Enzootic Bovine Leukosis Research Articles

Low proviral load in the Kumamoto strain of Japanese Brown cattle infected with the bovine leukemia virus

BackgroundThe Kumamoto strain of Japanese Brown (JBRK) cattle is a sub-breed of Wagyu and has a different genetic background than that of Japanese Black (JB) cattle. Bovine leukemia virus (BLV) is the pathogen causing enzootic bovine leukosis (EBL), the predominant type of bovine leukosis (BL). EBL is one of the most common bovine infectious diseases in dairy countries, including Japan. Some host genetic factors, including the bovine leukocyte antigen (BoLA)-DRB3 gene, have been associated with the proviral load (PVL) of BLV and/or onset of EBL. Here, we determined the number of BL cases by analyzing prefectural case records in detail. We measured the PVL of BLV-infected JBRK cattle and compared it with that obtained for other major breeds, JB and Holstein-Friesian (HF) cattle. Finally, the relationship between PVL levels and BoLA-DRB3 haplotypes was investigated in BLV-infected JBRK cattle.ResultsWe determined the number of BL cases recorded over the past ten years in Kumamoto Prefecture by cattle breed. A limited number of BL cases was observed in JBRK cattle. The proportion of BL cases in the JBRK was lower than that in JB and HF. The PVL was significantly lower in BLV-infected JBRK cattle than that in the JB and HF breeds. Finally, in BLV-infected JBRK cattle, the PVL was not significantly affected by BoLA-DRB3 alleles and haplotypes. BoLA-DRB3 allelic frequency did not differ between BLV-infected JBRK cattle with low PVL and high PVL.ConclusionsTo our knowledge, this is the first report showing that BL occurred less in the JBRK population of Kumamoto Prefecture. After BLV-infection, the PVL was significantly lower in JBRK cattle than that in JB and HF breeds. The genetic factors implicated in maintaining a low PVL have yet to be elucidated, but the BoLA-DRB3 haplotypes are likely not involved.

Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Seroprevalence and detection of bovine leukosis virus in semen from breeding bulls in Costa Rica

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a persistent infection and the most important neoplastic disease in cattle. It is spread primarily by transferring infected lymphocytes through blood from carriers to healthy animals. The present study is aimed at determining the seropositivity of BLV in breeding bulls from Costa Rica and at detecting for the first time in the country BLV DNA in bull semen. Between May 2011 and August 2018, 379 blood and 133 semen samples were collected from bulls distributed in 118 farms. The serum was analyzed by an enzymatic immunoassay and the semen by polymerase chain reaction and sequencing. BLV seropositivity was 43.5% (165/379), while 64.4% (76/118) of the farms had positive reactors. Holstein (75.7%) and Jersey (73.0%) breeds showed the highest seropositivity. In addition, Bos taurus bulls (68.1%), older than seven years (50.0%), and those belonging to dairy farms (75.5%) had higher seropositivity compared to Bos indicus (17.7%), younger than seven years (42.2%), and those from beef farms (15.5%), respectively. Moreover, Bos taurus bulls had a higher risk of being seropositive than Bos indicus (OR = 3.4; 95% CI: 1.7–6.8). BLV DNA was found in one semen sample (2.5%; 1/40) from a seropositive bull. The importance of serum and molecular BLV screening in semen samples and the potential role of some risk factors associated with the disease, such as the bull’s age, genotype, and type of livestock productive system, is argued in the present report.

Bovine leukosis virus, bovine viral diarrhea, and bovine neosporosis seroprevalence in specialized dairy herds in Antioquia-Colombia

