Space allowance influences individually housed Holstein male calves' age at feed consumption, standing behaviors, and measures of immune resilience before and after step-down weaning.

Many dairy calves in the southwestern regions of the United States are raised in wooden hutches with 1.23 m2 of free space that house 3 calves individually. Producers claim that calves in hutch systems are not ready to wean and be placed in groups at the country-average age of 6 wk. Calves may remain in this individual housing system until as late as 10 wk of age. The objective of this study was to modify space allowance of hutches and evaluate weaning readiness using age at solid feed consumption, standing behaviors, and measures of immunity. Calves were randomly assigned at 4 d of age to conventional (CONV; 1.23 m2 of space; n = 18), moderate (MOD; 1.85 m2 of space; n = 17), or maximized (MAX; 3.71 m2 of space; n = 19) space allowance in hutches. These modifications also changed the number of calves housed per hutch from 3 (CONV) to 2 (MOD) and 1 (MAX). Calves were fed milk replacer via bottle twice daily until weaning and offered ad libitum feed throughout the experiment. Step-down weaning was initiated (Wi) at age 53 or 54 d by withdrawal of the p.m. bottle and was completed (Wc) 11 d later by removal of the a.m. milk replacer. Accelerometer data for standing behaviors were collected relative to Wi (3 consecutive days to represent -4 wk, -3 d, 3 d, and 3 and 5 wk). Blood samples were collected in the a.m. just before Wi (d 0) and at d 3, 11, 14, and 18 after Wi. Calves provided with more space (MOD and MAX) compared with CONV calves consumed feed at an earlier age and had slightly healthier erythrocytes, greater circulating glucose, and fewer circulating eosinophils. The CONV calves had haptoglobin (Hp) responses to the stressors of both Wi and Wc and had more IFN-γ from whole blood stimulated with phytohemagglutinin-P. The MAX calves had the least active neutrophils (phagocytosis and oxidative burst), but MOD calves' leukocytes secreted the most TNF-α from whole blood stimulated with lipopolysaccharide. Just before and after weaning, MAX calves spent more time per day in the standing position than CONV and MOD calves and had an Hp response to Wc, but MOD calves did not have an Hp response to Wi or Wc. Based on these results, MOD calves were the most ready for weaning; therefore, they potentially can be moved to group housing at an earlier age than CONV calves, thus improving animal welfare concerns over space allowance and individual housing.

Space allowance influences individually housed Holstein bull calf innate immune measures and standing behaviors after castration at 3 weeks of age.

Dairy calves in the Southwest regions of the United States are typically raised individually in wooden hutches with 1.23 m2 of space. The objective of the study was to determine if increased space allowance in wooden hutches influences measures of innate immunity and behaviors of Holstein bull calves pre- and postcastration. Calves were randomly assigned at 4 d of age to conventional (CONV; 1.23 m2 of space; n = 18), moderate (MOD; 1.85 m2 space; n = 17), or maximized space allowance (MAX; 3.71 m2 space; n = 19) in hutches. Calves were surgically castrated at 24 d of age. Peripheral whole blood samples were collected at -1, +1, +5, and +12 d of castration. Accelerometer loggers (n = 16 calves per treatment) were used from -3 to +5 d of castration to assess standing behaviors. All calves decreased total standing duration the day of castration versus precastration. Overall, MAX spent the most time in the stand position postcastration versus CONV and MOD. Within treatments, MOD and MAX had increased plasma cortisol 1 d postcastration versus precastration. A treatment × time tendency was observed for cortisol at 12 d postcastration; MAX had the least circulating cortisol. A treatment × time tendency for circulating haptoglobin (Hp) was observed and Hp was greatest among CONV 1 d pre- and 12 d postcastration. Compared with precastration, CONV had increased Hp at 1, 5, and 12 d, whereas MOD had increased Hp at 5 d, and Hp remained similar within MAX. A treatment × time tendency for tumor necrosis factor-α (TNF-α) from lipopolysaccharide-stimulated whole blood was observed; at 1 d postcastration, MOD had the most TNF-α, whereas MAX had the least. Within MAX, calves had increased TNF-α from precastration to 5 d postcastration. A treatment × time interaction was observed for whole blood bactericidal activity against Escherichia coli (WB anti-E). The CONV tended to have the greatest WB anti-E at d -1, but at d 1 and 5 postcastration, CONV had the least WB anti-E. Overall, MAX had less intensity of neutrophil oxidative burst versus CONV and MOD. The lower response of neutrophil oxidative burst and slower Hp secretion after castration is indicative that the wound site likely had less microbial exposure. The findings of this study suggest that calves housed with more space are potentially at less risk of too much inflammation after castration, which may likely be due to the effects of increased space on hide cleanliness and increased standing time.

