Bovine Leukemia Research Articles

HIV-1 and BLV are insensitive to SERINC5 restriction under the cell-cell infection.

Serine incorporator 5 (SERINC5, SER5) suppresses viral cell-free infection. However, its antiviral potency under viral cell-cell infection is not examined yet. Here, we established the in vitro cell-cell infection systems to assess SER5's antiviral activity on HIV-1 and bovine leukemia virus (BLV). Our results showed SER5 from different mammalian species, including Homo sapiens, Bos taurus, and Felis catus, was capable of significantly inhibiting HIV-1 cell-free infection. However, these SER5s were unable to restrict HIV-1 cell-cell infection. Intriguingly, Ebola virus glycoprotein-mediated pseudoviral cell-free infection was greatly enhanced by Homo sapiens and Bos taurus SER5. Notably, BLV was also insensitive to SER5-mediated restriction under the cell-cell infection. In addition, BLV envelope glycoproteins, which contained one glycosylation variation, were not restricted by SER5 in the model of cell-cell infection. Overall, our results indicated SER5 was ineffective against HIV-1 and BLV under the cell-cell infection, which was utilized by them to circumvent SER5-imposed restriction in vitro and may aid their transmission in vivo.IMPORTANCESER5 potently inhibits virus infection under the cell-free mode. However, few studies check whether SER5 keeps its restriction under the virus cell-cell transmission. In this work, we uncover SER5 loses its restriction to HIV-1 and BLV under the mode of cell-cell infection, demonstrating the viruses could employ this mode to overcome SER5 restriction and thus facilitate their transmission.

Phylogenetic and mutational analysis of bovine leukemia virus (BLV) tax gene in specialized dairy production systems in Antioquia, Colombia.

The bovine leukemia virus (BLV) is a pathogen of high importance for the dairy industry. Currently, twelve genotypes have been described worldwide with different pathogenicity and virulence, so it is critical to evaluate the circulating genotypes in each country/region to associate this information with risk situations. The aim of this work was to perform a phylogenetic and mutational analysis of the BLV tax gene in cows that belong to specialized dairies in the Department of Antioquia, Colombia. A conventional PCR for the tax gene was performed on 86 bovine samples. Sanger sequencing was carried out on 22 PCR products with a size of 959 bp. The sequences obtained were aligned and analyzed using the Maximum Likelihood and Bayesian phylogenetic approaches. A predictor was used to analyze the possible impact of amino acid substitution on the Tax structure and function. Although all sequences were found to belong to genotype 1, four of the 22 sequences were grouped into a different subclade G1A. Fifty percent of the samples showed punctual mutations in their amino acids. Mutation S104L was identified as "possibly harmful," while the V146A change found in all subclade G1A samples was identified as "possibly benign." Although further studies are necessary to determine whether there is an effect of these mutations on the development of the disease, this study presents part of the evolution of the virus and the changes at the amino acid level that are occurring in cattle from specialized dairy farms in Antioquia.

Investigating BoLA Class II DRB3*009:02 carrying cattle in Japan.

Enzootic bovine leukosis (EBL) is a malignant lymphoma of cattle that is mainly caused by bovine leukemia virus (BLV) infection. In this study, PCR-RFLP was used to investigate the frequency of the DRB3*009:02 allele in several farms with different herd management practices in Japan. A total of 742 Holsteins (384) and Japanese Blacks (230) were used as the sample size for the study, which was larger than the number of cattle in the study area with a confidence level of 95 % and a margin of error of 8. PBMCs isolated from whole blood from clinically healthy cattle were used for examination. The presence of BLV provirus infection was determined by qPCR targeting the env region. BLV antibodies were detected using a commercial ELISA kit. The results showed that 35 cattle were heterozygous for DRB3*009:02. The frequency on each farm varied between farms, and PCR analysis showed that the prevalence of BLV also varied between farms. The incidence rate (4.7 %) was lower than in previous studies. The BLV seroprevalence (14.4 %) in this study was lower than the BLV infection rate (35 %) in the study. Holstein dairy cows had low levels of BL resistance genes, confirming the spread of the virus within the farm's herd. Cattle on farms with low BLV-positive rates had a resistance gene-carrying rate of 16.9 %, meanwhile, those who did not carry the resistance gene had a rate of 91.4 %. BLV provirus levels vary between farmers, with herds carrying low BL-resistance genes tending to have higher levels of BLV provirus. In light of the current BLV epidemic, herd composition reform, along with aggressive breeding of BL-resistant sires, is a required component to increase the herd of BL-resistant sires and improve livestock productivity.

