Effects of TNF receptor blockade on in vitro cell survival and response to negative energy balance in dairy cattle.

BACKGROUND: Associative data and some controlled studies suggest that the inflammatory cytokine tumor necrosis factor (TNF) α can induce fatty liver in dairy cattle. However, research demonstrating that TNFα is a necessary component in the etiology of bovine fatty liver is lacking. The aim of this work was to evaluate whether blocking TNFα signaling with a synthetic cyclic peptide (TNF receptor loop peptide; TRLP) would improve liver metabolic function and reduce triglyceride accumulation during feed restriction. RESULTS: Capability of TRLP to inhibit TNFα signaling was confirmed on primary bovine hepatocytes treated with recombinant bovine TNFα and 4 doses of TRLP (0, 1, 10, 50 µmol/L) over 24 h. Next, 4 lactating Holstein cows (parity 1.4 ± 0.5, 433 ± 131 d in milk) in an incomplete Latin rectangle design (3 × 2) were subcutaneously administered with different TRLP doses (0, 1.5, 3.0 mg/kg BW) every 4 h for 24 h, followed by an intravenous injection of TNFα (5 µg/kg BW). Before and for 2 h after TNFα injection, TRLP decreased plasma non-esterified fatty acid (NEFA) concentration (P ≤ 0.05), suggesting an altered metabolic response to inflammation. Finally, 10 non-pregnant, non-lactating Holstein cows (3.9 ± 1.1 yr of age) were randomly assigned to treatments: control (carrier: 57% DMSO in PBS) or TRLP (1.75 mg TRLP /kg BW per day). Treatments were administrated every 4 h for 7 d by subcutaneous injection to feed-restricted cows fed 30% of maintenance energy requirements. Daily blood samples were analyzed for glucose, insulin, ß-hydroxybutyrate, NEFA, and haptoglobin concentrations, with no treatment effects detected. On d 7, cows completed a glucose tolerance test (GTT) by i.v. administration of a dextrose bolus (300 mg glucose/kg BW). Glucose, insulin, and NEFA responses failed to demonstrate any significant effect of treatment during the GTT. However, plasma and liver analyses were not indicative of dramatic lipolysis or hepatic lipidosis, suggesting that the feed restriction protocol failed to induce the metabolic state of interest. Injection site inflammation, assessed by a scorer blinded to treatment, was enhanced by TRLP compared to control. CONCLUSIONS: Although the TRLP inhibited bovine TNFα signaling and altered responses to i.v. administration of TNFα, repeated use over 7 d caused apparent local allergic responses and it failed to alter metabolism during a feed restriction-induced negative energy balance. Although responses to feed restriction seemed atypical in this study, side effects of TRLP argue against its future use as a tool for investigating the role of inflammation in metabolic impacts of negative energy balance.

Sodium salicylate treatment in early lactation increases whole-lactation milk and milk fat yield in mature dairy cows.

Multiple lines of inquiry have suggested that a high degree of inflammation in early lactation cows is associated with low productivity and increased disease incidence. In addition, some small studies have suggested that milk production increases in response to antiinflammatory treatment in the first week of lactation. Our objective was to determine if administration of sodium salicylate (SS), a nonsteroidal antiinflammatory drug (NSAID), in the first week of lactation changes whole-lactation productivity and retention in the herd. At calving, 78 cows [n=39 primiparous (1P); n=24 second parity (2P); n=15 third parity or greater (3P)] were alternately assigned to either control (CON) or SS treatments for 7 d postpartum. Sodium salicylate treatment was administered via individual water bowls at a concentration of 1.95 g/L, delivering a mean of 123.3±5.5 g of salicylate/d during the 7-d treatment. For the first 21 d of lactation, dry matter intake, water intake, milk yield, and health were monitored daily, and milk samples were collected twice weekly for milk component analysis. Monthly milk yield and component testing through the rest of the lactation provided data to assess long-term responses, and the effects of treatment on the risk of leaving the herd and on 305-d milk, fat, and protein yields were assessed. During the first 21 d of lactation, we observed no differences in morbidity, except for increased risk of metritis in 3P SS cows. Treatment interacted with parity to influence both 305-d milk and milk fat yields, and a tendency for an interaction was detected for 305-d milk protein yield. Milk yield was 2,469±646 kg greater over the lactation in 3P SS cows compared with 3P CON cows (21% increase) and tended to decrease by 8% in 1P cows treated with SS; no effects were detected in 2P cows. Furthermore, 3P SS cows produced 130±23 kg more milk fat over the lactation (30% increase), with no effects detected for 1P or 2P. Treatment with SS tended to increase 305-d milk protein yield in 3P cows by 14%, with no effects in 1P or 2P cows. A tendency for a treatment × parity interaction was also observed for the risk of leaving the herd. First-parity cows treated with SS tended to have greater risk of leaving the herd than controls (30 vs. 6% risk); however, treatment did not alter herd retention in 2P or 3P groups, and SS had no effect on the risk of leaving the herd overall. Results indicate that SS has long-term effects on lactation of mature dairy cows, particularly on fat yield, but may have negative effects for primiparous cows.

