Nutritional Supplement of Hatchery Eggshell Membrane Improves Poultry Performance and Provides Resistance against Endotoxin Stress.

Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of chickens to endotoxin.

Ascorbic Acid Differentially Affects Stress Response and Resistance to Colibacillosis in Turkeys from Genetic Lines Differing in Growth Rate.

Genetic selection for fast growth can affect the ability of male turkeys to cope with stressors, resulting in decreased immunity to opportunistic bacterial infection. The purpose of the current study was to compare the effects of ascorbic acid (AA) on the stress response and resistance to Escherichia coli challenge of birds selected for increased 16-wk body weight (BW; F-line) with their random-bred parent line (RBC2). Male turkeys were raised in duplicate floor pens in a two line×two AA treatment×two stress challenge (SC) design. At 5 wk of age, AA (1200 ppm) was provided in drinking water for a 24-hr period, during which all birds were weighed. After AA treatment, the SC group was subjected to a transport stress protocol. Six hours after the start of transport, SC birds were also inoculated in the thoracic air sac with 1×10(4) colony-forming units of E. coli. The following morning four birds from each pen were bled, and all birds were weighed and necropsied 2 days later. BW and gain after SC were decreased in the F-line but not the RBC2 line, and there were no AA effects on BW. The weight of the bursa of Fabricius relative to BW was higher in the RBC2 line than in the F-line, was decreased by SC, and was not affected by AA. The heterophil∶lymphocyte ratio was higher in the SC F-line as compared to the SC RBC2 and was decreased by AA only in the SC F-line. Corticosterone (C) levels were increased by SC only in the F-line, and AA decreased C levels only in the RBC2 line. Airsacculitis scores were increased in the F-line SC birds. The challenge strain of E. coli was only detected in the air sac and liver of the AA-treated F-line SC birds and in the liver of the no-AA F-line birds. These results suggest that SC at 5 wk of age had a more deleterious effect on the fast-growing F-line than on its parent line and that AA may have increased susceptibility to colibacillosis in the SC F-line birds.

Nutritional effects of egg shell membrane supplements on chicken performance and immunity.

Eggshell membranes (ESM) contain a variety of proteins and peptides which help in the development of embryo and provide protection to it. Many of the peptides and proteins associated with ESM have antimicrobial, immune-modulatory, and adjuvant properties. We hypothesized that the membrane byproducts from egg, provided as posthatch nutritional supplements to chickens, may improve their performance and immunity. To explore its effect, we fed 3 groups of broiler chicks with feed containing 0, 0.2, and 0.4% ESM from d 1 posthatch through 14 d and regular feed thereafter. The birds were individually weighed at the onset of the study and at weekly intervals until the termination at third wk when they were bled and euthanized. The relative weights of liver, spleen, bursa, and heart, hematology profiles, and clinical chemistry variables including serum IgM, IgG, and corticosterone concentrations were measured. The chickens in the ESM treated groups showed a statistically significant increase in BW with no impact on relative organ weights. Compared with controls, the WBC and lymphocyte percentage increased in chickens fed 0.4% ESM whereas the monocyte percentage decreased at both levels of ESM. Except for the serum protein which increased in ESM fed birds no other metabolic clinical chemistry variables showed any significant change. Both IgM and IgG(Y) levels were elevated and corticosterone levels reduced in chickens fed ESM supplemented diets. Our results suggest that ESM supplements during the early phases of growth may improve immunity and stress variables, and enhance their growth performance without any detrimental effect on other physiological parameters.

Efficacy of a novel prebiotic and a commercial probiotic in reducing mortality and production losses due to cold stress and Escherichia coli challenge of broiler chicks 1.

