Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle

Lectins, polyclonal antibodies and monoclonal antibodies (MAbs) were evaluated as markers for bovine lymphocytes obtained from healthy animals and from cattle infected with bovine leukemia virus (BLV). In the blood from healthy cattle the proportion of cells identified as T lymphocytes with the lectin Helix pomatia (HP) (67.8 +/- 6.2%) using the indirect immunofluorescence technique was similar to the proportion of cells identified by the MAbs P5 (66.1 +/- 3.8%) and BLT-1 (59.8 +/- 7.1%). The proportion of B cells in blood from healthy animals identified with a polyclonal antibody to bovine IgM (18.0%) was similar to that identified with a MAb to bovine IgM (16.2%). However, greater variation between individual values was detected with the MAb (SD = 8.2) than with the polyclonal antibody (SD = 4.0). In the blood from BLV-infected cattle with persistent lymphocytosis, both the polyclonal and the MAb revealed a threefold increase of B cells. A proportion of the B cells had an increased amount of immunoglobulin molecules in their plasma membrane as indicated by flow cytometry. The proportion of T lymphocytes, identified by the MAb P5, was reduced to one-third of that in non-infected cattle. The indirect HP labelling gave inconsistent results and seems not to detect solely T lymphocytes among blood lymphocytes from BLV-infected cattle.     

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.     

Monoclonal antibodies that distinguish bovine T and B lymphocytes

The specificities of three monoclonal antibodies (MAbs) were investigated using microcytotoxicity, fluorescence microscopy and laser flow cytometry (LFC) techniques. By microcytotoxicity, bovine thymocytes (n = 4) were estimated to be 85% B26A+, 4% TH21A+, and 1% H4+. Nylon wool enriched peripheral blood T lymphocytes (n = 3) were 90% B26A+, 10% TH21A+ and 10% H4+. Adherent B cell enriched fractions (n = 3) were 10% B26A+, 90% TH21A+ and 90% H4+. The two fluorochrome method was used to simultaneously identify lymphocytes that were sIg+ and MAb+. In these experiments, 92% of all sIg+ cells were H4+. An identical result was obtained for TH21A. 85% of all sIg- cells were B26A+. Using LFC, the mean percentages of sIg+, H4+ and TH21A+ PBL (n = 5) were not significantly different. B26A recognized a significantly greater population of cells, equivalent to the expected percentage of T lymphocytes. LFC also revealed two relatively discrete sizes of B26A+ PBL. The larger population overlapped the size range in which sIg+, H4+, TH21A+ PBL were found. The more numerous smaller B26A+ PBL were in a size range in which few sIg+, H4+ and TH21A+ PBL were found. In a study of MAb reactions with PBL of 185 cows, it was shown that in 92% of the animals H4 and TH21A were positively correlated (r = +.93), when H4 and TH21A were negatively correlated with B26A (r = -.94 and r = -.92, respectively). These correlation coefficients indicate a converse relationship between B26A and both H4 and TH21A. The remaining 8% of the animals were heterogeneous in their expression of the H4 and TH21A markers but not the B26A marker. These results provide strong evidence that: 1) B26A is a pan-T lymphocyte MAb in cattle, 2) a small but significant degree of heterogeneity exists in the expression of the epitopes recognized by H4 and TH21A. However, both MAbs recognize all B lymphocytes of most individuals, and 3) using a variety of immunological methods these three MAbs can now reliably be used to assay bovine T and B lymphocytes.     

Changes in B cell and T cell subsets in bovine leukaemia virus-infected cattle

Direct immunofluorescence and fluorescence-activated cell sorter techniques were used for the detection of surface immunoglobulin positive (SIg+) cells in peripheral blood lymphocytes (PBL's) of bovine leukaemia virus (BLV) infected cattle with or without persistent lymphocytosis (PL+, PL-) and in BLV-free cattle. The percentage of SIg+ cells was more than twice as high in BLV+PL+ cattle than in BLV-free and BLV+PL- cattle. Bovine T cells, and T cell subsets were identified indirectly by the same techniques using three monoclonal antibodies (MAb's) specific for all T cells (IL-A43), T helper (BoT4) cells (IL-A12) and T cytotoxic (BoT8) cells (IL-A17). The major histocompatibility complex (MHC) determinants of both class II (BoT4) and class I (BoT8) as well as all T cells were significantly reduced in BLV+PL+ compared to BLV-free cattle. The actual decrease in the BoT8 cell subset or the dilution effect that would change effector:target cell ratio suggests that a resultant decrease in cytotoxic activity in BLV+PL+ cattle may play an important role in the progress of BLV infection in cattle.     

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.     

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.     

