Analyzing Cattle Activity Patterns with Ear Tag Accelerometer Data.

In this study, we equip two breeds of cattle located in tropical and temperate climates with smart ear tags containing triaxial accelerometers to measure their activity levels across different time periods. We produce activity profiles when measured by each of four statistical features, the mean, median, standard deviation, and median absolute deviation of the Euclidean norm of either unfiltered or high-pass-filtered accelerometer readings over five-minute windows. We then aggregate the values from the 5 min windows into hourly or daily (24 h) totals to produce activity profiles for animals kept in each of the test environments. To gain a better understanding of the variation between the peak and nadir activity levels within a 24 h period, we divide each day into multiple equal-length intervals, which can range from 2 to 96 intervals. We then calculate a statistical measure, called daily differential activity (DDA), by computing the differences in feature values for each interval pair. Our findings demonstrate that patterns within the activity profile are more clearly visualised from readings that have been subject to high-pass filtering and that the median of the acceleration vector norm is the most reliable feature for characterising activity and calculating the DDA measure. The underlying causes for these differences remain elusive and is likely attributable to environmental factors, cattle breeds, or management practices. Activity profiles produced from the standard deviation (a feature routinely applied to the quantification of activity level) showed less uniformity between animals and larger variation in values overall. Assessing activity using ear tag accelerometers holds promise for monitoring animal health and welfare. However, optimal results may only be attainable when true diurnal patterns are detected and accounted for.

Postnatal maternal behaviour expression depends on lambing difficulty in Merino ewes.

Dystocia, a prolonged or non-progressive birth event, is the main contributor to lamb mortality in Australia and across the world. Dystocia can cause neonatal hypoxia, central nervous system (CNS) damage leading to increased risk of starvation, exposure and mismothering, and death. These prolonged birth events can also cause fatigue, injury and death in the ewe. Dystocia may interrupt the expression of maternal behaviour and the strength of the ewe-lamb bond, and consequently lamb survival. This study focused on the effect of dystocia on ewe behaviour in the 2 h post-lambing. A total of 18 ewes were chosen for continuous behaviour annotation and analysis (dystocic (n = 9) and eutocic (n = 9)) based on the quality of video recordings, length of stage 2 parturition and classification by a single experienced observer. Dystocic ewes showed significantly lower expression of maternal behaviours and a significantly greater expression of avoidance behaviours compared to eutocic ewes. Additionally, dystocic ewes performed fewer behaviours in total compared to eutocic ewes. Dystocia can significantly affect the quality and quantity of ewe maternal behaviour expression, leading to increased avoidance of the lamb, increased risk of maternal disinterest, and increased risk of death for the lamb. If dystocic events can be identified quickly and accurately, measures can be taken to ensure the ewe and lamb recover successfully.

Selection of Genome-Wide SNPs for Pooled Allelotyping Assays Useful for Population Monitoring.

Parasitic worms are serious pests of humans, livestock, and crops worldwide. Multiple management strategies are employed in order to reduce their impact, and some of these may affect their genome and population allelic frequency distribution. The evolution of chemical resistance, ecological changes, and pest dispersal has allowed an increasing number of pests to become difficult to control with current management methods. Their lifestyle limits the use of ecological and individual-based management of populations. There is a need to develop rapid, affordable, and simple diagnostics to assess the efficacy of management strategies and delay the evolution of resistance to these strategies. This study presents a multilocus, equal-representation, whole-genome pooled single nucleotide polymorphisms (SNPs) selection approach as a monitoring tool for the ovine nematode parasite Haemonchus contortus. The SNP selection method used two reference genomes of different quality, then validated these SNPs against a high-quality recent genome assembly. From over 11 million high-quality SNPs identified, 334 SNPs were selected, of which 262 were species-specific, yielded similar allele frequencies when assessed as multiple individuals or as pools of individuals, and suitable to distinguish mixed nematode isolate pools from single isolate pools. As a proof-of-concept, 21 Australian H. contortus populations with various phenotypes and genotypes were screened. This analysis confirmed the overall low level of genetic differentiation between populations collected from the field, but clearly identifying highly inbred populations, and populations showing genetic signatures associated with chemical resistance. The analysis showed that 66% of the SNPs were necessary for stability in assessing population genetic patterns, and SNP pairs did not show linkage according to allelic frequencies across the 21 populations. This method demonstrates that ongoing monitoring of parasite allelic frequencies and genetic changes can be achieved as a management assessment tool to identify drug-treatment failure, population incursions, and inbreeding signatures due to selection. The SNP selection method could also be applied to other parasite species.

Using Pen-Side Measurable Blood Parameters to Predict or Identify Dystocic Lambing Events.

