Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus.

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.

Cytokine TNF-α and its receptors TNFRI and TNFRII play a key role in the in vitro proliferative response of BLV infected animals.

Bovine leukemia virus (BLV) main host cells are B lymphocytes. Infected animals can be classified into high or low proviral load (HPL or LPL respectively), regarding the number of proviral copies infected lymphocytes they carry. After infection, there is an overexpression of several cytokines, particularly TNF-α, which has a delicate regulation mediated by receptors TNFRI and TNFRII; the first one involved with apoptosis, while the other stimulates cell proliferation. The study aimed to quantify TNF-α and its receptors mRNA expression, and in which extent in vitro proliferation was affected, in peripheral blood mononuclear cells (PBMC) from BLV-infected animals with different proviral loads, after the addition or not of synthetic TNF-α (rTNF-α) for 48 h. PBMC from BLV-infected animals showed spontaneous proliferation after 48 h in culture but did not show changes in proliferation rates after 48 h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48 h culture without exogenous stimulation was significantly lower, regardless of the proviral load of the donor, compared to non-infected animals. In the LPL animals, the expression of TNF-α mRNA was significantly lower with respect to the control group while the expression of TNFRI mRNA was significantly increased. The HPL animals showed a significant decrease in the expression of TNF-α and TNFRII mRNA respect to the control group. After 48 h incubation with rTNF-α, PBMC from infected animals had different responses: TNF-α and TNFRI mRNA expression was reduced in PBMC from the LPL group compared to the BLV negative group, but no differences were observed in PBMC from the HPL group. TNFRII mRNA expression showed no differences between HPL, LPL, and BLV negative groups, though HPL animals expressed 10.35 times more TNFRI mRNA than LPL. These results support the hypothesis that LPL animals, when faced with viral reactivation, present a pro-apoptotic and anti-proliferative state. However, complementary studies are needed to explain the influence of TNFRII on the development of the HLP profile. On the other hand, exogenous stimulation studies reinforce the hypothesis that BLV infection compromises the immune response of the animals.

Increased T-cell responses that control bovine leukemia virus proviral load in beef cattle under dietary vitamin A restriction for marbling.

Bovine leukemia virus (BLV) proviral load is controlled by T-cell responses, which require vitamin A (VA) derived from food. However, whether dietary VA restriction for marbling impairs the T-cell responses that control BLV proviral load in beef cattle is unknown. We assessed T-cell subsets, interferon (IFN)-γ gene expression, and BLV proviral load in naturally BLV-infected Japanese Black cattle that were fed a diet with decreased VA levels. We found that the percentage of CD4+ T cells increased over time during dietary VA restriction. In addition, BLV proviral load was negatively correlated with the percentage of CD4+ T cells and with the level of IFN-γ gene expression. These observations suggest that dietary VA restriction for marbling enhances T-cell responses that control BLV proviral load and thus does not promote leukemogenesis in fattening beef cattle.

Autotransporter-based surface expression and complementation of split TreA fragments utilized for the detection of antibodies against bovine leukemia virus.

Bovine Leukemia Virus (BLV) is an oncogenic virus which is the etiological agent of a neoplastic disease in infected cattle called enzootic bovine leukemia (EBL). The most common and sensitive diagnostic methods for EBL like enzyme-linked immunosorbent assay (ELISA) is time-consuming and requires manual handling which makes it unsuitable as an on-farm diagnostic test. Hence, there is a need for an alternative test with rapid detection and reduced manual labour. We have previously reported the use of E. coli periplasmic trehalase (TreA) in a split enzyme sensor diagnostic technology to detect immunoglobulins and antigen-specific antibodies. In the current study, a more sensitive detection was attempted by bacterial surface display of split TreA fragment by fusion with the autotransporter AIDA-I. The split TreA fragments fused to antigens require antigen-specific antibodies for complementation and to trigger trehalase activity. This surface complementation strategy was used to detect anti-BLV antibodies in clinical serum by incorporating the antigenic BLV capsid protein in the fusion proteins. To validate this assay, a panel of serum samples obtained from BLV positive and negative cattle were tested in comparison with ELISA results. Evaluation of this panel resulted in positive detection of all true positive samples. We further demonstrated that this assay can be enhanced by pre-adsorption of clinical serum samples using E. coli cells to increase the specificity and help reduce nonspecific binding. In conclusion, the p24 antigen specific BLV assay is a potential tool for simple and rapid diagnosis of BLV infection, which is compatible with both lab-based and a more user friendly on-farm format.

