Phenotypic Selection of Dairy Cattle Infected with Bovine Leukemia Virus Demonstrates Immunogenetic Resilience through NGS-Based Genotyping of BoLA MHC Class II Genes.

Characterization of the bovine leukocyte antigen (BoLA) DRB3 gene has shown that specific alleles associate with susceptibility or resilience to the progression of bovine leukemia virus (BLV), measured by proviral load (PVL). Through surveillance of multi-farm BLV eradication field trials, we observed differential phenotypes within seropositive cows that persist from months to years. We sought to develop a multiplex next-generation sequencing workflow (NGS-SBT) capable of genotyping 384 samples per run to assess the relationship between BLV phenotype and two BoLA genes. We utilized longitudinal results from milk ELISA screening and subsequent blood collections on seropositive cows for PVL determination using a novel BLV proviral load multiplex qPCR assay to phenotype the cows. Repeated diagnostic observations defined two distinct phenotypes in our study population, ELISA-positive cows that do not harbor detectable levels of provirus and those who do have persistent proviral loads. In total, 565 cows from nine Midwest dairy farms were selected for NGS-SBT, with 558 cows: 168 BLV susceptible (ELISA-positive/PVL-positive) and 390 BLV resilient (ELISA-positive/PVL-negative) successfully genotyped. Three BoLA-DRB3 alleles, including one novel allele, were shown to associate with disease resilience, *009:02, *044:01, and *048:02 were found at rates of 97.5%, 86.5%, and 90.3%, respectively, within the phenotypically resilient population. Alternatively, DRB3*015:01 and *027:03, both known to associate with disease progression, were found at rates of 81.1% and 92.3%, respectively, within the susceptible population. This study helps solidify the immunogenetic relationship between BoLA-DRB3 alleles and BLV infection status of these two phenotypic groupings of US dairy cattle.

Tracing Viral Transmission and Evolution of Bovine Leukemia Virus through Long Read Oxford Nanopore Sequencing of the Proviral Genome.

Bovine leukemia virus (BLV) causes Enzootic Bovine Leukosis (EBL), a persistent life-long disease resulting in immune dysfunction and shortened lifespan in infected cattle, severely impacting the profitability of the US dairy industry. Our group has found that 94% of dairy farms in the United States are infected with BLV with an average in-herd prevalence of 46%. This is partly due to the lack of clinical presentation during the early stages of primary infection and the elusive nature of BLV transmission. This study sought to validate a near-complete genomic sequencing approach for reliability and accuracy before determining its efficacy in characterizing the sequence identity of BLV proviral genomes collected from a pilot study made up of 14 animals from one commercial dairy herd. These BLV-infected animals were comprised of seven adult dam/daughter pairs that tested positive by ELISA and qPCR. The results demonstrate sequence identity or divergence of the BLV genome from the same samples tested in two independent laboratories, suggesting both vertical and horizontal transmission in this dairy herd. This study supports the use of Oxford Nanopore sequencing for the identification of viral SNPs that can be used for retrospective genetic contact tracing of BLV transmission.

Diagnostic Measures of Disease Progression in Cattle Following Natural Infection with Bovine Leukemia Virus.

This study describes the longitudinal changes in bovine leukemia virus (BLV) ELISA antibodies, proviral load (PVL), and blood lymphocyte counts (LC) observed over a 2.5-year period in naturally infected cattle. The dataset utilized was from a BLV intervention field trial on three Midwestern dairy herds. Our analysis showed ELISA false negatives were more likely to occur in cattle with low PVL and normal LC. On average, negligible changes in LC were observed during six-month intervals. Periods of lymphocytosis, defined as >10,000 lymphocytes per uL of blood, were observed in 31.5% (68/216) of BLV test-positive cattle. In BLV test-positive cows, an average increase of 2900 to 3100 proviral copies per 100,000 cells was observed during each subsequent six-month sampling interval. The difference between the minimum and maximum PVL observed for an ELISA-positive cow with 3 or more observations ranged from 0 to 115,600 copies per 100,000 cells (median: 12,900; mean: 19,200). Therefore, following the identification of ELISA-positive cattle and the assessment of PVL and LC, subsequent semiannual tests to assess disease progression may not be needed. Further work is needed to determine how available diagnostic tests can be optimized to design cost-effective testing schemes for BLV control programs.