Dystocia is the greatest contributor to neonatal lamb mortality in Australia and poses significant welfare and economic concerns worldwide. In this study, we set out to investigate whether pen-side analysis technology could be employed to detect blood parameters predictive of dystocic labour events in sheep. In a pilot trial, we collected and analysed blood samples in pen-side assays for glucose, lactate, pH, pCO2, pO2, base excess, HCO3, TCO2, sO2, lactate, sodium, potassium, chloride, calcium, urea nitrogen, creatinine, haematocrit, haemoglobin and anion gap. From the pilot data, we identified creatinine, TCO2, chloride and calcium as potentially useful markers. To develop a time course and to establish variability of the selected blood parameters, a time series of samples was collected from 12 ewes, from mid-gestation to 48 h after birth. For the main trial, blood samples were collected at mid- and late gestation for glucose determination and for the full set of blood parameters at three time points before, at and after birth. Possible predictors of lambing difficulty were chloride, haematocrit and haemoglobin, sampled one week before birth; creatinine, sampled at birth; and blood pH and base excess after birth. In conclusion, we found that pen-side analysis of blood markers showed promise in identifying dystocic lambing events.

Quantification of differences in resistance to gastrointestinal nematode infections in sheep using a multivariate blood parameter.

Breeding for resistance to gastrointestinal nematodes (GIN) in sheep relies largely on the use of worm egg counts (WEC) to identify animals that are able to resist infection. As an alternative to such measures of parasite load we aimed to develop a method to identify animals showing resistance to GIN infection based on the impact of the infection on blood parameters. We hypothesized that blood parameters may provide a measure of infection level with a blood-feeding parasite through perturbation of red blood cell parameters due to feeding behaviour of the parasite, and white blood cell parameters through the mounting of an immune response in the host animal. We measured a set of blood parameters in 390 sheep that had been exposed to an artificial regime of repeated challenges with Trichostrongylus colubriformis followed by Haemonchus contortus. A simple analysis revealed strong relationships between single blood parameters and WECs with correlation coefficients -0.54 to -0.60. We then used more complex multi-variate methods based on supervised classifier models (including Bayesian Network) as well as regression models (Lasso and Elastic Net) to study the relationships between WECs and blood parameters, and derived algorithms describing the relationships. The ability of these algorithms to classify sheep GIN resistance status was tested using the WEC and blood parameters collected from a different group of 418 sheep that had acquired natural infections of H. contortus from pasture. We identified the most resistant and most susceptible animals (10% percentiles) of this group based on WECs, and then compared the identities of these animals to the identities of animals that were predicted to be most resistant and most susceptible by our algorithms. The models showed varying abilities to predict susceptible and resistant sheep, with up to 65% of the most susceptible animals and 30% of the most resistant animals identified by the Elastic Net model algorithms. The prediction algorithms derived from female sheep data performed better than those for male sheep in some cases, with the predicted animals accounting for up to 50-60% of the actual resistant and susceptible female animals. Heritability values were calculated for blood parameters and the aggregate trait descriptions defined by the novel prediction algorithms. The aggregate trait descriptions were moderately heritable and may therefore be suitable for use in genetic selection strategies. The present study indicates that multivariate models based on blood parameter data showed some ability to predict the resistance status of sheep to infection with H. contortus.

Analysis of antibody levels in egg yolk for detection of exposure to Ascaridia galli parasites in commercial laying hens.

Ascaridia galli is one of the most abundant nematode parasites in poultry. A. galli infections can significantly impact the profitability of egg farms and have negative implications for bird health and welfare. The main objectives of this study were to determine whether A. galli specific antibodies in egg yolks can be used to detect prior or current exposure to A. galli in laying hens, and to distinguish between eggs obtained from caged and free-range hens. Twenty-two laying hen flocks from different production systems (10 free-range, 2 barn-housed, and 9 caged flocks) were enrolled in the study. An in-house enzyme-linked immunosorbent assay was used to analyze levels of A. galli specific antibodies in yolk. The numbers of A. galli eggs in hen excreta were also determined in a subset of farms. Free-range flocks had higher and also more variable levels of anti-A. galli antibodies in the egg yolk compared to those of the cage flocks (0.50 ± 0.39 vs. 0.16 ± 0.13 OD units) (P < 0.001). Results also confirmed that excreta from free-range and barn-housed flocks contained higher numbers of A. galli eggs than did excreta from caged flocks in which no A. galli eggs were detected. In conclusion, analysis of anti-A. galli antibodies in the egg yolk can be used to detect worm exposure in commercial layer flocks. However, the method used in this study cannot be used in isolation to distinguish between eggs from cage and free-range production systems as anti-A galli antibodies were detected in egg yolk samples from all production systems, and the range of antibody levels overlapped between production systems.

Across-Experiment Transcriptomics of Sheep Rumen Identifies Expression of Lipid/Oxo-Acid Metabolism and Muscle Cell Junction Genes Associated With Variation in Methane-Related Phenotypes.