Prebiotics consisting of resistant starch may alter intestinal ecology, thus modulating inflammation and increasing intestinal health through increased cecal production of short-chain fatty acids (SCFA). Probiotics may directly alter the intestinal microbiome, resulting in the same effects. We hypothesize that adding prebiotics and probiotics to feed may protect the gut of young chicks under stress. Studies 1, 2, and 3 evaluated treatments in a cold stress (CS) and Escherichia coli (EC) oral challenge to 430 day-old broiler chicks for 3 wk. In study 1, prebiotics were administered as 15% of the diet during the first week only and consisted of the following: Hi-Maize resistant starch (HM), potato starch (PS), or raw potato (RP). In studies 2 and 3, the PS treatment was identical to study 1, and an additional probiotic treatment (PRO) was administered in feed and water. In study 1, PS protected BW during the first week and decreased the mortality of CS/EC-challenged birds during the first week and wk 3, while RP decreased the mortality of warm-brooded birds challenged with EC during the first week. In study 2, PS decreased and PRO increased the main effect mean (MEM) of the first week BW. PS and PRO numerically decreased the feed conversion ratio (FCR) by 23 and 29 points, respectively, in CS/EC-challenged birds with no effects on mortality. In study 3, PS decreased and PRO increased the first week and wk 3 MEM BW. PS numerically increased FCR by 16 points, while PRO decreased FCR by 2 points. Both PS and PRO tended to increase overall mortality, and PRO significantly increased mortality in the CS/EC challenge. These results suggest that the effects of PS may be too variable in this challenge model for further study; however, the PRO treatment improved production values and may have potential as an alternative to antibiotics during the first weeks after hatch.

Isolation and characterization of chicken bile matrix metalloproteinase.

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.

The effects of yeast feed supplementation on turkey performance and pathogen colonization in a transport stress/Escherichia coli challenge.

A commercial yeast culture feed supplement (YC; Celmanax SCP, Vi-COR, Mason City, IA) was provided to turkeys throughout a 16-wk grow-out to determine if it would prevent the effects of stress on production and pathogen colonization. The YC was provided either continuously at 100 g/t (YC-CS) or intermittently during times of stress at 200 g/t (YC-IS). Birds were stressed with an environmental challenge of Escherichia coli and by transporting them in a vehicle for 3 h after which they were penned in new social groups, without feed or water, for an additional 9 h. Turkeys were transported and challenged at 6, 12, and 16 wk of age to model the movement of birds within a 3-stage housing system. The YC-IS was provided only for the first week after hatch and for a 1-wk period encompassing each challenge. At wk 7 and 9, a decrease in BW of challenged birds was prevented by YC-IS but not YC-CS. There were no significant differences in BW due to either challenge or YC during wk 11 and 13. At wk 16, the challenge decreased BW, but there was no improvement in either of the YC treatments. Overall feed conversion ratio (FCR) was increased by transport/E. coli (P < 0.0001). The YC-CS improved FCR of challenged birds by 21 points, whereas YC-IS improved FCR by 36 points and this effect was significant (P = 0.013). The YC-CS tended to decrease both Salmonella and Campylobacter isolation from the ceca of stressed birds (P > 0.05). The YC-IS also tended to decrease Salmonella isolation (P > 0.05) with no effect on Campylobacter isolation. These data suggest that the practice of transporting turkeys decreases performance and that YC-IS may be more effective than YC-CS for alleviating the effects of this stressor on feed efficiency.

Effects of dietary yeast extract on turkey stress response and heterophil oxidative burst activity.

1. Effective nutritional approaches to counteract the negative effects of stress may provide food animal producers with useful alternatives to antibiotics. In this study, turkeys were fed on a standard diet, or the same diet supplemented with yeast extract (YE), to determine if YE would improve disease resistance in a stress model. 2. At 16 weeks of age, half of the birds were exposed to a bacterial challenge using a coarse spray of the pen environment. A subset of control and challenged birds was also treated with dexamethasone (Dex) prior to challenge (Dex/challenge). At 18 weeks, another subset was subjected to a 12?h transport stress protocol (Challenge/transport). All birds were bled and necropsied the morning after transport. The numbers and proportions of blood cells and the heterophil oxidative burst activity (OBA) were determined. Serum corticosterone (Cort) levels of male birds were measured using a commercial ELISA kit. Body weight and gain were increased by YE during week 1. 3. YE decreased mortality and bacterial isolation following Dex/challenge only in females. Cort levels in male turkeys were decreased by YE and Dex treatment. OBA was higher in males and in birds given YE and was reduced by challenge and transport. 4. These results suggest there may be gender differences in the turkey stress response and that dietary YE has potential for modulating the impact of stress on innate immunity of turkeys.