Liver mitochondrial oxygen consumption and proton leak kinetics in broilers supplemented with dietary copper or zinc following coccidiosis challenge.

Mitochondrial respiration was assessed in sixteen 7-day-old broilers as a subset of a larger study assessing the effects of Cu and Zn supplementation above requirements with a coccidiosis challenge on gain/feed ratio. The birds were selected from four treatments (four birds/treatment): a control diet (Cu 15 mg/kg and Zn 60 mg/kg) + coccidiosis challenge (CC), a Cu diet with 245 mg/kg Cu from tribasic copper chloride (TBCC) + CC, a negative control diet (Cu 15 mg/kg and Zn 60 mg/kg) - CC and a Zn diet with 2000 mg/kg Zn from ZnO. The diets were composed of 49% corn, 40% soybean meal, 6.2% vegetable oil (diet dry matter = 90.62%, crude protein = 21.37%, fat = 7.7%, metabolizable energy = 12.1 MJ/day) and were fed for 14 days. Birds were dissected, and approximately one gram of liver tissue was used for mitochondrial oxygen consumption and proton leak kinetics assays. Respiratory control ratio and mitochondrial proton leak assessed by calculating rates of oxygen consumption at 175mV membrane potential were greater for the negative control group, but there were no differences in average gain/feed among treatments. In summary, broilers that did not undergo coccidiosis challenge had lower proton leak and higher respiratory control ratio. However, the impact of supplementation of Cu and Zn above requirements did not appear to prevent changes in respiratory control ratio and proton leak kinetics with coccidiosis challenge.

Wooden hutch space allowance influences male Holstein calf health, performance, daily lying time, and respiratory immunity.

Dairy calves in the western United States are commonly raised individually in wooden hutches with a space allowance of 1.23m(2)/calf. Recent legislative initiatives in California and across the United States were passed regarding concern over space allowance for farm animals. The objective of this study was to determine if rearing male Holstein calves in wooden hutches modified to increase space allowance would influence measures of performance, lying time per day, health, and respiratory immunocompetence. At 4d of age, 60 calves were randomly assigned to 1 of 3housing treatments: (1) conventional housing (CONV; 1.23m(2)/calf), (2) 1.5 × CONV (MOD; 1.85m(2)/calf), or (3) 3 × CONV (MAX; 3.71m(2)/calf). Intakes of milk and solid feed were recorded daily and body weight was measured at 0, 3, 6, 10, and 12 wk of age. For the first 3 wk of the trial, calves were scored daily for fecal consistency, hydration, and hide cleanliness. In addition, calves were scored for respiratory health (i.e., nasal and eye discharge, ear position) until 7 wk of age. The total lying duration per day was recorded using data loggers at 3, 6, and 10 wk of age. Eight clinically healthy calves from each treatment were sensitized with subcutaneous ovalbumin (OVA) and then challenged with aerosolized OVA to assess calf respiratory immunity at 11 wk of age. Bronchoalveolar lavage fluid (BALF) was collected 4d after the OVA challenge and analyzed for leukocyte differentials and OVA-specific IgG, IgG1, IgA, and IgE. Calf average daily gain and body weight were positively associated with space allowance at approximately 3 wk before weaning and throughout postweaning, respectively. A greater space allowance decreased lying time after 46d. Space allowance did not influence fecal consistency, but there was a tendency for MAX calves to take 1d longer to recover from loose feces than MOD calves. The MAX calves had the fewest (%) observations with feces on their body compared with CONV or MOD. At 3 wk of age, peripheral eosinophil concentrations decreased with increased space allowance. However, observations (%) of eye discharge increased with greater space allowance. Among calves challenged with OVA, MOD calves had the least BALF OVA-IgE, and the percent of BALF eosinophils decreased with increased space allowance. Increased space allowance for calves raised in wooden hutches may improve some measures of calf performance, health, and respiratory immunocompetence.

Effects of conjugated linoleic acid and lutein on the growth performance and immune response of broiler chickens.

