Carvacrol attenuates Campylobacter jejuni colonization factors and proteome critical for persistence in the chicken gut.

Campylobacter jejuni is a major foodborne pathogen that causes gastroenteritis in humans. Chickens act as the reservoir host for C. jejuni, wherein the pathogen asymptomatically colonizes the ceca leading to contamination of carcasses during slaughter. The major colonization factors in C. jejuni include motility, intestinal epithelial attachment, acid/bile tolerance, and quorum sensing. Reducing the expression of the aforementioned factors could potentially reduce C. jejuni colonization in chickens. This study investigated the efficacy of subinhibitory concentration (SIC; compound concentration not inhibiting bacterial growth) of carvacrol in reducing the expression of C. jejuni colonization factors in vitro. Moreover, the effect of carvacrol on the expression of C. jejuni proteome was investigated using liquid chromatography-tandem mass spectrometry. The motility assay was conducted at 42°C, and the motility zone was measured after 24 h of incubation. For the adhesion assay, monolayers of primary chicken enterocytes (∼105 cells/well) were inoculated with C. jejuni (6 log cfu/well) either in the presence or absence of carvacrol, and the adhered C. jejuni were enumerated after 90 min of incubation at 42°C. The effect of carvacrol on C. jejuni quorum sensing and susceptibility to acid/bile stress was investigated using a bioluminescence assay and an acid-bile survival assay, respectively. The SIC (0.002%) of carvacrol reduced the motility of C. jejuni strains S-8 and NCTC 81-176 by ∼50 and 35%, respectively (P < 0.05). Carvacrol inhibited C. jejuni S-8 and NCTC 81-176 adhesion to chicken enterocytes by ∼0.8 and 1.5 log cfu/mL, respectively (P < 0.05). Moreover, carvacrol reduced autoinducer-2 activity and increased the susceptibility of C. jejuni to acid and bile in both the strains (P < 0.05). Liquid chromatography-tandem mass spectrometry revealed that the SIC of carvacrol reduced the expression of selected C. jejuni colonization proteins critical for motility (methyl-accepting chemotaxis protein), adhesion (GroL), growth and metabolism (AspA, AcnB, Icd, Fba, Ppa, AnsA, Ldh, Eno, PurB-1), and anaerobic respiration (NapB, HydB, SdhA, NrfA) (P < 0.05). Results suggest the mechanisms by which carvacrol could reduce C. jejuni colonization in chickens.

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.

Protein profiles of hatchery egg shell membrane.

BACKGROUND: Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). METHODS: Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. RESULTS: Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. CONCLUSION: The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

Poultry Femoral Head Separation and Necrosis: A Review.

Femoral head separation (FHS) is a degenerative skeletal problem in fast-growing poultry wherein the growth plate of the proximal femur separates from its articular cartilage. At its early phase, FHS may remain asymptomatic but lead to epiphyseal breakage, infection, and femoral head necrosis (FHN). Healthy femoral head is viewed as a positive trait for genetic selection. However, the etiology of FHS is poorly understood for use in noninvasive diagnosis and genetic selection. Focal cell death and atrophic changes are likely associated with separation of tissues and necrotic changes. Fibrotic thickening of the articular surface can also impair free movement of the proximal epiphysis in the acetabulum, leading to FHS, under strain. The major limitation to understanding the pathophysiology of FHN is the lack of suitable experimental models and biomarkers to diagnose the problem. In this review, we discuss the possible etiologic factors, anatomic features of the chicken femoral head, biomarkers, and molecular mechanisms relevant to FHN.

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.

Effect of dexamethasone on bacteriostatic activity of turkey monocytes and implications for food safety.