BLV-CoCoMo Dual qPCR Assay Targeting LTR Region for Quantifying Bovine Leukemia Virus: Comparison with Multiplex Real-Time qPCR Assay Targeting pol Region.

The proviral load (PVL) of the bovine leukemia virus (BLV) is a useful index for estimating disease progression and transmission risk. Real-time quantitative PCR techniques are widely used for PVL quantification. We previously developed a dual-target detection method, the "Liquid Dual-CoCoMo assay", that uses the coordination of common motif (CoCoMo) degenerate primers. This method can detect two genes simultaneously using a FAM-labeled minor groove binder (MGB) probe for the BLV long terminal repeat (LTR) region and a VIC-labeled MGB probe for the BoLA-DRA gene. In this study, we evaluated the diagnostic and analytical performance of the Dual-CoCoMo assay targeting the LTR region by comparing its performance against the commercially available Takara multiplex assay targeting the pol region. The diagnostic sensitivity and specificity of the Liquid Dual-CoCoMo assay based on the diagnostic results of the ELISA or original Single-CoCoMo qPCR were higher than those of the Takara multiplex assay. Furthermore, using a BLV molecular clone, the analytical sensitivity of our assay was higher than that of the Takara multiplex assay. Our results provide the first evidence that the diagnostic and analytical performances of the Liquid Dual-CoCoMo assay are better than those of commercially available multiplex assays that target the pol region.

Direct TaqMan assay for the detection and genotyping of bovine viral diarrhea virus types 1 and 2

Bovine viral diarrhea (BVD), caused by bovine viral diarrhea virus (BVDV), has a significant economic impact on affected farms worldwide. For effective disease control, it is crucial to select an appropriate vaccine based on the specific genotype of BVDV. Therefore, developing a rapid and reliable assay to detect and genotype BVDV is imperative for controlling the spread of disease. In this study, we developed a TaqMan assay to detect and genotype BVDV types 1 and 2 directly in bovine serum without extraction of RNA. The direct BVDV TaqMan assay effectively detected both BVDV1 and BVDV2 with confirmed specificity and showed no cross-reactivity with any of the other viruses tested, including bovine respiratory syncytial virus, bovine coronavirus, Akabane virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine immunodeficiency virus, and bovine leukemia virus. The assay could detect the virus in serum samples with a titer as low as 102 TCID50/mL in two out of three trials for BVDV1 and all three trials for BVDV2, indicating that its sensitivity is equivalent to that of virus isolation. Our findings represent a significant advancement in BVDV detection and typing directly from bovine serum.

BoLA-DRB3 alleles influence proviral load and peripheral blood lymphocyte distribution in bovine leukaemia virus-infected cattle.

