J.A. Ellis

Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies

Five monoclonal antibodies (MAb) produced against cell surface antigens on bovine mononuclear phagocytes (MPh) were characterized. None of the MAb recognized erythrocytes, thrombocytes, B lymphocytes or resting or activated T lymphocytes. Two MAb (IL-A22 and IL-A24) reacted with the majority of monocytes and granulocytes in peripheral blood, with 20-40% bone marrow cells comprising myelo-monocytic cells, and with a proportion of mature macrophages. Reactivity of the remaining three MAb was restricted to MPh: one of these (IL-A25) was apparently specific for pulmonary macrophages, whereas the molecules recognized by the other two (IL-A23 and CH16A) were expressed on subpopulations of blood monocytes and tissue macrophages. None of the MAb inhibited adherence of MPh to plasma-coated gelating surfaces or Fc-mediated rosette formation. One of the MAb, IL-A24, which reacts with MPh and granulocytes, inhibited antigen-specific proliferative response or peripheral blood mononuclear leukocytes (PBM) to the soluble antigen, keyhole limpet hemocyanin (KLH) but did not inhibit responses to concanavalin A or allogeneic leukocytes. This MAb was shown to react with two polypeptides of approximately 75 kD and 110 kD on the surface of peripheral blood monocytes.

Identification and characterization of monoclonal antibodies reactive with bovine, caprine and ovine T-lymphocyte determinants by flow microfluorimetry

Comparative flow microfluorimetric (FMF) analysis was used to identify and characterize 27 monoclonal antibodies (MoAbs) reactive with bovine T-lymphocytes. Determinants present on all circulating T-lymphocytes were recognized by 11 MoAbs, 8 of which blocked E rosette formation. Determinants present on only the BoCD4+ T-lymphocyte subset were detected by 9 MoAbs, while determinants restricted to the BoCD8+ T-lymphocyte subset were recognized by 7 MoAbs. Competitive labeling experiments demonstrated that determinants recognized by subset-specific MoAbs were present on BoCD4 or BoCD8 molecules. Comparative studies revealed that some determinants, both pan-T specific and subset-specific, were conserved on homologous (orthologous) molecules expressed on leukocytes from other species of ruminants. Polymorphism was evident with several determinants.

Capture immunoassay for ruminant tumor necrosis factor-α: comparison with bioassay

Abstract Monoclonal antibodies and IgG purified from rabbit polyclonal antiserum, raised against recombinant bovine tumor necrosis factor-alpha (TNF-α), have been employed in ELISA procedures to quantitate bovine TNF-α. These antibodies were potent in neutralizing the biological activity of recombinant as well as natural bovine TNF-α. The monoclonal antibodies were used as capture antibodies and were either passively adsorbed or covalently linked to ELISA plates. Polyclonal rabbit anti-TNF IgG was used as the detecting antibody in combination with a biotinylated anti-rabbit serum and a streptavidin-horseradish peroxidase conjugate. The detection limit for recombinant TNF-α medium was 10 pg ml−1 and in bovine or ovine serum was 35 pg ml−1. A good correlation was found between the ELISA and the WEHI-164 Clone 13 biologic assay when TNF-α was measured in medium containing serum or in serum. This capture ELISA was also capable of detecting ovine, but not porcine. TNF in supernatants from cultures of lipopolysaccharide-stimulated pulmonary alveolar macrophages.

Cell-mediated immune responses of cattle to Theileria parva.

Theileria parva is a protozoan parasite that infects lymphocytes of cattle and African buffalo. As is the case with certain viruses, the parasite causes antigenic changes on the cell surface against which the host mounts cytotoxic T-cell. Precise definition of the cells participating in these response and their specificity has been facilitated by the recent identification of markers for bovine T-cell subpopulations and functional analyses of bovine lymphocytes at the clonal level. In this paper Ivan Morrison and his colleagues discuss current information on the parasite specificity and MHC restriction of anti-Theileria cytotoxic T cell, in relation to their role in protective immunity.

BOVINE MONONUCLEAR PHAGOCYTIC-CELLS - IDENTIFICATION BY MONOCLONAL-ANTIBODIES AND ANALYSIS OF FUNCTIONAL-PROPERTIES

Monoclonal antibodies (mAb) which react with bovine monocytes have been produced. These include three mAb (P8, IL-A22 and IL-A24) that recognize the majority of monocytes and granulocytes in peripheral blood; two of these mAb were also shown to react with 30-40% of cells in bone marrow, including both monocytic and granulocytic cells, and with variable percentages of tissue macrophages. Thus these mAb can act as markers for myeloid cells in haemopoietic tissues and for monocytes in cell populations devoid of granulocytes. A further two mAb (IL-A23 and IL-A25) recognize monocytes and/or macrophages. The reactivity of one of these mAb (IL-A25) appears to be mainly restricted to pulmonary macrophages. The other mAb reacts with a variable proportion of blood monocytes and generally with a higher percentage of tissue macrophages, suggesting that its expression may relate to activation or maturation of monocytes. In order to study the functional properties of peripheral blood monocytes, techniques were developed for obtaining populations of peripheral blood mononuclear cells (PBM) depleted of monocytes to less than 0.2% and monocyte populations of greater than 97% purity. Removal of monocytes from PBM abrogated the capacity of the cells to proliferate in response to Con A and PBS, although addition of 2-mercaptoethanol to the cultures restored proliferation. In both allogeneic and autologous mixed leukocyte cultures (MLC), monocytes were required in the stimulator cell populations for induction of the proliferative responses, and both responses could be elicited with purified monocytes. However, proliferation in the autologous MLC occurred only with responder cell populations that were depleted of monocytes. Moreover, it was shown that addition of more than 5% unirradiated monocytes to the autologous MLC suppressed proliferation. These findings indicate that monocytes play an important role in the induction and regulation of cellular immune responses in cattle. Two of the mAb that react with monocytes and granulocytes were tested for their capacity to inhibit proliferative responses of PBM to mitogens, alloantigens or the soluble antigen, KLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymphocyte alterations in zinc-deficient calves with lethal trait A46

