The Effect of Probiotics in a Milk Replacer on Leukocyte Differential Counts, Phenotype, and Function in Neonatal Dairy Calves.

Probiotics have been investigated for many health benefits; however, few studies have been performed to determine the effects of oral probiotics on peripheral blood and respiratory immune cells in cattle. Our objectives were to determine changes in health and growth status, differential blood cell counts and function, and blood and lung cell function using flow cytometry and PCR in dairy calves fed a milk replacer with (PRO, N = 10) or without (CON, N = 10) the addition of probiotics to the milk replacer and dry rations from birth to weaning. Performance and clinical scores were not different between the treatment groups. Treatment-by-day interactions for peripheral blood leukocyte populations differed in cell number and percentages. A greater percentage of leukocytes expressed the cell surface markers CD3, CD4, CD8, CD11b, and CD205 on d 21 in CON animals. Lung lavages were performed on five animals from each treatment group on d 52. There were no differences between treatment groups for the expression of cytokines and Toll-Like Receptors as measured using Polymerase Chain Reaction, possibly due to the small sample size. Oral probiotics appear to affect peripheral blood immune cells and function. Their effect on overall calf health remains to be determined.

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Introduction: Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. Methods: A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Results: Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). Discussion: In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Potential improvements of the cognition of piglets through a synbiotic supplementation from 1 to 28 days via the gut microbiota.

The influence of feed supplements on behavior and memory has been recently studied in livestock. The objectives of the study were to evaluate the effects of a synbiotic on: an episodic-like (SOR: Spontaneous Object Recognition), a working (BARR: Fence barrier task), a long-term (TMAZE: Spatial T-maze task) memory test and on gut microbiota composition. Eighteen female piglets were supplemented from 1 to 28 days of age with a synbiotic (SYN), while 17 served as control (CTL). Feces were collected on days 16, 33 and 41 for 16S rRNA gene composition analyses. In the SOR, SYN piglets interacted more quickly with the novel object than CTL piglets. In the BARR, SYN piglets had shorter distances to finish the test in trial 3. In the TMAZE, SYN piglets were quicker to succeed on specific days and tended to try the new rewarded arm earlier during the reversal stage. Difference of microbiota composition between treatments was nonexistent on D16, a tendency on D33 and significant on D41. The synbiotic supplement may confer memory advantages in different cognitive tasks, regardless of the nature of the reward and the memory request. Difference in memory abilities can potentially be explained by differences in microbiota composition.

Replacing dietary antibiotics with 0.20% l-glutamine and synbiotics following weaning and transport in pigs.

Dietary antibiotic use has been limited in swine production due to concerns regarding antibiotic resistance. However, this may negatively impact the health, productivity, and welfare of pigs. Therefore, the study objective was to determine if combining dietary synbiotics and 0.20% l-glutamine would improve pig growth performance and intestinal health following weaning and transport when compared with traditionally used dietary antibiotics. Because previous research indicates that l-glutamine improves swine growth performance and synbiotics reduce enterogenic bacteria, it was hypothesized that supplementing diets with 0.20% l-glutamine (GLN) and synbiotics (SYN; 3 strains of Lactobacillus [1.2 × 10^9 cfu/g of strain/pig/d] + ß-glucan [0.01 g/pig/d] + fructooligosaccharide [0.01 g/pig/d]) would have an additive effect and improve pig performance and intestinal health over that of dietary antibiotics. Mixed-sex pigs (N = 226; 5.86 ± 0.11 kg body weight [BW]) were weaned (19.4 ± 0.2 d of age) and transported for 12 h in central Indiana. Pigs were blocked by BW and allotted to one of two dietary treatments (5 to 6 pigs per pen): antibiotics (positive control [PC]; chlortetracycline [441 ppm] + tiamulin [38.5 ppm]), no antibiotics (negative control [NC]), GLN, SYN, or the NC diet with both the GLN and SYN additives (GLN + SYN) fed for 14 d. From day 14 post-weaning to the end of the grow-finish period, all pigs were provided common antibiotic-free diets. Data were analyzed using PROC GLIMMIX and PROC MIXED in SAS 9.4. Overall, haptoglobin was greater (P = 0.03; 216%) in NC pigs compared with PC pigs. On day 13, GLN and PC pigs tended to have reduced (P = 0.07; 75.2% and 67.3%, respectively) haptoglobin compared with NC pigs. On day 34, the jejunal goblet cell count per villi and per millimeter tended to be greater (P < 0.08; 71.4% and 62.9%, respectively) in SYN pigs compared with all other dietary treatments. Overall, jejunal mucosa tumor necrosis factor-alpha (TNFα) gene expression tended to be greater (P = 0.09; 40.0%) in NC pigs compared with PC pigs on day 34. On day 34, jejunal mucosa TNFα gene expression tended to be greater (P = 0.09; 33.3%, 41.2%, and 60.0%, respectively) in GLN pigs compared with SYN, GLN + SYN, and PC pigs. Although it was determined that some metrics of pig health were improved by the addition of GLN and SYN (i.e., haptoglobin and goblet cell count), overall, there were very few differences detected between dietary treatments and this may be related to the stress load incurred by the pigs.