Bovine leukaemia virus (BLV)-infected Holstein cattle carrying certain bovine leukocyte antigen (BoLA)-DRB3 alleles were previously shown to be resistant to BLV provirus multiplication, while those carrying other alleles were susceptible. This study aimed to determine whether the BoLA-DRB3 alleles carried by BLV-infected cattle could predict proviral load (PVL) and peripheral blood lymphocyte (PBL) count distribution (PVL/PBL distribution). Blood samples from Holstein cattle on four dairy farms were tested for the presence of BLV antibodies using a commercial ELISA. The PVL and PBL levels of the BLV-infected cattle were also measured, and genotyping was performed to identify the BoLA-DRB3 alleles they carried, impact of the various BoLA-DRB3 alleles on the PVL/PBL distribution was then investigated. Of the 316 cattle tested, 114 were positive for BLV. BLV-infected cattle carrying BoLA-DRB3 alleles DRB3*009:02, DRB3*002:01 and DRB3*014:01:01 were classified as resistant (n=43), those carrying DRB3*012:01 and DRB3*015:01 alleles were classified as susceptible (n=42) and the remaining cattle were classified as nonsusceptible/nonresistant (n=29). Multiple regression analysis revealed that PVL was positively correlated (p = 2.1×10-23) with PBL count and age was negatively correlated (p=1.9×10-6) with PBL count. Cattle with DRB3*014:01:01 tended to have a lower PBL count (p=0.031). The effects of the BoLA-DRB3 alleles DRB3*002:01, DRB3*009:02, DRB3*012:01 and DRB3*015:01 on PVL/PBL distribution were unclear due to the small numbers of BLV-infected animals carrying these alleles. The BLV transmission risk in cattle can be estimated by examining their BoLA-DRB3 alleles.

The transmission of bovine leukemia virus to calves occurs mostly through colostrum and milk

Background and Aim: Enzootic bovine leukemia is highly prevalent in most dairy farms, and strategies to reduce both vertical and horizontal transmission are being investigated. In this study, we aimed to investigate the rate of in utero infection, transmission of bovine leukemia virus (BLV) to calves through colostrum and milk, and the effectiveness of colostrum and milk pasteurization in reducing BLV transmission to calves. Materials and Methods: This study included four groups of calves from seropositive and seronegative cows. Group 1: Calves from BLV-positive cows (n = 11) were fed pasteurized colostrum and milk; Group 2: Calves from BLV-negative cows (n = 9) were fed pasteurized colostrum and milk; Group 3: calves from BLV-positive cows (n = 16) were fed unpasteurized colostrum and milk; and Group 4: calves from BLV-negative cows (n = 9) were fed unpasteurized colostrum and milk. In utero infection was evaluated using blood samples collected from calves before colostrum ingestion (day 0), and BLV transmission through colostrum and/or milk was evaluated by collecting blood samples after colostrum ingestion (days 1, 7, and 30). Samples seropositive on days 0 and/or 30 were also analyzed for the presence of viral DNA by nested polymerase chain reaction (nPCR). Results: All calves born to BLV seronegative cows (Groups 2 and 4) tested negative on days 0 and 30, indicating a lack of virus transmission via tank milk. Among the calves from Group 1, we found one in utero infection, and among the nine calves serologically positive on day 30, we found four positives by nPCR. Within Group 3, we found one in utero infection, and among the 10 calves serologically positive on day 30, we found 7 also positive by nPCR. Conclusion: The transmission of BLV through colostrum is central to the persistence of this virus in dairy cattle. Molecular detection of BLV in seropositive calves during the 1st month of life, followed by culling, may be a valuable eradication strategy. Keywords: diagnosis, epidemiology, retrovirus, vertical transmission.

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Metabolic Fingerprinting of Blood and Urine of Dairy Cows Affected by Bovine Leukemia Virus: A Mass Spectrometry Approach.