Proteomic profiling of milk small extracellular vesicles from bovine leukemia virus-infected cattle.

Milk small extracellular vesicles (sEV) contain proteins that provide potential information of host physiology and immunology. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. In this study, we aimed to explore the proteomic profile of milk sEV from BLV-infected cattle compared with those from uninfected cattle. Milk sEV were isolated from three BLV-infected and three uninfected cattle. Proteomic analysis was performed by using a comprehensive nanoLC-MS/MS method. Furthermore, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to evaluate the candidates for uniquely or differentially expressed proteins in milk sEV from BLV-infected cattle. Proteomic analysis revealed a total of 1330 common proteins in milk sEV among BLV-infected cattle, whereas 118 proteins were uniquely expressed compared with those from uninfected cattle. Twenty-six proteins in milk sEV were differentially expressed proteins more than two-fold significant difference (p < 0.05) in BLV-infected cattle. GO and KEGG analyses indicated that the candidates for uniquely or differentially expressed proteins in milk sEV had been involved in diverse biological activities including metabolic processes, cellular processes, respond to stimulus, binding, catalytic activities, cancer pathways, focal adhesion, and so on. Taken together, the present findings provided a novel insight into the proteomes of milk sEV from BLV-infected cattle.

A TLR7 agonist activates bovine Th1 response and exerts antiviral activity against bovine leukemia virus.

Bovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7 agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells and increased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV infection.

IFN-γ mRNA expression is lower in Holstein cows infected with bovine leukemia virus with high proviral load and persistent lymphocytosis.

Bovine leukemia virus (BLV) is a retrovirus that affects primarily milky cows. Animals serologically positive to BLV show a Th1 cytokine profile with a predominance of interferon gamma (IFN-γ). IFN-γ has antiviral activity through mechanisms such as resistance to infection, inhibition of viral replication and apoptosis. The objective of this work was to determine the transcription levels of IFN-γ and its relationship with proviral load and persistent lymphocytosis in a population of Holstein cows of the province of Antioquia, Colombia. IFN-γ transcription levels were evaluated by qPCR in 140 Holstein cows. A one-way analysis of variance and a Student's t test were used to evaluate the differences between the means. The amount of IFN-γ mRNA found in BLV-positive cows was lower than in BLV-negative cows. Moreover, in the group of infected cows a lower level of IFN-γ mRNA expression was found in BLV and persistent lymphocytosis cows (BLV+PL) compared with BLV and aleukemia cows (BLV+AL). The level of IFN-γ mRNA expression was lower in cows with high proviral load (HPL) compared to cows with low proviral load (LPL). BLV infection is related to abnormal expression of IFN-γ mRNA, although IFN-γ has antiviral activity, its expression is affected by high proviral load. Keywords: cytokine; immune system; leukemia; bovine leukemia virus.

Changes in bovine leukemia virus serological status and lymphocyte count between dry-off and early lactation in Michigan dairy cows.

This study addresses how the serological status of bovine leukemia virus (BLV) and lymphocyte count fluctuate from dry-off to early lactation in dairy cattle. Very few studies have investigated how BLV antibody status and lymphocyte count of cows changes longitudinally during the lactation cycle. Blood samples were collected from dairy cattle (n = 149) on 5 commercial dairy herds in Michigan at dry-off, close-up, and 7 to 10 d after calving. Plasma was analyzed for anti-BLV antibodies using a BLV-ELISA and whole blood was analyzed for lymphocyte counts. We found that BLV seroprevalence increased from dry-off (38.9%) to close-up (43.6%), then slightly decreased from close-up to 7 to 10 d after calving (43.0%). However, the change in seroprevalence was only significant from dry-off to close-up. Cows of third or higher parity were more likely to seroconvert than cows of lower parity and had the highest ELISA-negative prevalence of BLV. Lymphocyte counts were significantly higher in ELISA-positive animals, but only among second and third or greater parity animals. These results indicate that the use of lymphocyte counts as a disease severity monitoring tool for BLV should differ by parity group. Future studies should investigate if changes in seroprevalence are due to new infections or natural changes in antibody concentrations as the cow prepares for colostrum production. More accurate lymphocyte guidelines to be used for monitoring the progression of BLV should be created that consider parity and lactation stage.

