Whole blood transcriptome analysis in dairy calves experimentally challenged with bovine herpesvirus 1 (BoHV-1) and comparison to a bovine respiratory syncytial virus (BRSV) challenge.

Bovine herpesvirus 1 (BoHV-1), is associated with several clinical syndromes in cattle, among which bovine respiratory disease (BRD) is of particular significance. Despite the importance of the disease, there is a lack of information on the molecular response to infection via experimental challenge with BoHV-1. The objective of this study was to investigate the whole-blood transcriptome of dairy calves experimentally challenged with BoHV-1. A secondary objective was to compare the gene expression results between two separate BRD pathogens using data from a similar challenge study with BRSV. Holstein-Friesian calves (mean age (SD) = 149.2 (23.8) days; mean weight (SD) = 174.6 (21.3) kg) were either administered BoHV-1 inoculate (1 × 107/mL × 8.5 mL) (n = 12) or were mock challenged with sterile phosphate buffered saline (n = 6). Clinical signs were recorded daily from day (d) -1 to d 6 (post-challenge), and whole blood was collected in Tempus RNA tubes on d six post-challenge for RNA-sequencing. There were 488 differentially expressed (DE) genes (p < 0.05, False Discovery rate (FDR) < 0.10, fold change ≥2) between the two treatments. Enriched KEGG pathways (p < 0.05, FDR <0.05); included Influenza A, Cytokine-cytokine receptor interaction and NOD-like receptor signalling. Significant gene ontology terms (p < 0.05, FDR <0.05) included defence response to virus and inflammatory response. Genes that are highly DE in key pathways are potential therapeutic targets for the treatment of BoHV-1 infection. A comparison to data from a similar study with BRSV identified both similarities and differences in the immune response to differing BRD pathogens.

Messenger RNA biomarkers of Bovine Respiratory Syncytial Virus infection in the whole blood of dairy calves.

Bovine Respiratory Syncytial Virus (BRSV) is a primary viral cause of Bovine Respiratory Disease (BRD) in young calves, which is responsible for substantial morbidity and mortality. Infection with BRSV induces global gene expression changes in respiratory tissues. If these changes are observed in tissues which are more accessible in live animals, such as whole blood, they may be used as biomarkers for diagnosis of the disease. Therefore, the objective of the current study was to elucidate the whole blood transcriptomic response of dairy calves to an experimental challenge with BRSV. Calves (Holstein-Friesian) were either administered BRSV inoculate (103.5 TCID50/ml × 15 ml) (n = 12) or sterile phosphate buffered saline (n = 6). Clinical signs were scored daily and whole blood was collected in Tempus RNA tubes immediately prior to euthanasia, at day 7 post-challenge. RNA was extracted from blood and sequenced (150 bp paired-end). The sequence reads were aligned to the bovine reference genome (UMD3.1) and EdgeR was subsequently employed for differential gene expression analysis. Multidimensional scaling showed that samples from BRSV challenged and control calves segregated based on whole blood gene expression changes, despite the BRSV challenged calves only displaying mild clinical symptoms of the disease. There were 281 differentially expressed (DE) genes (p < 0.05, FDR < 0.1, fold change > 2) between the BRSV challenged and control calves. The top enriched KEGG pathways and gene ontology terms were associated with viral infection and included "Influenza A", "defense response to virus", "regulation of viral life cycle" and "innate immune response". Highly DE genes involved in these pathways may be beneficial for the diagnosis of subclinical BRD from blood samples.

Electrochemical assay of sorbitol dehydrogenase at PEDOT modified electrodes - a new milk biomarker for confirmation of pregnancy in dairy cattle.

A robust electrochemical assay for sorbitol dehydrogenase (SORDH) activity in milk was developed using voltammetry and chronocoulometry at bare and polymer modified transducers. The motivation for the work was to evaluate the potential of SORDH as an early biomarker of bovine pregnancy using milk as sample matrix. SORDH is an enzyme involved in carbohydrate metabolism converting sorbitol, the sugar alcohol form of glucose, into fructose, with NAD+ as a cofactor being simultaneously reduced to NADH. The assay was optimised via direct NADH oxidation on glassy carbon and screen printed carbon electrodes followed by electropolymerisation of 3,4-ethylenedioxythiophene (EDOT) monomer to form an NADH responsive PEDOT surface which operated well in undiluted milk samples. Assay conditions such as incubation time and temperature were optimised resulting in a 3 min assay at 37 °C in the presence of 10 mM NAD+ and 20 mM sorbitol co-substrates, enabling NADH electro oxidation (linear range 0.25-5 mM, sensitivity 9.17 µC cm-2 mM-1 in undiluted milk). SORDH determination followed over the range 0.31-10 U mL-1 in milk samples with sensitivity 5.45 µC cm-2 U-1 mL with LOD 0.0787 U mL-1. The assay was applied to milk sample testing acquired as part of an approved animal study involving control and breeding cycles of dairy cows with focus on analysis at day 19 post artificial insemination. Significant differences between control and pregnant SORDH levels in whole milk animal samples were found (average values 2.57 and 4.07 ng mL-1 respectively), as verified using a commercial SORDH ELISA optical assay. Finally, progesterone monitoring over days 16-21 of the oestrous cycle employed an optical ELISA assay and confirmed maintenance of progesterone levels from day 19 onwards.