Bovine Leukemia Virus-infected Cows Research Articles

Development of a high-affinity anti-bovine PD-1 rabbit–bovine chimeric antibody using an efficient selection and large production system

Immune checkpoint molecules PD-1/PD-L1 cause T-cell exhaustion and contribute to disease progression in chronic infections of cattle. We established monoclonal antibodies (mAbs) that specifically inhibit the binding of bovine PD-1/PD-L1; however, conventional anti-PD-1 mAbs are not suitable as therapeutic agents because of their low binding affinity to antigen. In addition, their sensitivity for the detection of bovine PD-1 is low and their use for immunostaining PD-1 is limited. To address these issues, we established two anti-bovine PD-1 rabbit mAbs (1F10F1 and 4F5F2) and its chimeric form using bovine IgG1 (Boch1D10F1), which exhibit high binding affinity. One of the rabbit mAb 1D10F1 binds more strongly to bovine PD-1 compared with a conventional anti-PD-1 mAb (5D2) and exhibits marked inhibitory activity on the PD-1/PD-L1 interaction. In addition, PD-1 expression in bovine T cells could be detected with higher sensitivity by flow cytometry using 1D10F1. Furthermore, we established higher-producing cells of Boch1D10F1 and succeeded in the mass production of Boch1D10F1. Boch1D10F1 exhibited a similar binding affinity to bovine PD-1 and the inhibitory activity on PD-1/PD-L1 binding compared with 1D10F1. The immune activation by Boch1D10F1 was also confirmed by the enhancement of IFN-γ production. Finally, Boch1D10F1 was administered to bovine leukemia virus-infected cows to determine its antiviral effect. In conclusion, the high-affinity anti-PD-1 antibody developed in this study represents a powerful tool for detecting and inhibiting bovine PD-1 and is a candidate for PD-1-targeted immunotherapy in cattle.

Dairy Cows Experimentally Infected With Bovine Leukemia Virus Showed an Increased Milk Production in Lactation Numbers 3-4: A 4-Year Longitudinal Study.

Bovine leukemia virus (BLV) is widespread in global cattle populations, but the effects of its infection on milk quantity and quality have not been clearly elucidated in animal models. In this study, 30 healthy first-lactation cows were selected from ≈2,988 cows in a BLV-free farm with the same criteria of parity, age, lactation number, as well as milk yield, SCS, and composition (fat, protein, and lactose). Subsequently, these cows were randomly assigned to the intervention (n = 15) or control (n = 15) group, and reared in different cowsheds. Cows in the intervention group were inoculated with 1 × phosphate-buffered solution (PBS) resuspended in peripheral blood mononuclear cells (PBMC) from a BLV-positive cow, while the controls were inoculated with the inactivated PBMC from the same individual. From June 2016 to July 2021, milk weight (kg) was automatically recorded by milk sensors, and milk SCS and composition were originated from monthly performed dairy herd improvement (DHI) testing. Fluorescence resonance energy transfer (FRET)–qPCR and ELISA showed that cows in the intervention group were successfully infected with BLV, while cows in the control group were free of BLV for the entire period. At 45 days post-inoculation (DPI), the numbers of whole blood cells (WBCs) (P = 0.010), lymphocytes (LYMs) (P = 0.002), and monocytes (MNCs) (P = 0.001) and the expression levels of IFN-γ (P = 0.013), IL-10 (P = 0.031), and IL-12p70 (P = 0.008) increased significantly in the BLV infected cows compared to the non-infected. In lactation numbers 2–4, the intervention group had significantly higher overall milk yield (P < 0.001), fat (P = 0.031), and protein (P = 0.050) than the control group, while milk SCS (P = 0.038) and lactose (P = 0.036) decreased significantly. Further analysis indicated that BLV infection was associated with increased milk yield at each lactation stage in lactation numbers 3–4 (P = 0.021 or P < 0.001), but not with SCS and milk composition. Together, this 4-year longitudinal study revealed that artificial inoculation of BLV increased the milk yield in cows in this BLV challenge model.

Leukocytosis and Expression of Bovine Leukemia Virus microRNAs in Cattle.