Immune interference of bacteriophage efficacy when treating colibacillosis in poultry.

A study was conducted to determine if prior exposure with bacteriophage would limit the ability of the same bacteriophage to treat colibacillosis. There were 5 treatments with 3 replicate pens of 20 birds per pen. The treatments consisted of 1) control, 2) birds treated with bacteriophage at 10 and 17 d of age, 3) birds challenged with Escherichia coli at 17 d of age, 4) birds challenged with E. coli and treated with bacteriophage at 17 d of age, and 5) birds treated with bacteriophage at 10 d of age and challenged with E. coli and treated with bacteriophage at 17 d of age. Colibacillosis was induced by injecting 0.1 mL of E. coli into the thoracic airsac containing 1 x 10(6) cfu. The bacteriophage was administered by i.m. injection of 0.1 mL into the thigh, providing a dose of 6.8 x 10(8) pfu. The study was concluded 14 d after E. coli challenge. Mortality in the birds challenged with E. coli and not treated with bacteriophage was 55% (treatment 3), and bacteriophage therapy significantly (P < or = 0.05) reduced mortality to 8% (treatment 4), which was not significantly (P < or = 0.05) different from the 2 non-E. coli-challenged controls (3%, treatment 1, and 2%, treatment 2). However, mortality in the birds administered bacteriophage before challenge with E. coli and treated with bacteriophage (treatment 5) was 33%, which was not significantly different (P < or = 0.05) from the birds that were challenged with E. coli and untreated (55%, treatment 3). A kinetic in vitro assay of bacteriophage activity found that serum from birds pretreated with bacteriophage (treatment 5) inhibited bacteriophage activity. The IgG levels to the bacteriophage in serum from birds pretreated with bacteriophage (treatment 2) were significantly higher at all dilutions compared with control serum (treatment 1). These data demonstrate that prior exposure to bacteriophage will limit bacteriophage therapeutic efficacy and suggests that the reduced efficacy is due to an immune response to bacteriophage.

Bacterial clearance, heterophil function, and hematological parameters of transport-stressed turkey poults supplemented with dietary yeast extract.

Yeast extracts (YE) contain biological response modifiers that may be useful as alternatives to antibiotics for controlling pathogens in poultry production and mitigating the deleterious effects of production stressors. The objective of the present study was to determine the ability of a commercial dietary YE (Alphamune) to modulate the immune response in male turkey poults challenged with Escherichia coli and subjected to transport stress. Alphamune was added to turkey poult diets at 0, 500, or 1,000 g/ton. Poults were challenged by air sac injection with 60 cfu of E. coli at 1 wk of age. At 3 wk of age, these challenged birds were subjected to transport stress and birds were bled and necropsied the following morning. Blood cell numbers and percentages, hematological parameters, and clinical chemistry values were determined. Oxidative burst activity of isolated heterophils was measured using stimulation with phorbol myristate acetate and a 2',7'-dichlorofluorescein diacetate assay. Data were analyzed using GLM and least squares means procedures of the SAS program. The numbers and percentages of heterophils in peripheral blood were increased and their oxidative burst activity was stimulated by YE. The stress challenge dramatically increased oxidative burst and this increase was significantly modulated by YE treatment. Serum levels of calcium, phosphorus, and triglycerides were decreased and uric acid levels, erythrocyte numbers, hemoglobin, and hematocrit were increased by YE supplementation. Bacteria were isolated from the air sac and liver of a lower percentage of birds provided with YE. These results suggest that dietary YE has potential as a nonantibiotic alternative for decreasing bacterial pathogens in turkey production.

Bacteriophages for prophylaxis and therapy in cattle, poultry and pigs.