This study investigated metabolic changes associated with bovine leukemia virus (BLV) infection in dairy cows, focusing on pre-parturition alterations. Metabolite identification in serum and urine samples was performed using a targeted metabolomics method, employing the TMIC Prime kit in combination with flow injection analysis and liquid chromatography-tandem mass spectrometry. Of 145 cows examined, 42 (28.9%) were BLV-seropositive. Around 38% of infected cows showed high somatic cell counts indicative of subclinical mastitis, with 15 experiencing additional health issues such as ketosis, milk fever, and lameness. Despite these conditions, no significant differences in milk yield or composition were observed between the infected and control groups. Metabolomic analysis conducted at -8 and -4 weeks prepartum revealed significant metabolic differences between BLV-infected and healthy cows. At -8 weeks, 30 serum metabolites were altered, including sphingomyelins, lysophosphatidylcholines, amino acids, and acylcarnitines, suggesting disruptions in membrane integrity, energy metabolism, and immune function indicative of early neoplastic transformations. By -4 weeks, the number of altered metabolites decreased to 17, continuing to reflect metabolic disruptions in cows with leukemia. Multivariate analysis highlighted distinct metabolic profiles between infected and control cows, identifying key discriminating metabolites such as choline, aspartic acid, phenylalanine, and arginine. Urine metabolomics revealed significant prepartum shifts in metabolites related to glucose, asymmetric dimethylarginine, and pyruvic acid, among others. The research confirmed metabolomics' efficacy in defining a BLV infection metabolic profile, elucidating leukosis-associated metabolic disruptions. This approach facilitates the identification of BLV-infected cows and enhances understanding of infection pathophysiology, providing a foundation for advanced management and intervention strategies in dairy herds. The study underscores the profound impact of leukosis on metabolic processes and highlights urine metabolomics' utility in non-invasively detecting BLV infection, offering the potential for improved herd health management.

CRISPR-Cas13a-Based Lateral Flow Assay for Detection of Bovine Leukemia Virus.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), which presents worldwide prevalence. BLV caused substantial economic loss in China around the 1980s; then, it could not be detected for some time, until recently. Due to its latent and chronic characteristics, the efficient and accurate detection of BLV is of utmost significance to the timely implementation of control measures. Therefore, this study harnessed the recombinase-aided amplification (RAA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) technology, and lateral flow (LF) strips to develop an efficient method for detection of BLV. In this method, isothermal amplification of the targeted pol gene is performed at 37 °C with a detection threshold of 1 copy/µL, and the procedure is completed in 100 min. This assay demonstrated high selectivity for BLV, as indicated by the absence of a cross-reaction with six common bovine pathogens. Remarkably, 100 blood samples from dairy cows were tested in parallel with a conventional quantitative polymerase chain reaction (qPCR) and this method and the results showed 100% agreement. Furthermore, this method exhibited good repeatability. In conclusion, in this study, we established a sensitive and specific method for BLV detection, which shows promise for application in BLV surveillance.

Reduction of antisense transcription affects bovine leukemia virus replication and oncogenesis.

In sheep infected with bovine leukemia virus (BLV), transcription of structural, enzymatic, and accessory genes is silenced. However, the BLV provirus transcribes a series of non-coding RNAs that remain undetected by the host immune response. Specifically, three RNAs (AS1-L, AS1-S, and AS2) are consistently expressed from the antisense strand, originating from transcriptional initiation at the 3'-Long Terminal Repeat (LTR). To investigate the role of these non-coding RNAs in viral replication and pathogenesis, a reverse genetics approach was devised, capitalizing on a mechanistic disparity in transcription initiation between the 5' and 3' promoters. A two-nucleotide mutation (GG>TA) in the TFIIB-recognition element (BRE) impaired antisense transcription originating from the 3'-LTR. In the context of the provirus, this 2bp mutation significantly diminished the expression of antisense RNAs, while not notably affecting sense transcription. When inoculated to sheep, the mutated provirus was infectious but exhibited reduced replication levels, shedding light on the role of antisense transcription in vivo. In comparison to lymphoid organs in sheep infected with a wild-type (WT) provirus, the mutant demonstrated alterations in both the spatial distribution and rates of cell proliferation in the lymph nodes and the spleen. Analysis through RNA sequencing and RT-qPCR unveiled an upregulation of the Hmcn1/hemicentin-1 gene in B-lymphocytes from sheep infected with the mutated provirus. Further examination via confocal microscopy and immunohistochemistry revealed an increase in the amount of hemicentin-1 protein encoded by Hmcn1 in peripheral blood mononuclear cells (PBMCs) and lymphoid organs of sheep infected with the mutant. RNA interference targeting Hmcn1 expression impacted the migration of ovine kidney (OVK) cells in vitro. In contrast to the WT, the mutated provirus showed reduced oncogenicity when inoculated into sheep. Collectively, this study underscores the essential role of antisense transcription in BLV replication and pathogenicity. These findings may offer valuable insights into understanding the relevance of antisense transcription in the context of human T-cell leukemia virus (HTLV-1).