Bovine Leukemia Virus (BLV) is an established model for studying retroviral infections, in particular the infection by the human T-cell leukemia type 1 (HTLV-1) virus. Here, we quantified gene expression of several BLV-related genes: effector protein of T and NK-killer cells NK-lysin (Nklys), reverse BLV transcriptase pol, BLV receptor (blvr), and also key enzymes of the microRNA maturation, Dicer (dc1) and Argonaut (ago2). The differences in the expression of the above genes were compared between five groups: (1) BLV infected cows with high and (2) low lymphocyte count, (3) with and (4) without BLV microRNA expressions, and (5) cows without BLV infections (control group). As compared to control, infected cows with high lymphocyte count and BLV microRNA expression had significantly decreased Nklys gene expression and increased dc1 and ago2 gene expressions. Few infected animals without pol gene expression nevertheless transcribed BLV microRNA, while others with pol gene expression didn't transcribe BLV microRNA. Notably, Pol expression significantly (P < 0.05) correlated with dc1 expression. For infected animals, there were no direct correlations between the number of leukocytes and pol, Nklys, and BLV microRNA gene expressions. Blvr gene expression is typical for juvenile lymphocytes and decreases during terminal differentiation. Our data suggest that BLV infects primarily juvenile lymphocytes, which further divide into two groups. One group expresses BLV DNA and another one expressed BLV microRNA that decreases host immune response against cells, expressing BLV proteins. It is suspected that regulatory microRNAs play a significant role in the bovine leukemia infections, yet the precise mechanisms and targets of the microRNAs remain poorly defined. Vaccines that are currently in use have a low response rate. Understanding of microRNA regulatory mechanisms and targets would allow to develop more effective vaccines for retroviral infections.

Reduced IL-2 and IL-4 mRNA Expression in CD4 + T Cells from Bovine Leukemia Virus-Infected Cows with Persistent Lymphocytosis

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4 + T cells to the expression of mRNAs encoding interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-γ, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4 + T cells from cows seronegative to BLV (BLV −), seropositive without persistent lymphocytosis (BLV +PL −), and seropositive with PL (BLV +PL +) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-γ, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV +PL + cows as compared to BLV − cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4 + T cells from BLV +PL + cows. In contrast, Con A stimulated PBMCs and CD4 + T cells did not differ significantly in expression of IFN-γ, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4 + T cells and semiquantitative RT-PCR the frequencies of CD4 + T cells transcribing IFN-γ, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV −, BLV +PL −, and BLV +PL + cows were determined. There were no significant differences in the frequencies of CD4 + T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4 + T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4 + T cells, thus suggesting aberrant Th regulation in subclinically infected animals.

Bovine leukemia virus gp30 transmembrane (TM) protein is not tyrosine phosphorylated: examining potential interactions with host tyrosine-mediated signaling

Bovine leukemia virus (BLV) causes persistent lymphocytosis, a preneoplastic, polyclonal expansion of B lymphocytes. The expansion increases viral transmission to new hosts, but the mechanisms of this expansion have not been determined. We hypothesized that BLV infection contributes to B-cell expansion by signaling initiated via viral transmembrane protein motifs undergoing tyrosine phosphorylation. Viral mimicry of host cell proteins is a well-demonstrated mechanism by which viruses may increase propagation or decrease recognition by the host immune system. The cytoplasmic tail of BLV transmembrane protein gp30 (TM) has multiple areas of homology to motifs of host cell signaling proteins, including two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are homologous to B-cell receptor and inhibitory co-receptor motifs. Signaling by these motifs in B cells typically relies on tyrosine phosphorylation, followed by interactions with Src-homology-2 (SH2) domains of nonreceptor protein tyrosine kinases or phosphatases. Phosphorylation of tyrosine residues in the cytoplasmic tail of TM was tested in four systems including ex vivo cultured peripheral blood mononuclear cells from BLV infected cows, BLV-expressing fetal lamb kidney cell and bat lung cell lines, and DT40 B cells transfected with a fusion of mouse extracellular CD8α and cytoplasmic TM. No phosphorylation of TM was detected in our experiments in any of the cell types utilized, or with various stimulation methods. Detection was attempted by immunoblotting for phosphotyrosines, or by metabolic labeling of cells. Thus BLV TM is not likely to modify host signal pathways through interactions between phosphorylated tyrosines of the ITAM or ITIM motifs and host-cell tyrosine kinases or phosphatases.