Enzootic bovine leukosis (EBL) is a chronic infectious disease caused by the bovine leukosis virus (BLV), a Deltaretrovirus. Bovine viral diarrhea (BVD) is an infectious disease caused by a pestivirus. Bovine neosporosis is caused by the obligate intracellular parasite Neospora caninum (Nc). These pathogens can have horizontal (postnatal) or vertical (transplacental) transmissions and affect the productive and reproductive performance of infected bovines. This work aimed to detect BLV, BVD, and Nc seroprevalence in specialized dairy cattle from the north, east, and Aburrá Valley regions of the Department of Antioquia, the highest in milk production regions in Antioquia. A total of 599 blood samples, obtained from 53 specialized dairy cattle herds, were evaluated by the ELISA test. The results revealed a seroprevalence of 41.13% for BLV (242/599), 28.48% (163/599) for Nc, and 22.7% (132/599) for BVD. Regarding the regional seroprevalence evaluation, BLV was found in 47.02% of the samples from the east, 36.87% from the north, and 46.02% from the Aburrá Valley. Nc was found in 31.03% of the samples from the east, 24.26% from the north, and 36.63% from Aburrá Valley. BVD was found in 21.62% of the samples from the east, 25.03% from the north region, and 10.39% of the samples from the Aburrá Valley. It is highlighted by these results that the north region, with the highest milk production in Antioquia, had the lowest BLV and Nc seroprevalences but the highest seroprevalence of BVD. BLV has increased in Antioquia in recent years, and as an immunosuppressive infection, opportunities for other pathogens are created by it. A significant statistical difference was found in the average prevalence of the pathogens according to the municipality, cattle breed, and region of origin of the sample. The seroprevalence of these pathogens in specialized dairy herds in Antioquia can be classified as medium-low. However, it is recommended that biosecurity practices should be maximized to avoid the spread of these pathogens due to the variability detected in the region, municipality, breed group, and herd age. The rapid and efficient diagnosis of these three pathogens through reliable methodologies will allow for the control of dissemination in dairy herds.

Seroprevalence and risk factors associated with bovine Leukemia virus infection in argentine beef cattle

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, an endemic disease in dairy cattle of Argentina. However, little is known about the seroprevalence of BLV in beef cattle. In this study, we conducted a cross-sectional study including farms from thirteen provinces of Argentina. A total of 5827 bovine serum samples were collected from 76 farms and analyzed using an in-house developed enzyme-linked immunosorbent assay. Information about herd management was collected through a questionnaire, and univariate and multivariate analyses were performed to detect risk factors associated with BLV infection. Herd-level seroprevalence was 71.05%, while the mean animal-level seroprevalence was 7.23% (median = 2.69%; min = 0, max = 75). Only two provinces had no positive BLV samples. The other eleven provinces showed more than 50% of their farms infected with BLV. The multivariate model revealed that BLV prevalence was significantly associated with the use of animals raised in the same farm for cattle replacement (P = 0.005), breeding cows by natural mating with a bull (P < 0.001), and weaning calves after 6 months of age (P = 0.011). This extensive study revealed that BLV seroprevalence in Argentine beef farms has increased during the last years and allowed identifying some management practices associated with BLV prevalence. These data deserve special attention because BLV infection in beef cattle seems to lead to a dissemination pattern similar to that observed during the last decades in dairy cattle, especially considering that Argentina is the sixth beef producer in the world, with about 5% of global beef production.

Exploration of microRNA Biomarkers in Blood Small Extracellular Vesicles for Enzootic Bovine Leukosis.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). While most infected cattle show no clinical signs, approximately 30% of infected cattle develop persistent lymphocytosis (PL), and a small percentage may develop EBL. Currently, there is no method for predicting the possibility of EBL onset. In this study, we analyzed the microRNAs (miRNAs) encapsulated in small extracellular vesicles (sEVs) in the blood to explore the biomarkers of EBL. To identify candidate biomarkers, blood samples were collected from three BLV-uninfected and three EBL cattle. Total RNA was extracted from filtered serum and used for microarray analysis. Due to their association with cancer in human orthologs, we selected three miRNAs as candidate biomarkers, bta-miR-17-5p, bta-miR-24-3p, and bta-miR-210, which were more than twice as abundant in EBL cattle than in BLV-uninfected cattle. Quantitative real-time polymerase chain reaction (qPCR) using serum RNAs from six cattle used for the microarray analysis was carried out for the detection of the three selected miRNAs. Additionally, bta-miR-92a, whose ortholog has been associated with cancer in humans, was also examined by qPCR. bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were successfully detected, but bta-miR-210 was not. To further evaluate the utility of these three miRNAs as biomarkers, new blood samples were collected from 31 BLV-uninfected and 30 EBL cattle. The levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were significantly higher in EBL cattle than in BLV-uninfected cattle. These results suggest that increased levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a in the blood could be used as biomarkers for EBL. This study may contribute to the control of BLV infections and develop a prediction method of EBL onset.