Variation in the response of bovine alveolar lavage cells to diverse species of probiotic bacteria.

OBJECTIVE: Probiotics are fed to improve enteric health, and they may also affect respiratory immunity through their exposure to the upper respiratory tract upon ingestion. However, their effect on the respiratory system is not known. Our aim was to determine how probiotics affect functions and markers of bronchoalveolar lung lavage cells (BAL) isolated from lungs of calves at slaughter. RESULTS: Treatments consisted of ten probiotic species and one control treatment. Probiotics and BAL were incubated 1:1 for 2 h at 37 °C and 5% CO2. The cell surface markers measured included CD14, CD205, and CD18, and E. coli bioparticles were used to measure phagocytosis and oxidative burst. Differences were considered significant at P ≤ 0.05 and were noted for percent cells fluorescing and mean fluorescence intensity for CD14 and CD205. Additionally, oxidative burst was different as measured by both percentage of cells fluorescing and mean fluorescence intensity, and phagocytosis differed among species as measured by mean fluorescence intensity. Overall, probiotic species differed in their ability to suppress or increase leukocyte function showing that probiotic bacteria differentially modulate BAL.

Effects of orally administered cortisol and norepinephrine on weanling piglet gut microbial populations and Salmonella passage.

Stress and anxiety have been associated with changes in the microbiota of the gut and ultimately diminished resistance to pathogens. The objective of this study was to observe intestinal microbiota and susceptibility to Salmonella associated with stress hormones, cortisol (CORT), and norepinephrine (NE), in piglets. At weaning, 90 piglets (15 for a Salmonella challenge) were trained to take the carrier (apple juice) orally. At 2 wk after weaning, pens of piglets were assigned randomly to 1 of 3 treatments: control (CNT), NE, or CORT. Blood samples were collected prior to treatment, then piglets were dosed orally with treatments twice on day 0; at 0800 and 1600 h. Control piglets were administered 6.1 mL of the carrier only, NE pigs were administered 40 mg/mL of NE-bitartrate salt dissolved in the carrier, and CORT pigs were administered 12 mg/mL of hydrocortisone acetate dissolved in the carrier. Jugular blood samples were collected prior to necropsies (n = 5/treatment) at 0800 and 1600 h on day 1, and at 0800 h on days 2, 7, and 14 after treatments were started. A subset of pigs were subjected to a 24-h Salmonella challenge. Jejunal and ileal tissues and jejunal, ileal, cecal, and rectal contents were collected and colonies were counted. Microbial data and blood samples were analyzed using mixed models with fixed effects of treatment and day. Cortisol-treated piglets exhibited a spike in plasma CORT concentrations at 0800 h day 1 (P = 0.001) accompanied by greater concentrations of cecal Escherichia coli (P < 0.05) and a shift in intestinal environment to favor coliforms on day 2 (P < 0.05). Salmonella concentrations from rectal contents tended (P = 0.07) to be suppressed by CORT. Lactic acid-producing bacteria rectal concentrations were greater (P = 0.03) in CORT pigs on 0800 h on day 1 then NE pigs and tended to be greater than CNT (P = 0.09) and were greater on day 14 for both CNT (P = 0.003) and NE (P = 0.02). Norepinephrine spiked in NE piglets at 0800 h on day 1 (P = 0.001), 1600 h day 1 (P = 0.004), through day 2 (P = 0.04). Intestinal environment of NE pigs shifted to favor ileal anaerobes (P ≤ 0.05) and facultative anaerobes (E. coli; P = 0.01) compared to CNT. However, Salmonella concentrations in rectal contents were suppressed by NE compared to CNT (P = 0.05). Oral administration of NE and CORT had the desired effect of increasing concentrations of stress hormones and resulted in microbiome shifts throughout the intestines.