The successful use of virulent (lytic) bacteriophages (phages) in preventing and treating neonatal enterotoxigenic Escherichia coli infections in calves, lambs and pigs has prompted investigation of other applications of phage therapy in food animals. While results have been very variable, some indicate that phage therapy is potentially useful in virulent Salmonella and E. coli infections in chickens, calves and pigs, and in control of the food-borne pathogens Salmonella and Campylobacter jejuni in chickens and E. coli O157:H7 in cattle. However, more rigorous and comprehensive research is required to determine the true potential of phage therapy. Particular challenges include the selection and characterization of phages, practical modes of administration, and development of formulations that maintain the viability of phages for administration. Also, meaningful evaluation of phage therapy will require animal studies that closely represent the intended use, and will include thorough investigation of the emergence and characteristics of phage resistant bacteria. As well, effective use will require understanding the ecology and dynamics of the endemic and therapeutic phages and their interactions with target bacteria in the farm environment. In the event that the potential of phage therapy is realized, adoption will depend on its efficacy and complementarity relative to other interventions. Another potential challenge will be regulatory approval.

Effects of Escherichia coli challenge and transport stress on hematology and serum chemistry values of three genetic lines of turkeys.

Three lines of turkeys were compared for response to an Escherichia coli challenge followed by transport stress (transport). The turkey lines were a slow-growing line selected for increased egg production (egg line), a fast-growing line selected for increased 16-wk BW (F line), and a commercial line (Comm line). Birds were challenged at 14 wk of age with an air sac injection of 5,000 to 10,000 cfu of E. coli. At 8 d postchallenge, birds were subjected to a transport stress procedure that included 12 h of holding time in a transport vehicle. The following morning all birds (n = 10 to 19 birds/line) were bled. Whole blood was analyzed using the Cell-Dyn 3500 blood analysis system (Abbott Diagnostics), and serum chemistry was measured using the Express Plus analyzer (Ciba-Corning Diagnostics Corp.). Transport significantly decreased the levels of hematocrit, hemoglobin, mean cell volume, mean corpuscular hemoglobin, glucose, triglycerides, cholesterol, phosphorus, iron, albumin, and alkaline phosphatase (AP) and increased the levels of uric acid, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Line differences were variable, but the levels of both iron and AP were least in the fastest-growing Comm line birds and greatest in the slowest-growing egg-line birds with intermediate values in the F line. Iron and AP were also the only parameters influenced by sex, with males having greater levels of both compared with females. The creatine kinase levels were more than 6-fold greater in transported Comm line birds, and iron levels of transported Comm males were 3-fold less than controls. Previously, the growth rate of these lines was positively correlated with increased heterophil to lymphocyte ratios and susceptibility to colibacillosis. The differences seen in the Comm line for these commonly measured blood parameters suggest that they may be useful for profiling flocks to determine their response to transport stress and feed withdrawal.

Evaluation of the efficacy of vitamin D3 or its metabolites on thiram-induced tibial dyschondroplasia in chickens.

Two trials were conducted to determine if thiram-induced tibial dyschondroplasia (TD) in chickens was linked to a vitamin D deficiency and calcium homeostasis dysregulation, and whether feeding vitamin D fortified diets may prevent it. Day-old chickens were given grower diets containing different vitamin D products throughout the experiment until necropsy on day 16. Half of the birds in each feed group received thiram at levels of 100 ppm (trial 1) or 50 ppm (trial 2) between days 7-9 to induce TD. The birds were weighed, bled, and euthanized to determine TD incidences and severity by examining the growth plates. Tibial bones were used to measure biomechanical strength and ash content. Blood concentrations of 25-hydroxyvitamin D, Ca, P, alkaline phosphatase, and creatine kinase were measured in serum that showed no differences between different groups. Thiram reduced body weight and induced TD regardless of any vitamin D treatment to the same extent as untreated birds.

Therapeutic efficacy of bacteriophage and Baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers.