[Characterization of B1-cells during experimental leukomogenesis.]

BACKGROUND: Bovine leukemia causes a significant polyclonal expansion of CD5+, IgM+ B lymphocytes, known as persistent lymphocytosis (PL), in approximately 30% of infected cattle. However, it is not yet clear what happens to this subpopulation of B cells in the early period of infection of animals. PURPOSE: Quantitative characterization of IgM+ and CD5+ B cells during the immune response, which can provide important information on the mechanisms of lymphocyte priming in BLV infection. MATERIAL AND METHODS: The experiment used BLV-negative calves of black-motley breed at the age of 8 months (n = 11). Animals (n = 8) were intravenously injected with blood of a BLV-positive cow. Control calves (n = 3) were injected with saline. Studies were performed before and after infection on days 5, 7, 14, 21, 28 and 65 of the immune response. The determination of the number of B-lymphocytes in the blood was carried out by the method of immunoperoxidase staining based on monoclonal antibodies to IgM, CD5. RESULTS: As a result of the studies, it was found that the level of CD5+ B cells increases on the 14th day of the primary immune response, characterized by polyclonal proliferation of CD5+ B cells, which are the primary target for BLV. Our research data confirm that in the lymphocytes of experimentally infected cattle, surface aggregation of IgM and CD5 molecules on B-lymphocytes is absent. DISCUSSION: It is known that the wave-like nature of IgM synthesis, which was shown in previous studies, depends on a subpopulation of B1 cells. After 7 days of the immune response, IgM+ and CD5+ cells do not correlate, which shows their functional difference. The increase in CD5+ cells is probably not associated with B cells, but with T cells differentiating under the influence of the virus. CONCLUSIONS: A subset of B1 cells is the primary target of cattle leukemia virus. The 65th day of the immune response is characterized by the expansion of IgM+ B cells, a decrease in the number of CD5+ cells and a uniform distribution of receptors around the perimeter of the cells.

Serological evidence of enzootic bovine leukosis in the periurban dairy cattle production system of Al Ain, United Arab Emirates.

The serostatus of enzootic bovine leukosis (EBL) was determined at three dairy farms and the Al Ain Livestock Market (AALM), within the Al Ain region of Abu Dhabi, UAE. Of the 957 bovine sera tested by ELISA, 657 were from Holstein-Friesians from three dairy farms, and 300 from Bos indicus cattle at AALM. The chi-square homogeneity test (CSHT) and the Marascuilo multiple comparison procedure (MMCP) assessed the level of significance between the proportions of EBL-seropositive cattle (ESPC) across the study farms and AALM, and between the age groups at farms 1 and 3. Overall, the proportion of ESPC was 25.7% at dairy farms and AALM, 37.0% for farms and 1.0% for AALM. Furthermore, the proportions of ESPC at farms 1, 2 and 3 were 54.7%, 0.0% and 26.3% respectively, and statistically significant differences were seen across the farm/farm and farm/AALM comparisons, and between two age groups at farms 1 and 3. The 37-72-month-old age group showed the highest proportion of ESPC. This is the first serological evidence of EBL in the UAE. As previously reported, the ESPC are comparatively higher in dairy than Bos indicus cattle. Molecular and more extensive serological studies are needed to further corroborate the present data. Meanwhile, the UAE veterinary authorities will need to formulate national EBL control policies.

The chemokines CCL2 and CXCL10 produced by bovine endometrial epithelial cells induce migration of bovine B lymphocytes, contributing to transuterine transmission of BLV infection.