The effects of lutein and conjugated linoleic acid (CLA) on growth performance and immune response of broiler chickens were evaluated in the presence and absence of Salmonella lipopolysaccharide (LPS) immune challenge. Cobb chicks (360; 1 to 22 d of age) were used in a 3 × 2 factorial arrangement of CLA (0, 1, and 2%) and lutein (0 and 50 mg/kg) dietary levels. At d 8 and 15, birds were injected with BSA to assess IgY production. At d 20, birds were injected with LPS. Samples of liver, spleen, and duodenum were collected at 3 and 16 h post-LPS challenge for RT-qPCR analysis of RXRα, RXRγ, PPARα, PPARγ, TLR-4, IL-1ß, IL-2, IL-10, and IL-12 gene expression. CLA decreased BW, BW gain (BWG), and G:F from d 1 to 20, but these effects were reversed when lutein was included in the 1% CLA diet (P < 0.001). The production of IgY anti-BSA increased following a 2% CLA supplementation (P < 0.01). LPS increased the liver:BW ratio at 3 h post-injection (P < 0.001) and decreased BWG at 3, 16, and 40 h (P < 0.001). Lutein decreased plasmatic nitric oxide levels (P < 0.01). LPS downregulated PPARα mRNA in the duodenum (P = 0.02) and liver (P = 0.04), and PPARγ (P = 0.01) and RXRα (P = 0.08) in the spleen; these effects were not reversed by CLA or lutein as initially hypothesized. Although LPS upregulated IL-1ß (P = 0.02) and IL-12 (P = 0.07) expression, lutein downregulated these pro-inflammatory cytokines in the liver (P = 0.03 and P = 0.07, respectively). Lutein decreased splenic (P = 0.09) but increased hepatic (P = 0.06) TLR-4 mRNA. A dietary CLA supplementation of 2% increased hepatic RXRα (P = 0.10). In conclusion, CLA decreased broiler chicken growth performance, but lutein could prevent this negative effect (depending on CLA dose). Lutein had an anti-inflammatory effect, and a 2% CLA supplementation improved the humoral immune response.

Effect of weaning and in-feed high doses of zinc oxide on zinc levels in different body compartments of piglets.

High doses of Zn are widely used for prevention and treatment of diarrhoea in weaning piglets; however, the mechanism of action of Zn against diarrhoea is still not well understood. The objective of this study was to evaluate whether weaning induces Zn deficiency in piglets. Eight litters of primiparous sows were selected for the experiment, and 3 piglets presenting similar weights were selected within each litter. Two of the three selected piglets from each litter were weaned at 21d of age and fed two different diets: a commercial control diet (WCt) and the same diet plus 2000 ppm of Zn as ZnO (WZn). The third selected pig from each litter was kept unweaned (Uw) with the sow and the rest of the litter. All 24 selected animals were killed at 28 d of age, and blood, gastrointestinal content, liver, pancreas and spleen were sampled for Zn, Fe and Cu analysis (mg/kg or L of sample). Data were analysed using anova including treatment as a fixed factor. Weaned pigs fed WCt diet presented a lower Zn concentration in plasma than Uw animals (0.76 ± 0.091 vs. 1.10 ± 0.099 mg/L, p = 0.05). Zinc levels in liver, pancreas and spleen were not affected by weaning. Total concentration of Zn was higher in gastrointestinal contents of weaned animals fed WCt diet than in Uw pigs (p ≤ 0.001 for stomach, jejunum, ileum, caecum and colon). Supplementation with high doses of ZnO increased levels of Zn in gastrointestinal content (p < 0.001), liver (p < 0.001) and pancreas (p < 0.001) compared to WCt diet. It also increased plasma Zn to non-deficient levels (1.32 ± 0.086), but the increase was not as marked as in other locations and final concentration was not different than that in Uw animals (p = 0.231). Weaning creates a Zn deficiency situation in weaned pigs as observed by plasma Zn concentrations. ZnO supplementation counteracts Zn deficiency.

Domestic cats convert [2H8]-ß-carotene to [2H4]-retinol following a single oral dose.