Interaction of Bacillus species and Salmonella enterica serovar Typhimurium in immune or inflammatory signaling from swine intestinal epithelial cells.

Previous research evaluated a laboratory strain of Bacillus licheniformis (BL) in a model swine epithelium and found it exerted antiinflammatory effects on Salmonella enterica serovar Typhimurium (Sal)-induced secretion of IL-8. The current investigation evaluated the antiinflammatory actions of Bacillus bacteria available commercially as feed additives for the swine industry. Three isolates were obtained from the product, 2 Bacillus subtilis (BS1 and BS3) and 1 BL (BL2). Swine jejunal epithelial IPEC-J2 cells were seeded into wells on permeable membrane supports and allowed to form confluent monolayers. Treatments included apical pretreatment with BL, BS1, BL2, or BS3 for 17 h without Sal, and the same Bacillus treatments but with 10(8) cfu of Sal added in the final hour of Bacillus incubation. Two additional treatments included negative control wells receiving no bacteria (control) and positive control wells receiving only Sal (10 total treatments). After bacterial incubation, wells were washed and fresh medium containing gentamicin was added. Cells were incubated for an additional 5 h, after which apical and basolateral media were recovered for determination of IL-8 and bacitracin. In addition, inserts with epithelial cells that had received Sal were lysed and lysates were cultured to determine treatment effects on Sal invasion. Exposure to Sal alone provoked an increase in IL-8 secretion from IPEC-J2 cells compared with control wells (P < 0.001 for both the apical and basolateral directions). Pretreatment with each Bacillus isolate followed by challenge with Sal reduced Sal-induced IL-8 secretion in both the apical and basolateral compartments compared with wells receiving only Sal (P < 0.001; except for BS3 apical, P < 0.01). The residual presence of bacitracin could be detected only in BL2 and BL2+Sal. Fewer Sal colonies could be cultured from lysates of BL2+Sal than from the Sal, BS1+Sal, and BS3+Sal treatments (P < 0.001). Results indicate that B. subtilis and BL have the ability to intervene in secretion of the neutrophil chemoattractant IL-8 from swine intestinal epithelial cells. This effect on chemokine secretion by gastrointestinal epithelial cells in vitro could not be explained solely by reduced invasion of epithelial cells by Sal.

Oral inoculation with Salmonella enterica serovar Typhimurium or Choleraesuis promotes divergent responses in the somatotropic growth axis of swine.

Enteric disease and immune challenge are processes that have detrimental effects on the growth performance of young swine. The current study tested the hypothesis that salmonella-induced enteric disease would perturb the endocrine growth axis in a serovar-dependent fashion. Specifically, we evaluated the effects of Salmonella enterica serovar Typhimurium (Typhimurium) and serovar Choleraesuis (Choleraesuis) on critical regulatory components of growth in young swine. Weaned pigs were housed 2 per pen with ad libitum access to feed and water in a 14-d experiment. Pigs were then repeatedly fed 10(8) cfu of either Choleraesuis or Typhimurium in dough balls, with control pigs receiving dough without bacteria. Bacteria were refed twice weekly. Rectal temperatures were monitored daily from d 0 to 7 and ADFI was measured through d 14. Pigs were weighed and samples of serum were obtained for circulating IGF-I on d 0, 7, and 14. At the conclusion of the study, samples of semitendinosus muscle and liver were obtained and subsequently assayed for IGF-I, IGFBP-3, and IGFBP-5 mRNA. Rectal temperatures were elevated in pigs given Choleraesuis from d 2 through 7 (P < 0.05) when compared with control pigs and pigs fed Typhimurium. Pigs receiving Choleraesuis had a substantially decreased feed intake on d 2, 3, 4, 7, 8, 9, and 10 (P < 0.01), with a trend for a reduction on d 5 (P = 0.08), and they experienced an approximately 25% reduction in BW compared with control pigs and pigs given Typhimurium by the conclusion of the study. Pigs given Choleraesuis also experienced marked reductions in circulating IGF-I on d 7 (P < 0.01 vs. control and Typhimurium), with reductions of lesser magnitude on d 14 (P = 0.07 vs. control and P < 0.05 vs. Typhimurium). Inoculation tended to affect liver IGFBP-3 mRNA (P = 0.08), for which expression tended to be elevated in pigs given Typhimurium and Choleraesuis. In contrast, IGFBP-3 mRNA relative abundance was increased (P < 0.03) in pigs given Typhimurium compared with control pigs. Muscle IGF-I mRNA was reduced in pigs given Choleraesuis compared with control pigs and pigs given Typhimurium (P < 0.05). Treatment tended to affect muscle IGFBP-3 mRNA (P = 0.10). Oral inoculation of growing pigs with Choleraesuis disrupted feed intake and BW gain, and this was accompanied by decreases in circulating IGF-I and reduced muscle expression of mRNA for IGF-I and IGFBP-3.

Expression of porcine Toll-like receptor 2, 4 and 9 gene transcripts in the presence of lipopolysaccharide and Salmonella enterica serovars Typhimurium and Choleraesuis.

