Avian Lymphocytes Research Articles

Exploring the immunomodulatory and antioxidant capacities of Berberis aristata in avian lymphocytes

India is one of the leading producers for eggs and broilers globally and thus has witnessed significant growth in the poultry sector. Different poultry stressors can negatively impact the growth, gut health, immune function and production reproductive performances of poultry. Antibiotic growth promoters (AGPs) have been used in the poultry industry as they also combat stress in poultry along with growth promotion. But the use of antimicrobials as growth promoter raises the concern of antibiotic resistance and its potential hazardous effect on human health as well as the environment. Several other alternatives are available for AGPs such as acidifiers, probiotics, prebiotics, antibacterial peptides and phytobiotics. Phytobiotics are known to possess several benefits for poultry as well as livestock such as immunomodulation, anti-inflammatory effect, antioxidant effect, maintain intestinal health etc. One such important plant is Berberis aristata belonging to the family Berberidaceae which is used in traditional system of medicine for the management of various inflammatory disorders. In the present study aqueous root extract of Berberis aristata (BAE) was evaluated for its immunomodulatory and antioxidant activity. The extraction yield of the roots of Berberis aristata in water was about 9.78%. The BAE showed presence of phenolics, flavonoids, tannins, terpenoids and contained about 104.97 mg/gm of BAE and 83.14 mg/gm of BAE of phenolics and flavonoids, respectively. BAE showed significant antioxidant activity in DPPH free radical scavenging assay. Maximum non-cytotoxic dose (MNCD) of BAE for lymphocyte culture was determined to be 600 µg/ ml. Lymphocytes proliferation assay (LPA) showed that BAE enhanced lymphocyte proliferation of B-cells stimulated by lipopolysaccharide (LPS) and T-cells stimulated by Concanavalin A and Phytohaemagglutinin M (PHA). Treatment with BAE significantly reduced lipid peroxidation (LPO) and increased the level of reduced glutathione, superoxide dismutase, and catalase in chicken lymphocytes, thus indicating antioxidant potential of BAE. The study reveals significant immunomodulatory and antioxidant properties of BAE, thus suggesting that it could be a potential alternative to AGPs in poultry production. But further in vivo studies are required to explore its efficacy and safety for its application in commercial poultry farming.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed, paraffin-embedded tissues of 53 avian species using commercial antibodies.

Providing a method to detect avian lymphocytes by immunohistochemistry (IHC) would be helpful for analyzing immune function and diagnosing diseases in birds. In this study, we comprehensively examined the immunohistochemical identification of avian T and B lymphocytes in formalin-fixed, paraffin-embedded tissues from 53 avian species across 15 orders, using eight commercially available lymphocyte markers. T lymphocytes from all 53 avian species tested were specifically detected by IHC using the anti-CD3 antibody (clone F7.2.38). The appropriate antibody for detecting avian B lymphocytes in IHC varied depending on the avian species. B lymphocytes were specifically labeled by IHC in 46 of 53 avian species (86.8%) using any of seven B cell markers. The anti-PAX5 antibody (clone SP34) immunohistochemically detected B lymphocytes from the majority of avian species (41 out of 53 species), excluding those in the orders Falconiformes (falcons) and Passeriformes (oscines). The anti-BAFF-R antibody (clone 2C4) proved suitable for detecting B lymphocytes in the orders Galliformes (landfowls) and Anseriformes (waterfowls) in IHC. Caution is advised when using the anti-BLA36 (clone A27-42) and two anti-CD20 (clone L26 and product No. PA5-16701) antibodies, which are commonly used as B cell markers in mammals, for detecting avian B lymphocytes. These antibodies reacted with cells located in both T and B cell areas in certain avian species. The anti-Bu-1a/b (clone AV20) and anti-CD79a (clone HM57) antibodies were found not to bind to B lymphocytes in various avian species in IHC.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed paraffin-embedded chicken tissues using commercial antibodies

Immunohistochemical method to detect avian lymphocytes is an efficient and reliable tool for accurate diagnosis, and immunological analysis of avian diseases. However, there are scarce studies reporting immunohistochemistry (IHC) using commercially available antibodies against lymphocytes in formalin-fixed paraffin-embedded (FFPE) chicken tissues. In the present study, we established an immunohistochemical method to identify chicken T and B lymphocytes in FFPE chicken tissues using commercial antibodies against chicken or human antigens. For this IHC method, the five tested anti-T lymphocyte antibodies reacted with chicken T lymphocytes on the FFPE sections. Further, 10 commercial anti-B lymphocyte antibodies were tested; of these, three successfully detected chicken B lymphocytes for IHC. In particular, anti-human CD3 (clone F7.2.38) antibody was most suitable for the detection of chicken T lymphocytes, whereas anti-chicken B cell activating factor receptor (BAFF-R) antibody (clone 2C4) was most suitable for the detection of chicken B lymphocytes under our IHC staining conditions. These two antibodies reacted with numerous lymphocytes of all representative lymphoid tissues without problematic background staining and nonspecific reactions. Our results indicate that T and B lymphocytes in FFPE chicken tissues can be immunohistochemically detected using commercial antibodies.