Many animals convert ß-carotene to retinol to meet their vitamin A (VA) requirement. However, this pathway is inefficient in many carnivores. This study quantified the plasma response to a single oral dose of [(2) H(8)]-ß-carotene in adult domestic cats, including measurement of [(2) H(4)]-retinol derived from the dose. Cats were fed with either a control diet containing adequate VA (n = 5) or a VA-devoid diet (n = 5) for 28 days. An oral dose of either 5 mg/kg body weight (BW) (n = 4) or 10 mg/kg BW (n = 6) of [(2) H(8) ]-ß-carotene was administered on day 28. Plasma samples were collected prior to dosing and at 6, 12, 24, 32, 48, 72, 120, 168 and 216 h post-dose. Plasma retinoids and ß-carotene were measured using HPLC and [(2) H(4)]-retinol by GC-ECNCI-MS (gas chromatography/electron capture negative chemical ionization/mass spectrometry). ß-carotene was undetectable in plasma prior to dosing. Post-dose, mean peak plasma ß-carotene was 0.37 ± 0.06 nmol/ml at 9.0 ± 1.8 h following the dose, while [(2) H(4) ]-retinol peaked at 3.71 ± 0.69 pmol/ml at 55.2 ± 16.3 h. The ratio per cent of total area under the curve for [(2) H(4)]-retinol compared with the ß-carotene response was 4.6 ± 2.6%. There was little effect of diet or dose on the ß-carotene or [(2) H(4)]-retinol responses. The appearance of [(2) H(4)]-retinol in plasma indicates that cats are capable of converting ß-carotene to active VA. Conversion efficiency was not calculated in this study, but it is likely inadequate to meet cats' VA requirement without the inclusion of preformed VA in the diet.

Retinyl palmitate does not have an adjuvant effect on the antibody response of chicks to keyhole limpet hemocyanin regardless of vitamin A status.

To determine whether supplementing retinyl palmitate (RP) to vitamin A-deficient or -adequate chicks during vaccination increased the antibody response, an experiment was conducted that used a 2-generational model. White Leghorn chicks were hatched from eggs that contained 1.06 ± 0.13, 1.41 ± 0.14, or 1.90 ± 0.13 µg of retinol/g of egg yolk. Upon hatch, chicks were allotted to a diet that contained 70, 100, or 130% of the vitamin A requirement (1,500 IU/kg). At 14 d of age, chicks were challenged with keyhole limpet hemocyanin (KLH), Freund's incomplete adjuvant (FIA), KLH and FIA, KLH and RP administered in the same injection, or KLH and RP administered at different injection sites. The RP dosage was 7,000 IU/kg of BW, and dosage of the KLH was 1 mg/kg of BW. Chicks were bled and rechallenged at 19 d of age, and a final blood draw was taken at 29 d of age. The IgM and IgG antibody responses to KLH were determined by ELISA, and agglutination was used to determine natural antibody titers. When compared with chicks fed diets that contained 70 and 100% of the vitamin A requirement, the chicks fed 130% of the vitamin A requirement had a lower primary IgM and IgG antibody response (P < 0.02) and the secondary IgG response was significantly lower (P = 0.004). However, administering RP together or separate from KLH did not significantly change the secondary IgM or IgG responses compared with chicks challenged with KLH and FIA (P < 0.01). Moreover, natural antibody titers were not different among dietary treatments (P = 0.91). In conclusion, administration of RP with KLH did not improve antibody response in chicks.

Lutein exposure, in ovo or in the diet, reduces parameters of inflammation in the liver and spleen laying-type chicks (Gallus gallus domesticus).

These trials examined whether the demonstrated effects of embryonic and dietary carotenoid exposure on the inflammatory immune response in fast growing chickens also occur in slow growing chickens. The systemic and local inflammatory responses of chicks were examined in two experiments with two in ovo lutein levels (C+, carotenoid replete; or C-, carotenoid-deplete), two dietary lutein levels (0 or 40 mg lutein/kg diet), and two inflammatory challenges [no exposure or lipopolysaccharide (LPS)-vaccinated]. At 24 h after LPS vaccination, spleen weight was not affected by diet or in ovo lutein, but liver weight increased from C+ eggs (p < 0.01), and in LPS-vaccinated chicks fed 0 mg lutein (p < 0.05), but not in chicks fed 40 mg lutein. Plasma carotenoids and liver carotenoids were reduced post-LPS (p < 0.05). Splenic IL-6 mRNA abundance was the greatest post-LPS in C- chicks fed 40 mg lutein vs. C+ chicks fed 40 mg lutein (p < 0.05). Hepatic IL-6, iNOS and TGFß and splenic iNOS and TGFß were not affected by in ovo or dietary lutein. The systemic and local inflammatory results are similar to those observed in fast growing chickens, and support that lutein-depleted birds have greater inflammatory responses.