Lymphocyte numbers and activities were evaluated at 2, 4, 8 and 12 weeks of age in two calves with lethal trait A46 (A46), a genetic disorder affecting intestinal zinc absorption. Plasma zinc concentrations declined to subnormal by 3 weeks of age, after which anorexia, diarrhea, alopecia and hyperkeratosis occurred. Lymphocyte response to phytohemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) stimulation was variably reduced. CD4+ T-lymphocytes were subnormal on at least one observation period following onset of zinc deficiency, and relative numbers of B lymphocytes were decreased at 8 weeks. Secondary antibody responses to bacteriophage phi X 174 were significantly reduced. The results demonstrate that calves homozygous for the A46 trait have normal numbers of functional lymphocyte subpopulations at birth, and that the activity of their lymphocytes is altered once the calves become zinc deficient.

Antigen specificity and activity of ovine antibodies induced by immunization with Corynebacterium pseudotuberculosis culture filtrate.

Sheep were immunized three times with a vaccine composed of filtrate from a 36 h culture of Corynebacterium pseudotuberculosis and a block polymer adjuvant. Immunization resulted in the development of exotoxin-neutralizing antibody. This corresponded to the recognition of a 31.6 kDa protein on sequential immunoblots of ammonium sulfate-precipitated filtrate. In addition sera from vaccinated sheep recognized at least eight bacterial cellular antigens on immunoblots of ether-extracted C. pseudotuberculosis, including bands of 12, 25.1, 31.6, 36.3, 39.8, 63.1, 70, 75 or 79.4 kDa. Sera from these sheep altered the colony growth characteristics of C. pseudotuberculosis in vitro. These results indicate that immunization with soluble C. pseudotuberculosis in vitro. These results indicate that immunization with soluble C. pseudotuberculosis antigen preparations that have been used in toxoid vaccines induces antibody responses to numerous cellular antigens in addition to exotoxin and suggest that serologically mediated antibacterial effects could be an important component in the protection from disease that has been reported following immunization with C. pseudotuberculosis toxoids.

Antigen specificity of antibody responses to Corynebacterium pseudotuberculosis in naturally infected sheep with caseous lymphadenitis

Constituents of Corynebacterium pseudotuberculosis were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of sonicated whole bacterial cells and ether-extracted cells revealed more than 35 bands in silver-stained gels. SDS-PAGE analysis of concentrated culture filtrates with exotoxin activity demonstrated more than 15 bands. Sera from sheep with C. pseudotuberculosis-induced disease of variable severity were used to probe immunoblots of electrophoresed ether-extracted cells and culture filtrates. Twenty or more corynebacterial molecules, ranging in molecular weight from 20 to 112 kDa, in ether-extracted cells were recognized by antibodies in the sera of naturally exposed sheep with positive ELISA titers. These sera also recognized up to six molecules, ranging from, 20 to 68.1 kDa, on immunoblots of ammonium sulfate-concentrated culture filtrate. There was no apparent relationship between the stage of disease and the response to specific corynebacterial antigens in these animals.

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of the specificity and activity of serum antibodies.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and serum antibody responses were analyzed. Compared with preinculation values, at Day 14 after two biweekly immunizations with modified-live or inactivated vaccines there were significant increases in BRSV-specific titers in the sera of cattle that received both types of vaccines, as determined by a whole cell ELISA. Using a blocking ELISA and radioimmune precipitation it was determined that there was recognition of the fusion (F) protein by antibodies from cattle that received both types of BRSV antigens: however, virus neutralization assays revealed that only cattle that received modified live virus, either in monovalent or polyvalent vaccines, developed neutralizing antibodies to BRSV after two immunizations. These results indicate that inactivation of BRSV can lead to a dissociation between serological recognition of the F protein and virus neutralization in vaccinated cattle.

Phenotypic and functional characteristics of bovine T lymphocytes

Monoclonal antibodies have been derived which detect the bovine equivalents of the human pan-T cell marker CD2 and the T lymphocyte subpopulation markers CD4 and CD8. We refer to the bovine analogues as BoT2, BoT4 and BoT8. Monoclonal antibodies have also been derived which detect an antigen(s) with similarities to CD3, although the precise nature of the target molecule(s) in this instance remains to be elucidated. In general there is close similarity between the tissue distributions and, where these have been determined, the molecular masses of the BoT2, BoT4, BoT8 and putative BoT3 entities and their counterparts in other species. BoT2 is expressed on a majority of peripheral blood T lymphocytes and thymocytes and BoT2+ cells are found in both thymic cortex and medulla. In contrast, the putative BoT3 marker is expressed by a minority of thymocytes which are moreover, largely restricted to medulla. Monoclonal antibodies detecting BoT2 determinants have been shown to precipitate 55 kDa molecules. Antibodies to the BoT2 and BoT3 entities have been shown to induce proliferation in peripheral blood mononuclear cells of some cattle, and to be capable of inhibition of antigen-driven proliferative responses and cytolytic function. The BoT4 and BoT8 markers are expressed in a mutually exclusive manner by bovine peripheral blood mononuclear cells but they are coexpressed on a large population of thymocytes. Monoclonal antibodies have been used to precipitate molecules of 52 and 55 kDa in the case of those detecting BoT4 and 34 and 35 kDa in the case of an antibody reactive with a BoT8 determinant. The BoT4 and BoT8 markers have been associated with specificity for, and restriction by, MHC class II and class I molecules respectively.