Claire B. Andreasen

Chicken heterophil extracellular traps (HETs): Novel defense mechanism of chicken heterophils

Recent findings in mammals and fish have revealed that neutrophil nuclear material associated with cytoplasmic granular content is released in the form of neutrophil extracellular traps (NETs) that can trap and kill invading microorganisms in vitro and in vivo. To determine if a similar mechanism is present in chicken heterophils, hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA) were used for stimulation of blood-derived heterophils. Stimulated heterophils produced structures that were characterized using immunocytochemistry and confocal microscopy as heterophil extracellular traps (HETs). Released HETs contained DNA, histone-DNA complex and elastase from heterophil cytoplasmic granules. HETs released from chicken heterophils are structurally similar to NETs found in mammalian and fish neutrophils. Extracellular DNA released from heterophils was quantified by Picogreen assay. Stimulation with PMA or H(2)O(2) significantly increased the HET-DNA release index in vitro compared to non-stimulated heterophils (1.11+/-0.04 and 1.55+/-0.10, respectively), and H(2)O(2) stimulation induced significantly higher HET-DNA release than PMA (P<0.001). Thus, HETs are now characterized as an important heterophil-mediated defense mechanism in chickens.

Chicken heterophils from commercially selected and non-selected genetic lines express cytokines differently after in vitro exposure to Salmonella enteritidis.

Resistance to pathogens such as Salmonella enteritidis (SE) is a heritable trait important in maintaining the health of chickens and reducing bacterial contamination of poultry products. In chickens, heterophils act as the first responders to bacterial infections and are, therefore, responsible for initiating the immune response against SE challenge. This study measured mRNA expression of several immune response genes [interleukin-6 (IL-6), IL-10, transforming growth factor-beta4 (TGF-beta4), granulocyte macrophage-colony stimulating factor (GM-CSF), and Toll-like receptor-4 (TLR-4)] by heterophils from broiler, Leghorn, and Fayoumi chickens, either non-stimulated or stimulated in vitro with SE using quantitative reverse-transcriptase PCR. We found that heterophils of commercially selected broiler and Leghorn birds had differing early heterophil responses to SE in comparison with the native Fayoumi line. Heterophil stimulation with SE in vitro increased expression of pro- (IL-6 and GM-CSF) and anti-inflammatory cytokine mRNA (IL-10 and TGF-beta4) in the Fayoumi line, while the broiler and Leghorn line heterophils had decreased or no changes in the cytokine gene expression levels. The unique response of the Fayoumi line is in contrast to the lines with a history of genetic selection to increase growth or reproduction, a process which may favor reduced or suppressed inflammatory responses. The findings illustrate the potential value of native lines to provide biodiversity to enhance innate health in commercially selected poultry.

Separation of Avian Heterophils from Blood Using Ficoll-Hypaque Discontinuous Gradients

Rapid separation of avian heterophils from anticoagulated whole blood was achieved using Ficoll-Hypaque discontinuous gradients. An average of 14.4% of blood heterophils was harvested with a mean purity exceeding 99%. Heterophil viability, as determined by trypan blue dye exclusion, averaged 99.8%. The integrity of isolated heterophils was evaluated by cytochemical staining and ultrastructural examination. Cytochemical staining reactions of heterophils in whole blood and of isolated cell suspensions were similar. No ultrastructural abnormalities were observed. Using this procedure, viable intact heterophils were rapidly isolated from blood with an acceptable cell yield and purity for cell function studies.

Differential splenic cytokine responses to dietary immune modulation by diverse chicken lines