Ruminants are significant contributors to the livestock generated component of the greenhouse gas, methane (CH4). The CH4 is primarily produced by the rumen microbes. Although the composition of the diet and animal intake amount have the largest effect on CH4 production and yield (CH4 production/dry matter intake, DMI), the host also influences CH4 yield. Shorter rumen feed mean retention time (MRT) is associated with higher dry matter intake and lower CH4 yield, but the molecular mechanism(s) by which the host affects CH4 production remain unclear. We integrated rumen wall transcriptome data and CH4 phenotypes from two independent experiments conducted with sheep in Australia (AUS, n = 62) and New Zealand (NZ, n = 24). The inclusion of the AUS data validated the previously identified clusters and gene sets representing rumen epithelial, metabolic and muscular functions. In addition, the expression of the cell cycle genes as a group was consistently positively correlated with acetate and butyrate concentrations (p < 0.05, based on AUS and NZ data together). The expression of a group of metabolic genes showed positive correlations in both AUS and NZ datasets with CH4 production (p < 0.05) and yield (p < 0.01). These genes encode key enzymes in the ketone body synthesis pathway and included members of the poorly characterized aldo-keto reductase 1C (AKR1C) family. Several AKR1C family genes appear to have ruminant specific evolution patterns, supporting their specialized roles in the ruminants. Combining differential gene expression in the rumen wall muscle of the shortest and longest MRT AUS animals (no data available for the NZ animals) with correlation and network analysis, we identified a set of rumen muscle genes involved in cell junctions as potential regulators of MRT, presumably by influencing contraction rates of the smooth muscle component of the rumen wall. Higher rumen expression of these genes, including SYNPO (synaptopodin, p < 0.01) and NEXN (nexilin, p < 0.05), was associated with lower CH4 yield in both AUS and NZ datasets. Unlike the metabolic genes, the variations in the expression of which may reflect the availability of rumen metabolites, the muscle genes are currently our best candidates for causal genes that influence CH4 yield.

Gene network analysis identifies rumen epithelial cell proliferation, differentiation and metabolic pathways perturbed by diet and correlated with methane production.

Ruminants obtain nutrients from microbial fermentation of plant material, primarily in their rumen, a multilayered forestomach. How the different layers of the rumen wall respond to diet and influence microbial fermentation, and how these process are regulated, is not well understood. Gene expression correlation networks were constructed from full thickness rumen wall transcriptomes of 24 sheep fed two different amounts and qualities of a forage and measured for methane production. The network contained two major negatively correlated gene sub-networks predominantly representing the epithelial and muscle layers of the rumen wall. Within the epithelium sub-network gene clusters representing lipid/oxo-acid metabolism, general metabolism and proliferating and differentiating cells were identified. The expression of cell cycle and metabolic genes was positively correlated with dry matter intake, ruminal short chain fatty acid concentrations and methane production. A weak correlation between lipid/oxo-acid metabolism genes and methane yield was observed. Feed consumption level explained the majority of gene expression variation, particularly for the cell cycle genes. Many known stratified epithelium transcription factors had significantly enriched targets in the epithelial gene clusters. The expression patterns of the transcription factors and their targets in proliferating and differentiating skin is mirrored in the rumen, suggesting conservation of regulatory systems.

Real-time PCR quantification of infectious laryngotracheitis virus in chicken tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals high levels in faeces and dust.

Infectious laryngotracheitis (ILT) is an important disease of chickens caused by ILT virus (ILTV). We used the Australian SA2 and A20 vaccine strains of ILTV to determine tissue distribution and excretion characteristics of ILTV in specific-pathogen-free chickens and to determine whether ILTV is readily detectable in environmental samples such as faeces, bedding material and dust using real-time quantitative PCR. Three groups of 10 freshly hatched chicks were placed in isolators and infected orally with high doses of the two strains of vaccine virus or left unchallenged as controls. Over a 28-day post-infection (p.i.) period, faecal and serum samples were collected at frequent intervals from six individually identified chickens in each group. Dust and litter samples from the isolators were collected less frequently. Tissue samples were collected from three to four sacrificed or dead/euthanized birds at 6, 14 and 28 days p.i. Infection resulted in clinical ILT, a pronounced antibody response and sustained qPCR detection of the viral genome in the trachea, Harderian gland, lung and kidney up to 28 days p.i. A high level of the viral genome was also detected in faeces between 2 and 7 days p.i., declining by about approximately four orders of magnitude to low, but detectable, levels at 21 and 28 days p.i. The finding of high-level shedding of ILTV in faeces warrants further investigation into the epidemiological role of this, and the sustained high levels of ILTV observed in dust suggest that it may be a useful sample material for monitoring ILTV status in flocks.

DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces.

The presence of gastrointestinal nematode eggs in faecal samples is diagnostic of infection by these parasites. However, this technique cannot be used to distinguish between species of importance. The faecal culture technique and subsequent microscopic analysis of developed larvae is currently used to determine which parasite species are present in the samples, but these techniques take a week to perform and have inherent limitations. To overcome these parasite detection and identification problems, we have developed a DNA extraction method for sheep faeces, and a quantitative multiplex PCR (qPCR) test which can both enumerate and identify Haemonchus, Trichostrongylus and Teladorsagia. We demonstrate that the technique is sensitive to 10 eggs per gram and that dilution of DNA to 0.1 fold can overcome PCR inhibition issues for samples obtained from the field, while maintaining assay sensitivity. Further development of these tests for commercial use is warranted, given their potential to provide consistently faster and more accurate diagnoses of these parasites using simple sample collection and laboratory methods which can be easily adapted for the detection of a variety of pathogens from the same faecal sample.

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design.

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials.

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae.

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.