Bovine leukemia virus (BLV) causes a lymphoproliferative disease in cattle and is transmitted horizontally and vertically via infected lymphocytes. Although transplacental infection is considered the predominant route of vertical transmission of BLV, the molecular mechanisms of this process remain to be elucidated. Notably, how BLV passes through the blood-placental barrier remains unclear, given that BLV is transmitted primarily by cell-to-cell contact. One hypothesis is that B cell migration to the placenta may be induced by certain endometrium-expressed chemokines. To test this hypothesis, we performed an in vitro cell migration assay using bovine B cell lines and endometrial epithelial cells. Cell migration assays showed that two bovine B cell lines, BL2M3 and BL3.1 cells, were attracted to the supernatant of bovine endometrial epithelial cells (BEnEpCs). Quantitative real-time RT-PCR showed that expression levels of mRNAs encoding the chemokines CCL2 and CXCL10 were higher in BEnEpCs than in MDBK cells. Additionally, an inhibition assay using immune serum against CCL2 and CXCL10 showed suppression of migration of bovine B cell lines. A syncytium assay showed that cells expressing BLV envelope (Env) protein fused with BEnEpCs. Here we found that bovine B cells are attracted by chemokines produced in the endometrium and that cells expressing BLV Env protein fused with endometrium epithelial cells. These results explain part of the molecular mechanism of transplacental transmission during BLV infection, although further analysis will be required. Advances in these areas are expected to contribute to controlling the spread of BLV.

Seroprevalence of bovine leukemia virus in cattle, buffalo, and camel in Egypt.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. It causes significant economic losses associated with losses due to slaughter and eradication of infected animal from infected area and other indirect economic losses such as restriction on importation of animals and semen from infected area. The main objective of this study was to determine the seroprevalence of BLV antibodies in cattle, buffaloes, and camels in Egypt using ELISA test. Serum samples were collected from 350 cattle, 100 buffaloes, and 100 camels during 2018. The seropositivity for BLV-specific antibody was 20.8%, 9%, and 0% in cattle, buffaloes, and camels, respectively. The result revealed significant association (p < 0.05) between age and seroprevalence of BLV infection in cattle > 4 years (24%) compared with those < 4 years (13%). We found no significant association between pregnancy and herd size and seroprevalence of BLV infection in this study (p > 0.05). Furthermore, the age, pregnancy state, and herd size had significant effect on seroprevalence of BLV infection in buffaloes. This study contributes that BLV is detected in cattle and buffaloes in Egypt and confirms that the camels has resistance against BLV infection. Hence, the control measures are very necessary to combat the transmission of the disease and reduce its economic impact.

Mapping of CD4+ T-cell epitopes in bovine leukemia virus from five cattle with differential susceptibilities to bovine leukemia virus disease progression.

BACKGROUND: Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia virus, is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly prolonged course involving persistent lymphocytosis and B-cell lymphoma. The bovine major histocompatibility complex class II region plays a key role in the subclinical progression of BLV infection. In this study, we aimed to evaluate the roles of CD4+ T-cell epitopes in disease progression in cattle. METHODS: We examined five Japanese Black cattle, including three disease-susceptible animals, one disease-resistant animal, and one normal animal, classified according to genotyping of bovine leukocyte antigen (BoLA)-DRB3 and BoLA-DQA1 alleles using polymerase chain reaction sequence-based typing methods. All cattle were inoculated with BLV-infected blood collected from BLV experimentally infected cattle and then subjected to CD4+ T-cell epitope mapping by cell proliferation assays. RESULTS: Five Japanese Black cattle were successfully infected with BLV, and CD4+ T-cell epitope mapping was then conducted. Disease-resistant and normal cattle showed low and moderate proviral loads and harbored six or five types of CD4+ T-cell epitopes, respectively. In contrast, the one of three disease-susceptible cattle with the highest proviral load did not harbor CD4+ T-cell epitopes, and two of three other cattle with high proviral loads each had only one epitope. Thus, the CD4+ T-cell epitope repertoire was less frequent in disease-susceptible cattle than in other cattle. CONCLUSION: Although only a few cattle were included in this study, our results showed that CD4+ T-cell epitopes may be associated with BoLA-DRB3-DQA1 haplotypes, which conferred differential susceptibilities to BLV proviral loads. These CD4+ T-cell epitopes could be useful for the design of anti-BLV vaccines targeting disease-susceptible Japanese Black cattle. Further studies of CD4+ T-cell epitopes in other breeds and using larger numbers of cattle with differential susceptibilities are required to confirm these findings.

Prostaglandin E2-Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti-PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection.

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies.

Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Lymphocyte proliferation and apoptosis of lymphocyte subpopulations in bovine leukemia virus-infected dairy cows with high and low proviral load.