Investigation of bovine gut associated lymphoid tissue (GALT) using monoclonal antibodies against bovine lymphocytes

Gut associated lymphoid tissue of the small and large intestine of calves and cows has been compared morphologically and quantitatively using monoclonal antibodies to bovine lymphocytes. B cells were significantly decreased in the ileum of the cow compared to the calf. Significantly increased numbers of T cells were present in cell suspensions of all lymphoid areas of the cow compared to the calf. T lymphocyte subsets were quantified into cryostat sections of lymphoid tissues expressing BoT4, and BoT8 antigens demonstrated increased numbers in follicular and dome areas of the discrete Peyer's patches of the small and large intestine of the cow. BoT4+, BoT8+, and the non-BoT4/BoT8+ T cell subsets were increased in the mucosa of the cow as compared to the calf. Similarities in structure and lymphocyte composition of the discrete Peyer's patches of the small intestine, cecum and colon and isolated single follicles in the large intestine suggest similar functional properties.     

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.     

A monoclonal antibody that identifies mature T lymphocytes of cattle

Hybridomas to bovine leukocytes were produced by immunization of BALB/C mice with bovine lymphoblasts and fusion of the mouse spleen cells with mouse myeloma cells. Monoclonal antibodies (MABs) were tested against various cell populations by indirect fluorescent microscopy using fluorochrome conjugated antibodies to mouse immunoglobulins. MAB-15, one of the resulting MABs obtained after cloning antibody-producing hybridomas, reacted with 56.8 +/- 8.4% of peripheral blood mononuclear cells (PBMC). MAB-15 did not react with monocytes or B cells, but did react with T cells (fluorescein isothiocyanate-conjugated peanut agglutinin positive cells). MAB-15 reacted with 3.2% of thymocytes from adult cattle. In addition to reacting with T cells, MAB-15 reacted with neutrophils and eosinophils. MAB-15 was characterized as an IgM antibody that was unable to lyse PBMC in the presence of complement. Thus, MAB-15 is a useful marker of mature T cells in the mononuclear cell population.     

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.     

Use of monoclonal antibodies against human HLA II antigens for the detection of bovine B lymphocytes and macrophages]

The crossreactivity of mouse monoclonal antibodies (MoAbs) (Tab. I) prepared against human HLA-DR and HLA-DP antigens was studied in various bovine cells: lymphocytes from lymph nodes and peripheral blood, adherent (B) and nonadherent (T) lymphocytes, monocytes, granulocytes and platelets. In the immunofluorescence test, MoAbs Bra13, Bra14, Bra20, Bra22, Bra30, Bra70, HL-38 reacted with bovine B lymphocytes and monocytes, but not with other tested cells (Tab. III, IV). These antibodies, except Bra22, were positive with B lymphocytes in the complement dependent cytotoxic test (Tab. II). The similarity of the bovine antigens and HLA-DR antigens determined by used MoAbs was also proved by immunoblotting. Monoclonal antibodies Bra38 and BraFB6 did not react with the bovine cells and separated antigens. The epitope (HLA-DR) recognized by the antibody Bra38 is probably absent in cattle. The presence of HLA-DP analogue determined by the antibody BraFB6 has not been confirmed. The crossreactive MoAbs could be used for the detection of B lymphocytes and macrophages in veterinary immunology.     

Evaluation of labelling methods for bovine T and B lymphocytes

Two lectins, one from Helix pomatia (HP) and one from Peanut (PN), were evaluated as bovine T cell markers. HP attaches to about 40% of the bovine peripheral blood lymphocytes (PBL). With an indirect technique about 60% of PBL are HP positive, while PN attaches to a slightly higher proportion ot the PBL.In double labelling experiments, HP was shown to bind only to Ig negative cells while 2% of the PBL were double labelled with PN and anti-Ig.The influence of different pretreatments of the PBL or the antibodies for labelling of the membrane bound Ig was studied. Preincubation for detaching cytophilic antibodies was important to avoid false Ig positive cells.     

Effects of bovine viral diarrhea virus on the percentages and absolute numbers of circulating B and T lymphocytes in cattle

The percentage and absolute numbers of circulating B and T lymphocytes were determined for 10 healthy cattle by labeling mononuclear cells with anti-bovine immunoglobulin or peanut agglutinin. The cattle were then inoculated with a cytopathogenic isolate of bovine viral diarrhea (BVD) virus, and B- and T-lymphocyte populations were again quantitated at given intervals. Seemingly, BVD virus caused a decrease in the absolute numbers of B and T lymphocytes and in the percentage of T lymphocytes. Although these effects lasted through 7 days, all of the cattle recovered from infection and had detectable BVD virus-neutralizing antibodies in their sera 17 days after exposure.