Salmonella enterica serovar Typhimurium (ST) and Choleraesuis (SC) are among the most frequently isolated salmonellae serovars causing enteric disease in swine. Enteric disease in young pigs is of major concern in modern production systems due to the negative implications on animal health, food safety and economic return. Epithelial cells express Toll-like receptors (TLR) that recognize conserved microbial structures and act as mediators of innate and adaptive immune responses. However, little is known about the expression of TLR gene transcripts in swine. The objective of the current study was to characterize the relative abundance of porcine TLR2, 4 and 9 gene transcripts in vitro in a porcine jejunal epithelial cell line (IPEC-J2) and in porcine mononuclear phagocytes (pMP) in the presence of ST or SC, as well as in vivo in the distal ileum of pigs orally challenged with ST. Our results indicate that TLR2, 4 and 9 are constitutively expressed in vitro in IPEC-J2 cells and pMP and in vivo in the distal ileum. Additionally, transient modulation of porcine TLR was observed in vitro and in vivo in the presence of ST and SC. Further investigation is warranted to determine the effects of ST and SC on functional TLR.

Board-invited review: porcine mucosal immunity of the gastrointestinal tract.

The gastrointestinal tract (GIT) constitutes one of the largest immunological organs of the body. The GIT must permit absorption of nutrients while also maintaining the ability to respond appropriately to a diverse milieu of dietary and microbial antigenic components. Because of the diverse population of antigenic components within the GIT, a sophisticated mucosal immune system has evolved that relies on collaboration between the innate and adaptive arms of immunity. The collaborative, mucosal immune effort offers protection from harmful pathogens while also being tolerant of dietary antigens and normal microbial flora. Knowledge with respect to porcine mucosal immunity is important as we strive to understand the interrelationships among GIT physiology, immunology, and the resident microbiota. The aim of this review is to provide a descriptive overview of GIT immunity and components of the mucosal immune system and to highlight differences that exist between the porcine species and other mammals.

Effects of feeding L-carnitine to gilts through day 70 of gestation on litter traits and the expression of insulin-like growth factor system components and L-carnitine concentration in foetal tissues.

We investigated the influence of supplemental L-carnitine on foetal blood metabolites, litter characteristics, L-carnitine concentration in skeletal muscle and insulin-like growth factor (IGF) axis components in foetal hepatic and skeletal muscle tissues at day 40, 55 and 70 of gestating gilts. A total of 59 gilts (body weight = 137.7 kg) received a constant feed allowance of 1.75 kg/day and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length. Foetuses from the gilts fed diets with L-carnitine tended to be heavier (p = 0.06) and the circulating IGF-II tended to be lower (p = 0.09) at day 70, compared with the foetuses from the control gilts. Insulin-like growth factor-I messenger RNA (mRNA) was lower (p = 0.05) in hepatic tissue in the foetuses collected from gilts fed L-carnitine. Free and total carnitine concentration increased (p < 0.05) in the skeletal muscle from the foetuses collected from gilts fed supplemental L-carnitine. This study showed that L-carnitine had beneficial effects on the average foetal weight at day 70 of gestation, associated with changes in the foetal IGF system.

Source of dietary lipid may modify the immune response in stressed feeder cattle.

Five studies were conducted to evaluate the effects of lipid source on performance and health of stressed feeder cattle. A total of 332 heifers (195 +/- 2.37 kg initial BW) in trial 1 and 336 heifers (206 +/- 1.70 kg initial BW) in trial 2 were fed diets containing ground flaxseed (FLAX), rolled full-fat soybeans (SOY), or tallow (TAL) at 13, 20, or 4%, respectively (DM basis). All diets were formulated to be isonitrogenous and isocaloric. The ADG and G:F for the first 7 d and for the entire feeding period were greater (P < 0.05) for TAL and FLAX than for SOY. Percentage of animals treated and retreated for bovine respiratory disease did not differ among dietary treatments. The FLAX treatment increased (P < 0.05) total n-3 PUFA concentrations in the plasma, whereas SOY increased (P < 0.05) plasma concentrations of total n-6 PUFA. In trial 3, 18 steers were individually fed diets containing TAL and 18 steers were fed a diet containing SOY (20% of DM). In trials 4 and 5, 18 steers were individually fed diets containing TAL and 18 steers were fed diets containing FLAX (12.9% of DM). On d 14 and 17 of study 3, 4, and 5, 16 steers from each dietary treatment were injected i.v. with Escherichia coli O55:B5 lipopolysaccharide (LPS), and 2 steers from each diet were injected with saline. Rectal temperatures after LPS challenge were lower (P < 0.05) for SOY and FLAX than for TAL, and plasma TNF was greater (P < 0.05) for SOY than for TAL. Serum haptoglobin and blood fibrinogen increased and white blood cell count decreased in response to LPS, but none of these variables was affected by treatment. Although this research failed to measure an effect of lipid source on feedlot morbidity or mortality, these studies indicate that altering the source and type of dietary fatty acids may modify the immune response in stressed feeder cattle and that performance may be hindered by feeding full-fat soybeans to receiving cattle.

