Association of tumor-associated antigen on the proliferation of bovine leukemia virus-infected lymphoblastoid B-cell lines.

The association of tumor-associated antigen (TAA) on the proliferation of BLV-infected lymphoblastoid B-cell lines (BL2M3 and BL312) was investigated. Flow cytometric analysis of the expression of TAA with monoclonal antibody (mAb) c143 showed high expression of TAA on the surfaces of BL2M3 and BL312 cells. A large amount of TAA was found in the culture supernatant of BL2M3 and BL312 cells as well as in the lysates of BL2M3 and BL312 cells. Culture supernatant but not lysates of BL2M3 and BL312 cells promoted the growth of either BL2M3 cells or BL312 cells. Furthermore, this growth promoting activity in culture supernatants of BL2M3 and BL312 cells was inhibited in a dose-dependent manner when cultured with mAb c143. These results suggested that TAA may be involved in the growth factor-mediated cell growth of bovine B-lymphoblastoid cell lines expressing TAA on their cell surface.     

Bovine costimulator. II. Generation and maintenance of a bovine costimulator-dependent bovine lymphoblastoid cell line

Bovine peripheral blood leukocytes, activated with conconavalin A, were cultured in bovine costimulator-containing conditioned medium prepared in a totally defined, serum-free medium. A population of leukocytes subsequently grew exponentially. These bovine cells had the morphology of lymphoblasts, were negative for chloroacetate esterase, slightly positive for nonspecific esterases, and highly peanut agglutinin-positve. These data suggested that the bovine leukocytes were of the T-cell lineage and that the active factor in the costimulator-containing conditioned medium might be the bovine equivalent of Interleukin 2. A quantitative microassay, subsequently developed, revealed that the lymphoblastoid cell line was costimulator-dependent and lectin-independent. Further utilization of the microassay supported this contention and strengthened the concept of a bovine Interleukin 2-dependent bovine T-cell line: Phytohemagglutin-M, phytohemagglutinin-P, and concanovalin A induced active factor from peripheral blood leukocytes, while lipopolysaccharide, a potent inducer of Interleukin 1 in other systems, failed to induce activity; and both T-cells and macrophages were required for optimal factor activity. Finally, a means by which to optimize production of the active moiety, utilizing lymph node cells, as opposed to peripheral blood leukocytes, was examined.     

Levamisole does not affect the virological and serological responses of bovine leukemia virus-infected cattle and sheep.

Levamisole, a compound that has been used widely as an anthelmintic in man and domestic animals, has also been found to be an immunomodulator. It was, thus, of interest to determine whether treatment with levamisole would affect bovine leukemia virus infections in cattle and sheep or the results of serological and virological tests routinely used to identify infected animals. Studies of cattle and sheep given either the recommended anthelmintic dose of levamisole or repeated larger doses of the drug failed to provide evidence of significant changes in antibody titer or virus replication. It is, therefore, concluded that levamisole neither potentiated nor repressed bovine leukemia virus replication or the associated immunological responses.     

Tumor necrosis factor alpha and its receptors in experimentally bovine leukemia virus-infected sheep

To examine whether tumor necrosis factor alpha (TNF alpha) contributes to the pathogenesis of bovine leukemia virus (BLV) infection, the mRNA expression patterns of TNF alpha and its receptors, type 1 (TNF R1) and type 2 (TNF R2) were investigated. Sheep inoculated with BLV were divided into two groups; one was BLV-positive and the other BLV-negative based on the detection in peripheral blood mononuclear cells (PBMC). Expression of TNF R1 mRNA was down-regulated in PBMC from the BLV-positive compared to BLV-negative sheep. No difference was shown in the expression levels of TNF R2 mRNA between the two groups. Furthermore, proliferative responses of PBMC in the presence of TNF alpha were observed from the BLV-positive, but not BLV-negative sheep. Membrane-bound TNF alpha (mTNF alpha) is thought to be one of the ligands, inducing B-cell activation. Flow cytometric analysis demonstrated that the number of PBMC, that were positive for mTNF alpha expression, was increased in the BLV-positive sheep. Thus, the expression of TNF alpha and its receptors may be closely associated with lymphocytosis induced by BLV.     

Effects of Pasteurella haemolytica leukotoxic culture supernatant on bovine neutrophil aggregation.

Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to cause aggregation of bovine peripheral neutrophils. Neutrophils were isolated by a hypotonic lysis method and incubated with zymosan-activated plasma (ZAP), leukotoxic culture supernatant, antileukotoxin serum, calcium and magnesium-free media, p-bromophenacyl bromide and protein kinase C inhibitors. Aggregation was evaluated by changes in infrared light transmittance. Leukotoxic culture supernatant caused neutrophils to aggregate, and this effect was significantly removed by preincubation with antileukotoxin serum. Aggregation to ZAP and leukotoxin was dependent on the presence of extra-cellular calcium. Activation of protein kinase C by phorbol myristate acetate induced aggregation which was reduced by staurosporine; however, aggregation to leukotoxin did not involve protein kinase C activation. Phospholipase A2 inhibition did not alter the aggregation response to ZAP or to leukotoxin. The in vitro measurement of neutrophil aggregation induced by the leukotoxin of P. haemolytica reflects cytoskeletal and other activation events that may contribute to the intense inflammatory process which this organism induces in the lungs of cattle.     

Selected phenotypic and cloning properties of a bovine lymphoblastoid cell line, BL20

A bovine lymphoblastoid cell line, BL20, was shown to express a BoLA antigen and surface IgM, implying that it was probably of B-cell origin. At low density (less than 10(5)/ml), the cells failed to grow. Inclusion of growth factor-containing supernatants from concanavalin A or pokeweed mitogen (PWM)-activated bovine lymphocytes or from thymus fibroblast-like cells did not improve cloning of the cells. Feeder cells also did not enhance cloning of the cells except for bovine thymus fibroblast-like cell monolayer.     

Development of a cell culture line of bovine fetal endometrial cells.

Monolayers of bovine fetal endometrial cells were established as primary culture cells within 1 to 2 weeks. After the 2nd passage, these cells were inoculated with bovine viral diarrhea virus. Effects of the virus were observed each day with a light microscope. Specific cytopathic effects consisting of degeneration and sloughing of the cells and a well-defined pattern of cytoplasmic vacuolation were observed at 5 days after inoculation.     

Identification of two independent MHC class II antigens in a bovine lymphoblastoid cell line

Bovine major histocompatibility complex (MHC) class II antigens were investigated using monoclonal antibodies (MoAbs) with known MHC class II specificities in other species. Thirty-four MoAbs were tested for reactivity with bovine peripheral blood mononuclear (PBM) cells and the bovine lymphoblastoid cell line, BL3, by flow cytometry. Twenty-seven of 31 MoAbs tested, reacted with BL3 cells, and 22 of 25 MoAbs tested with PBM cells were reactive. MoAbs that reacted with BL3 cells were used to immunoprecipitate class II molecules from BL3 lysate labeled with [35S]methionine. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, many MoAbs were found to immunoprecipitate a single band of approximately 31,000 relative mass (Mr). MoAbs yielding successful immunoprecipitations and with known antigen specificity in other species were then used in sequential immunoprecipitations and two dimensional (2-D) non-equilibrium pH gradient electrophoresis (NEPHGE). The HLA-DR specific MoAb H4 and the predominantly HLA-DQ specific MoAb CC11.23 were used to identify the presence of two independent antigens in BL3 cell lysate. These class II molecules consist of alpha and beta chains.     

Spontaneous and mitogen-induced RNA synthesis by blood lymphocytes from bovine leukemia virus-infected and normal cows

Peripheral blood lymphocytes (PBL) from normal and bovine leukemia virus (BLV)-infected cattle were prepared by density gradient technique and incubated with and without phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). RNA synthesis was determined at different periods of incubation by 3H-uridine incorporation. PBL from BLV-infected cows with persistent lymphocytosis (PL) showed the highest spontaneous RNA synthesis. PBL from BLV-infected cows with normal lymphocyte counts synthesized more RNA than cells from normal animals. Decreased mitogen responses were observed in PBL from infected cows with PL in comparison to normal and BLV-infected cattle without PL. PHA and PWM did not show significant differences in their degree of stimulation of RNA synthesis.     

Antioxidant status and biomarkers of oxidative stress in bovine leukemia virus-infected dairy cows

Bovine leukemia virus (BLV) is among the most widespread livestock pathogens in many countries. Despite advances in understanding the pathogenesis of this disease, little is known about the involvement of oxidative stress. Therefore, this study examined the antioxidant status and the markers of oxidative stress in BLV-infected dairy cows. BLV infection was associated with an increase in triacylglycerol levels, a decrease in glutathione peroxidase (GSH-Px) activity and a tendency toward lower superoxide dismutase activity in the infected animals. No significant difference was observed in other markers of oxidative stress (i.e., conjugated dienes, hydroperoxides and malondialdehyde) in the infected animals compared to controls. A novel method for the analysis of oxidative stress, Z-scan based on the measurement of the mean-value of θ in low density lipoprotein indicated that the infected animals had low-density lipoprotein particles that were slightly less modified than those from the healthy group. Thus, we conclude that BLV infection is associated with a selective decrease in GSH-Px activity without any alteration in the common plasma markers of oxidative stress.     

