Dayle Johnston

Characterisation of haematological profiles and whole blood relative gene expression levels in Holstein-Friesian and Jersey bull calves undergoing gradual weaning.

Haematological profiles indicate the health status of an animal and can be used to identify sub-clinical stress responses. The objectives of the study were to examine (i) the effect of breed and plane of nutrition, on haematological profiles of artificially reared Holstein-Friesian and Jersey bull calves in response to gradual weaning, and (ii) the effect of breed on immune response genes in bovine whole blood using real-time quantitative PCR. Holstein-Friesian and Jersey bull calves were group housed indoors and individually fed using an automatic feeder. They were allocated to a high, medium or low plane of nutrition, based on milk replacer (MR) and concentrate. The nutrition treatments were calculated using National Research Council guidelines in order to achieve a high, medium or low growth rate for each respective breed. During the weaning phase MR was gradually reduced over a 14-day (d) period (d -13 to d 0). Calves were blood sampled on d -14, -6, -3, 0, 1, 3, 8 and 14 relative to weaning (d 0) for subsequent haematological analysis. On d -14, 1 and 8, a subset of eight Holstein-Friesian calves randomly selected from the medium nutrition treatment and eight Jersey calves randomly selected from the high nutrition treatment, were blood sampled for gene expression profiling, targeting biomarkers of weaning stress. These two treatment groups were chosen to examine the effect of breed on expression of the genes of interest, as energy intake and animal performance were similar. There was no effect of breed×plane of nutrition interaction nor effect of plane of nutrition on any variable measured (P>0.05). Gradual weaning produced differential biological responses in the two breeds evidenced by breed×time interactions for lymphocyte, monocyte and red blood cell number, plasma haemoglobin and haptoglobin concentrations (P<0.05). The typical stress response consisting of neutrophilia and lymphopaenia was not observed for any treatment. An immune response to gradual weaning was observed as the relative gene expression level of the pro-apoptotic gene, Fas, increased on d 1 relative to d -14 (P<0.05). Relative gene expression levels were greater in Jersey calves compared with Holstein-Friesian for the pro-inflammatory cytokine CXCL8 (P=0.05) and the glucocorticoid receptor, GRα (P<0.05). The increased levels of these transcripts suggest that Jersey calves may have a more sensitive immune system compared with Holstein-Friesian.

Effect of milk replacer and concentrate intake on growth rate, feeding behaviour and systemic metabolite concentrations of pre-weaned bull calves of two dairy breeds

Early-life nutrition affects calf development and thus subsequent performance. The aim of this study was to examine the effect plane of nutrition on growth, feeding behaviour and systemic metabolite concentrations of artificially reared dairy bull calves. Holstein-Friesian (F; n=42) and Jersey (J; n=25) bull calves with a mean±SD age (14±4.7 v. 27±7.2 days) and BW (47±5.5 v. 33±4.7 kg) were offered a high, medium or low plane of nutrition for 8 weeks using an electronic feeding system which recorded a range of feed-related events. Calves were weighed weekly and plasma samples were collected via jugular venipuncture on weeks 1, 4 and 7 relative to the start of the trial period. The calves offered a high plane of nutrition had the greatest growth rate. However, the increased consumption of milk replacer led to a reduction in feed efficiency. Holstein-Friesian calves offered a low plane of nutrition had the greatest number of daily unrewarded visits to the feeder (P<0.001). β-hydroxybutyrate (BHB) concentrations were greater in F calves on a low plane of nutrition (P<0.001). Although there was no effect of plane of nutrition, BHB concentrations in F calves increased before weaning, concomitant with an increase in concentrate consumption. Urea concentrations were unaffected by plane of nutrition within either breed. Jersey calves on a low plane of nutrition tended to have lower triglycerides than those on a high plane (P=0.08), but greater than those on a medium plane (P=0.08). Holstein-Friesian calves offered a high plane of nutrition tended to have greater triglyceride concentrations than those on a medium plane (P=0.08). Triglycerides increased from the start to the end of the feeding period (P<0.05), across both breeds. A medium plane of nutrition resulted in a growth, feeding behaviour and metabolic response comparable with a high plane of nutrition in pre-weaned bull calves of both F and J breeds.

Early recognition of bovine respiratory disease in calves using automated continuous monitoring of cough sounds

Abstract Bovine respiratory disease (BRD) complex in calves impairs health and welfare and causes severe economic losses for the Stockperson. Early recognition of BRD should lead to earlier veterinary (antibiotic/anti-inflammatory) treatment interventions thereby reducing the severity of the disease and associated costs. Coughing is one of the clinical manifestations of BRD. It is believed that by automatically and continuously monitoring the sounds within calf houses, and analysing the coughing frequency, early recognition of BRD in calves is possible. Therefore, the objective of the present study was to develop an automated calf cough monitor and examine its potential as an early warning system for BRD in artificially reared dairy calves. The coughing sounds of 62 calves were continuously recorded by a microphone over a three-month period. A sound analysis algorithm was developed to distinguish calf coughs from other sounds (e.g. mechanical sounds). During the sound recording period the health of the calves was assessed and scored periodically per week by a trained human observer. Calves presenting with BRD received antibiotic and/or anti-inflammatory treatment and the dates of treatment were recorded. This treatment date reference served as a comparison for the investigation of whether an increase in coughing frequency could be related to calves developing BRD. The calf cough detection algorithm achieved 50.3% sensitivity, 99.2% specificity and 87.5% precision. Four out of five periods, where coughing frequency was observed to be increased, coincided with the development of BRD in more than one calf. This period of increased coughing frequency was always observed before the calves were treated. Therefore, the calf cough monitor has the potential to identify early onset of BRD in calves.

Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue

BackgroundBovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences.ResultsLeptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences.ConclusionsThe microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant Leptotrichiaceae OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.

Characterisation of the Whole Blood mRNA Transcriptome in Holstein-Friesian and Jersey Calves in Response to Gradual Weaning

Weaning of dairy calves is an early life husbandry management practice which involves the changeover from a liquid to a solid feed based diet. The objectives of the study were to use RNA-seq technology to examine the effect of (i) breed and (ii) gradual weaning, on the whole blood mRNA transcriptome of artificially reared Holstein-Friesian and Jersey calves. The calves were gradually weaned over 14 days (day (d) -13 to d 0) and mRNA transcription was examined one day before gradual weaning was initiated (d -14), one day after weaning (d 1), and 8 days after weaning (d 8). On d -14, 550 genes were differentially expressed between Holstein-Friesian and Jersey calves, while there were 490 differentially expressed genes (DEG) identified on d 1, and 411 DEG detected eight days after weaning (P < 0.05; FDR < 0.1). No genes were differentially expressed within breed, in response to gradual weaning (P > 0.05). The pathways, gene ontology terms, and biological functions consistently over-represented among the DEG between Holstein-Friesian and Jersey were associated with the immune response and immune cell signalling, specifically chemotaxis. Decreased transcription of several cytokines, chemokines, immunoglobulin-like genes, phagocytosis-promoting receptors and g-protein coupled receptors suggests decreased monocyte, natural killer cell, and T lymphocyte, chemotaxis and activation in Jersey compared to Holstein-Friesian calves. Knowledge of breed-specific immune responses could facilitate health management practices better tailored towards specific disease sensitivities of Holstein-Friesian and Jersey calves. Gradual weaning did not compromise the welfare of artificially-reared dairy calves, evidenced by the lack of alterations in the expression of any genes in response to gradual weaning.

Electronic feeding behavioural data as indicators of health status in dairy calves

Abstract The objectives of this study were (i) to characterise clinical health in dairy calves on an Irish research farm during the artificial calf-rearing period and (ii) to determine whether calves’ pre-weaning intakes and feeding behaviour, recorded by electronic calf feeders, changes in response to incidents of bovine respiratory disease (BRD). Holstein-Friesian (H-F) and Jersey (J) calves were fed by automatic milk replacer (MR) and concentrate feeders. Feeding behaviour, including MR consumption, drinking speed, number of rewarded and unrewarded visits to the feeder as well as concentrate consumption, was recorded by the feeders. A modified version of the Wisconsin calf health scoring criteria chart was used to score calves’ clinical measurements and identify incidences of BRD. Thus, 40% of calves were found to have at least one incident of BRD. Feeding behaviour was altered during incidents of BRD. The number of unrewarded visits to the feeder was reduced, by approximately four visits, for calves with BRD during the 3 d prior to the identification of BRD (P < 0.05) and tended to be reduced during the 7 d following the identification of BRD (P = 0.05), compared with healthy calves. Additionally, calves with BRD had a tendency for reduced net energy intake (approximately 8%) during the 3 d prior to the identification of BRD, compared with healthy calves. Therefore, calf feeding behavioural data, recorded by electronic feeders during the pre-weaning period, can indicate cases of BRD.

Simultaneous detection of DNA and RNA virus species involved in bovine respiratory disease by PCR-free rapid tagmentation-based library preparation and MinION nanopore sequencing

The Oxford Nanopore MinION Mk1B is a portable 90 g device that sequences DNA directly at 450 bases/second generating sequence reads in excess of 400 kb. Recent improvements in error rate and speed of library preparation mean that this device has considerable potential for rapid molecular bovine pathogen diagnostics. We tested the MinION for rapid untargeted detection of viral pathogens associated with bovine respiratory disease (BRD), an economically important disease often involving primary infection of the lung by one or more of a number of DNA and/or RNA viruses. We combined three foetal lung cell cultures which were infected with either Bovine Respiratory Syncytial Virus (BRSV), Bovine Herpes Virus 1 (BoHV1) or Bovine Parainfluenza Virus 3 (BPI-3). BoHV1 is a DNA virus and BPI-3 and BRSV are RNA viruses. The cell cultures were treated with DNase and RNase to deplete bovine nucleic acid prior to viral nucleic acid extraction and double-stranded cDNA synthesis. Sequencing libraries were generated by PCR-free tagmentation in under 10 minutes and loaded onto a MinION sequencer. Approximately 7,000 sequencing reads were generated and analysed using high-throughput local BLAST against the NCBI nr/nt database. The top BLAST hit for 2,937 of these reads was identified as a virus. Of these, 2,926 (99.6%) were correctly identified either as BoHV1, BRSV or BPI-3.