Lairage during transport of eighteen-kilogram pigs has an impact on innate immunity and commensal bacteria diversity in the intestines.

Long distance transportation may affect the health of pigs; thus, adding a rest stop (lairage) during long journeys may improve their well-being. The objective of this study was to determine whether a mid-journey lairage influenced swine innate immunity and intestinal microbial populations after a 16-h transport. Four replications were conducted, 1 in each of 4 seasons. Eighteen-kilogram pigs were housed in 16 pens (13 to 16 pigs/pen) with 8 pens/treatment. Lairage pigs were transported for 8 h, given a rest with food and water for 8 h, then transported for an additional 8 h. Continuous pigs were continuously transported for 16 h. Jugular blood samples and intestinal tissue and contents were collected from 16 pigs (8/treatment) on d 1, 3, 7, and 14 posttransport. Hematocrit and white blood cell counts were determined and neutrophil cell functions, including phagocytosis/oxidative burst and phagocytosis of latex beads and leukocyte phenotypic cell markers (CD14 and CD18), were analyzed using flow cytometry. Expression of toll-like receptors 2, 4, and 5; IL-8 (a cytokine that is a chemoattractant for neutrophils); CCL20 (a chemokine that is a chemoattractant for dendritic cells); and the antimicrobial peptide PR39 were determined from ileal and jejunal total RNA. Denaturing gradient gel electrophoresis was used to determine shifts in intestinal microbial populations. Total white blood cell and granulocyte counts in continuous pigs were greater (P < 0.01) on d 1 than in lairage pigs. Phagocytosis of microbeads was greater in continuous (P < 0.05) than in lairage pigs on d 7. Expression of IL-8 in jejunum was greater (P < 0.05) for continuous than for lairage pigs on d 1. Expression of CCL20 in the ileum was greater (P < 0.05) on d 14 for the continuous pigs. Expression of PR39 was greatest (P < 0.05) in the jejunum of lairage pigs on d 3. Lairage pigs had a greater (P < 0.05) variation in microbial populations (lower similarity coefficient) in the jejunum contents on d 1 and 7, in the cecum contents and tissue on d 3, and in the jejunum contents and tissue on d 14. However, continuous pigs had greater (P < 0.05) variation in the ileal tissues on d 14. This study indicates that adding a lairage to an extended transport alters immune functions, receptor, cytokine and chemokine expression, and gut microbiota compared with pigs transported for 16 h without lairage.

Influence of dietary L-carnitine and chromium picolinate on blood hormones and metabolites of gestating sows fed one meal per day.

Gestating sows (n = 44; parity = 2.0; BW = 208 kg) were used to determine the effects of dietary L-carnitine and Cr picolinate (CrP) on daily blood hormone and metabolite profiles. Diets were formulated as a 2 x 2 factorial with L-carnitine (0 or 50 ppm) and CrP (0 or 200 ppb) and were fed from breeding through gestation, lactation, and 28 d into the subsequent gestation, at which time blood collection occurred. Sows were fed 1 meal per day during gestation (2.04 kg from breeding until d 100 and 2.95 kg from d 100 until farrowing) and ad libitum during lactation. Sows were fitted with indwelling venous catheters, and blood (plasma) was collected at feeding, then once every 15 min for the first 3 h after feeding, and at 6, 9, 15, 20, and 24 h after feeding. Postfeeding and overall insulin and connecting peptide of insulin (c-peptide) was decreased for sows fed diets with CrP or L-carnitine and was greatest for sows fed the control diet; however, sows fed both L-carnitine and CrP had an intermediate response (L-carnitine x CrP, P < 0.01). Postfeeding glucose peak was decreased (P < 0.05) in sows fed diets with L-carnitine, CrP, or both, vs. the control, and mean glucose concentration was decreased (P < 0.01) for sows fed diets with CrP. L-Carnitine decreased (P < 0.04) the NEFA concentration. Sows fed diets with CrP exhibited increased (P < 0.03) postfeeding and overall NEFA and greater (P < 0.02) fasting and overall glycerol. Overall plasma urea N was lowest for sows fed the diet with L-carnitine; however, diets containing CrP had intermediate responses compared with the control (L-carnitine x CrP, P < 0.005). Sows fed diets with L-carnitine had greater (P < 0.008) IGF-I from 3 to 24 h after feeding and tended to exhibit greater (P < 0.06) overall IGFBP-3. Sows fed the diets with CrP had greater (P < 0.05) IGFBP-3 from 2 to 20 h after feeding. No differences were observed for glucagon or triacylglycerol (P > 0.10). The changes in metabolites and metabolic hormones indicate that both L-carnitine and CrP influence energy metabolism of gestating sows; however, their effects on blood hormones and metabolites differ. Thus, the improvement in energy status from adding both L-carnitine and CrP may have an additive effect on reproductive performance of sows.

Growth characteristics, blood metabolites, and insulin-like growth factor system components in maternal tissues of gilts fed L-carnitine through day seventy of gestation.