Stress has been shown to affect the immune system of turkeys making them more susceptible to bacterial infections. Five-week-old male and female turkeys were treated with 3 intra-muscular injections of dexamethasone (Dex) at 0, 0.5 and 2.0mg/kg body weight. Twenty-four hours after the third injection birds were bled and white blood cell (WBC) differentials and bacteriostatic activity of monocytes were measured. Dex at both 0.5 and 2.0mg/kg decreased phagocytic activity in females only. Bacteriostatic activity was decreased at both concentrations of Dex at 8 and 16 h post-infection in both sexes and was lower in males as compared to females. Total WBC counts were increased in females at both concentrations of Dex whereas male total WBC counts were unaffected. Both males and females had an increase in the heterophil to lymphocyte ratio. Within the same study, replicate pens of turkeys were challenged with intra-air sac inoculation of 100 cfu of Escherichia coli. Isolation of E. coli was significantly increased by both Dex and E. coli challenge, but there were no differences between sexes. These results suggest that stress can compromise the bacteriostatic activity of turkey monocytes and increase bacterial colonization of blood and tissues, potentially affecting food safety.

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.

Effect of butyrate on immune response of a chicken macrophage cell line.

Butyric acid is a major short chain fatty acid (SCFA), produced in the gastrointestinal tract by anaerobic bacterial fermentation, that has beneficial health effects in many species including poultry. To understand the immunomodulating effects of butyrate on avian macrophage, we treated a naturally transformed line of chicken macrophage cells named HTC with Na-butyrate in the absence or presence of Salmonella typhimurium lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA), a metabolic activator, evaluating its various functional parameters. The results demonstrate that, butyrate by itself had no significant effect on variables such as nitric oxide (NO) production and the expression of genes associated with various inflammatory cytokines but it inhibited NO production, and reduced the expression of cytokines such as IL-1ß, IL-6, IFN-γ, and IL-10 in LPS-stimulated cells. Butyrate decreased the expression of TGF-ß3 in the presence or absence of LPS, while it had no effect on IL-4, Tß4, and MMP2 gene expression. In addition, butyrate augmented PMA induced oxidative burst indicated by DCF-DA oxidation and restored LPS induced attenuation of tartrate resistant acid phosphatase (TRAP) activity. Although butyrate had no significant effect on phagocytosis or matrix metalloproteinase (MMP) activities of resting macrophages, it significantly suppressed the effects induced by their respective stimulants such as LPS induced phagocytosis and PMA induced MMP expression. These results suggest that butyrate has immunomodulatory property in the presence of agents that incite the cells thus, has potential to control inflammation and restore immune homeostasis.

Environmental augmentation with bacteriophage prevents colibacillosis in broiler chickens.

Bacteriophages are viruses that kill bacteria. They are plentiful in nature; are safe, having no known activity to human or animal cells; and are an attractive alternative to antibiotics. The objectives of this research were to establish an experimental model of colibacillosis induced by indirect exposure to Escherichia coli and to determine if bacteriophage could protect the birds from developing colibacillosis. In study 1 there were 6 treatments with 2 replicate pens of 25 birds. The treatments were control warm brooded; control cold stressed; litter inoculated with E. coli, warm brooded; litter inoculated with E. coli, cold stressed; seeder birds (5 per pen) challenged with E. coli, warm brooded; and seeder birds (5 per pen), cold stressed. The study concluded when the birds were 3 wk of age. Body weights at 1, 2, and 3 wk of age were significantly decreased (P ≤ 0.05) by cold stress, decreased at 1 and 2 wk of age by both the litter and seeder bird treatments compared with the control treatment and by the seeder bird treatment at 3 wk of age. Study 2 consisted of 8 treatments with 2 replicate pens of 20 birds per treatment. The treatments were control, warm brooded; control, cold stressed; litter inoculated with E. coli, cold stressed; and seeder birds (5/pen) challenged with E. coli, cold stressed with and without bacteriophage treatment. In the bacteriophage treatments the bacteriophages were sprayed on the litter. The study was concluded at 3 wk of age. Body weights at 1 wk of age were significantly (P ≤ 0.05) decreased from the control treatment by the seeder bird treatment and were significantly (P ≤ 0.05) higher in all the bacteriophage treatments compared with their matched untreated treatments, except in the control cold stressed treatment. Mortality was significantly (P ≤ 0.05) decreased by bacteriophage in the litter challenged treatment. These data suggest that augmentation of the environment with bacteriophage is a practical and efficacious way to prevent colibacillosis in broiler chickens.