Development of a chicken ileal explant culture model for measurement of gut inflammation induced by lipopolysaccharide.

Gut mucosa holds a single layer of epithelial cells and the largest mass of lymphoid tissue in the body. Although the epithelial cell culture model is widely used to assess intestinal barrier function, it has limitations for studying cellular interactions, in particular those of the immune system. In this study, a chicken ileal explant culture model was developed for investigating short-term gut inflammatory and secretory responses in an ex vivo environment. Initially, ileal explants from broilers at 21 d of age were cultured ex vivo up to 6 h. Explants cultured for a maximum of 2 h remained over 90% viable, based on lactate dehydrogenase (LDH) release assay. Morphologically, explants cultured for 2 h displayed normal morphology compared to those cultured longer, further confirming that short-term culture for up to 2 h duration is an acceptable model for studying ex vivo regulation of inflammation. Subsequently, lipopolysaccharide (LPS) dose-related responses were determined for explants cultured for 2 h. Results from LDH activity assay showed that the viability of explants was decreased (P ≤ 0.05) at an LPS dose higher than 50 µg/mL. A significant (P ≤ 0.05) nitric oxide release was observed at LPS concentrations of 10 and 20 µg/mL. In addition, the highest inflammatory and secretory responses were detected at 20 µg/mL LPS based on gene expression of TLR-4, IL-1ß, IL-8, MUC2, IgA, and pIgR (P ≤ 0.05). However, the gene expression of claudin-1 and claudin-4 were not increased at the determined LPS concentrations (P > 0.05). These results demonstrated the potential usefulness of this intestinal explant culture model for short-term study of biological factors in gut inflammatory and secretory responses, but not a sufficient duration for evaluation of tight junction responsiveness.

Effect of threonine deficiency on intestinal integrity and immune response to feed withdrawal combined with coccidial vaccine challenge in broiler chicks.

For this study, threonine (Thr) deficiency was hypothesised to exacerbate the intestinal damage induced by feed withdrawal with coccidial infection because of its high obligatory requirement by the gut; two dietary Thr treatments (0·49 and 0·90 %) were applied to chicks from 0 to 21 d of age. At 13 d of age, feed was withdrawn for 24 h from one-half of birds of each dietary treatment with subsequent gavage of a 25× dose of coccidial vaccine. Overall, there were four treatments with eight replicate cages per treatment. Under combined challenge, birds fed the Thr-deficient diet had 38 % lower 13-21-d body weight gain (P≤0·05) compared with birds fed the Thr-control diet. At 21 d, the challenged group fed low Thr had higher number of oocysts (+40 %, P=0·03) and lower crypt depth (-31 %, P0·05). Overall, Thr deficiency worsened the detrimental effects of combined feed withdrawal and coccidial infection on growth performance and oocyst shedding by impairing intestinal morphology, barrier function, lymphocyte profiles and their cytokine expressions.

Susceptibility of Broiler Chickens to Coccidiosis When Fed Subclinical Doses of Deoxynivalenol and Fumonisins-Special Emphasis on the Immunological Response and the Mycotoxin Interaction.

Deoxynivalenol (DON) and fumonisins (FB) are the most frequently encountered mycotoxins produced by Fusarium species in livestock diets. The effect of subclinical doses of mycotoxins in chickens is largely unknown, and in particular the susceptibility of birds to pathogenic challenge when fed these fungal metabolites. Therefore, the present study reports the effects of DON and FB on chickens challenged with Eimeria spp, responsible for coccidiosis. Broilers were fed diets from hatch to day 20, containing no mycotoxins, 1.5 mg DON/kg, 20 mg FB/kg, or both toxins (12 pens/diet; 7 birds/pen). At day 14, six pens of birds per diet (half of the birds) were challenged with a 25×-recommended dose of coccidial vaccine, and all birds (challenged and unchallenged) were sampled 6 days later. As expected, performance of birds was strongly affected by the coccidial challenge. Ingestion of mycotoxins did not further affect the growth but repartitioned the rate of reduction (between the fraction due to the change in maintenance and feed efficiency), and reduced apparent nitrogen digestibility. Intestinal lesions and number of oocysts in the jejunal mucosa and feces of challenged birds were more frequent and intense in the birds fed mycotoxins than in birds fed control feed. The upregulation of cytokines (interleukin (IL) IL-1ß, IL-6, IL-8 and IL-10) following coccidial infection was higher in the jejunum of birds fed mycotoxins. Further, the higher intestinal immune response was associated with a higher percentage of T lymphocytes CD4⁺CD25⁺, also called Tregs, observed in the cecal tonsils of challenged birds fed mycotoxins. Interestingly, the increase in FB biomarker of exposure (sphinganine/sphingosine ratio in serum and liver) suggested a higher absorption and bioavailability of FB in challenged birds. The interaction of DON and FB was very dependent on the endpoint assessed, with three endpoints reporting antagonism, nine additivity, and two synergism. In conclusion, subclinical doses of DON and FB showed little effects in unchallenged chickens, but seem to result in metabolic and immunologic disturbances that amplify the severity of coccidiosis.

