2 cells grown on six different permeable support systems, were investigated. The Transwell(®)-COL collagen-coated membrane (1.12 cm(2)) showed the best results concerning time efficiency and TEER values. The optimum seeding density of 12 × 10(5) cells/mL ensured that after 9 days of differentiation a confluent monolayer was formed. The decrease in TEER values after a maximum had been reached, coincided with the ultrastructural development of apical microvilli. We conclude that IPEC-J2 cells grown on collagen-coated membranes represent a valuable in vitro model system for the small intestinal epithelium which can be of great interest for intestinal research.

Transcriptomics of enterotoxigenic Escherichia coli infection. Individual variation in intestinal gene expression correlates with intestinal function.

Acute secretory diarrhea is a major cause of morbidity and mortality in young animals and humans. Deaths result from excessive fluid and electrolyte losses. The disease is caused by non-invasive bacteria such as Vibrio cholerae and Escherichia coli which produce enterotoxins, however, much less is known about the role of individual host responses. Here we report the response of intact porcine small intestinal mucosa to infection with enterotoxigenic E. coli (ETEC). Jejunal segments in four piglets were infused with or without ETEC, and perfused for 8h, and net absorption measured. Microarray analysis at 8h post-infection showed significant differential regulation of on average fifteen transcripts in mucosa, with considerable individual variation. Differential net absorption varied between animals, and correlated negatively with the number of up regulated genes, and with one individual gene (THO complex 4). This shows that quantitative differences in gene regulation can be functionally linked to the physiological response in these four animals.