BoLA-DRB3*15:01 allele is associated with susceptibility to early enzootic bovine leukosis onset in Holstein-Friesian and Japanese Black cattle

Enzootic bovine leukosis (EBL) is typically observed in cattle older than 3 years, but some cases of onset in cattle younger than 3 years have been reported in Japan. BoLA-DRB3 polymorphisms are associated with susceptibility to EBL onset. However, little is known about the relationship between the polymorphisms and EBL onset in young cattle. In the present study, we performed BoLA-DRB3 genotyping in 59 EBL cattle younger than 3 years (25 Holstein-Friesian and 34 Japanese Black) and compared the results with those of 69 EBL cattle older than 3 years (38 Holstein-Friesian and 31 Japanese Black). The BoLA-DRB3*15:01 allele was detected at a frequency of 37.3 % (48.0 % and 29.4 % in Holstein-Friesian and Japanese Black, respectively) and was identified as an early EBL onset susceptibility allele. Nine EBL cattle younger than 3 years (5 Holstein-Friesian and 4 Japanese Black), but only 1 EBL cattle older than 3 years (1 Holstein-Friesian), had a BoLA-DRB3*15:01/*15:01 homozygous genotype. The frequency of the BoLA-DRB3*15:01 allele occurring with a different allele (BoLA-DRB3*015:01/other) in cattle younger than 3 years was 44.1 % (56.0 % Holstein-Friesian and 35.3 % Japanese Black) and significantly higher than that in cattle older than 3 years (28.9 % Holstein-Friesian and 9.7 % Japanese Black) (P = 0.0013). These results suggest that BoLA-DRB3*15:01/*15:01 and BoLA-DRB3*15:01/other genotypes are early EBL onset susceptibility genotypes. The present findings may contribute to cattle breeding selection.

Development of a novel fluorogenic assay method for screening inhibitors of bovine leukemia virus protease and identification of mitorubrinic acid as an anti-BLV compound.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a fatal cattle disease that leads to significant economic losses in the livestock industry. Currently, no effective BLV countermeasures exist, except testing and culling. In this study, we developed a high-throughput fluorogenic assay to evaluate the inhibitory activity of various compounds on BLV protease, an essential enzyme for viral replication. The developed assay method was used to screen a chemical library, and mitorubrinic acid was identified as a BLV protease inhibitor that exhibited stronger inhibitory activity than amprenavir. Additionally, the anti-BLV activity of both compounds was evaluated using a cell-based assay, and mitorubrinic acid was found to exhibit inhibitory activity without cytotoxicity. This study presents the first report of a natural inhibitor of BLV protease-mitorubrinic acid-a potential candidate for the development of anti-BLV drugs. The developed method can be used for high-throughput screening of large-scale chemical libraries.

Multizentrisches B-Zell-Lymphom bei einem 8 Wochen alten Kalb

The presented report describes a case of sporadic bovine leukosis and its disease progression in an 8-week old, male cross-breed calf (Red Holstein Fleckvieh). The calf was initially presented due to suspect pulmonary infection. However, generalized enlargement of the subcutaneous lymph nodes was noticed, which is untypical for this disease. Based on the hematologic findings of highly increased numbers of lymphoblasts in peripheral blood as well as the sonographic examination of the lymph nodes, sporadic bovine leukosis was suspected. The calf died suddenly, three weeks after initial presentation. Pathohistological examination revealed a high-degree enlargement of all lymph nodes as well as an infiltration of nearly all organs and tissues with a monomorphic round cell population. These cells were also detected in bone marrow cytology. Immunhistochemical examination was performed and the cells reacted positive for the B-cell markers Pax 5 and CD20. Virologic examination for enzootic bovine leukosis was negative. In conjunction with the diagnosis of multicentric B-cell lymphoma, the test results indicated a juvenile form of sporadic bovine lymphoma.