Up-regulation of IL-2 receptor α and MHC class II expression on lymphocyte subpopulations from bovine leukemia virus infected lymphocytotic cows

Infection with bovine leukemia virus (BLV) leads to a persistent lymphocytosis (PL) characterized by a marked increase in circulating B lymphocytes that express the orthologue of CD5. To gain insight into the factors accounting for lymphocytosis, experiments were conducted to determine the fuctional activation status of lymphocytes from BLV seronegative and BLV infected aleukemic cows with PL. Stimulation with the B lymphocyte mitogen Staphylococcus aureus Cowan strain I (SAC), recombinant human interleukin-2 (rIL-2), or pokeweed mitogen (PWM), a T lymphocyte-dependent B lymphocyte mitogen, revealed differences in the pattern of expression of IL-2 receptor α (IL-2Rα) and major histocompatibility (MHC) class II molecules on B and T lymphocytes from uninfected and BLV infected PL cows. rIL-2 induced expression of IL-Rα on B lymphocytes from PL cows but not B lymphocytes from BLV seronegative cows. SAC alone, or in combination with rIL-2, had no effect on B lymphocytes from BLV seronegative cows. However, rIL-2 alone or in combination with SAC induced expression of IL-2Rα on B lymphocytes from PL cows. PWM stimulated expression of IL-2Rα on bovine B lymphocytes regardless of BLV status, and induced a significantly higher level of expression on B lymphocytes from PL cows. Mitogens and rIL-2 had a similar stimulatory effect on induction of IL-2Rα expression on CD4 T lymphocytes regardless of BLV status. Only PWM induced expression of IL-2Rα on bovine CD8 T lymphocytes and induced a significantly higher level of expression on this T lymphocyte subset from PL cows. Examination of freshly isolated B lymphocytes from PL cows revealed increased spontaneous expression of the MHC class II molecule compared to B lymphocytes from control cows. None of the culture conditions examined induced MHC-II expression on CD4 and CD8 T lymphocytes from BLV seronegative cows. In contrast, SAC + IL-2 and PWM induced MHC-II expression on CD4 and CD8 T lymphocytes from BLV infected PL cows, resulting in a significantly greater proportions of these lymphocyte subsets expressing this molecule compared to CD4 and CD8 T lymphocytes from control cows. The data indicate that infection with BLV affects the response of B and T lymphocytes to signals of activation, up-regulating the expression of surface molecules involved in both direct contact and cytokine-mediated T lymphocyte-dependent B lymphocyte activation.

Milk and fat production in dairy cattle influenced by advanced subclinical bovine leukemia virus infection.

Genetic potentials (pedigree-estimated breeding value) for milk and for fat were compared in cows grouped according to subclinical stage of bovine leukemia virus infection. Genetic potential for milk production was significantly greater in seropositive cows with persistent lymphocytosis (622 +/- 72 kg) and in seropositive hematologically normal cows (554 +/- 34 34 kg) than in seronegative herdmates (418 +/- 53 kg). When 305-day twice-daily-milking mature equivalent milk production records for the current lactation were adjusted for genetic potential, bovine leukemia virus-infected cows that were hematologically normal had significantly greater milk production than did seronegative herdmates, suggesting that early bovine leukemia virus infection was positively associated with milk yield. Genetic potential for fat production was significantly greater for cows with persistent lymphocytosis (21 +/- 2 kg) than for other seropositive (16 +/- 1 kg) and seronegative herdmates (13 +/- 2 kg); however, 305-day twice-daily-milking mature equivalent fat production for the current lactation was not significantly different between the groups. Thus, cows with persistent lymphocytosis did not produce fat according to their genetic potential. As an apparent consequence of tendencies for greater milk yield and less fat production, milk fat percentage was significantly reduced in cows with persistent lymphocytosis (3.33 +/- 0.09%) and other seropositive cows (3.48 +/- 0.05%) relative to seronegative herdmates (3.67 +/- 0.07%). These results suggest a need to reevaluate the economic impact of bovine leukemia virus infection on the dairy industry.