Evaluation of Immunotoxicity and Oxidative Stress due to in vitro Exposure of Dimethoate in Chicken Lymphocytes

Pesticide hazards have been amplified by the sharp rise in their agricultural, industrial and domestic use. Though there is considerable contribution of pesticides in various agriculture, domestic and industrial applications; however, continuous exposure of pesticides even at low dose levels can exert adverse effects on mammalian immune system. Dimethoate is an insecticide and acaricide of moderate mammalian toxicity which is used in housefly control and against a broad range of agricultural insect and mite pests. Present communication reports dimethoate induced immunotoxic effects due to enhanced oxidative stress and down regulation of interleukin (IL) levels in avian lymphocyte cell culture system employing lymphocyte proliferation assay, cytokine assay and oxidative stress assay. There was a marked reduction in B and T cells proliferation in avian lymphocytes due to the exposure of thousand times dilution of No Observable Effect Level (NOEL/103) dose of dimethoate. Cytokine assay revealed significant reduction in IL-1 and IL-2 levels. There was a significant increase in oxidative stress in dimethoate treated cells in comparison with controls cells as revealed by nitric oxide estimation. Thus, from our findings, it is revealed that in vitro exposure of low dose of dimethoate induces significant immunotoxic effects and higher oxidative stress in avian lymphocytes.

Interactive mechanism between avian infectious bronchitis S1 protein T cell peptide and avian MHC I molecule

This study aims to construct a 3D structure of the avian major histocompatibility complex (MHC)–β2M complex through homology modelling technology, perform molecular docking of the predicted infectious bronchitis virus (IBV) S1 protein potential epitope peptide Sp6 (NQFYIKLT) and the avian MHC–β2M complex, and demonstrate the interactive mechanism between Sp6 and MHC using molecular dynamical simulations. The peptide Sp6 and the non-related peptide NP89–97 (PKKTGGPIY) were used to stimulate in vitro recombinant plasmid (pCAGGS-S1) avian splenic lymphocytes. Flow cytometric results show that CD8+ T lymphocytes reproduce stimulated by the Sp6 and the nonrelated peptide proliferate by 34.8% and 2.6%, respectively. Meanwhile, fluorescent quantitative PCR results show that the secretion of IFN-γ in avian splenic lymphocytes increases after Sp6 stimulation. These data suggest that Sp6 can induce the activated avian lymphocytes in vitro to produce CTL, which is the CTL epitope in IBV S1.

Study on T and B-cell proliferation induced by lindane and quinalphos insecticides underin-vitrocondition in avian lymphocytes

Lindane and quinalphos insecticides were evaluated in avian lymphocytes for in-vitro effect on T and B-cell proliferation. The lymphocytes were treated with NOEL/102, NOEL/103, NOEL/104, NOEL/105, NOEL/106 and NOEL/107 dose of insecticides for 30, 60 and 90 min and a group of cells were kept untreated control. The cells were collected for detection of T and B- lymphocyte proliferation. The proliferation of T- and B-lymphocytes was markedly reduced in insecticide treated groups in comparison to control indicating the down regulation of not only the number of the variable lymphocytes but also their proliferative activity required to mount immune response in birds. Results showed that insecticides even in very low doses can reduce proliferation of avian lymphocytes leading to a state of immunosuppression.

Detection of cell-mediated immune response to avian influenza viruses.

The measurement of cell-mediated immunity (CMI) is critical to understanding the role and regulation of avian lymphocytes following avian influenza virus (AIV) infection. While these different cell types have distinctly different modes of action in terms of contributions to protection, they account for the majority of adaptive immunity induced following infection or vaccination. Although the ability to measure CMI has steadily improved over the last few years, few studies have examined its role in protection of birds against AIV. The increasing availability of monoclonal antibodies recognizing various avian cell-associated antigens has made this technique more specific and informative.