The effect of light intensity on the behavior, eye and leg health, and immune function of broiler chickens.

Broilers are typically raised commercially in dim lighting. It has been suggested that providing brighter light intensity could improve health and provide opportunities for more normal behavioral rhythms. We examined the effects of 3 photophase light intensities (5, 50, and 200 lx) on activity patterns, immune function, and eye and leg condition of broilers (n = 753; 6 replicate pens/treatment). Broilers were reared with one of these intensities from 1 to 6 wk of age; photoperiod consisted of 16L:8D with 1 lx intensity during the scotophase. Broilers reared with 5 lx were less active (P = 0.023) during the day than 50 or 200 lx and showed less (P < 0.0001) change in activity between day and night than 50 or 200 lx. There was no difference between treatments for final BW (2.30 +/- 0.02 kg) or for most immune parameters (IgG primary and secondary responses to keyhole limpet hemocyanin, B and T lymphocyte proliferation, plasma lysozyme, haptoglobin, NO, whole blood killing of Escherichia coli and Staphylococcus aureus), but there was a trend (P = 0.072) for a greater IgM response in 50 lx (6.21 titer) than 5 lx (5.78 titer), with 200 lx (5.92 titer) intermediate. There was no effect of light intensity on back-to-front (1.13 +/- 0.01 cm) or side-to-side (1.48 +/- 0.01 cm) diameter of the eyes or on corneal radii (0.82 +/- 0.01 cm), but 5 lx (2.33 +/- 0.07 g) had heavier eyes (P = 0.002) than 50 lx (2.09 +/- 0.04 g) or 200 lx (2.11 +/- 0.04 g). There were no differences in gait score, although 200 lx broilers had more hock and footpad bruising (P = 0.038) but fewer erosions (P = 0.006) than 5 or 50 lx. Increased daylight intensity had little effect on broiler health but resulted in more pronounced behavioral rhythms.

Dietary ß-hydroxy-ß-methylbutyrate supplementation influences performance differently after immunization in broiler chickens.

Dietary addition of the leucine metabolite ß-hydroxy-ß-methylbutyrate (HMB) promotes growth in various species. In addition, HMB is described to enhance immune responses which might be associated with metabolic costs. We elaborated further on the role of HMB in growth, metabolism and immunity of meat-type chickens using the following parameters: zootechnical performance, blood chemistry and a specific immune responses after immunization with a human serum albumin (HSA)/Freund's (in) complete adjuvant combination. The chickens received commercial feeds either unsupplemented or supplemented with 300 mg HMB/kg feed. ß-hydroxy-ß-methylbutyrate-supplemented chickens were significantly heavier at 2 weeks of age but this difference was attenuated at later ages. Compared with their unsupplemented controls, cumulative feed conversion was significantly lower in HMB-supplemented chickens. There were no differences in blood chemistry between both dietary treatments. After immunization, HMB significantly attenuated the acute phase protein response at day 1 of post-immunization compared with that of their unsupplemented counterparts. After day 7 post-immunization, body weight gain of the immuno-challenged HMB-supplemented chickens was significantly depressed, but their specific anti-HSA IgG response was significantly enhanced compared with that of their immuno-challenged unsupplemented counterparts. The underlying mechanisms and signalling pathways for these phenomena need to be elucidated. Nevertheless, we are able to conclude that HMB is beneficial for performance under normal circumstances. On the other hand, HMB stimulates the immune response after an immunological challenge, though at the cost of reduced growth.

The effects of vaccination with keyhole limpet hemocyanin or oral administration of Salmonella enterica serovar Enteritidis on the growth performance of immunoglobulin knockout chickens.

