Proliferative responses of a bovine leukemia virus-infected lymphoblastoid B-cell line by its culture supernatant and cytokines.

The growth-promoting activity in the culture supernatant of bovine lymphoblastoid B-cell lines (BL2M3 and BL312) were examined. BL2M3 cells proliferated well in response to conditioned medium (CM) obtained from BL2M3 and BL312 cell cultures. These BL2M3 and BL312 CM were used as sources of BL2M3 cell growth-promoting factor (BL2M3-GPF). BL2M3-GPF was sensitive to acid (pH 2) and alkali (pH 10) and was heat-labile. Proliferative responses of BL2M3 cells were not induced by human recombinant (r)IL 1, rIL 2, rIL 6, granulocyte-colony stimulating factor (rG-CSF) or tumor necrosis factor (rTNF)-alpha. Human low molecular weight B cell-growth factor (LMW-BCGF) was, however, capable of augmenting the proliferation of BL2M3 cells. BL2M3 cells formed clusters in response to LMW-BCGF, whereas they showed single and discete appearance in the presence of BL2M3-GPF. These results suggested that bovine lymphoblastoid B-cell lines might release and respond to the growth-promoting factor for in vitro proliferation of its own cell line, BL2M3.     

Porcine lymphoblastoid cell lines of B-cell origin

Two lymphoblastoid cell lines were isolated from different pigs and were maintained in culture for over 100 passages or 20 months. These cell lines were characterized by their cell surface antigens, ability to stimulate a mixed lymphocyte reaction and production of immunoglobulin. When tested against a panel of monoclonal anti-cell surface antigen antibodies, only those monoclonal antibodies which detect porcine class I or II molecules reacted against the lymphoblastoid cell lines in a microcytotoxicity assay. The two pig cell lines could stimulate peripheral blood mononuclear cells in a mixed lymphocyte reaction. P-SC(1) and P-16(2) also demonstrated a dependency upon the presence of 2-mercaptoethanol for cell division. The secretion of pig immunoglobulin by P-SC(1) or P-16(2) was first demonstrated by ELISA using a polyclonal anti-swine IgG (heavy and light chain) serum. By the use of monoclonal anti-IgA, IgG or IgM antibodies in an enzyme-linked assay on Western blots of P-SC(1) or P-16(2) lysate/supernatant, the two cell lines were demonstrated to be producing a whole monomeric IgA molecule and a mu chain.     

Tumor-associated antigen on bovine leukemia virus-induced bovine lymphosarcoma

Specific tumor-associated antigen (TAA) was detected on enzootic bovine leukosis (EBL) cells by monoclonal antibodies against TAA. One of the monoclonal antibodies, c143, reacted with all EBL tumor cells tested but not with bovine leukemia virus (BLV) antigens. c143 reacted slightly with bovine fetal thymus and mitogen-stimulated lymphocytes from BLV-free cows but not with normal bovine lymphoid cells. TAA may be a good tumor marker of EBL tumor cells. We sacrificed eight TAA-positive but clinically normal animals and examined them in order to elucidate whether or not they had gross or histological tumors. At necropsy, four animals had tumors macroscopically. Three animals had no tumors histologically but had initial lesions showing follicular hyperplasia and the TAA on affected lymph nodes. The one remaining showed medullary hyperplasia in the spleen but there were no findings of tumors. Thus, c143 is a useful tool not only for diagnosing EBL, but also for screening of BLV-infected cattle with potential to develop tumors in the future.     

Inactivation of bovine leukemia virus-infected lymphocytes in milk

The survival of bovine leukemia virus (BLV)-infected lymphocytes in milk was studied to determine whether treatments similar to those on a dairy farm would inactivate BLV. Bovine leukemia virus was found in milk stored for 72 hours at 1.1 C (34 F); milk constituents, such as protein, total solids, minerals, fat, and somatic cell concentration did not affect the presence of BLV. Infectivity also was found in the cream layer of milk. Pasteurization at 63 C for 30 minutes did inactivate BLV-infected lymphocytes.     

Properties of nine continuous B-cell lines established from enzootic bovine leukosis tumors.

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.     

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.     

Induction of the 68 kDa major heat-shock protein in different Theileria annulata- and virus-transformed bovine lymphoblastoid cell lines.

Expression of the major inducible heat-shock protein of 68 kDa (hsp68) has been analyzed in peripheral blood mononuclear cells (PBMC) from cattle and in six Theileria annulata- and two bovine leukemia virus-transformed bovine lymphoblastoid cell lines (BoLCL). By metabolic labeling, hsp68 could be detected in PBMC and BoLCL only after heat-shock, but not under normal culture conditions. Immunoblot analysis with an hsp68 reactive monoclonal antibody similarly revealed a strong hsp68 response after heat-shock in BoLCL, and no hsp68 expression under normal culture conditions. Normally kept PBMC, however, were weakly positive with the antibody. The data are discussed with respect to the constitutive expression of hsp68 seen in several other cell lines.     

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.     

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.     

Stable transfection of reticuloendotheliosis virus-transformed lymphoblastoid cell lines.

Lymphoblastoid T cell lines were established by infection of chicken splenocytes with reticuloendotheliosis virus (REV). The target cells first were cultured in interleukin-containing conditioned medium or were stimulated by concanavalin A, or both. Most cell lines were T cells expressing CD3 and one of the T cell receptors, and all cell lines were positive for major histocompatibility complex (MHC) class II antigens. Several REV-transformed cell lines were stably transfected using electroporation with a selectable plasmid, pNL1, containing the neor gene. Transfected cell lines were selected using G418 and were maintained for periods up to 137 days. Transfected cell lines were susceptible to MHC class-I restricted lysis by cytotoxic T lymphocytes from REV-infected chickens.     

Association between the strength of serologic recognition of bovine leukosis virus and lymphocyte count in bovine leukosis virus-infected cows

OBJECTIVE: To determine whether strength of serologic recognition of bovine leukosis virus (BLV) by use of ELISA is associated with blood lymphocyte counts. DESIGN: Prospective study. ANIMALS: 161 cows with positive results of ELISA for BLV. PROCEDURE: Sample-to-positive ratio (S:P), which is the ratio between the test sample and a positive control sample, was compared among lymphocytotic and nonlymphocytotic cows. A regression model was constructed to evaluate the association between blood lymphocyte concentration and S:P, age, and the interaction of these terms. RESULTS: Mean S:P differed significantly between lymphocytotic (2.58 +/- 0.36) and nonlymphocytotic (2.38 +/- 0.39) cows. Age and S:P were significantly associated with lymphocyte count. CONCLUSIONS AND CLINICAL RELEVANCE: Sample-to-positive ratio and lymphocyte count were related; however, cows with high S:P were not always lymphocytotic. Culling cows on the basis of S:P will reduce the herd load of infectious virus faster than random culling of ELISA-positive cows; however, culling on the basis of lymphocyte count will eliminate a greater proportion of the reservoir of infection.     

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.