Viability and Function in Lymphocytes Cultured from the Horse, Chicken, and Mouse: Effects of Different Leukocyte Enrichment Techniques

Methods of lymphocyte enrichment tend to vary across species, with the most common techniques employed being density-gradient separation and erythrocyte lysis buffer enrichment. In this study, we assessed lymphocyte viability and proliferation of avian, equine, and murine lymphocytes enriched by a commercial density-gradient technique and under identical, standardized culture conditions. The results of this study clearly show that, under identical enrichment and culture conditions, lymphocyte viability and function can be quite different among the equine, bird, and mouse species. Secondly, the type of enrichment technique employed in the mouse can impact the quality of the immune data generated.

Nucleic acid distribution pattern in avian erythrocytes and mammalian lymphocytes: Comparative studies by fluorescence microscopy and digital imaging analytical techniques

Nucleated erythrocytes of healthy domestic chicken and ducks, and lymphocytes of healthy Sprague Dawley rats were evaluated for nucleic acid distribution pattern, employing light and fluorescence microscopy procedures, as well as digital imaging analytical methods. The results demonstrate a unique organization of nuclear DNA of mature chicken and duck erythrocytes, as well as immature duck erythrocytes, as delineated spherical nuclear bodies that mostly corresponded with euchromatin zones of the cells in routine Wright-stain blood smears. The nuclear DNA of the rat lymphocytes, on the other hand, was observed as a more diffuse green fluorescing nuclear areas, with punctate variably-sized diffuse areas of RNA red fluorescence. RNA red color fluorescence was also evident in the narrow cytoplasm of the lymphocytes, especially in large lymphocytes, in comparison with the cytoplasm of the mature avian erythrocytes that completely lacked any nucleic acid fluorescence. Nuclear RNA fluorescence was lacking in the mature chicken erythrocytes, compared with those of the mature and immature duck erythrocytes as well as lymphocytes of both avian and rats blood. The significance of these findings lies in the establishment of normal benchmarks for the nuclear and cytoplasmic nucleic acid pattern in eukaryotic cells. These normal benchmarks become valuable in rapid diagnostic situations associated with pathologies, such as the presence of viral nuclear and cytoplasmic inclusion bodies that can alter the nucleic acid pattern of the host cells, and in conditions of cellular abnormal protein aggregations. Variability of cellular nucleic acid pattern can also aid in prognostic assessments of neoplastic conditions.

Modulatory effects of several herbal extracts on avian peripheral blood cell immune responses

Standardized ethanol extracts of Allium sativum (garlic), Glycyrrhiza glabra (licorice), Plantago major (plantain) and Hippophae rhamnoides (sea buckthorn) were assessed for their effects on cellular immunity in laying hens. Birds (n = 25) had blood samples taken and both specific and non-specific immune cell responsiveness were evaluated by a leukocyte proliferation assay, carbon clearance test and SRBC phagocytosis in monocyte-derived macrophage cultures. Licorice and sea buckthorn (50 microg/mL) clearly enhanced the macrophage membrane function (p < 0.05 and p < 0.01, respectively). Dual effects on circulating phagocytes were revealed for plantain and sea buckthorn, while garlic at 200 microg/mL impaired the phagocytic capacity of blood cells. None of the tested extracts showed mitogenic properties, but high concentrations of sea buckthorn (400 microg/mL) inhibited leukocyte proliferation. Small concentrations (20 microg/mL) of licorice proved the co-mitogenic potential for both T and B avian lymphocytes (p < 0.05). Certain extracts definitely enhanced the fowl innate and/or specific cell immunity and may therefore improve host resistance in poultry. Considering the chicken as an important non-mammalian model that also serves as an available laboratory approach for some human diseases, herbs exerting immunomodulatory properties may find relevant clinical applications.

Use of Avian Lymphocytes to Detect Toxicity: Effects of a Commonly Utilized Deltamethrin Preparation

In building upon the increasing utility of avian immunology as an alternative to the use of mammalian systems for assessing immunotoxic potentials of environmentally and/or occupationally encountered chemicals, in vitro studies were performed to determine if easily obtainable freshly harvested cells from White Leghorn chickens could be used to detect effects on a critical immune cell type by any given toxicant. These studies also sought to determine if these cells could also potentially provide a vehicle to elucidate potential mechanisms underlying effects previously described for the test agent in in vivo investigations. Here, specifically, the toxic effect (and thus, ultimately, the potential to be an immunotoxicant in situ) of a commonly used commercially available form of deltamethrin on Leghorn lymphocytes was examined. The toxicity of this synthetic pyrethroid was evaluated by assaying both lymphoproliferation and apoptosis after exposure. A significant (p < 0.05) inhibitory effect of deltamethrin on mitogen-stimulated lymphocytes was observed at 10− 5 M. Apoptosis was detected using DNA ladder analyses; annexin-V binding assays incorporating an immunoperoxidase technique were used to identify translocated phosphatidylserine; and, electron microscopy, including both scanning and transmission types, were used to detect ultrastructural changes (e.g., chromatin condensation, membrane blebbing and phagocytosis) in deltamethrin-treated cells. Our results indicated that deltamethrin, even at a very low dose, could give rise to potential immunotoxicities in situ by inducing toxicity (i.e., apoptosis) in immunogenic cells. Our results also show that this particular in vitro system may be used as an alternative to standard laboratory animals for the performance of immunotoxicity experiments involving pesticides.