Current Developments in the Epidemiology and Control of Enzootic Bovine Leukosis as Caused by Bovine Leukemia Virus.

Enzootic Bovine Leukosis (EBL) caused by the bovine leukemia virus (BLV) has been eradicated in over 20 countries. In contrast, the U.S. and many other nations are experiencing increasing prevalence in the absence of efforts to control transmission. Recent studies have shown that BLV infection in dairy cattle has a greater impact beyond the long-recognized lymphoma development that occurs in <5% of infected cattle. Like other retroviruses, BLV appears to cause multiple immune system disruptions, affecting both cellular and humoral immunity, which are likely responsible for increasingly documented associations with decreased dairy production and decreased productive lifespan. Realization of these economic losses has increased interest in controlling BLV using technology that was unavailable decades ago, when many nations eradicated BLV via traditional antibody testing and slaughter methods. This traditional control is not economically feasible for many nations where the average herd antibody prevalence is rapidly approaching 50%. The ELISA screening of cattle with follow-up testing via qPCR for proviral load helps prioritize the most infectious cattle for segregation or culling. The efficacy of this approach has been demonstrated in at least four herds. Breeding cattle for resistance to BLV disease progression also appears to hold promise, and several laboratories are working on BLV vaccines. There are many research priorities for a wide variety of disciplines, especially including the need to investigate the reports linking BLV and human breast cancer.

Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells.

Death-associated protein (DAP) undergoes substantial changes in expression during turkey skeletal muscle development, decreasing from the 18 day embryonic stage to 1 day posthatch, and again from 1 day posthatch to 16 weeks of age. These changes suggest that DAP plays an important role at critical stages of the developmental process. The objective of this study was to elucidate the role of DAP in muscle development by examining the effect of reduced DAP expression on global gene expression in proliferating and differentiating turkey pectoralis major muscle satellite cells. Small interfering RNA was used to knock down expression of DAP and the transcriptome was subsequently profiled using a turkey skeletal muscle long oligonucleotide microarray. Microarray data were corroborated using quantitative real-time PCR. In proliferating cells, 458 loci, resulting in 378 uniquely annotated genes, showed differential expression (false discovery rate, FDR < 0.05). Pathway analysis highlighted altered eukaryotic translational initiation factors (eIFs) signaling, protein ubiquitination, sirtuin signaling, and mechanistic target of rapamycin (mTOR) signaling as the primary pathways affected in the knockdown proliferating cells. The findings underpinned the potential DAP involvement in cell proliferation of turkey satellite cells through the coordination between protein synthesis and cell cycle. In differentiating cells, 270 loci, accounting for 189 unique genes, showed differential expression (FDR < 0.05). Decreased expression of genes encoding various myofibrillar proteins and proteins involved in sarcoplasmic reticulum calcium flux suggests that DAP may affect regulation of calcium homeostasis and cytoskeleton signaling. This study provides the first evidence that reduced expression of DAP significantly alters the transcriptome profile of pectoralis major muscle satellite cells, thereby reducing proliferation and differentiation.

T and B cell activation profiles from cows with and without Johne's disease in response to in vitro stimulation with Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) is a chronic wasting disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). JD is particularly problematic on US dairy farms: estimates show that over 50% of farms are MAP-contaminated and as many as 91% of dairy herds could be infected. Although estimates vary widely, JD may cost the dairy industry between $200 million and $1.5 billion every year. One major obstacle to JD management is that JD is difficult to detect in many animals, in part due to the variable immunity against MAP demonstrated by JD+ cattle. To characterize the diversity of immune responses against MAP, peripheral blood mononuclear cells from 154 JD test negative and 96 JD test positive cows from the same dairy herds were stimulated with MAP in vitro. The activation of CD4+, CD8+ and γδ T cells and surface IgM+ B cells was measured using flow cytometry. CD4+CD45R0+ T cells, γδ+MHCII+ and γδ+MHCII- T cells and SIgM+ B cells from JD test positive cows all exhibited increased proportions expressing CD25 after MAP stimulation, while CD8+ T cells did not demonstrate increased CD25 expression in response to MAP.