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.

Method of administration affects the ability of bacteriophage to prevent colibacillosis in 1-day-old broiler chickens.

Bacteriophages are viruses that kill bacteria. They are plentiful in nature with no known activity in human or animal cells, making them an attractive alternative to antibiotics. The objective of this research was to determine if a coarse or a fine spray of bacteriophage would prevent colibacillosis induced by an intratracheal (IT) challenge with Escherichia coli. Two studies were conducted with 6 treatments: untreated control, birds treated with a spray administration of bacteriophage and not challenged, birds administered bacteriophage IT and not challenged, birds not treated and challenged IT with E. coli, birds sprayed with bacteriophage and IT challenged with E. coli, and birds administered bacteriophage IT and challenged IT with E. coli. There were 3 replicate pens of 10 birds per pen, per treatment, and all treatments were administered at 1 d of age. Study 1 was concluded when the birds were 19 d of age, and study 2 was concluded when the birds were 21 d of age. In both studies, neither a coarse nor a fine spray protected the birds from an IT E. coli challenge; however, when bacteriophage was administered IT there was complete protection. This research demonstrates the necessity for the administration of bacteriophage therapeutics to deliver high bacteriophage titers to the site of a bacterial infection.

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.

Dexamethasone immunosuppression resulting in turkey clostridial dermatitis: a retrospective analysis of seven studies, 1998-2009.

We have studied the etiology of turkey osteomyelitis complex (TOC) for the past 20 yr and have determined that this syndrome is caused by the inability of some fast-growing male turkeys to cope with production stressors. Although immunosuppressive viruses have often been associated with susceptibility to gangrenous dermatitis (cellulitis), we hypothesize that production stressors alone can also undermine resistance to opportunistic pathogens by both increasing bacterial translocation from the intestine and disrupting the skin's antimicrobial barrier, resulting in subcutaneous lesions referred to as cellulitis and recently named turkey clostridial dermatitis (CD). Some common characteristics between TOC and CD are that they are both caused by opportunistic bacterial species that are prevalent in the environment and are both most common in adolescent male birds. In both diseases the affected birds are often large, healthy, and from the best-performing flocks. Our TOC studies using dexamethasone immunosuppression result in a high incidence of cellulitis lesions in dead turkeys that were given either Escherichia coli or Staphylococcus aureus respiratory challenges. The natural presence of Clostridium spp. in the poultry intestine and environment suggests that they may also have been concomitant pathogens. We suggest that a useful and repeatable model for CD can be developed by focusing on the ability of stress to increase diuresis and wet litter conditions and undermine both intestinal and cutaneous bacterial resistance in fast-growing male turkeys.

Effects of high fat diets or prednisolone treatment on femoral head separation in chickens.

1. The effects of high fat diets and prednisolone treatment were studied to understand the etiology of femoral head separation (FHS) in fast growing broiler chickens. Dietary effects on production parameters such as growth, feed conversion ratio (FCR) and blood chemistry were also measured. 2. Three groups of chickens, consisting of 30 birds each, in two replicate pens, were fed isonitrogenous diets containing 40 (control), 60, or 80 g poultry fat supplements per kg feed. The birds were fed a starter diet containing the fat supplements for the first three weeks, then switched to a grower diet containing the same supplements for the rest of the experimental period. Two groups of birds were also raised with the control diets, but were administered either cholesterol or prednisolone intramuscularly at 30 and 32 days of age to evaluate their effects on FHS incidences. 3. The chickens were euthanised and necropsied at 37 d of age. The presence of femoral head weakness was determined by applying mild pressure on the pelvic joint to cause the growth plate to become detached from its articular cartilage in affected cases. 4. High fat diets did not change FHS incidences, but increased 28 d body weights (BW) and FCR. At 37 d of age the BW differences were not significant but the FCR (gain: feed ratio) remained higher in high fat fed groups. Prednisolone treatment, by contrast, resulted in decreased BW, decreased feed efficiency, increased FHS index, and elevated blood lipid levels. 5. The results suggest that high dietary fats do not affect FHS incidence in broilers. Prednisolone treatment causes hyperlipidaemia and increases FHS index, and may therefore provide a suitable experimental model of FHS pathogenesis in growing chickens.