The nutritional cost of activating B cell-mediated immunity is thought to be low in chickens. However, this assumption is incompletely characterized. Immunoglobulin knockout (Ig-KO) chickens lacking B cells and immunoglobulin may potentially be a robust model to investigate the nutritional cost of immunity. Specifically, comparing the growth of Ig-KO chickens to immunocompetent birds (WT) following activation of the immune system should indicate costs associated with B cell-mediated immunity. A total of 3 experiments were conducted to determine if (1) an antibody response to keyhole limpet hemocyanin (KLH), (2) an intra-abdominal injection with Salmonella enterica serovar Enteritidis (SE), or (3) an oral challenge of SE would affect body weight gain (BWG) in homozygous Ig-KO (KO) chickens and their immunocompetent counterparts. In Experiment 1, a significant genotype*vaccination status interaction was observed (P = 0.03) during the period from 0 to 6 D after initial vaccination in which KLH injection reduced BWG in WT birds, but not in KO birds. A genotype*vaccination status interaction was present (P = 0.002) from 0 to 7 D after the first booster in which KLH injection due to reduced BWG in KO birds, but not WT birds. In Experiment 2, both KO and immunocompetent birds lost body weight during the period from 0 to 2 D after the SE injection, with no significant differences due to genotype (P = 0.92). Experiment 3 demonstrated that KO birds gained less weight than immunocompetent birds, with a tendency for less weight gain after an initial challenge (P = 0.07) and significantly so after a secondary challenge (P = 0.03). The results from these experiments collectively demonstrate that B cell-mediated immunity can affect growth performance in chickens. Furthermore, these effects can either preserve or impair growth performance, likely via mechanisms related to the immune response rather than the discrete nutritional cost of B cell-mediated immunity.

Constitutive immune defences correlate with life-history variables in tropical birds.

1. It has been suggested that immune defences are shaped by life history and ecology, but few general patterns have been described across species. We hypothesized that 'fast' life-history traits (e.g. short development times, large clutch sizes) would be associated with developmentally inexpensive immune defences, minimizing the resource demands of young animals' immune systems during periods of rapid growth. Conversely, 'slow' life histories should be associated with well developed antibody-mediated defences, which are developmentally costly. 2. We therefore predicted that 'fast-living' species would exhibit higher levels of complement proteins, a component of non-specific innate defence, but lower levels of constitutive ('natural') antibodies. Additionally, we predicted that constitutive immune defences in general would be higher in species with ecological characteristics that might increase exposure to pathogens, such as open nests, omnivorous diets, gregariousness, and closed forested habitat. 3. Across 70 Neotropical bird species, we found a strongly positive relationship between incubation period and natural antibody levels in adult birds, suggesting that longer developmental times might allow the production of a more diverse and/or more reactive adaptive immune system. Complement activity was positively, although weakly, correlated with clutch size, providing some support for the hypothesis that faster-living species rely more on innate defences, such as complement. Unexpectedly, solitary species had higher natural antibody titres than species that frequently join flocks. 4. Our results suggest that, despite probably widespread differences in the intensity and diversity of pathogen exposure, species-level variation in constitutive immune defences is understandable within the context of life-history theory.

Effects of immunomodulatory nutrients on growth performance and immune-related gene expression in layer chicks challenged with lipopolysaccharide.

Immunomodulatory nutrients alter the immune response to pathogens. This study was conducted to determine the effects of immunomodulatory nutrients on the immune response to a lipopolysaccharide (LPS) challenge of layer chicks fed supplemental corn oil (control; 3%), fish oil (3%), conjugated linoleic acid (CLA; 1%), lutein (0.05 g/kg), or vitamin E (90 I.U./ kg). Four-week-old layer chicks were allotted to 10 treatment groups arranged as a 2 × 5 factorial with 2 sexes and 5 dietary treatments (TRT; 5 replicate pens of 2 male and 2 female chicks per TRT). After a 2-week diet adaptation, all birds were injected intraperitoneal with 1.5 mg/kg BW LPS. Twelve hours post challenge, samples were collected. Fish oil fed birds had greater (P = 0.03) spleen weight (% final BW) than the CLA fed birds. In the liver, the fish oil TRT had higher (P = 0.040) IL-12 expression than the corn oil TRT, but the corn oil TRT had greater (P = 0.001) IL-4 expression than the CLA, lutein, and vitamin E TRT. There was a main effect of sex of the birds on growth parameters at 12 h post LPS challenge in which male birds had greater beginning BW (P < 0.001), final BW (P < 0.001), and greater 12-hour BW loss (P = 0.020) than the female birds, but not relative weight loss. There were also main effects of sex on immune-related gene expression with the females having greater gene expression than the males in the duodenal mucosal scrapings [IL-1ß, IL-12, and TLR-4 gene expression (P = 0.026, 0.011, and 0.002, respectively)]; liver [IL-10, IL-4, and iNOS gene expression (P = 0.017, 0.032, and 0.006, respectively)]; and spleen [IL-1ß, IL-10, IL-4, and iNOS gene expression (P = 0.001, 0.001, 0.001, and 0.005, respectively)]. Therefore, each immunomodulatory nutrient added to the diets of layer chickens resulted in different immune responses to an LPS challenge.

