M. A. Qureshi

Differential expression of inducible nitric oxide synthase is associated with differential Toll-like receptor-4 expression in chicken macrophages from different genetic backgrounds

The purpose of this study was to examine iNOS gene expression and activity in macrophages from different chicken genetic lines against various bacterial LPS. Furthermore, the possible involvement of surface LPS receptors as candidates for differential iNOS gene induction in these genetic lines of chicken was also examined. Sephadex-elicited abdominal macrophages (1 x 10(6)) as well as iNOS hyper-responder macrophages from a transformed chicken macrophage cell line, MQ-NCSU, were exposed to 5 microg/ml LPS from E. coli, Shigella flexneri, Serratia marcensces, and Salmonella typhimurium. Nitrite levels were quantitated in the culture supernatant fractions of macrophages after 24h by the Griess method. The results showed that macrophages from K-strain (B(15)B(15)) (range from two separate trials: 31-89 microM) and MQ-NCSU (22-81 microM) were high responders whereas macrophages from both GB1 (B(13)B(13)) (15-38 microM) and GB2 (B(6)B(6)) (7-15 microM) chickens were low responders against all LPSs used. Northern blot analysis revealed that K-strain macrophages expressed higher intensity of 4.5Kb iNOS mRNA (iNOS/beta-actin ratio) than macrophages from GB2 regardless of the LPS source. To elucidate possible molecular mechanism(s) involved in iNOS gene expression in these two strains of chickens, the constitutive expression of LPS-related macrophage cell surface receptors, CD14, Toll-like receptor-2 (TLR2), and Toll-like receptor-4 (TLR4), was examined via flow cytometry using anti-human CD14, TLR2 and TLR4 antibodies. CD14 surface expression and intensity was not different between macrophages from K-strain or GB2 chickens. In contrast, while the overall percentage of TLR4-positive macrophages was the same (K-strain, trial 1=92%, trial 2=62%; GB2, trial 1=91%, trial 2=64%), the mean fluorescence intensity (MFI), an indicator of receptor number, was significantly higher (P=0.05) in K-strain macrophages (MFI: trial 1=145; trial 2=131) than GB2 macrophages (MFI: trial 1=101; trial 2=98). Furthermore, TLR2 (a previously thought candidate as LPS signaling molecule) positive cell numbers were higher in K-strain than the GB2 macrophages in one of the two trials with no difference in the intensity of TLR2 expression in either trial. These findings suggest that the observed differences in iNOS expression and activity among the K-strain (hyper-responder) and GB2 (hypo-responder) chickens are, at least in part, due to differential expression of TLR4 (an LPS signaling molecule), leading to more intense LPS-mediated activation of K-macrophages.

Dietary Spirulina Platensis Enhances Humoral and Cell-Mediated Immune Functions in Chickens

Cornell K-strain White Leghorns and broiler chicks were raised to 7 wks and 3 wks of age respectively, with diets containing various levels (0, 10, 100, 1,000 and 10,000 ppm) of Spirulina platensis from day of hatch. Chicks in all treatment groups had comparable body weights. While bursal and splenic weights did not change, the K-strain chicks had larger thymuses (P < or = .05) over the controls (0 ppm group). No differences were observed in anti-sheep red blood cells antibodies during primary response. However, during secondary response, K-strain chicks in all Spirulina-dietary groups had higher total anti-SRBC titers with 10,000 ppm group being the highest (6.8 Log2) versus the 0 ppm (5.5 Log2) group. In broiler chicks, a one Log increase in IgG (P < or = .05) was observed in 10,000 ppm group over the controls. Similarly, chicks in 10,000 ppm Spirulina group had a higher PHA-P-mediated lymphoproliferative response over the 0 ppm controls. Macrophages isolated from both K-strain (10,000 ppm group) and broilers from all Spirulina groups had higher phagocytic potential than the 0 ppm groups. Spirulina supplementation at 10,000 ppm level also increased NK-cell activity by two fold over the controls. These studies show that Spirulina supplementation increases several immunological functions implying that a dietary inclusion of Spirulina at a level of 10,000 ppm may enhance disease resistance potential in chickens.