A total of 59 gilts (BW = 137.7 kg) from 3 breeding groups were used to assess the effects of feeding l-carnitine during gestation on gilt growth characteristics, blood metabolites, and uterine and chorioallantoic expression of IGF axis components at d 40, 55, and 70 of gestation. Experimental treatments were arranged in a 2 x 3 factorial, with main effects of added l-carnitine (0 or 50 ppm) and day after initial breeding (d 40, 55, or 70 of gestation). All gilts received a constant feed allowance of 1.75 kg/d and a top-dress containing 0 or 50 ppm of l-carnitine beginning on the first day of breeding through the assigned day of gestation. No dietary treatment differences were observed for gilt BW, backfat, or estimated protein or fat mass at any day of gestation. No differences were observed in circulating total and free carnitine at breeding, but concentrations increased (P < 0.01) as day of gestation increased for gilts fed diets containing l-carnitine compared with those fed the control diet. Maternal IGF-I concentration decreased (P < 0.01) from d 0 to 70 for all gilts, with no differences between treatments. Insulin-like growth factor binding protein-3 mRNA (P = 0.05) and IGFBP-5 mRNA (P = 0.01) increased in the endometrium of gilts supplemented with l-carnitine. These data demonstrate that l-carnitine supplementation and day of gestation alter the expression of the IGF axis by changing the expression of IGFBP at the fetal-maternal interface in swine. These changes in the IGF axis at the fetal maternal interface may aid in determining the reasons for the effects of l-carnitine on reproductive traits.

Effects of melengestrol acetate on the inflammatory response in heifers challenged with Mannheimia haemolytica.

Previous research from our laboratory has indicated that melengestrol acetate (MGA) added to the diet during the first 35 d after arrival in the feedlot improves growth rates and tends to reduce chronic respiratory disease in heifers naturally challenged with bovine respiratory disease. The current study was conducted to provide further insight into the possible immunomodulatory effects of MGA. Crossbred heifers (n = 48; 232 +/- 5.5 kg of BW) were used in a randomized complete block design to determine the effects of MGA on lung pathology and markers of inflammation after Mannheimia haemolytica challenge. On d 0, cattle were blocked by BW and randomly assigned, within block, to diets (54% concentrate) that provided 0 or 0.5 mg of MGA per heifer daily for the duration of the experiment. Inoculum containing from 1.3 x 10(9) to 1.7 x 10(9) cfu of M. haemolytica (20 mL) was instilled at the bifurcation of the trachea on d 14. Blood samples were collected, clinical observations were made, and rectal temperatures were recorded for each animal at 0, 12, 24, 48, 72, 96, 120, and 138 h after inoculation. Heifers fed MGA had greater circulating concentrations of eosinophils and postchallenge concentrations of segmented neutrophils and white blood cells (P < 0.01) than controls, as well as elevated plasma protein, serum haptoglobin, and fibrinogen after M. haemolytica challenge (P < 0.01). Heifers fed MGA had lower plasma glucose (P < 0.01), greater plasma urea N (P = 0.02), and elevated respiratory indices (P < 0.01) compared with controls. Necropsies performed on d 6 after inoculation suggested that M. haemolytica challenge was relatively mild, because lesions were confined to a small portion of the lungs. On a 0 to 100 scale, average lung lesion scores were 3 and 1 for MGA-fed and control groups, respectively (P < 0.06). Heifers fed MGA before mild M. haemolytica challenge were more susceptible to infection, as evidenced by a greater number of heifers fed MGA exhibiting pulmonary lesions 138 h after inoculation than controls (14 out of 23 vs. 6 out of 24 for MGA and controls, respectively; P < 0.02).

Effects of feeding Salmonella enterica serovar Typhimurium or serovar Choleraesuis on growth performance and circulating insulin-like growth factor-I, tumor necrosis factor-alpha, and interleukin-1beta in weaned pigs.

The most common Salmonella serovars causing clinical disease in pigs are Salmonella enterica serovars Typhimurium (Typhimurium) and Choleraesuis. Given that the swine host-adapted serovar Choleraesuis has been reported to cause systemic disease, a different disease outcome from that of Typhimurium, our working hypothesis was that this serovar would likely engage systemic immune-inflammatory mechanisms, resulting in elevated systemic cytokine secretion. Forty-eight weaned pigs were blocked by BW and sex, and randomly allotted to 1 of 3 treatments in a 14-d study. Each treatment had 8 replicates (pens), with 2 pigs/pen. The treatments consisted of a negative control and pigs repeatedly fed 10(8) cfu of Typhimurium or Choleraesuis. On d 0, the pigs were fed Choleraesuis or Typhimurium in dough balls, and the bacteria were refed twice weekly throughout the experiment. Control pigs received dough balls without bacteria. All pigs were housed in temperature-controlled rooms under constant lighting and were fed a standard corn-soybean meal-based nursery diet. Pig BW and feed disappearance were used to determine ADG, ADFI, and G:F. Rectal temperatures were obtained daily from 1 pig/pen beginning 2 d before the first bacterial feeding through d 7 using rapid-response digital thermometers. Serum was collected on d 0, 7, and 14 from a single pig/pen for analysis of IGF-I, tumor necrosis factor-alpha , and IL-1beta. There was no change in the rectal temperature of the control or the Typhimurium-challenged pigs (compared with d 0) or when comparing Typhimurium-challenged pigs with control animals. In contrast, pigs fed Choleraesuis had increased rectal temperatures beginning on d 2 and continuing through d 7 (P < 0.05), with the greatest elevation on d 3 (P < 0.001) compared with the control pigs. Average daily gain and ADFI of pigs challenged with Typhimurium did not differ from those of the control animals. Pigs fed Choleraesuis had a 25% reduction in ADG (P < 0.0001) and ADFI (P < 0.002) compared with the control pigs. On d 7, pigs fed Choleraesuis had reduced serum IGF-I compared with control (P < 0.01) or Typhimurium-challenged pigs (P = 0.01). Bacterial feeding did not affect serum tumor necrosis factor-alpha or IL-1beta compared with control pigs at any time throughout the experiment. We conclude that repeated exposure of weaned pigs to Choleraesuis reduced growth performance in the absence of changes in systemic inflammatory cytokines.

Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis.

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (CCL20) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P

Effects of Salmonella enterica serovar Typhimurium, or serovar Choleraesuis, Lactobacillus reuteri and Bacillus licheniformis on chemokine and cytokine expression in the swine jejunal epithelial cell line, IPEC-J2.

Direct-fed microbials, including Lactobacillus and Bacillus spp., are potential replacements for low dose in-feed antibiotics for swine and other livestock. To understand the function of these microbes in the gut, the current study used pig jejunal epithelial cells (IPEC-J2) to evaluate how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from Salmonella enterica serovars Typhimurium (ST) or Choleraesuis (SC) in their ability to regulate, stimulate, or modify the proinflammatory mediators, interleukin 8 (IL8), CC chemokine 20 (CCL20), and tumor necrosis factor-alpha (TNFalpha). To optimize the positive control to drive IL8 secretion by IPEC-J2 cells, cells were treated apically with various concentrations of ST (versus control (CTL)) for 1h, followed by a wash. Media containing gentamicin was added and collected at 6h post-treatment. Compared to CTL, 10(8) ST produced maximal IL8 secretion in both the apical and basolateral directions, with significant basolateral polarization (P<0.0001). We next evaluated the time course of IL8 secretion, and IL8, CCL20, and TNFalpha mRNA expression by IPEC-J2 cells treated apically with 10(8) ST, SC, LR, and BL versus CTL. Media and RNA were collected at 1.5, 3.0, and 6.0 h post treatment. Only ST stimulated an increase in IL8 secretion at any time point, with increases in IL8 mRNA at both 3 and 6h (P<0.05). However, BL increased IL8 mRNA at 1.5h (P<0.0001). Neither LR nor SC affected IL8 mRNA expression. CCL20 mRNA was strongly upregulated by ST (P<0.05) and BL (1.5 and 3.0 h; P<0.05), but not LR or SC. Only ST increased TNFalpha mRNA relative to CTL (P<0.05). Two experiments were conducted to determine if pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. Confluent cells were treated apically overnight with various levels of LR or BL (in separate experiments) followed by ST challenge. Media were collected at 4 (LR experiment) or 5h (BL experiment) post ST. In the LR study, IL8 secretion was increased by ST as compared to CTL (P<0.0001), reduced by LR (P<0.05), and LR+ST co-treatments failed to alter ST stimulated secretion. In the BL experiment, secretion of IL8 was increased by ST (P<0.0001), but blunted basolaterally in BL+ST co-treated wells. The data demonstrate that IPEC-J2 cells increase IL8 secretion in response to ST, and IL8 mRNA in response to ST and BL, but not LR. Furthermore, ST stimulated secretion of IL8 is inhibited basolaterally in the presence of BL.

Effects of Salmonella enterica serovars Typhimurium (ST) and Choleraesuis (SC) on chemokine and cytokine expression in swine ileum and jejunal epithelial cells.

The gastrointestinal epithelium represents a barrier to potentially invasive enteric pathogens, maintains a role in innate immune surveillance, and is a source of both chemokine and cytokine chemotactic mediators in response to bacterial invasion. In the current study, we evaluated cytokine and chemokine mediators known to regulate movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (IL8), dendritic cells (CCL20), and epithelial remodeling (osteopontin; OPN) in response to invasive swine enteropathogens Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). For the in vivo experiment, weaned pigs served as uninfected controls (0 h) or were given 3 x 10(9) CFU ST orally. Pigs were sacrificed at 8, 24, 48, and 144 h after inoculation and total RNA was extracted from defined segments of proximal (PI) and distal (DI) ileum. Relative expression of MIF and OPN were not affected by ST. IL8 expression was increased numerically (P = 0.17 for the interaction term) at 24 and 144 h in the PI and these increases accounted for greater expression in the PI relative to the DI (P < 0.05). Relative expression of CCL20 was increased at 24 h after ST (P < 0.05). Next, we evaluated the time course of MIF, IL8, CCL20, and OPN mRNA expression induced by application of lipopolysaccharide (LPS), ST or SC in vitro using pig jejunal epithelial cells (IPEC-J2). Cells were grown to confluency on permeable membranes, and treated apically with LPS (10 ng/mL), ST or SC (10(8)/well). After 1 h, cells were washed to remove LPS or extracellular bacteria, and media containing gentamicin was added to kill remaining extracellular bacteria. Media and RNA were collected at 1.5, 3, and 6 h after treatment. MIF mRNA was not affected by LPS or bacterial treatment. Similarly, IL8 expression was not affected by LPS, but was increased by ST and SC relative to controls at 1.5 and 3 h post exposure (P < 0.05 for all comparisons). Treatment with SC increased CCL20 mRNA relative to controls at 3 h (P < 0.05), while ST increased CCL20 at 1.5, 3, and 6h with maximal expression at 6 h (P < 0.05 for all comparisons). ST and SC increased polarized IL8 secretion. Our data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected cytokine and chemokine mediators, but serovars of Salmonella elicited differing patterns of activation in vitro.

