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.     

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.     

Production and related variables in bovine leukaemia virus-infected cows

A newly developed milk dot blot test was used to detect anti-bovine leukaemia virus (BLV) antibody in milk samples from 2079 lactating adult cows from among 61 herds. The milk dot blot test was highly repeatable; the concordance rate, compared with the agar gel immunodiffusion test performed on serum, was 83.5%. All herds contained BLV-positive cows; the prevalence rate was 36%. BLV-positive cows tended to come from larger herds and were older and more often later in lactation. Fourteen production and related variables (herd size, age, days open, days in milk, milk somatic cell count, milk, fat, and protein produced in the current lactation, projected production of milk, fat, and protein, and breed class average deviations for milk, fat, and protein) were compared between BLV-positive and BLV-negative cows. Although somatic cell count, milk produced, and projected production of milk and protein were related significantly to BLV status using simple tests of association, once the variables herd size, age and days in milk were controlled, these differences were removed. Further analyses using logistic (outcome: individual cow BLV status) and least-squares regression (outcome:herd proportion of BLV-positive cows) failed to show an association between any of the measured production or related variables and BLV-positivity. We concluded that the effect of BLV on production and related variables in dairy cows was below the sensitivity of our analytical techniques or was non-existent.Abbreviations ABCA herd average breed class average for milk, fat, and protein production - AVGAGE average age of the herd - ADIM herd average for days in milk - AGID agar gel immunodiffusion - AVGSCC herd average milk somatic cell count - BCA breed class average, a milk, fat and protein production index calculated by comparing a cow's actual 305-day lactation production to the corresponding BCA standard for the same breed, age, and month of calving - BLV bovine leukaemia virus - CALVINT calving interval - COWAGE cow age - DBCA breed class average deviation for milk, fat, and protein production, the difference between an individual cow's BCA and the herd average - DIM days in milk - HS herd size corresponding to the number of lactating cows in a herd - LACT actual amount of milk, fat, and protein produced in a cow's lactation - ODHIC Ontario Dairy Herd Improvement Corporation - PCTPOS percentage of herd that is BLV-positive - PROJ projected 305-day production for milk, fat, and protein by fitting to a standard lactation curve adjusted for days in milk and age at calving - RHBCA rolling herd average for breed class average for milk, fat, and protein production, the average for all cows that completed a lactation (cows must have completed a 305-day lactation) during the previous 12 months - SCC milk somatic cell count     

Differential cytokine expression in T cell subsets from bovine peripheral blood

Although distinct cytokine expression in T cell subsets is well understood in mice and humans, limited information is available on bovine T cell subsets. In the present study, we analyzed the mRNA expression of 10 kinds of cytokines and CD25 expression in CD4⁺, CD8⁺, WC1⁺ and WC1^-γδ T cell subsets in bovine peripheral blood by Concanavalin A (Con A) stimulation. CD25 expression was significantly increased in CD4⁺, CD8⁺ and WC1⁺γδ T cells, but not in WC1^-γδ T cells by Con A stimulation. In CD4⁺ T cells, the mRNAs of Interleukin (IL)-2, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β and transforming growth factor (TGF)-β were expressed in control cultures, and IL-3, IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) were newly expressed when the cells were stimulated with Con A. CD8⁺ T cells expressed the mRNAs of IL-6, TNF-α, TNF-β and TGF-β in control cultures, and newly expressed those of IL-2, IFN-γ and GM-CSF, but did not express those of IL-3, IL-4 or IL-10 after Con A stimulation. The cytokine expression profile of WC1⁺γδ T cells was similar to that of CD8⁺ T cells. However, WC1^-γδ T cells did not express any cytokine mRNA except TGF-â mRNA. These results will contribute to elucidate the participation of T cell subsets in immune responses against infectious disease in cattle.     

Alteration in lymphocyte subpopulations in bovine leukosis virus-infected cattle.

Alterations in peripheral blood lymphocyte subpopulations were examined in bovine leukosis virus (BLV)-infected cattle using antibodies specific for differentiation antigens in conjunction with analytical flow cytometry. Animals considered to be aleukemic and lymphocytotic were included in the study. Significantly fewer numbers of circulating B-lymphocytes (surface Ig-positive) and T-helper lymphocytes (BoCD4-positive) were identified in BLV-infected aleukemic cattle compared to non-infected controls while no significant differences were established for T-cytotoxic/suppressor lymphocytes (BoCD8-positive). In contrast, BLV-infected animals with persistent lymphocytosis had elevated numbers of circulating B-lymphocytes with no significant perturbation in circulating T-lymphocyte subsets identified when compared as a group with the negative control cattle. Application of regression analysis to data from individual lymphocytotic cattle demonstrated a significant correlation between absolute numbers of B- and T-lymphocytes. Increased numbers of B-lymphocytes were correlated with increased numbers of T-helper and T-cytotoxic/suppressor lymphocytes.     

Age-related changes in leukocytes and T cell subsets in peripheral blood of Japanese Black cattle

It is well known that the immune system changes with age during development and maturation in Holstein cattle. But age-related changes in leukocytes and T cell subsets in peripheral blood of Japanese Black cattle still remain unclear. The aim of the present study was to investigate comparative changes of leukocytes (granulocytes, monocytes, B cells and T cells) and T cell subsets (CD4⁺, CD8⁺, γδ, CD8⁺γδ and WC1⁺γδ T cells) in Japanese Black cattle aged 0.5, 1, 2, 6, 18 and 36–41 (adult) months on flow cytometry using specific monoclonal antibodies for the cell surface markers. T cell proportion was approximately 40% in 2-month-old cattle and decreased to 20.6% in adults. In contrast, B cell proportion significantly increased from 7.4% to 28.2% with age. In T cell subsets the percentage of CD4⁺ T cells significantly increased from 40.5% to 60%, but that of WC1⁺γδ T cell subset significantly decreased with age. The percentages of CD8⁺ and CD8⁺γδ T cells did not change. The present study details the proportional changes in leukocyte and T cell subsets with age in the peripheral blood of Japanese Black cattle and these findings are similar to those described for Holstein cattle.     