Enzootic bovine leukosis in a cow

Clinical History: A 5-year-old, Holstein-Friesian dairy cow was evaluated by a veterinary practitioner for a 30-day history of unilateral exophthalmos (Fig. 1). After 15 days, the cow presented lameness followed by progressive weight loss and pelvic limbs paresis, culminating in persistent sternal recumbency (Fig. 2). The superficial inguinal lymph nodes were enlarged. Due to the poor prognosis, the cow was euthanized and submitted to a postmortem examination. Gross Findings: The cow was in poor body condition with mild amounts of subcutaneous and visceral fat stores. The oral and conjunctival mucous membranes were pale. There was severe exophthalmos in the right eye, caused by a soft, homogenous, white to yellow mass (6 cm in diameter) (Fig. 3) in the retrobulbar space. Similar irregular masses were seen in the left renal pelvis, partially effacing the renal parenchyma, and in the epidural space, circumferentially surrounding the pachymeninges (extradural location) (Fig. 4) of the lumbar segment of the spinal cord. The superficial inguinal lymph nodes (supramammary) were markedly enlarged and, on the cut surface, had homogenous white to yellow discoloration and loss of the corticomedullary junction. Multifocal areas of the abomasum wall were moderately thickened and expanded by a soft, homogenous, white to yellow masses. No significant alterations were observed in other organs.

Influence of BoLA-DRB3 Polymorphism and Bovine Leukemia Virus (BLV) Infection on Dairy Cattle Productivity

Enzootic bovine leukosis caused by the bovine leukemia virus (BLV) results in substantial damage to the livestock industry; however, we lack an effective cure or vaccine. BoLA-DRB3 polymorphism in BLV-infected cattle is associated with the proviral load (PVL), infectivity in the blood, development of lymphoma, and in utero infection of calves. Additionally, it is related to the PVL, infectivity, and anti-BLV antibody levels in milk. However, the effects of the BoLA-DRB3 allele and BLV infection on dairy cattle productivity remain poorly understood. Therefore, we investigated the effect of BLV infection and BoLA-DRB3 allele polymorphism on dairy cattle productivity in 147 Holstein dams raised on Japanese dairy farms. Our findings suggested that BLV infection significantly increased milk yield. Furthermore, the BoLA-DRB3 allele alone, and the combined effect of BLV infection and the BoLA-DRB3 allele had no effect. These results indicate that on-farm breeding and selection of resistant cattle, or the preferential elimination of susceptible cattle, does not affect dairy cattle productivity. Additionally, BLV infection is more likely to affect dairy cattle productivity than BoLA-DRB3 polymorphism.

Upregulation of host genes during disease progression in bovine leukemia virus infection is independent of overexpression of viral transcriptional regulators in vitro.

Bovine leukemia virus (BLV) is a member of the genus Deltaretrovirus within the family Retroviridae that infects bovine B cells, causing persistent lymphocytosis and enzootic bovine leukosis (EBL) in a small fraction of infected cattle. As changes in the transcriptome of infected cells are important for BLV disease progression, comprehensive analysis of gene expression in different disease states is required. In this study, we performed an RNA-seq analysis using samples from non-EBL cattle with and without BLV infection. Subsequently, a transcriptome analysis was conducted in combination with previously obtained RNA-seq data from EBL cattle. We found several differentially expressed genes (DEGs) between the three groups. After screening and confirmation of target DEGs using real-time reverse transcription polymerase chain reaction, we found that 12 target genes were significantly upregulated in EBL cattle compared to BLV-infected cattle without lymphoma. In addition, the expression levels of B4GALT6, ZBTB32, EPB4L1, RUNX1T1, HLTF, MKI67, and TOP2A were significantly and positively correlated with the proviral load in BLV-infected cattle. Overexpression experiments revealed that these changes were independent of BLV tax or BLV AS1-S expression in vitro. Our study provides additional information on host gene expression during BLV infection and EBL development, which may be helpful for understanding the complexity of transcriptome profiles during disease progression.