Conceptus and maternal responses to increased feed intake during early gestation in pigs.

Maternal diet influences fetal growth and postnatal development. We hypothesized that conceptuses gestated in sows provided ad libitum vs. restricted feed intake would differ in the milieu of hormones, growth factors, nutrients, and metabolites associated with growth and metabolism. This hypothesis was tested in two experiments by providing fourth-parity sows (Pig Improvement Co. C15 bred to Line 326 boars) with either 1.81 kg/d (as-fed basis; control) or ad libitum access to gestation diet. In Exp. 1, control (n = 6) or ad libitum (6.4 +/- 0.11 kg/d; n = 9) treatments were provided from d 29 to 45 (onset of estrus is d 0), and sows were slaughtered on d 46. Ad libitum sows gained more weight from d 29 to 45 than controls (34.0 vs. 4.32 kg, respectively; P < 0.01). No differences were observed on d 46 for the number of fetuses, conceptus attachment length, allantoic + amniotic fluid volume, placental weight, fetal weight, and fetal crown-to-rump length. Variation in fetal crown-to-rump length was less (P < 0.03) in sows fed ad libitum. Sows fed ad libitum had greater (P < 0.01) IGF-I and insulin concentrations in plasma than controls on d 43. In Exp. 2, sows were fed 1.81 kg/d (n = 6) or ad libitum (7.0 +/- 0.11 kg/d; n = 4) from d 30 to 56 of gestation, when sows were anesthetized and samples were collected surgically from their gravid uteri. Sows fed ad libitum gained more weight (P < 0.01) than did controls and had more (P < 0.06) IGF-I in their plasma and the plasma collected from umbilical veins of their fetuses. No differences were found for concentrations of insulin or glucose in plasma of sows or fetuses, but urea N concentrations were greater (P < 0.05) in maternal plasma and in the plasma, and allantoic and amniotic fluids of conceptuses from sows fed ad libitum. Combined data from Exp. 1 and 2 revealed a treatment x fetal number interaction (P < 0.05) for average fetal weight. The expected negative relationship between within-litter average fetal weight and the number of fetuses per uterus was observed for control sows (y = 115.4 -1.75 x fetal number; P < 0.05), but litters of ad libitum sows did not show this effect. The hypothesis that providing feed in excess of established requirements in early gestation affects the in utero milieu is supported by these results. Data further reveal that, at least at mid-gestation, the restraint to fetal growth that is exhibited when fetal number increases in control sows is not exhibited when sows are fed ad libitum.

Effect of dietary mannanoligosaccharide and sodium chlorate on the growth performance, acute-phase response, and bacterial shedding of weaned pigs challenged with Salmonella enterica serotype Typhimurium.

A 28-d experiment evaluated the growth, acute-phase response, and bacterial shedding patterns in pigs (n = 96; initially 6.8 +/- 1.3 kg) fed mannanoligosaccharides (MANNAN) and sodium chlorate (CHLORATE) before and after oral challenge with Salmonella enterica serotype Typhimurium (ST). The negative control diet contained no antimicrobial (CON), and the positive control contained carbadox (CARB; 55 ppm). Test diets contained (as-fed basis) MANNAN (1,500 ppm) or CHLORATE (800 ppm). Pigs were fed diets for 14 d and then given ST orally. Pigs fed CARB had greater ADG over the entire study than pigs from other treatments (P < 0.05). During wk 1 to 2, before ST challenge, feed intake (as-fed basis) was lower for pigs fed MANNAN and CHLORATE than pigs fed CARB (P < 0.05). During the final 2 wk, pigs fed CARB had greater feed intake than pigs on other treatments (P < 0.05). Gain/feed was greater for pigs fed CARB in the 2 wk before ST (P < 0.05); however, in wk 3 to 4 after ST, gain/feed was reduced for CON pigs compared to pigs on other treatments (P < 0.05). Serum IGF-I was decreased at 2 and 4 d after ST (P < 0.001), and, overall, IGF-I was greater in pigs fed CARB than CON or CHLORATE (P < 0.05). Serum haptoglobin concentrations were greater (P < 0.001) for all treatments at d 6 compared with d 13 after ST. Overall, haptoglobin was greater for MANNAN than for CARB and CHLORATE (P < 0.05) and tended to be increased (P < 0.06) relative to CON. Interleukin-6 was not affected by treatment or day post-ST challenge. Fecal shedding of salmonellae organisms was less for CHLORATE (P < 0.05) than all other treatments at 7 d after ST. Shedding scores decreased from d 7 to 14 after ST (P < 0.05) for the CON, CARB, and MANNAN treatments. We conclude that feeding MANNAN and CHLORATE before acute enteric disease challenge may support improved gut function as evidenced by improved gain/feed, and that CHLORATE may decrease bacterial shedding. But neither MANNAN nor CHLORATE enhanced growth relative to the absence of dietary antimicrobials, nor was either treatment as effective as CARB following ST challenge.