Bovine leukemia virus (BLV) is one of the most important virus in dairy cattle. The infection behavior follows what we call the iceberg phenomenon: 60% of infected animals do not show clinical signs; 30% develop persistent lymphocytosis (PL); and the remaining 10%, die due to lymphosarcoma. BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms by which cattle governs the control of viral dissemination will be desirable for designing effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. It is known that BLV affects cellular homeostasis: proliferation and apoptosis. It is also known that the BLV tropism is directed towards B lymphocytes, and that lymphocytotic animals have elevated amounts of these cells. Usually, when an animal is infected by BLV, the B markers that increase are CD21, CD5 and CD11b. This increase could be related to the modulation of apoptosis in these cells. This is the first work in which animals infected with BLV are classified according to their proviral load and the subpopulations of B and T lymphocytes are evaluated in terms of their percentage in peripheral blood and its stage of apoptosis and viability. PBMCs from HPL animals proliferated more than LPL and non-infected animals. CD11b+/CD5+ lymphocytes in LPL animals presented greater early and late apoptosis than HPL animals and cells of HPL animals had increased viability than LPL animals. Our results confirm that BLV alters the mechanism of apoptosis and proliferation of infected cells.

Short communication: Bovine leukemia virus infection in adult cows does not interfere with foot-and-mouth disease vaccination.

Bovine leukemia virus (BLV) is a widespread infection that can affect innate and adaptive immunity; however, little information exists on how BLV infection affects foot-and-mouth disease virus (FMDV) vaccination programs. Vaccination for FMDV is compulsory in many regions of the world, and vaccine efficacy is monitored by measuring total antibodies against this virus. In a previous study, we observed that BLV-infected heifers produced a lower amount of antibodies in response to FMDV at first vaccination. In this follow-up study, we show that BLV status does not interfere with the total level and avidity of anti-FMDV-specific antibodies induced after repetitive routine vaccination in adult cattle. This is relevant information for the proficiency of vaccine-based FMDV control programs in BLV-endemic regions.

Cooperation of PD-1 and LAG-3 in the exhaustion of CD4+ and CD8+ T cells during bovine leukemia virus infection.

Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle.

Short communication: Evaluation of 5 different ELISA for the detection of bovine leukemia virus antibodies.

Although Canadian dairy herds have been infected with bovine leukemia virus (BLV) for years, recent research has put new emphasis on the potential negative effects of this infection. Consequently, BLV control is becoming more favorable; however, BLV control cannot be successful without identifying infected animals. Bovicheck BLV (Biovet, Saint-Hyacinthe, QC, Canada) is currently the only assay licensed by the Canadian Centre for Veterinary Biologics. The first goal of this study was, therefore, to determine the reproducibility of the Bovicheck BLV assay for serum samples derived from Canadian cattle. The second goal was to evaluate and compare 5 different ELISA and determine their test characteristics using serum samples from Canadian herds. The considered ELISA were Bovicheck BLV, ID Screen BLV Competition (IDvet, Grabels, France), Idexx Leukosis Serum X2 Ab Test (Idexx Europe B.V., Hoofddorp, the Netherlands), Svanovir BLV gp51-Ab (Svanova, Uppsala, Sweden), and the Serelisa BLV Ab Mono Indirect (Synbiotics, Lyon, France). Eighty serum samples from Canadian cattle provided by Prairie Diagnostic Services (PDS; Saskatoon, SK, Canada) and an additional 80 serum samples from Canadian dairy and beef herds were used for the study. The Bovicheck BLV assay yielded the same results for all PDS-derived samples, implying a high level of reproducibility and robustness of this assay. Additionally, the comparison of the assays' results showed high agreement between assays, with Cohen's kappa values between κ = 0.91 and κ = 1. Furthermore, using original test results of the field samples as true status, relative diagnostic sensitivity and specificity were calculated. Relative diagnostic sensitivity of all tests was 100%. False-positive results were probable; therefore, the following relative diagnostic specificities were determined: 100% for Bovicheck BLV, Idexx Leukosis Serum X2, and Svanovir BLV; 95% for ID Screen BLV; and 97% for Serelisa BLV. When considering other test characteristics, ID Screen BLV is exceptional due to considerable practical advantages.