Zinc metabolism with special reference to its role in immunity

A major goal of many poultry producers is to attain good flock liveability. Historically, most poultry producers have manipulated environmental conditions and management to maximize bird health. In the past two decades there has been much research into nutritional regimes that improve bird health through immunomodulation. Commercial poultry environments contain ubiquitous micro-organisms that continuously challenge the immune system. Nutritional supplements that enhance immune system function may improve flock performance and be economically advantageous. This paper reviews the literature on zinc-methionine and the avian cellular immune system. Current knowledge of the effects of zinc on many animal models is reviewed and a hypothetical mechanism for the action of zinc-methionine on this system is discussed.

Establishment and characterization of a chicken mononuclear cell line.

A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Mareks disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.

Involvement of lipopolysaccharide related receptors and nuclear factor κB in differential expression of inducible nitric oxide synthase in chicken macrophages from different genetic backgrounds

Macrophages from Cornell K-strain chickens (B(15)B(15)) are hyper and from GB2 chickens (B(6)B(6)) are hypo-responders to LPS-mediated inducible NOS (iNOS) expression and activity. The molecular mechanism(s) responsible for this differential expression is not yet fully understood. We have previously reported that macrophages from K (iNOS hyper-responder) and GB2 (iNOS hypo-responder) chickens differ in constitutive expression of TLR4 but not in CD14 molecules. The objectives of the current study was to determine if the iNOS differences between K and GB2 macrophages are possibly due to differential expression of LPS-induced TLR4, CD14 and/or nuclear factor kappa B (NF kappa B). The results showed that Sephadex-elicited, adherence purified K macrophages expressed more inducible TLR4 and CD14 receptors (P<0.05) at 6 and 12h post-LPS stimulation than GB2 macrophages as measured by flow cytometry. In addition, pre-incubation of macrophages from a transformed chicken macrophage cell line, MQ-NCSU, with 50 microg/ml anti-CD14 and anti-TLR4 antibodies significantly reduced where as pre-incubation with 100 microg/ml completely blocked LPS-mediated iNOS activity as measured by nitrite levels. Furthermore, the amount of nuclear bound NF kappa B was found to be significantly greater in K than in GB2 macrophages at 3 min post-LPS stimulation. This nuclear localization of NF kappa B as well as iNOS activity was completely inhibited by pretreatment of macrophages with 50 micro M MG132, a proteosome inhibitor, both in K and GB2 macrophages. Taken together, these findings suggest that a differential and perhaps more stronger LPS-mediated signaling via CD14, TLR4 and NF kappa B is responsible for the heightened iNOS gene induction in K-strain (hyper-responder) macrophages than in GB2 (hypo-responder) chickens.

Viral Agents Associated with Poult Enteritis and Mortality Syndrome: The Role of a Small Round Virus and a Turkey Coronavirus

Intestinal samples from turkey poults affected with poult enteritis and mortality syndrome (PEMS) were examined for viruses by immune electron microscopy and double-stranded RNA virus genome electropherotyping. Turkey coronavirus (TCV), avian rotaviruses, reovirus, and a yet undefined small round virus (SRV) were detected. The SRV and TCV were isolated and propagated in turkey embryos. Challenge of specific-pathogen-free turkey poults with SRV, TCV, or both resulted in mortality and clinical responses similar to those of natural PEMS. Our experiments indicate that SRV and TCV are possibly important agents in the etiology of PEMS and the combination of these infections might result in outbreaks with high mortality. The severity of clinical signs and mortality of PEMS are postulated to be partly related to the virus agents involved in individual outbreaks.