Dietary L-carnitine increases plasma leptin concentrations of gestating sows fed one meal per day.

Thirty-four sows (parity=1.8; BW=206 kg) were used to determine the influence of L-carnitine and/or chromium tripicolinate on plasma leptin concentrations of gestating sows fed one meal daily. Treatments were arranged in a 2 x 2 factorial with main effects of carnitine (0 or 50 ppm) and chromium (0 or 200 ppb). Diets were fed for approximately 167 days (through one gestation, the following lactation, the interval from weaning to estrus, and 28 days into the following gestation) prior to blood collection. Leptin concentration was determined in plasma that was collected at feeding, every 15 min for the first 3h after feeding, and at 6, 9, 15, 20, and 24h after feeding. Sows fed diets containing carnitine had greater (P<0.02) overall mean plasma leptin concentrations and greater (P<0.05) leptin concentrations at 2.25, 3, 6, 15, 20, and 24h after feeding compared to sows fed either the control diet or the diet containing chromium. Leptin concentrations of sows fed diets containing carnitine also were greater (P<0.05) than control sows at 2.5 and 2.75 h postprandial and greater than (P<0.05) sows fed diets with both carnitine and chromium at 6h after feeding. Chromium had no effect (P>0.10) on plasma leptin concentration. These results suggest that dietary carnitine, but not chromium, increases circulating leptin in gestating sows fed one meal per day. These results may help to explain the improvements in reproductive function previously observed from feeding sows diets containing carnitine.

Changes in circulating insulin-like growth factor-I, insulin-like growth factor binding proteins, and leptin in weaned pigs infected with Salmonella enterica serovar Typhimurium.

One of the hallmarks of the pathophysiology of enteric disease in young pigs is reduced growth performance. This reduction in growth is associated with changes in the endocrine somatotropic growth axis. Our laboratory previously demonstrated that circulating insulin-like growth factor-I (IGF-I) was reduced in pigs infected with Salmonella enterica serovar Typhimurium (S. typhimurium) while circulating growth hormone remained unchanged. The objective of the current study was to determine if infection with S. typhimurium also was associated with changes in circulating IGF binding proteins (IGFBP). In addition, pigs experiencing active enteric disease have reduced feed intake. Because this inappetence may be related to systemic appetite reduction signals, we also evaluated circulating leptin in pigs undergoing active S. typhimurium-induced enteric disease. Crossbred pigs were penned in environmentally controlled rooms with free access to feed and water. Following an acclimation period, pigs were gavaged with 10(10) cfu of S. typhimurium (SAL; n=6) or were given a similar volume of sterile growth media (CON; n=6). Rectal temperatures and feed intakes were measured daily through 168 h to track the time course of the response to S. typhimurium infection. Samples of serum were obtained by jugular venipuncture at 0, 24, 48, 96 and 168 h after infection. Sera were frozen until evaluation for IGF-I by immunoradiometric assay (IRMA). In addition, sera were subjected to western ligand blotting utilizing 125I-IGF-I and 125I-IGF-II. Images were evaluated for total IGFBP and IGFBP-3 by densitometric analyses. Rectal temperature was increased in SAL pigs 24h post-infection (P<0.001) but not at other times. Feed intake was reduced in SAL pigs during the intervals 24-72 h (P<0.001) and 96-144 h (P<0.05) after infection. Serum IGF-I, expressed as a percentage of the 0 h concentration, was reduced in SAL pigs versus CON pigs at 48 h (28.1+/-18.7% versus 102.2+/-17.1%; P<0.01) and 96 h (20.0+/-18.7% versus 128.4+/-17.0%; P<0.0001) post-infection. Both total IGFBP and IGFBP-3, as estimated by ligand blotting, also were reduced in infected pigs at 48 h postchallenge (P<0.05). IGFBPs were similar between the two treatments at other sampling times. Concentrations of IGFBP-3 also were estimated utilizing an IRMA for human IGFBP-3. Serum IGFBP-3 was reduced in S. typhimurium-infected pigs at 24 h (P<0.01), 48 h (P<0.001), 96 h (P<0.001), and 168 h (P<0.05). Serum leptin levels were similar between SAL and CON pigs. The data suggest that swine enteric disease is associated with reduced circulating IGF-I and reductions in total IGFBP and IGFBP-3. However, serum leptin was not affected by enteric disease challenge.