Use of blood meals from stable flies to evaluate the bovine leukemia virus infection status in cattle herds: a pilot study.

The incidence of enzootic bovine leukosis (EBL), a type of B-cell lymphoma, is increasing in Japan. EBL is caused by bovine leukemia virus (BLV; Retroviridae, Deltaretrovirus bovleu) infection and is diagnosed by detecting antibodies against BLV in milk and blood or BLV DNA in blood. We assessed the feasibility of using stable flies (Stomoxys calcitrans) as a sampling tool to assess BLV infection status in cattle herds. First, we collected blood from 3 cattle herds and, based on the measurement of BLV-proviral load (PVL) by quantitative real-time PCR (qPCR), identified 1) a BLV-free herd, 2) a herd with a low prevalence of BLV-infected cattle and low PVL, and 3) a herd wherein half of the cattle were BLV-infected with low-to-high PVLs. Next, we collected stable flies from the 3 herds, extracted DNA from their blood meals, analyzed it for BLV DNA, and measured the BLV PVL. Cattle DNA and BLV DNA, but not other mammalian DNA, were successfully detected by digestion of the flies. Based on fly blood meal qPCR, we identified one herd as BLV-free and the other 2 herds as having <50% prevalence of BLV-infected cattle with low PVLs. Our fly results were not consistent with preliminary BLV-PVL measurements on cattle blood. Our pilot study indicated that, to assess the feasibility of a stable fly blood meal test as an alternative technique for evaluating BLV infection status in dairy and beef cattle, additional investigations involving more cattle herds and stable flies are needed.

Effect of C-to-T transition at CpG sites on tumor suppressor genes in tumor development in cattle evaluated by somatic mutation analysis in enzootic bovine leukosis.

Oncogenic transformation of normal cells is caused by mutations and chromosomal abnormalities in cancer-related genes. Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma caused by bovine leukemia virus (BLV) infection in cattle. Although a small fraction of BLV-infected cattle develops EBL after a long latent period, the mechanisms for oncogenesis in EBL cattle remain largely unknown. In this study, we analyzed the types and patterns of somatic mutations in cancer cells from 36 EBL cases, targeting 21 cancer-related genes. Various somatic mutations were identified in eight genes, TP53, KMT2D, CREBBP, KRAS, PTEN, NOTCH1, MYD88, and CARD11. In addition, TP53 gene was found to be mutated in 69.4% of EBL cases, with most being biallelic mutations. In some cases, associations were observed between the ages at which cattle had developed EBL and somatic mutation patterns; young onset of EBL possibly occurs due to high impact mutations affecting protein translation and biallelic mutations. Furthermore, nucleotide substitution patterns indicated that cytosine at CpG sites tended to be converted to thymine in many EBL cases, which was considered to be the result of spontaneous deamination of 5-methylcytosine. These results demonstrate how somatic mutations have occurred in cancer cells leading to EBL development, thereby explaining its pathogenic mechanism. These findings will contribute to a better understanding and future elucidation of disease progression in BLV infection.IMPORTANCEEnzootic bovine leukosis (EBL) is a malignant and lethal disease in cattle. Currently, there are no effective vaccines or therapeutic methods against bovine leukemia virus (BLV) infection, resulting in severe economic losses in livestock industry. This study provides a renewed hypothesis to explain the general mechanisms of EBL onset by combining the previous finding that several integration sites of BLV provirus can affect the increase in survival and proliferation of infected cells. We demonstrate that two additional random events are necessary for oncogenic transformation in infected cell clones, elucidating the reason why only few infected cattle develop EBL. Further exploration of somatic mutation and BLV integration sites could support this hypothesis more firmly, potentially contributing to the development of novel control methods for EBL onset.