The Effect of Cooled Perches on Immunological Parameters of Caged White Leghorn Hens during the Hot Summer Months.

The objective of this study was to determine if thermally cooled perches improve hen immunity during hot summer. White Leghorn pullets at 16 week of age were randomly assigned to 18 cages of 3 banks at 9 hens per cage. Each bank was assigned to 1 of the 3 treatments up to 32 week of age: 1) thermally cooled perches, 2) perches with ambient air, and 3) cages without perches. Hens were exposed to natural ambient temperatures from June through September 2013 in Indiana with a 4 h acute heat episode at 27.6 week of age. The packed cell volume, heterophil to lymphocyte (H/L) ratio, plasma concentrations of total IgG, and cytokines of interleukin-1ß and interleukin-6, plus lipopolysaccharide-induced tumor necrosis factor-α factor were measured at both 27.6 and 32 week of age. The mRNA expressions of these cytokines, toll-like receptor-4, and inducible nitric oxide synthase were also examined in the spleen of 32 week-old hens. Except for H/L ratio, thermally cooled perches did not significantly improve currently measured immunological indicators. These results indicated that the ambient temperature of 2013 summer in Indiana (24°C, 17.1 to 33.1°C) was not high enough and the 4 h heat episode at 33.3°C (32 to 34.6°C) was insufficient in length to evoke severe heat stress in hens. However, cooled perch hens had a lower H/L ratio than both air perch hens and control hens at 27.6 week of age and it was still lower compared to control hens (P < 0.05, respectively) at 32 week of age. The lowered H/L ratio of cooled perch hens may suggest that they were able to cope with acute heat stress more effectively than control hens. Further studies are needed to evaluate the effectiveness of thermally cooled perches on hen health under higher ambient temperatures.

The influence of dietary zinc source and coccidial vaccine exposure on intracellular zinc homeostasis and immune status in broiler chickens.

Coccidia are protozoal parasites which compromise mucosal integrity of the intestine, potentiating poultry morbidity. The host's Zn status influences the course of infection. Therefore, two experiments were designed to determine how supplemental Zn regimens impacted jejunal and caecal immune status and Zn transporter expression. Coccivac®-B was administered weekly at ten times the recommended dose as a mild coccidial challenge (10 CV). Zn was provided through a basal diet, supplemental zinc sulfate (ZnSO4), or a supplemental 1:1 blend of ZnSO4 and Availa®-Zn (Blend). Mucosal jejunum (Expt 1) and caecal tonsils (Expt 2) were evaluated for intracellular Zn concentrations and phagocytic capacity. Messenger expression of Zn transporters ZnT5, ZnT7, Zip9 and Zip13 were investigated to determine Zn trafficking. With 10 CV, phagocytic capacity was decreased in jejunal cells by 2%. In the caecal tonsils, however, phagocytic capacity increased with challenge, with the magnitude of increase being more pronounced with higher dietary Zn (10 CV × Zn interaction; P = 0.04). Intracellular Zn within caecal tonsils was found significantly reduced with 10 CV (27%, P = 0.0001). 10 CV also resulted in an overall increase in the ratio of Zip:ZnT transporters. With the exception of Zip13 transporter expression, dietary Zn source had little impact on any of the measured cellular parameters. Thus, intestinal mucosal tissues had reductions in intracellular free Zn during coccidial challenge, which was coupled with an upregulation of measured Zip transporters. This suggests that under coccidial challenge, intestinal cells attempt to compensate for the drop in intracellular Zn.