A study was conducted to evaluate the therapeutic efficacy of bacteriophage and the antibiotic enrofloxacin individually and in combination to treat colibacillosis. The experimental design was a 2 x 2 x 2 factorial with 8 treatments and 4 replicate pens of 10 birds. The treatments were 1) control, 2) unchallenged birds treated with bacteriophage, 3) enrofloxacin, or 4) the combination; 5) birds challenged with Escherichia coli, and birds challenged with E. coli and treated with 6) bacteriophage, 7) enrofloxacin, or 8) the combination of bacteriophage and enrofloxacin. Birds in the E. coli challenged treatments were challenged at 7 d of age by injecting 10(4) cfu of E. coli into the thoracic air sac. The antibiotic treatment was initiated immediately after the birds were challenged and consisted of 50 ppm enrofloxacin in the drinking water for 7 consecutive days. The bacteriophage treatment consisted of a single intramuscular injection of 2 different bacteriophage (10(9) pfu) administered immediately after the E. coli challenge. Mortality in the birds challenged with E. coli and untreated was 68%, and the bacteriophage and enrofloxacin treatments significantly decreased mortality to 15 and 3%, respectively. There was total protection in birds that received both the bacteriophage and enrofloxacin representing a significant synergy. The decrease in mortality with enrofloxacin (3%) was significantly better than the decrease in mortality with bacteriophage (15%). Airsacculitis lesion scores and lesion incidence in surviving birds were significantly less in the enrofloxacin treatment compared with the bacteriophage treatment. Both bacteriophage and enrofloxacin provided effective treatments of colibacillosis, and the synergy between these 2 treatments suggests that bacteriophage combined with antibiotic treatment has significant value.

The effects of water supplementation with vitamin E and sodium salicylate (Uni-Sol) on the resistance of turkeys to Escherichia coli respiratory infection.

The objective of this study was to determine the prophylactic efficacy of two commercial products, soluble vitamin E and soluble sodium salicylate (Uni-Sol), in an Escherichia coli respiratory challenge. The drinking water of male turkey poults was nonsupplemented or supplemented with either vitamin E or Uni-Sol or a combination of both at dosages recommended by the manufacturer. There were 110 birds in each of the four treatments, housed in four floor pens per treatment. At 5 wk of age, birds in half of the pens were challenged with an air sac inoculation of approximately 50 colony-forming units of E. coli. Water treatment commenced 5 days before challenge and continued for 2 wk after challenge, when birds were necropsied. All water treatments prevented the decrease in body weight due to E. coli challenge; however, either vitamin E or Uni-Sol alone, but not the combination of the two, decreased body weight in nonchallenged controls. Either vitamin E or Uni-Sol treatment alone, but not the combination of the two, significantly decreased mortality and air sacculitis scores of challenged birds, and all treatments decreased the isolation rates of E. coli from the liver. All treatments protected liver, spleen, and bursa weights (relative to body weight) from the effects of E. coli challenge, and Uni-Sol alone or vitamin E with Uni-Sol protected relative heart weights from the effect of challenge. Uni-Sol treatment alone increased the main effect mean total leukocyte counts and the number and percent of lymphocytes. Uni-Sol in combination with vitamin E increased the number of lymphocytes of challenged birds. Uni-Sol alone decreased the main effect mean heterophil/lymphocyte ratio (H/L) ratio, whereas vitamin E alone increased the H/L ratio of challenged birds. These results indicate that treatment of turkey poults with vitamin E or Uni-Sol prior to and during the stressful events that can lead to colisepticema may decrease disease incidence and mortality.

Effect of dietary supplementation with vitamin D metabolites in an experimental model of turkey osteomyelitis complex.