An acute inflammatory response alters bone homeostasis, body composition, and the humoral immune response of broiler chickens.

To quantify the effects of an acute phase response in broilers, chicks were injected with 1 mg/kg Escherichia coli lipopolysaccharide (LPS) at 15 and 23 d. Lipopolysaccharide injection increased feed/gain (P = 0.03), increased liver weight (P = 0.09), and decreased tibia calcium (P = 0.05) and breaking strength (P < 0.04) by d 28. In a second experiment, 3 d postinjection of chicks at d 31, LPS decreased BW (P < 0.01), breast weight (P = 0.08), and tibia breaking strength (P = 0.05), and increased liver weight (P < 0.01), mortality (P = 0.05), and titers to bronchitis and Mycoplasma gallisepticum that were induced by vaccination at hatch or by field exposure, respectively (P = 0.04). For experiment 3, chicks were challenged with LPS at 23d and 27d. Lipopolysaccharide-injected chicks had decreased BW (P = 0.06), feed consumption (P = 0.05), tibia weight (P< 0.01), and breaking strength (P < 0.01), and increased feed/gain (P < 0.01), liver weight (P < 0.01), and plasma ionized calcium level (P = 0.08). For experiment 4, chicks were injected with 0, 0.33, 0.66, 1.00, or 4.25 mg of LPS/kg of BW. There was an inverse relationship between LPS level and BW or bone breaking strength. Experiment 5 compared 4 broiler strains. Strain x LPS interactions were found for bone breaking strength (P = 0.01). Mortality before LPS challenge was inversely correlated to liver weight (r2 = 0.95, P = 0.02) and bone breaking strength (r2 = 0.99, P = 0.01) only after an LPS challenge.

Divergence of the inflammatory response in two types of chickens.

We compared inflammatory responses to lipopolysaccharide (LPS) injection in laying type (Brown Nick) to broiler type (Avian x Avian) chicks. Rectal temperature was measured at 0, 1, 2, 4, 6, 12, and 24h after LPS injection (0, 0.1, 0.3, 0.6, 1, 2.5, or 5mg/kg bw). In layers, rectal temperature increased from 41.31+/-0.19 degrees C to a maximum 42.27+/-0.41 degrees C at 4h after 1mg/kg LPS. Relative to layers, the febrile response in broilers was considerably lower, delayed in onset, and required higher levels of LPS (5mg/kg). Proliferation of spleen cells from un-injected chicks in response to LPS, PHA, and Con A was evaluated in vitro. IFNgamma, TGFbeta(2), MGF and IL-1beta relative to beta-actin mRNA expression were analyzed in spleen cells stimulated with LPS. Splenocytes from layers had a higher proliferative response to LPS (P=0.045), but lower proliferative response to PHA (P=0.004) and Con A (P=0.004) than broilers. Expression of mRNA for MGF, IL-1beta and IFNgamma was lower in broilers than in layers (P<0.001). Reduced production of the pro-inflammatory cytokines in broilers could have resulted from the observed increased production of the immunosuppressive cytokine TGFbeta(2.) These differences in cytokine expression may explain the blunted febrile response in broilers compared to layers. Because the acute phase response of inflammation causes decreased food intake, the blunted inflammatory response of broilers may permit faster growth.

Influence of cell sources, stimulating agents, and incubation conditions on release of interleukin-1 from chicken macrophages.

Experiments were conducted to determine the cell source, stimulating agents, and incubation conditions that maximize interleukin-1 (IL-1) release by chicken macrophages/monocytes. Thymocyte co-mitogen proliferation was used to assay IL-1 activity of conditioned or partially purified supernatants. Monolayers of a transformed chicken macrophage cell line, HD11, released greater amounts of IL-1 than adherent cells isolated from peripheral blood, peritoneal cavity, or spleen. E. coli endotoxin and heat-killed S. aureus induced greater release of IL-1 by HD11 and splenic macrophages than latex or a super induction protocol with mezerien. Blocking macrophage eicosanoid synthesis with indomethacin did not influence IL-1 release from HD11 macrophages. Removing low molecular weight compounds from conditioned supernatants by dialysis did not influence IL-1 activity. IL-1 release was increased by incubating macrophages at 42 C compared to 39 C. Thymocyte co-mitogenic activity of IL-1 was increased by incubating thymocytes at 42 C compared to 39 C. Species cross reactivity between chicken and mammalian IL-1 was also investigated. Chicken IL-1 had slight co-stimulation activity on murine thymocytes, but murine and human IL-1 were without activity on chicken thymocytes.