Differential innate immune responses of bovine peripheral blood leukocytes to Salmonella enterica serovars Dublin, Typhimurium, and Enteritidis.

The majority of Salmonella serovars cause no clinical disease in cattle, while some are associated with severe disease. The objective of the current study was to determine the innate immune responses of bovine peripheral blood leukocytes exposed to Salmonella enterica serovar Dublin (bovine-specific), Salmonella typhimurium (murine adapted, but zoonotic), and Salmonella enteritidis (poultry host-adapted) in 3-week-old calves. All Salmonella exposures increased cell surface CD14 and CD18 regardless of serovar. The greatest CD14 marker mean fluorescence was in monocytes and the greatest mean fluorescent of the marker mean was in neutrophils. Phagocytosis increased with all serovars, but was not different among them. Neutrophils had the greatest marker mean fluorescence for phagocytosis, with all serovars being equal. Oxidative burst increased in all serovars compared to control cells, but were not different among the serovars. Neutrophils and monocytes were similar in the oxidative burst, with limited oxidative burst detected in the primarily lymphocyte population. mRNA expression of TNF-α, IL-8, and IL-12, increased above the control cells whereas none of these serovars affected mRNA expression of TLR4. TNF-α was greatest in S. enterica and S. typhimurium, compared to Salmonella dublin. In contrast, IL-8 was expressed more in S. dublin than S. typhiurium, with S. Enteriditus intermediary. These results show while cell surface markers, phagocytosis, and oxidative burst were largely unaffected by serovar, cytokine and chemokine expression differed among the Salmonella serovars. It appears that internal responses of the cells differ, rather than cell recognition, creating pathogenicity differences among of the serovars, even in the neonate with developing immunity.

Development of intestinal mucin 2, IgA, and polymeric Ig receptor expressions in broiler chickens and Pekin ducks.

Intestinal mucin 2 (MUC2), a major gel-forming mucin, represents a primary barrier component of mucus layers and a target site for secretory IgA. Polymeric Ig receptor (pIgR) expressed on the basolateral surface of epithelium is used to transport polymeric IgA from the lamina propria into luminal mucins to establish the first lines of intestinal defense. To determine the spatio-temporal expression of MUC2, IgA, and pIgR in broiler chickens and Pekin ducks, intestinal tissues (n=6/age) were dissected from late embryonic days up to 21 d posthatch. In the intestinal tissues, MUC2 was expressed with a rapid increase at hatching, followed by steady expression through 21 d posthatch both in chickens and ducks. IgA expression was low during the first week following hatching for both species. From the second week posthatch, IgA was rapidly expressed in the chickens, arriving at steady expression in the third week after hatching. However, in ducks, IgA expression during the 2 to 3 wk posthatch period was relatively slow. The expression of pIgR was greatly increased after hatching for both species, but its expression in ducks was relatively delayed. In addition, intestinal pIgR expression was highly correlated with MUC2 and IgA expressions in chickens but just moderately correlated in ducks. The relatively slow and late expression of IgA and pIgR as well as their moderate correlation may or may not account for the susceptibility of ducklings to mucosal pathogens at a young age.

Supplemental dietary L-arginine attenuates intestinal mucosal disruption during a coccidial vaccine challenge in broiler chickens.

The present study investigated the effects of dietary arginine (Arg) supplementation on intestinal structure and functionality in broiler chickens subjected to coccidial challenge. The present study was a randomised complete block design employing a 3 × 2 factorial arrangement (n 8) with three dietary concentrations of Arg (11·1, 13·3 and 20·2 g/kg) with or without coccidial vaccine challenge (unchallenged and coccidial challenge). On day 14, birds were orally administered with coccidial vaccine or saline. On day 21, birds were killed to obtain jejunal tissue and mucosal samples for histological, gene expression and mucosal immunity measurements. Within 7 d of the challenge, there was a decrease in body-weight gain and feed intake, and an increase in the feed:gain ratio (P< 0·05). Jejunal inflammation was evidenced by villus damage, crypt dilation and goblet cell depletion. Coccidial challenge increased mucosal secretory IgA concentration and inflammatory gene (iNOS, IL-1ß, IL-8 and MyD88) mRNA expression levels (P< 0·05), as well as reduced jejunal Mucin-2, IgA and IL-1RI mRNA expression levels (P< 0·05). Increasing Arg concentration (1) increased jejunal villus height (P< 0·05) and linearly increased jejunal crypt depth (P< 0·05); (2) quadratically increased mucosal maltase activity (P< 0·05) and linearly decreased mucosal secretory IgG concentration (P< 0·05) within the coccidiosis-challenged groups; and (3) linearly decreased jejunal Toll-like receptor 4 (TLR4) mRNA expression level (P< 0·05) within the coccidiosis-challenged groups. The mRNA expression of mechanistic target of rapamycin (mTOR) complex 1 pathway genes (mTOR and RPS6KB1) and the anti-apoptosis gene Bcl-2 quadratically responded to increasing dietary Arg supplementation (P< 0·05). These results indicate that dietary Arg supplementation attenuates intestinal mucosal disruption in coccidiosis-challenged chickens probably through suppressing TLR4 and activating mTOR complex 1 pathways.