Supplementation with vitamin D3 was previously shown to protect Escherichia coli challenged birds that underwent two dexamethasone (DEX) treatments at 5 and 12 wk of age in an experimental model of turkey osteomyelitis complex (TOC). The purpose of the present study was to determine the effects of dietary supplementation with 10 microg of 1,25 dihydroxyvitamin D3 (1,25D)/ kg feed or 99 microg of 25-hydroxyvitamin D3 (25D)/kg feed on disease resistance in the same model. Birds were fed the supplemented diets continuously and ad libitum. Seven hundred twenty turkey poults were placed into 24 floor pens in a 3 x 2 x 2 design (three vitamin D treatments, two DEX treatments, two E. coli treatments, with two replicate pens per treatment). At 5 wk of age, half of the birds were treated with DEX, and half of the DEX-treated birds and half of the nontreated birds were challenged with E. coli. All mortalities and lame birds were necropsied. At 9 wk, all of the DEX- or E. coli-treated birds were given another series of DEX injections; 2 wk later 10 birds per pen were necropsied. At 12 wk, survivors of the previous challenges were given a third DEX treatment, and all birds were necropsied 2 wk later. After the first series of DEX injections, mortality was increased in the 25D-supplemented birds that were given the DEX treatment and the E. coli challenge. After the second series of DEX injections, the main effect mean BW was significantly lower in birds given 1,25D as compared to controls and 25D-supplemented birds. Mortality was higher in 1,25D-supplemented birds that were challenged with E. coli at 5 wk and treated with DEX at 9 wk as compared to 25D-supplemented birds. The 1,25D-treated birds that were treated with DEX at 5 and 9 wk and challenged with E. coli at 5 wk had higher mortality and air sacculitis scores as compared to controls and 25D-treated birds. The main effect mean mortality was significantly higher in birds given 1,25D as compared to controls and 25D-treated birds. The percentage of birds with TOC lesions was decreased from 27% to 0 by 25D and 1,25D in the groups given two DEX treatments and E. coli challenge. After the third DEX treatment, BW of 1,25D-suppplemented birds was decreased, and mortality and air sacculitis scores were increased. Bone strength was generally increased by supplementation with 1,25D, whereas 25D supplementation increased bone strength only in birds challenged at 5 wk and treated with DEX at Weeks 9 and 12. In this study, supplementation with vitamin D metabolites decreased TOC incidence in E. coli-challenged birds given two DEX treatments. However, toxic effects were observed in most supplemented DEX-treated birds and may be attributed to an additive effect of DEX treatment, E. coli septicemia, and vitamin D supplementation.

Changes in serum ovotransferrin levels in chickens with experimentally induced inflammation and diseases.

A competitive enzyme immunoassay was developed to measure the changes in serum levels of ovotransferrin (OTF) during inflammation and infectious diseases in chickens. The assay is based on the competition of serum OTF with a fixed concentration of biotin-labeled OTF to bind to a rabbit anti-chicken transferrin antibody immobilized on microtiter wells. After several washing steps, the antibody-bound biotinylated OTF is probed with streptavidin-horseradish peroxidase conjugate (HRP) followed by a colorimetric detection of the HRP activity. The relative changes in the optical density of color are plotted against the competing concentrations of OTF with logarithmic regression to generate a standard curve that is used to determine the concentrations of OTF in unknown samples. Serum had no effect on the measurement of OTE By this method, the time course changes of serum OTF levels in 4-wk-old male broiler chickens that were subjected to inflammation by croton oil injection were measured. The results showed croton oil-induced inflammation elevated serum OTF levels at 16 hr postinjection. OTF levels reached a peak by 72 hr, remained high through 120 hr, and returned to a basal level of olive oil-injected controls by 240 hr. There were no changes in serum OTF levels at any of the above time points in olive oil-injected control chickens. For studies with poultry diseases, specific-pathogen-free (SPF) male chickens were challenged with known bacterial and viral pathogens, and serum was collected at the height of the infection, i.e., 7 days after the challenge. Compared with uninjected controls, the SPF chickens challenged with Escherichia coli, fowl poxvirus, respiratory enteric orphan virus, infectious bursal disease virus, infectious bronchitis virus, or infectious laryngotracheitis virus had higher levels of OTF in serum. Inflammation-induced changes in serum OTF levels were also evident in the changes in the density of a 65-kD band protein corresponding to OTF. These results demonstrate that serum OTF may be a nonspecific clinical marker of inflammation associated with traumatic or infectious avian diseases.