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

A clinical case of enzootic bovine leukosis diagnosed by using clonal analysis of peripheral B lymphocytes in a Japanese Black cow

A 7-year-old Japanese Black cow with anorexia was presented at a local veterinarian. As rectal palpation revealed an enlarged induration of the uterus, lymphoma was suspected, and the patient was introduced to the Veterinary Teaching Hospital of Obihiro University of Agriculture and Veterinary Medicine. Although lymphadenopathy and lymphocytosis were not found, a high proviral load of bovine leukemia virus (BLV) with 464 copies/10 ng DNA was recorded, and lymphocytes with constricted or flower-like nuclei were found occasionally in the peripheral blood; accordingly, enzootic bovine leukosis (EBL) was suspected. According to PCR for clonality analysis of B cells based on immunoglobulin heavy chain (IGH) gene rearrangement, monoclonal proliferation of peripheral blood B cells was evident, suggesting the presence of tumorigenic B cells in the peripheral blood and onset of EBL. Pathological examination including necropsy and histopathological examination confirmed the diagnosis of EBL. Clonality analysis for B cells in the peripheral blood is thought to be useful for diagnosing B-cell tumors such as EBL, especially in cases lacking lymphadenopathy and lymphocytosis. This is the first clinical case of EBL diagnosed by demonstrating monoclonal proliferation of peripheral blood B cells using IGH gene rearrangement-based PCR.

Phylogenetic analysis based on whole genome sequence of bovine leukemia virus in cattle under 3 years old with enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is one of bovine neoplasms caused by bovine leukemia virus (BLV). Although EBL is typically observed in cattle over 3 years old, several cases of EBL onset in cattle under 3 years old have been reported in Japan. The mechanism for EBL onset in young cattle remains unclear. Although genetic variation of BLV is limited, the variations could affect viral properties relating to BLV pathogenesis. The purpose of this study was to clarify relationship between early onset of EBL and BLV groups. Moreover, we also aimed to characterize BLV that cause early onset of EBL. Whole genome sequences of BLV in 72 EBL cattle under 3 years old and 50 EBL cattle over 3 years old were identified. Phylogenetic analysis showed that BLV was divided into 4 groups (A, B-1, B-2 and Other). The BLV from EBL cattle under 3 years old were mainly classified as group A and B-1, while those from EBL cattle over 3 years old were mainly included in group B-2. Common sequence of group A and B-1 was compared with those of group B-2. Specific sequences in LTRs, gag-pro-pol, env and tax gene regions were identified in these groups. Amino acid substitutions of Pro and Tax protein were predicted in those nucleotide sequences. Those genetic variations might contribute to the early onset of EBL.

Diagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil.

Enzootic bovine leukosis (EBL) is a chronic viral disease of wide distribution in cattle herds and may take several years for the first manifestation of clinical signs. Most animals do not present clinical signs. However, the economic losses are underestimated due to this disease. Thus, this work aimed to detect and characterize BLV in dairy cattle in the Maranhão state, northeastern Brazil. Blood samples were collected from 176 animals from 8 municipalities in the southeastern state of Maranhão. Bovine blood samples were subjected to DNA extraction and molecular diagnosis using nested PCR assays for BLV, targeting gp51 gene. Positive samples were then sequenced and then subjected to phylogenetic inferences. BLV DNA was detected in 16 cattle (16/176, 9.09%) in 4 municipalities. Phylogenetic analyzes showed that the sequence obtained clustered in a clade containing BLV sequences classified as genotype 6, with a high degree of support. Our data shows BLV occurrence in the Northeast of Brazil and the identification of genotype 6 in this region. These findings contribute to the molecular epidemiology of this agent in Brazil.

Correlation between the Biodistribution of Bovine Leukemia Virus in the Organs and the Proviral Load in the Peripheral Blood during Early Stages of Experimentally Infected Cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis. However, the propagation and distribution of BLV after primary infection still need to be fully elucidated. Here, we experimentally infected seven cattle with BLV and analyzed the BLV proviral load (PVL) in the blood and various organs. BLV was first detected in the blood of the cattle after one week, and the blood PVL increased for three weeks after infection. The PVL was maintained at a high level in five cattle, while it decreased to a low or medium level in two cattle. BLV was distributed in various organs, such as the heart, lung, liver, kidney, abomasum, and thymus, and, notably, in the spleen and lymph nodes. In cattle with a high blood PVL, BLV was detected in organs other than the spleen and lymph nodes, whereas in those with a low blood PVL, BLV was only detected in the spleen and lymph nodes. The amount of BLV in the organs was comparable to that in the blood. Our findings point to the possibility of estimating the distribution of BLV provirus in organs, lymph nodes, and body fluids by measuring the blood PVL, as it was positively correlated with the biodistribution of BLV provirus in the body of BLV infection during early stages.