The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes

Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4 + and CD8 + T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4 + and CD8 + T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.

Induction of telomerase activity in avian lymphoblastoid cell line transformed by Marek's disease virus, MDCC-MSB1.

Telomerase has been studied extensively in human and murine tumors, but little is known about the role of telomerase in the tumor biology of other vertebrate species such as the chicken. We studied the telomerase activity of the lymphoblastoid cell line derived from lymphomas induced by Marek's disease virus (MDCC-MSB1) compared with another avian cell line (PA5) and peripheral blood lymphocytes (PBL) using the telomeric repeat amplification protocol (TRAP) Assay. Telomerase activity in MDCC-MSB1 was 4.5 times greater than in the PA5 cell line and normal avian lymphocytes. These results demonstrate for the first time that telomerase is more intense in one transformed cell line than in normal cells, suggesting a potential role for telomerase in carcinogenesis induced by an avian virus.

Molecular cloning and characterization of the mouse thymocyte protein gene

Chicken thymocyte protein (cThy28) has recently been identified and implicated to be involved in apoptosis of avian lymphocytes, while its functional role remains undefined. To elucidate the role of Thy28, we have molecularly cloned the mouse Thy28 ( mThy28) cDNA and clarified its genomic organization in the present study. The mThy28 cDNA encodes a 226 amino acid protein, whose sequence is highly conserved among bacteria, yeast and plants as well as vertebrates. Northern blot analysis revealed abundant expression of approximately 1 kb mRNA in testis, liver, brain and kidney with lower levels of the expression in thymus, spleen, heart and stomach. The mThy28 gene spans at least 7 kb of genomic DNA, and contains eight exons and seven introns. The 5′-flanking region of the mThy28 gene does not contain a typical TATA box, but contains a putative CCAAT box and presumably multiple transcription initiation sites. Potential Sp1, GATA-1, -2, -3, AP-1, and p300 binding sites were found in the 5′-flanking region of the mThy28 gene. The gene was mapped to mouse chromosome 9.

The analysis of cThy28 expression in avian lymphocytes.

During the course of a screening procedure designed to isolate gene products that mediate avian lymphocyte apoptosis, a 1070 bp cDNA encoding a 242 amino acid protein, cThy28 (GenBank accession number U34350), was isolated. Based on the deduced amino acid sequence, cThy28 is a conserved protein that shares greater than 90% amino acid similarity with several putative mammalian homologues. A structural analysis of the protein suggests that is a nuclear-localized, phosphoprotein with potential glycosylation and myristolation sites. Compared to other non-lymphoid tissues, the cThy28 protein and its transcript are present in immune organs at elevated levels. When the expression of cThy28 was analyzed in cultured bursal lymphocytes undergoing apoptosis, the cellular levels of this protein and its transcript declined and underwent degradative processes as a function of the apoptotic process. Treatment of lymphocytes with phorbol esters inhibited apoptosis and protected the cellular levels of the cThy28 protein and its transcript from undergoing this degradative process; however, analysis of transcripts of a "housekeeping" gene, GAPDH (glyceraldehyde phosphate dehydrogenase), revealed a similar response to this apoptosis treatment regimen. Thus, the apoptotic-mediated alterations in cThy28 expression and its role in lymphocyte function remain undefined; however, the potential task of this gene in apoptosis may be related to the putative functions of the cThy28 mammalian homologues identified in this report.

Chemotactic activities of avian lymphocytes

Chicken lymphocytes, enriched chicken T and B lymphocytes, and a turkey B-lymphoblastoid cell line, the RP-9 cells, are used in the studies of chemotaxis in a Boyden-type chamber assay. The chemoattractants used are lipopolysaccharide, fMLP, interleukin-8, MIP1-beta, rabbit anti-chicken IgG and IgM. The results indicate that all these cells can migrate into the polycarbonate membranes in the absence of chemoattractants. When the chemoattractants are present, the numbers of migrating cells are greatly increased. It is, therefore, concluded that avian lymphocytes have the ability to migrate, and can respond to chemical signals which result in chemotaxis and accumulation of lymphocytes at the sites where the signals originate.