Identification of ovotransferrin as an acute phase protein in chickens.

Inflammation is homeostatic process associated with a variety of cellular injuries resulting from infections, toxicosis, and physical trauma. The studies on inflammation in avian species are limited. To understand the inflammation-induced changes, 4-wk-old male broiler chickens were subjected to experimental inflammation by a subcutaneous injection of croton oil (inflammatory) with changes in serum measured over time and were compared with birds treated similarly with olive oil (injected control). Croton oil treatment significantly elevated serum interleukin (IL)-6 concentrations and heterophil counts by 6 and 16 h postinjection, respectively, which returned to the basal levels of controls at 16 and 24 h, respectively. Croton oil treatment affected the serum protein profiles of chickens as assessed by SDS-PAGE and densitometric analyses. Compared with olive oil-injected or noninjected chicken sera, there were increases in the density of protein bands corresponding to molecular weights (MW) of 42, 65, 200, and 219 kDa and decreases in bands corresponding to 49 kDa (serum albumin) and a 56-kDa protein in chickens treated with croton oil. Most of these changes were evident at 24 h and lasted through 48 h. The protein band corresponding to 65 kDa was further characterized using two-dimensional gel electrophoresis and N-terminal sequence analyses. A sequence similarity search in the Genbank database using the first 22 amino acids yielded a complete homology with chicken ovotransferrin. Western blot analysis using antichicken serum transferrin or antichicken ovotransferrin antibodies also confirmed the 65-kDa protein band to be ovotransferrin. Under nonreducing conditions, the ovotransferrin standard also showed an apparent MW corresponding to 65 kDa, like the serum transferrin. The serum ovotransferrin was found to be glycosylated using a glycoprotein stain. Although the significance of ovotransferrin in avian inflammation is not clear, these results show that it is a major acute phase protein (APP) in chickens.

Turkey osteomyelitis complex.

Turkey osteomyelitis complex (TOC) is defined by the US Food Safety Inspection Service (FSIS) to include normal-appearing processed turkey carcasses that contain lesions including green discoloration of the liver, arthritis/synovitis, soft-tissue abscesses, and osteomyelitis of the proximal tibia. The lesions are associated with many different opportunistic organisms, mainly Staphylococcus aureus and Escherichia coli, suggesting that TOC incidence may be influenced more by deficiencies in the host immune response rather than by the virulence of any one organism. This syndrome is primarily a disease of adolescent male turkeys, and birds with TOC lesions have decreased indices of cell-mediated immunity, leading to the hypothesis that defects in the immune response of individuals within flocks of male turkeys may be responsible for the occurrence of these opportunistic infections. We have developed an experimental model for this disease in which treatment with dexamethasone (DEX), either with or without air sac inoculation with Escherichia coli, produces all of the lesions associated with TOC. These studies suggest that TOC is a result of stress-induced immunosuppression in a subpopulation of male turkeys that respond to the stressors in modern poultry production in a detrimental manner. Supplemental vitamin D3 treatment protected male turkeys from the immunosuppression induced by multiple treatments with DEX and resulted in decreased incidence of mortality, TOC, green liver, and isolation of bacteria from tissues, lower air sacculitis scores, and lower heterophil to lymphocyte ratios than nonsupplemented controls. Vitamin D3 also protected BW; relative weights of the liver, heart, spleen, and bursa; and clinical chemistry values from the effects of DEX treatment. The ability of vitamin D3 supplementation to protect turkeys from the immunosuppressive effects of severe stress emphasizes its role as a prohormone that affects health and disease resistance in turkeys and suggests that variation in the vitamin D receptor genotype may be involved in this disease process. This model has potential value in the identification of other nutritional and physiological immunomodulators that can decrease TOC incidence and will provide a means for the divergent selection of birds more resistant to the stressors of turkey production. In addition, this model will provide justification for management options designed to minimize stress.

