N. C. Rath

Characterization of a spontaneously transformed chicken mononuclear cell line

We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 or 41 degrees C in culture medium containing either 5% FBS or 2% chicken serum. The HTC cells are acid phosphatase positive, show expressions of both class I and class II major histocompatibility complex (MHC), CD44, K1, and K55 cell surface antigens, and engulf latex beads, produce nitrite and interleukin-6 on stimulation with bacterial lipopolysaccharide (LPS). Treatment with phorbol myristate acetate (PMA) induces respiratory burst in HTC cells and the secretion of matrix metalloproteinase (MMP) into culture medium. Using gene-specific primers and reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of mRNA trancripts for interferon-gamma (IFN-gamma), interleukin-1 (IL-1), interleukin-6 (IL-6), nitric oxide synthase (NOS), matrix metalloproteinase-2 (MMP-2), and transforming growth factor-beta (TGF-beta) were detected. Lipopolysaccharide (LPS) treatment of HTC cells modulated IL-1, IL-6, IFN-gamma, NOS mRNA levels as detected by RT-PCR analyses. Using different avian tumor virus gene-specific primers and PCR, the HTC cells were positive for the presence of avian leukosis virus (ALV) and Mareks disease virus (MDV) but negative for reticuloendothelial virus (REV), chicken infectious anemia virus (CIAV), and herpes virus of turkeys (HVT). The production of ALV antigens by HTC cells was further confirmed using p27 gag protein ELISA. Collectively, these results show that the HTC cells belong to myeloid/macrophage lineage and were likely transformed by ALV and MDV but retain many interesting and useful biological activities.

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.

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.

Fluorescein isothiocyanate staining and characterization of avian heterophils

Fluorescein isothiocyanate (FITC) was found to stain cytoplasmic granules of avian heterophilgranulocytes. In tissue sections, the fluorescent granulocytes were predominantly distributed adjacent to trabecular bones. The fluorescein stained granulocytes were abundant in synovial fluids of chickens with synovitis. A significant correlation was observed in the percent of fluorescein labeled granulocytes in blood smears and the percent of heterophils determined using an automated counting method, in unstained blood from normal and Escherichia coli-infected turkeys. The fluorescein-binding heterophils purified from chickens showed a time dependent increases in the oxidation of 2,7-dichlorofluorescin diacetate (DCF-DA) and the reduction of nitroblue tetrazolium (NBT) which were indicative of changes in oxidative burst in response to phorbol 12-myristate 13-acetate (PMA), Salmonella typhimurium lipopolysaccharide (LPS), and zymosan A (ZA). These heterophil-activating agents, also, caused significant degranulation at 16 h post-treatment, as indicated by the loss fluorescence. There were microscopically visible alterations in the cell shapes and a decrease in the density of granules due to treatment with LPS, PMA or ZA. In addition, these cells also showed phagocytic response which was evident at 30 min of incubation with fluorescent latex particles. Both chicken and turkey heterophils produced interleukin-6 in vitro at 24 h in response to LPS but not to PMA, FMLP or ZA. The chicken heterophils showed spontaneous production of matrix metalloproteinases (MMP) which was significantly enhanced by treatment with LPS, PMA, and ZA; however, LPS appeared to be most effective in inducing MMP production. These results demonstrate that the functions of heterophils can be differentially regulated by different activating agents and the fluorescein binding property of these cells may be useful for their histochemical identification.

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 12u2009h 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.

Direct screening identifies mature β-defensin 2 in avian heterophils

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to screen avian heterophils in the m/z range of 1 to 20 kDa with an objective to identify specific cell-associated peptides that may be reflective of their functional physiology. The MALDI-TOF-MS profiles of crude heterophil extract showed a high intensity peak with average mass of m/z 3916.1 for chicken and m/z 4129.6 for turkey. To identify these peaks, we first purified m/z 3916.1 from chicken bone marrow extract using reverse-phase high performance liquid chromatography (RP-HPLC). Edman sequencing and peptide mass fingerprinting exclusively confirmed this peptide as beta-defensin 2 (BD2) or gallinacin-2, a broad-range antimicrobial peptide. A Uniprot database search followed by the MASCOT sequence query revealed m/z 4129.6 to be the corresponding turkey ortholog of avian beta-defensin 2 (AvBD2), also called turkey heterophil peptide 2. Both AvBD2 peptides are 36 amino acids long including a highly conserved region with 6 invariant cysteines forming the 3 disulfide bonds characteristic of defensins. The method confirmed the existence of the complete mature peptide sequence of the turkey heterophilic BD2 previously proposed based on cDNA analysis. These results demonstrate that screening of the crude extract by MALDI-TOF-MS can identify cell- or tissue-associated peptides in their functional or mature forms, raising the possibility that such peptides can be used as biomarkers in their altered physiological states.

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 80u2009g 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 37u2009d 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 28u2009d body weights (BW) and FCR. At 37u2009d 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.

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.

Cellular component of lavage fluid from broilers with normal versus aerosol-primed airways

Previously, we reported that intratracheal administration of lipopolysaccharide elicited pulmonary hypertension (PH) in broilers reared under commercial conditions and in broilers reared in environmental chambers and pretreated with aerosolized red food colorant # 3 and propylene glycol (Red#3+PG), but not in control broilers reared in environmental chambers. The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers. Birds were aerosolized for 40 min with a saturated mixture of Red#3+PG. After 24 h, a blood sample was taken, the broilers were killed, and a pulmonary lavage process was conducted in each bird. Leukocyte concentration (white blood cells/microL) and differential leukocyte counts (%) were measured in blood and lavage fluid. Leukocyte concentration in blood did not differ between groups, but the percentage of blood lymphocytes was lower in broilers from the Red#3+PG group compared with birds from the control group (52.4+/-2.9 and 56.9+/-2.9%, respectively). Cells recovered from the lavage fluid from both groups were primarily heterophils. The concentration of leukocytes was greater in the lavage fluid of broilers from the Red#3+PG group compared with broilers from the control group (763.2+/-158.7 and 402.9+/-62.6 white blood cells/microL, respectively), but the proportions among leukocytes were not different between the 2 groups. We propose that the increased concentration of leukocytes present within the airways was one of the components that enabled broilers pre-treated with aerosolized Red#3+PG to exhibit PH responses to intratracheal lipopolysaccharide.