A prospective investigation of bovine leukemia virus infection in young dairy cattle, using survival methods.

The authors studied bovine leukemia virus infection in cattle born between July 1, 1979, and June 30, 1981, and followed up to 27 months in the University of Florida Dairy Research Unit herd, a 200-cow milking herd. Cattle were tested monthly for antibodies to the virus by agar-gel immunodiffusion with the glycoprotein-51 antigen. Of 473 live calves entering the study at birth, 54 became infected during the course of the study. Rates of detection of bovine leukemia virus infection were examined using survival methods. Plotted cumulative hazard rates revealed a nonlogarithmic survivorship function and three age-specific detection phases. These phases were 0-6 months, 7-16 months, and 17-26 months of age with estimated incidence rates of 5.25, 2.14, and 14.37 new detections per 10,000 cattle-days at risk, respectively. Significant differences were found between incidence rates of the first and second phase (p = 0.0168) and between those of the second and third phase (p less than 0.0001); corresponding relative risks were 0.41 and 6.71, respectively. The reduced incidence rate of the second phase coincided with movement of calves from a crowded calf barn to pastures. The increased incidence rate of the third phase coincided with mixing of bred heifers with bovine leukemia virus-infected dry cows. Increased incidence rates did not coincide with common-needle vaccination or artificial insemination. Evidence was not found for milk-borne infection. Age-specific detection rates were not associated with dam parity (p = 0.93), dam age (p = 0.79), breed (p = 0.60), or consumption of colostrum from cows infected with the virus (p = 0.23). However, detection rates were lower in cattle born to bovine leukemia virus-infected cows than in those born to noninfected cows (p = 0.03).

Circulating immune cells and immune complexes in peripheral blood of healthy and of bovine leukemia virus-infected cows and lymphosarcomatous calves

The peripheral blood (lymphocytes and immune complexes (IC)) from 20 healthy cows, 5 healthy calves, 10 bovine leukemia virus-infected cows and 4 lymphosarcomatous calves was investigated using 4 different immunological techniques. A highly significant increase in the percentage of surface immunoglobulin (SIg)-bearing peripheral blood lymphocytes of persistent lymphocytosis-cows could be demonstrated while the percentage was decreased in the lymphosarcomatous calves. Percentages of spontaneous sheep red blood cells (SRBC)-rosettes (E-rosettes) were not elevated. Antibodies to bovine leukemia virus-antigen were detected in the sera (immuno-diffusion test) of all the leukotic cows but not of the lymphosarcomatous calves. Although there seems to be an increased level of IC in the advanced stage, as compared to the beginning of the disease, there was no statistically significant difference in comparison with the control individuals. In the lymphosarcomatous calves as well as in the leukemic cows there was no statistical difference in the IC values between diseased animals and controls.

Somatic cell hybridization between bovine leukemia virus-infected lymphocytes and murine plasma cell tumors: Cell fusion studies with bovine cells

Hybridization of peripheral blood lymphocytes from bovine leukemia virus-infected cows with murine myeloma cells resulted in the generation of hybrid cells secreting immunoglobulins composed of various combinations of heavy and light chains of both bovine and murine Ig origin. Some hybrid cells derived from the light-chain producer, but non-secretor murine myeloma NS1 cell line, secreted IgM molecules composed of bovine μ-chain linked to bovine and/or murine light chains. Other hybridomas secreted mouse and bovine light-chain dimers and/or monomers, or failed to secrete any Ig polypeptide chain whatsoever. Immunoglobulins secreted by hybridomas obtained upon hybridization of bovine cells with the IgG-secreting murine myeloma P 3X63 cell line, contained bovine μ-chain in one of the seven hybridomas obtained, and bovine light chain in two of them. All the cell lines secreted murine light- and γ-chains.