The effect of vitamin D3 on resistance to stress-related infection in an experimental model of turkey osteomyelitis complex.

Male turkeys immunosuppressed by injection with dexamethasone (DEX) were given supplemental vitamin D3 in their drinking water in two experiments. In Experiment 1, vitamin D3 was supplemented at a dosage of either 2,064 IU/kg (low level) or 4,128 IU/kg (high level) in drinking water provided ad libitum only from Days 1 through 5 after hatch. In Experiment 2, vitamin D3 was provided at the low dosage for the first 5 d after hatch, followed by treatment with the high dosage for 12 h before and 12 h after each stressful event, which included weekly weighings and two DEX treatments. In both experiments, at 5 wk of age half of the birds were given intramuscular injections of 2 mg/kg DEX on 3 alternating d. In Experiment 1, 100 cfu of Escherichia coli was inoculated into the left thoracic airsac at the time of the third DEX injection. All mortalities were examined, and 10 birds per pen were necropsied 2 wk after treatment and examined for lesions of airsacculitis and turkey osteomyelitis complex (TOC). Four birds per pen were bled before necropsy, and white blood cell total counts, differential white blood cell counts, and clinical chemistry values were determined. In Experiment 2, healthy surviving birds were grown for an additional 5-wk period, after which the DEX-treated birds were given a second series of DEX injections and were bled and necropsied 2 wk later. There were no significant effects of vitamin D3 treatment in combined general linear models analysis of Experiment 1; however, when birds not treated with DEX or E. coli were compared with those treated with both DEX and E. coli, supplementation with the low level of vitamin D3 significantly decreased TOC incidence. There were no significant effects of vitamin D3 treatment in birds treated with DEX at 5 wk of age in Experiment 2. However, when surviving birds were given a second DEX treatment at 12 wk, vitamin D3 treatment resulted in significantly lower incidence of mortality, TOC, green liver, isolation of bacteria from tissues, and lower airsacculitis scores and heterophil-to-lymphocyte ratios than controls. Vitamin D3 also improved BW, relative weights of the liver and heart, and serum levels of glucose and alanine aminotransferase (ALT) of birds receiving two treatments with DEX. The ability of vitamin D3 supplementation to protect turkeys from the immunosuppressive effects of multiple DEX treatments emphasizes the role of vitamin D3 as a prohormone that affects health and disease resistance in turkeys.

Research on the probable cause of an outbreak of field rickets in turkeys.

An outbreak of field rickets in turkeys prompted studies on the cause. In Experiment 1, there were four treatments with two replicate pens of 10 poults per pen. The treatments consisted of poults fed newly manufactured feed (control), poults fed a diet containing control feed and 5% clean litter, poults fed control feed and 5% litter from the pens of affected poults, and poults challenged with an intestinal homogenate by gavage. Field rickets did not develop with these treatments. The feed was suspect, and, in Experiment 2, poults were either fed the suspect feed or newly manufactured feed. There were four replicate pens of 25 poults per pen. Poults fed the suspect feed had a decrease (P < or = 0.05) in BW at 1, 2, 3, and 4 wk of age; an increase in the relative weight of the liver, pancreas, kidney, and bursa of Fabricius; and a decrease in bone ash. There were changes in clinical chemistries. In the third study, there were five dietary treatments with two replicate pens of 25 poults per treatment. The treatments consisted of poults fed newly manufactured feed; new feed mixed with 25, 50, or 75% suspect feed; or 100% suspect feed. Body weights of poults fed 100% suspect feed were decreased at 2, 3, and 4 wk as was the relative weight of the liver, pancreas, and bursa of Fabricius. The relative weight of the kidney increased. Lameness, a decrease in bone ash, and changes in hematology and blood chemistry were observed in the poults consuming 100% suspect feed. These data demonstrate that feed from the original outbreak could induce field rickets and was toxic. Because the feed contained adequate vitamin D, calcium, and phosphorus, the cause of this outbreak of field rickets is thought to be a toxic feed contaminant affecting bone development.