A threshold nuclear level of the v-Rel oncoprotein is required for transformation of avian lymphocytes.

The net distribution of eukaryotic transcription factors between the cytoplasm and the nucleus provides an effective mechanism for controlling gene expression. We have utilized cis-acting signals for both nuclear import and nuclear export to experimentally manipulate the distribution of the v-Rel oncoprotein between the nucleus and the cytoplasm. The respective abilities of the v-Rel oncoprotein to localize to the nucleus in chicken embryo fibroblasts, to activate kappaB-dependent transcription in yeast, and to transform avian lymphoid cells were each markedly reduced by the fusion of a cis-acting nuclear export signal onto v-Rel. Our results demonstrate that a threshold nuclear function of v-Rel is required for manifestation of its oncogenic properties. In contrast, while increased expression of the avian IkappaB-alpha protein was able to prevent nuclear localization of v-Rel in chicken embryo fibroblasts, coexpression of IkappaB-alpha with v-Rel in the target cell for v-Rel mediated transformation did not reduce the ability of v-Rel to transform avian lymphoid cells or alter the distribution of v-Rel between the nucleus and the cytoplasm in v-Rel-transformed cells. Our results suggest that the ability of IkappaB-alpha to inhibit nuclear localization of v-Rel is affected by cell-type specific differences between fibroblasts and lymphoid cells.

Apoptosis induced by infectious bursal disease virus

Chicken peripheral blood lymphocytes (PBLs) show morphological and biochemical features of apoptosis (programmed cell death) when infected in vitro with infectious bursal disease virus (IBDV). DNA extracted from IBDV-infected lymphocytes displayed an intense laddering pattern when visualized after agarose gel electrophoresis. IBDV-infected PBLs had significantly higher apoptotic and necrotic indices measured by acridine orange-ethidium bromide staining than did control lymphocytes. Electron micrographs of the IBDV-infected PBLs revealed features typical of apoptosis such as peripheral condensation of chromatin, blebbing of the plasma membrane and fragmentation of the nucleus and of the cell leading to the formation of apoptotic bodies. These findings indicate that IBDV, in addition to causing necrosis, can also induce apoptosis in avian lymphocytes in vitro.

Pathology of lymphoid organs with emphasis on immunosuppression

A review of the literature is presented to include recent diagnostic and research histopathology accessions from poultry produced on the Delmarva peninsula region of the U.S.A. Emphasis is placed on immunosuppression (IS) and pathology of primary lymphoid organs of the chicken. The evolutionary relationship of the avian and mammalian lymphoid systems is based on a common reptilian past with 160 million years of evolutionary dichotomy. Four methods used to assess IS anatomically include: (1) recognition of marked reduction of lymphocytes in both primary and secondary lymphoid organs; (2) recognition of diseases commonly associated with IS; (3) recognition of known immunosuppressive diseases; (4) germinal center enumeration. An experimental method of qualifying and quantifying IS related to single or multiple insults is explored through the use of germinal center enumeration. The effect of stress and inanition on avian lymphocytes must be considered in histological evaluations as chickens are a steroid sensitive species.

Size and frequency characteristics of αβ and γδ T cells in the spleens of normal and cyclophosphamide-suppressed virus-infected chickens

The characteristics of avian lymphocytes expressing surface CD8 (CT8) and T cell receptor (TCR) glycoproteins have been monitored by two-color flow microfluorimetry. Exposure of 1-month-old birds to a lethal influenza A virus, which is known to be lympholytic, significantly decreased the frequency of both the αβTCR2+CT8+ and γδTCR1+CT8- subsets in spleen. However, all categories of T cells showed evidence of greater mean cell size, indicating that they are responding. Inoculation of baby chicks with fowl pox virus induced a response more typical of specific immunity in the TCR2+CT8+ set, in that the lymphocytes increased in both frequency and mean cell size. Greater numbers of lymphoblasts were also found for the TCR2+CT8-, TCR1+CT8+, and TCR1+CT8- subsets, but the total cell counts for the minority TCR1+CT8- cells in spleen were consistently decreased. Immunosuppression with cyclophosphamide prior to infection eliminated 90% of the white blood cells from spleen, with the greatest effect being on the TCR1+ populations. The CT8+ αβ T cell response in chick spleen following exposure to a poxvirus is thus comparable to the situation observed for this subset of lymphocytes in mice infected with other viruses. However, although the γδ T cells increase in size, their frequency in spleen either does not change (CT8+) or is significantly decreased (CT8-).