Performance of 5 commercial ELISA kits for the detection of antibody to bovine leukemia virus.

To prevent significant economic losses, some countries have successfully eradicated enzootic bovine leukosis (EBL), which is caused by bovine leukemia virus (BLV) infection. In Serbia, efforts to eliminate EBL commenced in the late 1990s. Recognizing the disparities in test selection among laboratories and variations in quality, we evaluated the diagnostic sensitivity and specificity of commercial ELISAs using field samples in Serbia. Using 5 commercial ELISA kits, we tested 138 cattle serum samples, submitted for confirmatory testing between 2020 and 2023, along with 100 serum samples from BLV-negative herds. We found 100% agreement of the ID Screen BLV Competition (IDvet), Svanovir BLV gp51-Ab (Svanova), and INgezim BLV Compac 2.0 (Ingenasa) ELISAs. We observed 93% agreement comparing these 3 kits to the Bovine Leukemia Virus Antibody test kit (VMRD). Agreements of 92% and 88.4% were determined between Idexx and IDvet, Svanova, and Ingenasa kits, and between Idexx and VMRD kits, respectively.

Peripheral Blood Mononuclear Cell Transcriptome of Dairy Cows Naturally Infected with Bovine Leukemia Virus.

The current literature has identified many abnormalities in the immune expression of cows infected with the bovine leukemia virus (BLV). These studies have focused on individual cell, gene, or protein expression, failing to provide a comprehensive understanding of the changes in immune expression in animals with BLV. To identify the overall alterations in immune expression during BLV infection, the transcriptomes of the peripheral blood mononuclear cells (PBMCs) of cows seropositive or seronegative for BLV antibodies were sequenced. Whole blood samples were collected from 20 dairy cows and screened for BLV antibodies and PCR was used to quantify the proviral load of the samples. PBMCs were separated from whole blood using density gradient centrifugation from which RNA was isolated and sequenced. Three seropositive samples (BLV+; n = 3), including one of each PVL category, low (n = 1), moderate (n = 1), and high (n = 1), and three seronegative samples (BLV-; n = 3) were sequenced for differential gene expression analysis. The results showed major differences in the transcriptome profiles of the BLV+ and BLV- PBMCs and revealed a wide variety of immunological pathways affected by BLV infection. Our results suggest that disease state and PBMC gene expression vary depending on BLV proviral load levels and that BLV causes the suppression of normal immune responses and influences B and T cell gene expression, resulting in immune dysfunction.

EXPERIENCE IN HEALING FARMS FROM LEUKEMIA OF CATTLE

This article presents the experience of rehabilitating two farms with initial infection with the bovine leukemia virus of 17.72% and 33.48%. A scheme for improving the health of a farm using the method of serological and hematological research, with the isolation of sero-positive animals and the subsequent elimination of animals with hematological leukemia, is described. This also shows the prevalence and incidence of oncornavirus infection in an uncontrolled epizootic process.

Screening for Japanese Black cattle herds at risk of bovine leukemia virus transmission based on the presence of persistent lymphocytosis

This study aimed to develop a screening method to identify Japanese Black (JB) cattle farms at high risk of bovine leukemia virus (BLV) transmission. We introduced a recently established lymphocyte count (LC) cut-off to detect cattle with persistent lymphocytosis (PL). To identify high-risk farms, we examined the relationship between the proportion of cattle with PL and the mean blood proviral loads (PVL) per farm. The results showed a strong correlation between the proportion of cattle with high LC, higher than the LC cut-off values for JB cattle, and the mean blood PVL per farm. This indicates that the mean blood PVL on a herd basis of JB cattle can be estimated from the proportion of cattle with high LC. Specifically, the mean blood PVL on farms with >30 % cattle having high LC was estimated to be greater than 100 copies/10 ng DNA. The higher the proportion of cattle with high LC per farm, the higher the proportion of cattle at risk as sources of BLV infection. The study demonstrated that the proportion of cattle with high LC, based on the new LC cut-off, serves as a practical index for screening high-risk farms with PL cattle that have high PVL. This finding is meaningful for prioritizing farms with a high proportion of cattle with high LC, necessitating BLV infection prevention measures, such as voluntary culling and segregation, in order to develop a regional, stepwise BLV eradication strategy.