Characterization of immune responses caused by bovine leukemia virus envelope peptides in sheep.

To study the immunomodulative activity caused by bovine leukemia virus envelope (BLV Env) peptide, sheep were immunized with two kinds of Th-epitope peptides, peptide 98 (BLV Env 98-117), and 61 (BLV Env 61-78). Four of eight immunized sheep showed specific proliferative responses against both of the peptide stimulations. To characterize the cells responding to the peptides, peptide-specific cells were established from the responding sheep by the continuous stimulation of peripheral blood mononuclear cells (PBMCs) with either peptide 98 or 61 in vitro. The peptide 98-specific cells consisted of CD4-positive cells, whereas the peptide 61-specific cells consisted of CD8-positive cells and MHC class II-positive cells. In addition, cytokine profile analysis indicated that the peptide 98-stimulated cells expressed IFN-gamma but not IL-10, although the peptide 61-stimulated cells expressed IL-10 but not IFN-gamma. These results show that BLV envelope peptides 98 and 61 can modulate immune responses of sheep lymphocytes in different ways and may contribute to the pathogenesis of BLV infection.     

Determination of proviral load in bovine leukemia virus-infected cattle with and without lymphocytosis

OBJECTIVE: To determine proviral load in bovine leukemia virus (BLV)-infected cattle with and without persistent lymphocytosis to assess the potential of transmitting the virus. ANIMALS: Cattle in 6 dairy herds. PROCEDURES: Blood samples from infected cows were evaluated 3 times at 6-month intervals for determination of proviral load via PCR assay, serologic results via ELISA, and hematologic status via differential cell counts. RESULTS: Infected cattle were classified into lymphocytotic and nonlymphocytotic groups. Lymphocytotic cattle consistently had > 100,000 copies of integrated provirus/mug of DNA (ie, high proviral load) in peripheral blood leukocytes. Titers of antibodies against BLVgp51 and BLVp24 indicated a strong immune response. Nonlymphocytotic cattle comprised 2 subgroups: a group with high proviral load and strong immune response, and a group with a weaker immune response, mostly against BLVp24, and a proviral load of < 100 copies/microg of DNA (ie, low proviral load). CONCLUSIONS AND CLINICAL RELEVANCE: Results emphasized the importance of characterizing nonlymphocytotic BLV-infected cattle during eradication programs. The risk of transmitting BLV infection from nonlymphocytotic cattle may differ depending on the proviral load. Nonlymphocytotic cattle with high proviral load could be efficient transmitters (as efficient as lymphocytotic cattle), whereas nonlymphocytotic cattle with low proviral load could be inefficient transmitters under standard husbandry conditions. Because most cattle with low proviral load do not develop anti-BLVp24 antibodies, it appears that lack of an anti-BLVp24 antibody response may be a good marker of this condition.     

Establishment of a novel feline leukemia virus (FeLV)-negative B-cell cell line from a cat with B-cell lymphoma

There is significant lack of basic hematologic and immunological data in adult sows. Therefore, aim of this study was to provide respective reference intervals. 32 clinically healthy multiparous Large White sows aged 33.5 ± 9.6 months and all of them two months postpartum were included in this study. Mean erythrocyte count was 5.5 ± 0.7 × 10(6)/μl and total leukocyte count was 12.1 ± 2.1 × 10(3)/μl. Proportion of lymphocytes was 44.7 ± 10.2% and of neutrophils 41.6 ± 11.0%. The ratio of na?ve T helper (Th) cells to memory Th cells was 1:3.1 and the ratio of Th cells to cytotoxic T cells (CTLs) was 1:4.2. Proportions of regulatory T cells, NK cells, and CD21(+) B cells were lower (3.1, 2.6, and 6.0%) than those of memory Th cells ranging from 8.8 to 27.5% depending on the activation status and CTLs with 37.3%. γδ T cells were found at comparably high numbers (19.1%). Flow cytometric measurement of intracellular cytokines in PBMCs revealed marginal levels for IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12p35, but remarkable levels for TNF-α and IFN-γ. Highest mRNA levels were found for IL-1, IL-10, and TNF-α, with TNF-α showing the least inter-individual variation.     

Effects of Pasteurella haemolytica A1 culture supernatant on mechanisms controlling bovine alveolar macrophage oxygen radical production.