Dairy Cows Naturally Infected with Bovine Leukemia Virus Exhibit Abnormal B- and T-Cell Phenotypes after Primary and Secondary Exposures to Keyhole Limpet Hemocyanin.

Bovine leukemia virus (BLV) is a retrovirus that is highly prevalent in US dairy herds: over 83% are BLV infected and the within-herd infection rate can be almost 50% on average. While BLV is known to cause lymphosarcomas, only 5% or fewer infected cattle will develop lymphoma; this low prevalence of cancer has historically not been a concern to dairy producers. However, more recent research has found that BLV+ cows without lymphoma produce less milk and have shorter lifespans than uninfected herdmates. It has been hypothesized that BLV infection interferes with normal immune function in infected cattle, and this could lead to reduced dairy production. To assess how naturally infected BLV+ cows responded to a primary and secondary immune challenge, 10 BLV+ and 10 BLV- cows were injected subcutaneously with keyhole limpet hemocyanin (KLH) and dimethyldioctadecylammonium bromide. B- and T-cell responses were characterized over the following 28 days. A total of 56 days after primary KLH exposure, cows were re-injected with KLH and B- and T-cell responses were characterized again over the following 28 days. BLV+ cows produced less KLH-specific IgM after primary immune stimulation; demonstrated fewer CD45R0+ B cells, altered proportions of CD5+ B cells, altered expression of CD5 on CD5+ B cells, and reduced MHCII surface expression on B cells ex vivo; exhibited reduced B-cell activation in vitro; and displayed an increase in BLV proviral load after KLH exposure. In addition, BLV+ cows had a reduced CD45R0+γδ+ T-cell population in the periphery and demonstrated a greater prevalence of IL4-producing T cells in vitro. All together, our results demonstrate that both B- and T-cell immunities are disrupted in BLV+ cows and that antigen-specific deficiencies can be detected in BLV+ cows even after a primary immune exposure.

Enhanced production of human influenza virus in PBS-12SF cells with a reduced interferon response.

Influenza is one of the most important infectious diseases in humans. The best way to prevent severe illness caused by influenza infection is vaccination. Cell culture-derived influenza vaccines are being considered in addition to the widely used egg-based system in order to support the increasing seasonal demand and to be prepared in case of a pandemic. Cell culture based systems offer increased safety, capacity, and flexibility with reduced downstream processing relative to embryonated eggs. We have previously reported a chick embryo cell line, termed PBS-12SF, that supports replication of human and avian influenza A viruses to high titers (>10(7) PFU/ml) without the need for exogenous proteases or serum proteins. Viral infections in cells are limited by the Interferon (IFN) response typified by production of type I IFNs that bind to the IFNα/ß receptor and activate an antiviral state. In this study, we investigated how neutralizing the interferon (IFN) response in PBS-12SF cells, via shRNA-mediated knock-down of IFNAR1 mRNA expression, affects influenza virus production. We were successful in knocking down ∼90% of IFNAR1 protein expression by this method, resulting in a significant decrease in the response to recombinant chIFNα stimulation in PBS-12SF cells as shown by a reduction in expression of interferon-responsive genes when compared to control cells. Additionally; IFNAR1-knock-down cells displayed enhanced viral HA production and released more virus into cell culture supernatants than parental PBS-12SF cells.

Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle.

BACKGROUND: Skeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16 wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16 wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage. RESULTS: A total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR. CONCLUSIONS: The current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products.