On the issue of prevention and eradication of minor viral bovine diseases in Ukraine

The study aimed to evaluate the epizootic status of livestock in Ukraine concerning the prevalence of bovine immunodeficiency virus and bovine foamy virus infections. A literature review was conducted to analyze the epizootic status of livestock farming in various countries regarding bovine immunodeficiency and spumavirus infections. To investigate this issue in Ukrainian livestock, blood samples were collected from 10–15 cows with further DNA extraction and studies via PCR, according to the developers’ recommendations. The biological characteristics of bovine foamy virus and bovine immunodeficiency virus were studied by infecting bovine fetal lung (LEK) and calf coronary vessels (KST) cell cultures, with each passage being visually monitored and examined through light microscopy. PCR was performed on the third and fifth passages to detect the genetic material. The genetic material of bovine leukemia virus, bovine immunodeficiency virus, and bovine foamy virus was confirmed in 12 farms across 8 regions of Ukraine. It was demonstrated that bovine immunodeficiency virus and bovine foamy virus can integrate into homologous cell cultures derived from cattle. The immunosuppressive effects of bovine foamy virus and its capability to inhibit components of the non-specific immune system were established on laboratory animal models. Emphasis is placed on the necessity to develop domestic tools for the retrospective diagnosis of bovine immunodeficiency and spumavirus infections and to implement a national anti-epizootic program

Cytotoxic T-lymphocyte antigen 4 and programmed cell death ligand 1 blockade increases the effectiveness of interleukin-15 immunotherapy in a bovine leukemia model.

Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4- and PD-L1-blocking algorithm. The codon-optimized bIL15 gene was synthesized under de novo conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent Escherichia coli BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the Bcl2, STAT3, and STAT5 genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells. Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the Bcl2, STAT3, and STAT5 genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production. The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections.

Estimation of genetic parameters and genome-wide association study for enzootic bovine leukosis resistance in Canadian Holstein cattle

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (leukosis) frequently observed in North American dairy herds. Infection with BLV can lead to persistent lymphocytosis and tumors, and is associated with decreased production, immunity and fertility. With no available treatment or vaccine, reducing the prevalence of leukosis through management and culling has not yet been successful. Genetic selection could contribute to permanent improvement in dairy cattle resistance to leukosis. This study aimed to examine the prevalence and impact of leukosis in Canada, and to assess the potential for including leukosis resistance in Canadian national genetic evaluations by characterizing the genetic architecture of leukosis resistance using pedigree and genomic information. A total of 117,349 milk enzyme-linked immunosorbent assay test records on 96,779 Holstein cows from 950 Canadian herds taken between 2007 and 2021 were provided by Lactanet Canada (Guelph, ON, Canada). Each cow was classified as test-positive for leukosis or test-negative for leukosis. Leukosis was present in approximately 77% of herds tested; within those herds, an average of 39% of cows tested were test positive for leukosis. Heritabilities of 0.10 (SE = 0.001) and 0.07 (SE <0.001) were estimated for leukosis resistance using a linear animal model and BLUP or single-step GBLUP methodology, respectively. Breeding value correlations were estimated between leukosis resistance and economically important and phenotypically relevant traits. Most correlations between leukosis resistance and traits already included in Canadian genetic evaluations were favorable, with the exception of somatic cell score. The candidate genes for leukosis resistance identified using a genome-wide association study, were on chromosome 23, with some being part of the major histocompatibility complex. This study showed that genetic evaluation for leukosis resistance is possible, and could be considered for inclusion in Canadian national selection indices.