Soluble type-I interleukin-1 receptor blocks chicken IL-1 activity.

The ligand-binding domain of the chicken type-I interleukin-1 (IL-1) receptor (soluble IL-1R(I); sIL-1R(I)) was cloned into a Pichia pastoris expression system and the resulting sIL-1R(I) binding protein was used to produce antisera in rabbits (anti-IL-1R(I)). Two experiments were conducted to determine the capacity of sIL-1R(I) or anti-IL-1R(I) to block the IL-1 bioactivity (thymocyte co-stimulation) in conditioned media (CM) from HD11 chicken macrophages stimulated with lipopolysaccharide. In the first experiment, pre-incubation of CM with unpurified sIL-1R(I) significantly decreased its thymocyte co-stimulation activity by 57%. Further purification of sIL-1R(I) from other proteins secreted or shed from P. pastoris expression system by size exclusion filtration or ammonium sulfate (60%) precipitation did not influence its capacity to neutralize IL-1 bioactivity. These partially purified sIL-1R(I) preparations significantly decreased thymocyte co-stimulation activity in CM by 70.7 and 77.3%, respectively. In the second experiment, pre-incubation of thymocytes with antisera against the sIL-1R(I) decreased IL-1 activity in CM by 70% relative to control thymocyte cultures that received no antibody and by 59% relative to thymocyte cultures incubated with pre-immune sera. Presumably anti-sIL-1R(I) diminished the IL-1 bioactivity in CM by blocking IL-1 binding to its type-I receptor on thymocytes. Thus, 30% of the IL-1-like activity released by LPS-stimulated HD11 macrophages is probably due to at least one other cytokine. Our data are consistent with the type-I receptor being the primary IL-1 receptor on chicken thymocytes that is capable of providing a signal for proliferation.

Cytokine-induced neutrophil chemoattractant production by primary rat alveolar type II cells.

This study was designed to determine the production of the chemokine cytokine-induced neutrophil chemoattractant (CINC) by primary rat alveolar type II (ATII) cells upon stimulation with exogenous and endogenous proinflammatory factors. Cultures of primary rat ATII cells were exposed to lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF alpha) over a 16 hour period and the production of CINC both apically and basolaterally was measured by ELISA. Compared to unstimulated (UNS) cultures, LPS, IL-1 beta and TNF alpha were found to significantly increase the level of CINC detected in culture by two, four and sixteen hours post stimulation, respectively. ATII cells also demonstrated a polar secretion of CINC. The accumulation of CINC basolaterally was significantly more than apically; 133%, 45%, 117% and 123% for UNS, IL-1 beta, LPS and TNF alpha respectively. We demonstrated that primary rat ATII cells may participate in the chemokine network during inflammation by the production of CINC upon stimulation with endogenous and exogenous factors.

The kinetics of hemopexin and alpha1-acid glycoprotein levels induced by injection of inflammatory agents in chickens.

The acute phase response to inflammation induces changes in the secretion of hepatic proteins. To examine the time course of an acute phase protein response in broiler chickens, the plasma levels of hemopexin (HX) and alpha1-acid glycoprotein (AGP) and liver HX mRNA were measured at various time points from 3 hr to 336 hr after an intraabdominal injection of either lipopolysaccharide (LPS), complete Freund's adjuvant, incomplete Freund's adjuvant, phytohemagglutin, or mineral oil. Uninjected chicks served as controls. The accumulation of liver HX mRNA began within 3 hr of stimulation and peaked at 12 hr. Relative to control levels, plasma HX and AGP levels increased by 6-12 hr postchallenge and peaked at 24 hr. Complete Freund's adjuvant and LPS treatments induced the greatest increase in plasma HX (threefold; P < 0.05). Plasma levels of HX and AGP returned to control levels at 336 and 168 hr postinjection, respectively. A second experiment demonstrated that turpentine induced a similar AGP response as LPS and that albumin is a negative acute phase protein. The results suggest that plasma levels of HX or AGP could be used as an indicator of the systemic component of a local inflammatory response in chickens.