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.

Tibial dyschondroplasia-associated proteomic changes in chicken growth plate cartilage.

Tibial dyschondroplasia (TD) is a poultry leg problem that affects the proximal growth plate of the tibia, preventing its transition to bone. To understand the disease-induced proteomic changes, we compared the protein extracts of cartilage from normal and TD-affected growth plates. TD was induced by feeding thiram to chickens 2 wk before tissue harvest. Proteins were extracted from whole tissues and from conditioned media (CM) prepared by incubating appropriate growth plate tissues in serum-free culture medium for 48 hr. The extracts were prefractionated to contain proteins ranging between 10 and 100 kD. Equal amounts of proteins were subjected to 2D gel electrophoresis with three individual samples per group. The gels were silver stained, and digital images were compared and analyzed with Melanie software to determine differentially expressed protein spots. On comparison of two sets of gels, 47 matching spots were detected in tissue extracts and 27 in CM extracts. Among the matching spots, 12 were determined to be down-regulated in tissue extracts (P < or = 0.05) and two in CM extracts (P < or = 0.05) of TD-affected growth plates. Altogether, 32 protein spots could be identified in both tissue and CM extracts by in-gel trypsin digestion, followed by peptide mass fingerprinting and mass spectrometry (MS)/MS fragmentation. The down-regulated proteins included alpha-enolase, G protein, origin recognition complex, peptidyl prolyl isomerase, calumenin, type II collagen precursor, and the expressed sequence tag pgm2n.pk014.f20, a protein with homology to human reticulocalbin-3 (RCN3). Most of the downregulated proteins are associated with signal transduction, energy metabolism, and secretory functions that are integral to cell viability. Consistent with our earlier findings that the TD chondrocytes are nonviable, the current results suggest that thiram very likely interferes with basic metabolic functions of chondrocytes, leading to their death and, consequently, to the pathogenesis of TD.

Serum ovotransferrin as a biomarker of inflammatory diseases in chickens.

Infectious and metabolic disorders are common in poultry and cause stress, poor performance, and mortality that results in considerable economic loss. Identifying the nature of stress in chickens will assist the development of appropriate measures to improve health and welfare. Acute phase proteins are hepatic proteins, the blood concentrations of which change significantly in the event of many health problems including inflammation and physical injuries. Thus, acute phase proteins are used as nonspecific diagnostic markers for various health disorders. Our previous studies showed that serum ovotransferrin (OVT) is an acute phase protein in chickens. Therefore, in the present study, we investigated whether OVT concentration can be a marker of physiological stress using sera from chickens with different infectious and metabolic disorders. A competitive enzyme immunoassay was developed to measure serum OVT concentrations. The results show that with experimentally induced pulmonary hypertension syndrome and tibial dyschondroplasia, there were no significant changes in OVT levels compared with matched controls. In contrast, when chickens were infected with microbes such as the bacterium Escherichia coli, or protozoan parasites such as Eimeria maxima and Eimeria tenella, there was a significant increase in the levels of OVT in the serum. Chickens with spontaneous autoimmune vitiligo also showed a significant increase in blood OVT levels. These studies suggest that blood OVT concentration is modulated under inflammatory and microbial stress and can therefore be used as a diagnostic marker of infection and inflammation in chickens.