Dietary L-arginine supplementation attenuates lipopolysaccharide-induced inflammatory response in broiler chickens.

In the present study, two experiments were conducted to investigate the effect of dietary L-arginine (Arg) supplementation on the inflammatory response and innate immunity of broiler chickens. Expt 1 was designed as a 2 × 3 factorial arrangement (n 8 cages/treatment; n 6 birds/cage) with three dietary Arg concentrations (1.05, 1.42 and 1.90%) and two immune treatments (injection of lipopolysaccharide (LPS) or saline) given at an interval of 48 h between 14 and 21 d of age. In Expt 2, correlation between dietary Arg concentration (0.99, 1.39, 1.76, 2.13 or 2.53%) and percentage of circulating B cells (percentage of circulating lymphocytes) was determined. In Expt 1, LPS injection decreased body-weight gain and feed intake and increased feed conversion ratio of the challenged broilers (14-21 d; P< 0.05). LPS injection suppressed (P< 0.05) the percentages of splenic CD11+ and B cells (percentages of splenic lymphocytes) and phagocytic activity of splenic heterophils and macrophages; Arg supplementation linearly decreased the percentages of CD11+, CD14+ and B cells in the spleen (P< 0.10). LPS injection increased (P< 0.05) the expression of IL-1ß and IL-6 mRNA in the spleen and caecal tonsils. Arginine supplementation decreased (P< 0.05) the expression of IL-1ß, Toll-like receptor 4 (TLR4) and PPAR-γ mRNA in the spleen and IL-1ß, IL-10, TLR4 and NF-κB mRNA in the caecal tonsils. In Expt 2, increasing dietary Arg concentrations linearly and quadratically reduced the percentage of circulating B cells (P< 0.01). Collectively, Arg supplementation attenuated the overexpression of pro-inflammatory cytokines probably through the suppression of the TLR4 pathway and CD14+ cell percentage. Furthermore, excessive Arg supplementation (1.76%) suppressed the percentages of circulating and splenic B cells.

Immunological, physiological, and behavioral effects of Salmonella enterica carriage and shedding in experimentally infected finishing pigs.

Finishing pigs infected with Salmonella pose significant food safety risks by carrying the pathogen into abattoirs. This study was conducted to determine the dynamics of Salmonella infection in finishing pigs, and associated immunological, physiological, and behavioral alterations, by longitudinally comparing infected to noninfected pigs during 6 weeks postinfection (p.i.). Bacteriological data revealed that all inoculated pigs started shedding Salmonella within 2 h p.i., and persistently shed the bacteria up to the end of the study. Ileal and cecal contents, as well as mesenteric lymph node samples, were all positive throughout the study, containing 3-4 log(10) cfu/g of Salmonella at 24 h p.i., and 4-5 log(10) cfu/g of Salmonella up to 4 weeks p.i. Levels of Salmonella dropped markedly (p < 0.05) in all samples at 5 weeks p.i. There was no difference between groups for blood cell counts. Tumor necrosis factor-α was greater (p < 0.05) in infected pigs: (1) in the mesenteric lymph nodes by 48 h p.i.; (2) at 24 h and 3 weeks p.i. in the ileum; and (3) in the cecum and spleen at 3 weeks p.i. Interleukin-12, interleukin-1 and its antagonist, and a porcine-specific antimicrobial peptide RNA expression in tissues changed over time, but were not different between groups. Infected pigs spent more time in ventral recumbency, standing, and sitting than controls (p < 0.01). Infected pigs were also more active (p < 0.01), and approached a novel object more quickly than control pigs (p < 0.05). No treatment differences were detected for rectal temperature or plasma cortisol (p > 0.10). This study shows that finishing pigs can carry high levels of Salmonella for up to 4 weeks p.i. in the gastrointestinal contents and mesenteric lymph nodes, shedding high levels of the bacteria without developing clinical symptoms, but developing an immune response throughout the intestinal tract. Moreover, subtle behavioral changes measured as postures were detected, and therefore warrant additional investigation.