Pasteurella haemolytica A1 culture supernatant containing leukotoxin, and modifiers of cyclic nucleotide and arachidonate metabolism, were evaluated for their ability to alter oxygen radical production by pulmonary alveolar macrophages obtained from seven Holstein calves. Calves were sedated, and underwent bronchoalveolar lavage to harvest macrophages, which were then incubated with culture supernatant and/or the drugs and toxins under study, and challenged with opsonized zymosan to induce oxygen radical generation. This was measured by a chemiluminescence technique. Pasteurella haemolytica A1 culture supernatant alone delayed the time to maximum oxygen radical production, although total production was increased. The cyclooxygenase inhibitor indomethacin and the phospholipase inhibitor p-bromophenacyl bromide significantly reduced maximum oxygen radical production, but their effects were diminished in the presence of culture supernatant. Although forskolin markedly inhibited oxygen radical generation, this effect was not altered by culture supernatant. Incubation of macrophages with pertussis toxin had no effect on oxygen radical production, while incubation with cholera toxin did inhibit production. This inhibitory effect was significantly lessened by concurrent incubation with P. haemolytica A1 culture supernatant.     

Enumeration of T and B lymphocytes in bovine leukemia virus-infected cattle, using monoclonal antibodies

Monoclonal antibodies and microfluorimetry were used to determine the absolute number of B and T lymphocytes in the blood of bovine leukemia virus (BLV)-infected cows. The blood lymphocyte populations from BLV-infected cows were significantly higher than those from BLV-negative cows. The increase in the lymphocyte population in 3 BLV-infected nonlymphocytotic cows was attributed to a significant increase in the number of T lymphocytes; in 3 BLV-infected persistently lymphocytotic cows, the increase was attributed to a significant increase in the number of B and T lymphocytes. One persistently lymphocytotic cow had a high lymphocyte count, and lymphocytes from this cow contained cells that appeared to stain with markers specific for bovine B and T lymphocytes. We concluded that infection of cattle with the B-cell lymphotropic retrovirus, BLV, not only affected B cells, but also T cells.     

Ultrastructural and immunologic characteristics of mouse x cattle xenogeneic hybridomas originating from bovine leukemia virus-infected cattle

Nine percent of xenogeneic hybridomas originating from a bovine leukemia virus (BLV)-infected cow secreted monoclonal IgM antibodies with multispecific reactivity. Similar reactivity was evident in some antibodies with an unusually long (> 50 amino acids) third complementarity-determining region of the heavy chain. Electron microscopy of hybridomas demonstrated the presence of c-type virus particles consistent with polymerase chain reaction detection of BLV env gene. Some hybridomas contained dilated rough endoplasmic reticulum and cisternae filled with moderately electron-dense granular substance compatible with plasma cells at presecretory stage. The number of chromosomes in xenogeneic hybridomas corresponded to the sum total of mouse and bovine chromosomes. None of the hybridomas showed polyploidy. The immunochemical and genetic analysis of stable bovine immunoglobulin-secreting xenogeneic hybridomas confirms that BLV infection causes polyclonal B cell activation regardless of antigen specificity. Presence of c-type particles in hybridomas suggests that T cell-derived cytokines are not required for sustained BLV expression.     

Detection of bovine leukemia virus by syncytium assay.

When various indicator cells, including virus transformed and nontransformed cells, were cocultivated with bovine leukemia virus-producing cells, strong positive syncytia formation was found in transformed cells one day after cocultivation. The results of comparison of bovine leukemia virus antibody titers and the detection of bovine leukemia by the syncytium assay showed 89% of serologically positive cows were positive for bovine leukemia virus, whereas no reactors were found in serologically negative cows. However, the frequency of bovine leukemia virus detection differed according to the difference of incubation periods in the syncytium assay. Therefore, it is important to choose the appropriate indicator cell and culture conditions for the detection of bovine leukemia virus in the syncytium assay.     

Mitogen and mixed lymphocyte culture responses of isolated bovine lymphocyte subpopulations

Examinations were made of the mitogen and mixed lymphocyte culture (MLC) responses of subpopulations of bovine lymphocytes isolated by density sedimentation following sequential E-rosetting with aminoethylisothiouronium bromide- and neuraminidase-treated sheep erythrocytes (EAET, EN). These procedures consistently separated 3 populations of E-rosetting T cells from a 4th population of non-E-rosetting cells (B and null cells). The isolated B and null cell population did not respond to phytohemagglutinin, concanavalin A, or pokeweed mitogen and responded minimally in mixed lymphocyte culture. Conversely, isolated T cells responded well to each of these stimuli. Moreover, differences in responsiveness were found among the 3 T-cell populations isolate by differential rosetting. T cells with receptors for both EAET and EN rosette formation were the most responsive to the mitogens used and demonstrated maximum activity in MLC. T cells with only EAET receptors had equivalent activity in MLC and less activity in response to mitogens. Cells with only EN receptors were the least active T-cell population in these assays. The different reactivities of these T-cell populations in lymphoproliferative assays indicated that they represent distinct subsets of bovine T cells.     

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.