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.

Bovine leukemia virus: a perspective insight into the infection and immunity.

Bovine leukemia virus (BLV) is a member of the Retroviridae family and belongs to the Deltaretrovirus genus. It has a close relationship with human T-cell leukemia virus type I. BLV is responsible for causing enzootic bovine leukosis (EBL), a contagious disease that affects the bovine lymphatic system. This virus poses challenges for the global cattle industry, as it impacts cattle populations all over the world. Despite being widespread and impactful, BLV often goes unnoticed, with many researchers unaware of its presence and the potential consequences it carries. BLV demonstrates varying levels of pathogenicity. The majority of cattle (around 70%) become seropositive asymptomatic carriers, displaying no noticeable clinical symptoms. However, a smaller proportion of infected animals experience persistent lymphocytosis, characterized by an elevated number of lymphocytes in the bloodstream. If not monitored and managed, a subset of these persistently infected cattle may advance to lymphosarcoma. This condition typically presents as tumors in different lymphoid tissues, impacting various organs and overall health and productivity. Furthermore, recent research has highlighted the potential association between the occurrence of breast and lung cancer in humans and the presence of BLV. This review will delve into the recent discoveries concerning BLV, specifically exploring its epidemiology, the economic impact it has on the global cattle industry, its implications for human medicine, and the association between different alleles of the major histocompatibility complex (MHC) and susceptibility or resistance to BLV. Bovine leukemia virus, Enzootic bovine leukosis, Major histocompatibility complex, Retroviridae.

Risk factors associated with enzootic bovine leukosis in Boyacá and Cundinamarca municipalities, Colombia.

Enzootic bovine leukosis (EBL) is a lymphoproliferative disorder caused by the bovine leukemia virus (BLV), a virus of the Retroviridae family. The infection is distributed worldwide, and a high percentage of animals infected by the BLV are asymptomatic and act as carriers of the virus in many cattle populations. To identify the risk factors associated with EBL in the municipalities of Boyacá and Cundinamarca (Colombia). A simple descriptive cross-sectional study with random sampling was conducted. A total of 1,140 blood samples were taken from cattle (females and males) from the municipalities of Chiquinquirá, Ubaté, and San Miguel de Sema of different breeds and age groups. The samples were processed using the commercial ELISA SERELISA® BLV Ab Mono Blocking kit (sensitivity 97%, specificity 98%). The data were processed with the statistical programs WinEpi and Epi Info® version 7.2.4.0, estimating the prevalence ratio, implementing the chi-square test (p ≤ 0.05) and logistic regression. A true prevalence (TP) and apparent prevalence (AP) of 23.61% and 22.7% in Ubaté, 19.22% and 18.1% in Chiquinquirá, and 15.61% and 14.3% in San Miguel de Sema, respectively, were established. Bovines 2-4 years old were the most prevalent in Ubaté and Chiquinquirá (37.5% and 21.21%, respectively), while in San Miguel de Sema individuals >4 years had the highest percentage of antibodies (18.3%). The Holstein breed had a higher prevalence in Ubaté and San Miguel de Sema (26.02% and 19.67%), and crossbreeds were more BLV-seroprevalence in Chiquinquirá (20.20%). In Ubaté, re-use of needles was identified as a risk factor, contaminated blood in needles is considered one of the main routes of transmission. On the other hand, manual milking was identified as a risk factor in San Miguel de Sema. The non-implementation of an individual needle per animal in Ubaté; the Holstein breed and manual milking in San Miguel de Sema were identified as risk factors for the presence of antibodies against the disease. EBL prevention and control plans should be established that focus on the implementation of management and sanitary practices based on herd biosecurity.