Tanner F. Scull

The bovine gastrointestinal (GI) epithelium plays a central role in nutrient absorption, barrier regulation, and host–microbe interactions; however, primary cell-based in vitro systems for bovine GI tissues remain limited. Isolation and maintenance of primary gastrointestinal epithelial cells are challenging due to tissue complexity, microbial contamination risk, and culture-associated phenotypic changes. This study aimed to isolate, culture, and perform a preliminary marker-based characterization of epithelial-enriched primary bovine gastrointestinal cell cultures derived from four GI tract segments: rumen, jejunum, ileum, and colon. Gastrointestinal tissues were collected from three 4-month-old Holstein calves. Luminal mucosal tissue was mechanically separated, enzymatically dissociated, and cultured in vitro. Initial culture performance outcomes, including post-cell isolation viability, were recorded. Epithelial cell-associated gene expression was evaluated using qPCR for CDH1, CLDN4, TJP1, KRT8, and KRT18 at three stages: whole tissue, post-cell isolation, and post-cell culture. Keratin 8 (KRT8) staining was assessed by immunofluorescence in pooled cultured cells, with descriptive quantification of KRT8-positive cells in the available images. Linear mixed-effects analysis accounting for repeated measurements within calf identified an effect of experimental stage for all five transcripts after false-discovery-rate correction. Overall effects of Segment and the Segment × Stage interaction were retained only for TJP1, indicating that stage-associated expression patterns were not uniform among gastrointestinal regions for this marker. KRT8 immunofluorescence demonstrated KRT8-positive cells in cultures derived from all four gastrointestinal segments. Across eight quantified fields, 244 out of 325 DAPI-defined cells (75.1%) were classified as KRT8-positive. These findings support the isolation and culture of epithelial-enriched primary bovine GI cultures under the conditions tested. While additional standardized culture-performance assessment, expanded characterization of epithelial and non-epithelial cell populations, passage stability analysis, and functional barrier assays are needed, this work provides a preliminary methodological framework for epithelial-enriched primary bovine GI cell cultures and supports their future application in studies of epithelial biology, host–microbe interactions, and molecular responses of the bovine gastrointestinal tract.

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.

During the periparturient period, cows undergo heightened energy demands at lactation onset, paired with reduced dry matter intake, leading to negative energy balance (NEB). Excessive lipolysis-driven adipose tissue remodeling, triggered by NEB, significantly contributes to ketosis in periparturient dairy cows. However, the role of peripheral blood mononuclear cells (PBMCs) in the pathogenesis of ketosis and in modulating adipose tissue function remains poorly understood. Here, we investigated how ketosis affects the transcriptional profile and secretome of PBMCs and its influence on preadipocyte function in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Twenty-one postpartum Holstein dairy cows were categorized as either subclinical ketosis (SCK; BHB ≥ 1.0 mM) or control (CON; BHB < 0.8 mM) based on blood beta-hydroxybutyrate (BHB) concentration screening. Blood samples were collected intravenously for the isolation of PBMCs and serum metabolic profiling. Ketosis elevated circulating NEFA and BHB levels but reduced total WBC and neutrophil counts. Isolated PBMCs were evaluated for gene expression and used to produce conditioned media (PBMC-CM), during which PBMCs were stimulated with 10 ng/mL LPS. The overall phenotype of PBMCs was largely consistent between SCK and CON cows, with minimal differences detected in immunomodulatory cytokine expression and PBMC-CM composition following stimulation. Preadipocytes isolated from non-ketotic cows were treated with PBMC-CM to assess the effect of PBMC secretomes on adipose cell function. Preadipocytes treated with SCK PBMC-CM showed reduced lipid accumulation compared to those treated with CON PBMC-CM regardless of the depot. SAT preadipocytes had heightened expression of lipid metabolism-related genes, including DGAT1, LIPE, and FASN, compared to VAT when treated with SCK PBMC-CM. Preadipocytes treated with CM from PBMC stimulated by LPS exhibited upregulation in IL1B and IL6 regardless of the depot or source of PBMCs. Together, these results indicate that although PBMC profiles showed minimal differences, preadipocytes treated with PBMC-CM may be influenced by additional factors, leading to altered preadipocyte function and gene expression that may contribute to adipose cellular dysfunction.