Nutritional modulation of the immune system is an often exploited but poorly characterized process. In chickens and other food production animals, dietary enhancement of the immune response is an attractive alternative to antimicrobial use. A yeast cell wall component, beta-1,3/1,6-glucan, augments the response to disease in poultry and other species; however, the mechanism of action is not clear. Ascorbic acid and corticosterone are better characterized immunomodulators. In chickens, the spleen acts both as reservoir and activation site for leukocytes and, therefore, splenic gene expression reflects systemic immune function. To determine effects of genetic line and dietary immunomodulators, chickens of outbred broiler and inbred Leghorn and Fayoumi lines were fed either a basal diet or an experimental diet containing beta-glucans, ascorbic acid, or corticosterone from 56 to 77 d of age. Spleens were harvested, mRNA was isolated, and expression of interleukin (IL)-4, IL-6, IL-18, macrophage inflammatory protein-1beta, interferon-gamma, and phosphoinositide 3-kinase p110gamma transcripts was measured by quantitative reverse transcription PCR. Effects of diet, genetic line, sex, and diet x genetic line interaction on weight gain and gene expression were analyzed. At 1, 2, and 3 wk after starting the diet treatments, birds fed the corticosterone diet had gained less weight compared with birds fed the other diets (P < 0.001). Sex affected expression of IL-18 (P = 0.010), with higher levels in males. There was a significant interaction between genetic line and diet on expression of IL-4, IL-6, and IL-18 (P = 0.021, 0.006, and 0.026, respectively). Broiler line gene expression did not change in response to the experimental diet. Splenic expression of IL-6 was higher in Leghorns fed the basal or ascorbic acid diets, rather than the beta-glucan or corticosterone diets, whereas the opposite relationship was observed in the Fayoumi line. Expression of IL-4 and IL-18 responded to diet only within the Fayoumi line. The differential splenic expression of birds from diverse genetic lines in response to nutritional immunomodulation emphasizes the need for further study of this process.

Immunohistochemical Demonstration of Desmin in Canine Smooth Muscle Tumors

Sections of formalin-fixed, paraffin-embedded canine leiomyomas, leiomyosarcomas, or fibrosarcomas were examined by immunohistochemical methods for the presence of desmin. Twenty-two leiomyomas and leiomyosarcomas were stained using the avidin-biotin complex technique, and 14 samples demonstrated positive staining for desmin. The eight negative results obtained may reflect differences in fixation or the affinity of the primary antibody for the tissues examined. Desmin was specific for myogenic tissues. Five canine fibrosarcomas examined immunohistochemically were all negative for desmin staining. The results indicate that desmin is a useful marker for immunohistochemical identification of canine leiomyomas and leiomyosarcomas.

Desmin as a marker for canine botryoid rhabdomyosarcomas

The intermediate filament desmin was present in three canine botryoid rhabdomyosarcomas. The use of desmin as a diagnostic tool may, as in these tumours in man, be of value in the classification of canine sarcomas where the origin of the tumour is not always apparent from routine histological sections.

Genetic control of chicken heterophil function in advanced intercross lines: associations with novel and with known Salmonella resistance loci and a likely mechanism for cell death in extracellular trap production

Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F13 advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease.

Determination of Chicken and Turkey Plasma and Serum Protein Concentrations by Refractometry and the Biuret Method

Plasma and serum protein concentrations were determined in chickens and turkeys by refractometry (with human and veterinary refractometers) and by the biuret method. Chicken and turkey serum protein values were significantly lower than respective plasma protein values according to both methods. Refractometer readings for both plasma and serum correlated closely with the results of the biuret test (r2 = 0.72 to 0.97). These findings indicate that plasma and serum protein values may be determined accurately in chickens and turkeys with a handheld refractometer.

The Effects of Ascorbic Acid on In Vitro Heterophil Function

As a feed additive, ascorbic acid has been shown to have a protective effect against bacterial and viral diseases and to reduce the impact of detrimental stress in chickens. This study examined the effect of ascorbic acid treatment on in vitro heterophil function by examining random migration and phagocytosis and bacterial killing of Staphylococcus aureus. Heterophils were evaluated in broiler chickens ranging from 5 to 16 wk of age, and age differences were seen. Significant increases in bacterial killing were found in heterophils treated with ascorbic acid, and this difference tended to be greater in chickens from 5 to 10.5 wk of age. No significant differences were found in phagocytosis or random migration, but ascorbic acid tended to decrease random migration. The most significant effect on in vitro heterophil function was an increase in bacterial killing.

Heterophil Chemotaxis in Chickens with Natural Staphylococcal Infections

Heterophil chemotaxis using heterophils isolated from the peripheral blood of five commercial broiler chickens naturally infected with staphylococcal bacteria was compared by the modified Boyden-chamber technique with chemotaxis of heterophils from two chickens from the same flock not infected with Staphylococcus (field controls) and from four healthy laboratory control broiler chickens. The infected chickens had gross and histologic lesions of staphylococcal tenosynovitis and osteomyelitis. Staphylococci were isolated from the lesions. Hematologic parameters and histologic lesions of infected chickens also were examined. Compared with field and laboratory controls, Staphylococcus-infected chickens had heterophilic leukocytosis. The heterophils of Staphylococcus-infected chickens had significantly lower chemotactic activity than both control groups in terms of random movement and directed chemotactic movement in response to stimulus. Toxic changes were observed in heterophils of some of the Staphylococcus-infected broilers.