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.     

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.     

Priming of multiple T cell subsets by modified-live and inactivated bovine respiratory syncytial virus vaccines

T cell activity is a critical component of immunity to bovine respiratory syncytial virus (BRSV). We tested the effects of immunization by modified-live and inactivated BRSV vaccines on cell-mediated and humoral immunity in young calves. The two forms of vaccine stimulated similar serum neutralizing antibody production, although the early kinetics of those responses differed. CD4+, CD8+, and gammadelta T cells were analyzed before and after immunization for BRSV-specific in vitro recall responses, as evaluated by CD25 upregulation measured by flow cytometry. Modified-live virus (MLV) primed each of the three subsets for statistically significant in vitro responses to antigen. Inactivated vaccine also primed each T cell population for significant antigen-driven CD25 upregulation, including responses by CD4+ and gammadelta T cells that were stronger and longer-lasting than those primed by MLV. Monoclonal antibody was used in additional assays to block MHC class I during incubation of BRSV antigen with peripheral blood mononuclear cells from an animal in the inactivated vaccine group. The recall response by CD8+ T cells was more inhibited by this treatment than the other subsets, further suggesting that the inactivated vaccine had primed antigen-specific CD8+ T cells. In summary, the data indicate that balanced BRSV-specific T cell responses can be induced by inactivated, as well as modified-live, conventional vaccines, which may implicate an alternative pathway of MHC class I antigen presentation.     

Evaluation of alternative methods for the detection of bovine leukaemia virus in cattle

AIM: To evaluate commercially available enzyme-linked immunosorbent assays (ELISAs) and the polymerase chain reaction (PCR) for their ability to detect antibodies against or nucleic acid of the bovine leukaemia virus (BLV), the causal agent of enzootic bovine leukosis (EBL), and to assess their usefulness in a national eradication programme. METHODS: Eighty-two well-defined sera (including 18 from an OIE reference laboratory) and 399 field sera from New Zealand cattle were tested in five ELISAs and the results compared with the agar gel immunodiffusion (AGID) test and electrophoretic immunoblotting (EIB) results. A polymerase chain reaction-based technique, which could detect BLV-RNA and proviral-DNA, was also evaluated on a subsample of the field cases. RESULTS: Two commercial ELISAs classified 99% of the defined sera correctly, with the other three ranging in their correct classification between 88% and 95%. The ELISAs agreed in their general classification on the majority of the 399 blood samples (91.7%), and with the AGID for more than 95 % of the sera. In a dilution series of the international reference serum E4, the highest dilution with a positive (or suspicious) result ranged from 1:80 to 1:5120. A dilution series of 202 field positive samples tested in the preferred ELISA detected 98% of positive sera at a 15 and 1: 10 dilution, reducing to 78% at a 1:80 dilution of the sera. Agreement between serological tests and PCR was poor, mainly due to failure of the PCR to detect a number of serologically positive animals. CONCLUSION: ELISA tests detected about 10% more reactors than the AGID and the EIB combined. Some ELISA-positive animals were not detected by PCR, raising doubts about the usefulness of PCR-based technology in EBL eradication programmes.     

Regulatory T cells in cattle and their potential role in bovine paratuberculosis

The intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in wild and domestic ruminants. Johne's disease presents as a chronic enteritis with severe inflammation of intestinal tissues, characterized by widespread infiltration of macrophages, the target cell of MAP. Clinical signs of Johne's disease are typically accompanied by a loss of peripheral CD4+ T cell responses to MAP antigens and an increase in anti-MAP serum IgG levels. Recently, it was proposed that regulatory T cells might develop over the lengthy course of subclinical MAP infection. In the past five years, significant progress in defining bovine regulatory T cells has been made. These studies grew out of observations that IL-10 is produced by PBMCs in response to MAP antigen stimulation and that neutralization of this IL-10 could enhance IFN-γ production from MAP-antigen reactive effector T cells. Depletion studies revealed that MAP responsive cell populations producing IL-10 were largely CD4+ and CD25+, although monocytes have also been shown to produce IL-10 in response to MAP. In addition, evidence for a regulatory population of γδ T cells has also begun to accumulate. We summarize current thinking regarding regulatory T cells in MAP infection and provide data suggesting a potential link between regulatory T cells, bovine leukemia virus, and MAP.     

B cell lymphoma and secondary leukaemia in a mule

A 20‐year‐old mule was evaluated for anorexia and fever. Haematological evaluation revealed a population of large, immature lymphoid cells and significant anaemia. Bone marrow aspiration was unsuccessful, and abdominal ultrasonographic evaluation was unremarkable. The mule was subjected to euthanasia due to poor prognosis, and necropsy and histological evaluation revealed multicentric B cell lymphoma. The leukaemic phase of lymphoma is rarely documented in equids, and is regarded as an indication of advanced or aggressive disease. To the authors' knowledge, lymphoma in a mule has been reported only once previously.     

Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro

Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1⁺ CD4⁺ T cells in blood and lymph node and PD-1⁺ CD8⁺ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.     

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.     

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.