Spirulina Platensis Exposure Enhances Macrophage Phagocytic Function in Cats

Bronchoalveolar lavage macrophages isolated from cats were cultured on glass coverslips. Macrophages were exposed to a water-soluble extract of Spirulina platensis in concentration range of 0 to 60 micrograms per mL for two hours. Spirulina-extract exposure did not cause significant macrophage cytotoxicity over untreated control cultures. Macrophage monolayers from treated and control cultures were incubated with sheep red blood cells (SRBC) as well as viable Escherichia coli. The percentages of phagocytic macrophages for both of these particulate antigens were higher (a two-fold increase in SRBC phagocytosis and over 10% increase in Escherichia coli uptake) in cultures treated with various concentrations of Spirulina-extract. However, the numbers of either types of particles internalized by phagocytic macrophage were not different between the control and treated cultures. These data which showed that Spirulina platensis extract enhances macrophage phagocytic function imply that dietary Spirulina supplementation may improve the disease resistance potential in cats.

Avian macrophage and immune response: an overview

ABSTRACT Macrophages belong to the mononuclear phagocytic system lineage. This cell type is unique in that it is a crucial player in both the innate and adaptive immune responses. The material described in this overview is a brief description of what I presented as a Worlds Poultry Science Association-sponsored lecture at the annual meetings of the Poultry Science Association in 2002. Therefore, I have not attempted to present an up-to-date review of literature on this topic. Rather, I have summarized some salient research accomplishments made by our research group over the years in the area of avian macrophage biology and function.

The Influence of β‐Glucan on Immune Responses in Broiler Chicks

Abstract Beta‐1,3/1,6‐glucan (β‐glucan) was tested as a possible immunomodulator. Chicken macrophages from a macrophage cell line MQ‐NCSU and from abdominal exudate of broiler chickens were exposed to various concentrations of β‐glucan in vitro. In addition, day‐old broiler chicks were fed a diet containing 0, 20, and 40 mg/kg β‐glucan in the starter and 0, 20, and 20 mg/kg in the grower diet. Several baseline immune parameters were examined following such exposures. The results showed that β‐glucan exposure increased nitrite and interleukin‐1 (IL‐1) production as well as induced macrophage to proliferate in culture. However, IL‐6 production was not affected. Dietary β‐glucan supplementation increased the macrophage phagocytic activity, anti‐sheep red blood cells antibody response post‐boost, as well as the PHA‐P‐mediated lymphoproliferative response measured as a toe‐web swelling. The percentage of CD4+, CD8+, and CD4+/CD8+ double positive lymphocytes in the intestinal intraepithelial leukocytes was increased in β‐glucan supplemented chicks. Furthermore, the primary and secondary lymphoid organs such as bursa of Fabricius, thymus and spleen were larger in β‐glucan‐supplemented chicks as compared to the chicks on basal diet. The findings of these studies which showed that β‐glucan improves several base‐line immune responses in the chicken imply that β‐glucan can be used as a possible immunomodulator in food animals such as the chicken.

Dietary zinc-methionine enhances mononuclear-phagocytic function in young turkeys. Zinc-methionine, immunity, and Salmonella.

The ability of dietary zinc-methionine (Zn-Met) to enhance mononuclear-phagocytic function againstSalmonella arizona andenteritids was investigated in young turkeys. Feed/gain and body wt gain at 21 d of age were not affected by Zn-Met. The addition of 30 or 45 ppm Zn from Zn-Met to a Zn adequate diet significantly increased cutaneous basophil hypersensitivity to phytohemagglutinin-P. The clearance of intravenously administeredS. enteritidis from blood was not affected by 30 ppm of supplemental Zn from Zn-Met. However, 30 ppm Zn from Zn-Met increased the reduction of intravenously administeredS. arizona from spleen. Percentages of myeloid and mononuclear-phagocytic cells before and afterS. enteritidis infection were not affected by supplemental Zn-Met. Turkeys supplemented with Zn-Met showed enhanced in vitro phagocytosis ofS. enteritidis by Sephadex-elicited abdominal exudate cells. The phagocytosis ofS. arizona was unaffected by Zn-Met.