Influence of milk yield, stage of lactation, and body condition on dairy cattle lying behaviour measured using an automated activity monitoring sensor.

Time spent lying by lactating Holstein-Friesian cows of varying body condition scores (BCS) and milk yield was measured using an animal activity monitor. A 3-week average BCS was calculated for each cow; and in total, 84 cows were selected with 28 cows each among three BCS categories (Thin: BCS<2.75; Moderate: 2.75 > or = BCS<3.25; Heavy: BCS> or = 3.25) and two stage of lactation categories (<150 days in milk or >150 days in milk). Cows were kept in two management systems: parlour/freestall (n=60) or automated milking system/freestall (n=24). Behaviour was recorded for 5.3+/-0.1 d for each cow. Production levels were considered using a 28-d rolling average of daily milk production. Cows that exhibited clinical lameness before or during the observation period were excluded from analyses. For cows exhibiting oestrus, the day prior to, day of, and day following breeding were removed. The final analysis included 77 cows (408 d of observation). A mixed model was fitted to describe average daily hours spent lying. Results demonstrated that lying time increased as days in milk (DIM) increased (P=0.05). Variables that were tested but not significant (P>0.05) were BCS category, parity category (1 or 2) and 28-d rolling average daily milk production. Although a numerical trend for increasing hours spent lying with increasing BCS was observed, after accounting for other factors in the mixed model, BCS did not significantly impact lying time. Continued investigation of these management factors that impact lying time and bouts, using new technologies, more cows, and more herds will help dairy owners better manage facilities and cow movements to optimize this essential behaviour.

Prepartum milking effects on parlour behaviour, endocrine and immune responses in Holstein heifers.

Transition of primiparous heifers to the milking herd is a period with multiple stressors. The objective of these studies was to determine effects of parlour experience and prepartum milking (pre-milking) on behavioural and physiological indicators of stress after calving. Two experiments were conducted, one was in a free-stall housing confinement system and the second was in a modified grazing system. Forty-eight first-calf heifers were assigned to three treatments: control; experienced heifers taken through the parlour without milking; or pre-milk heifers milked for 3 weeks prior to estimated parturition. Blood was collected within 24 h of parturition and on days 3, 5, 7, 10 and 14 following parturition for cortisol and acute phase protein determination. In the grazing system, 20 heifers were assigned to a prepartum milked or control group as in the confinement system and behaviour observations included days -21, -14, -7, -5, -3 and -1 relative to calving and days 1, 3, 7, 9, 14, and 16 post-calving. Milk production was greatest for prepartum milked heifers in both housing systems. However, somatic cell score was reduced by prepartum milking only in the confinement system. Balking occurred least in parlour-experienced heifers. In confinement housing, shifting while in the parlour was the only behaviour that was greater at first milking in control heifers. Kicking was most frequent for parlour experienced heifers on day 2. Grazing system pre-milked heifers shifted more at their first milking (day -21) than did the controls at their first milking (day 1). Shifting within cow was greatest on day -21 compared with day -5 (P<0.05). Pre-milked heifers shifted more on day 1 post-calving than did the control heifers (P<0.05). These results showed that shifting was the most indicative behaviour of restlessness, was transient, and decreased by day 5 prior to calving. Cortisol and alpha1-acid glycoprotein concentrations were not different; however, haptoglobin increased for all treatments up to and including day 3 and haptoglobin concentrations of pre-milked heifers began to decrease by day 5 post-calving. Pre-milked heifers had lower haptoglobin concentrations than the control heifers and tended to have lower concentrations than experienced heifers on day 10 post partum. By day 14 post partum, all haptoglobin concentrations were <200 microg/ml, but the haptoglobin concentration of control heifers was greater than that of pre-milked and experienced heifers. These results showed that prepartum milking and parlour experience shorten some acute phase protein responses, but minimally affect early parlour behaviours.