Comparison of traditional copromicroscopy with image analysis devices for detection of gastrointestinal nematode infection in sheep.

Sustainable parasite control practices are necessary to combat the negative effects of gastrointestinal nematodes on animal health and production while reducing the selection pressure for anthelmintic resistance. Parasite diagnostic tests can inform treatment decisions, the timing and effectiveness of treatment and enable livestock breeding programmes. In recent years new diagnostic methods have been developed, some incorporating machine learning (ML), to facilitate the detection and enumeration of parasite eggs. It is important to understand the technical characteristics and performance of such new methods compared to long standing and commonly utilised methods before they are widely implemented. The aim of the present study was to trial three new diagnostic tools relying on image analysis (FECPAKG2, Micron and OvaCyte) and to compare them to traditional manual devices (McMaster and Mini-FLOTAC). Faecal samples were obtained from 41 lambs naturally infected with gastrointestinal nematodes. Samples were mixed and separated into 2 aliquots for examination by each of the 5 methods: McMaster, Mini-FLOTAC, FECPAKG2, Micron and OvaCyte. The techniques were performed according to their respective standard protocols and results were collected by trained staff (McMaster and Mini-FLOTAC) or by the device (FECPAKG2, Micron and OvaCyte). Regarding strongyle worm egg count, McMaster values varied from 0 to 9,000 eggs per gram (EPG). When comparing replicate aliquots, both the Mini-FLOTAC and Micron methods displayed similar repeatability to McMaster. However, we found FECPAKG2 and OvaCyte significantly less precise than McMaster. When comparing parasite egg enumeration, significant positive linear correlations were established between McMaster and all other methods. No difference was observed in EPG between McMaster and Mini-FLOTAC or FECPAKG2; however, Micron and OvaCyte returned significantly higher and lower EPG, respectively, compared to McMaster. The number of eggs ascribed to other parasite species was not sufficient for performing a robust statistical comparison between all methods. However, it was noted that FECPAKG2 generally did not detect Strongyloides papillosus eggs, despite these being detected by other methods. In addition, Moniezia spp and Trichuris spp eggs were detected by OvaCyte and Mini-FLOTAC, respectively, but not by other methods. The observed variation between traditional and new methods for parasite diagnostics highlights the need for continued training and enhancing of ML models used and the importance of developing clear guidelines for validation of newly developed methods.

Immunoproteomic analysis of the secretome of bovine-adapted strains of Staphylococcus aureus demonstrates a strain-specific humoral response.

Staphylococcus aureus is a common pathogen associated with bovine intramammary infection. A number of distinct S. aureus lineages are associated with such infections although there is a dearth of knowledge regarding the major immunogenic antigens associated with each lineage and whether these antigens provide protection against heterologous strains. Identification of the major immunogenic antigens of the predominant bovine-adapted S. aureus lineages would assist in the design of effective vaccines and diagnostic tests to control intramammary infections caused by S. aureus. The aim of this study was to characterise the serum IgG response to S. aureus extracellular proteins in cows infected with strains from different lineages, as well as to identify antigenic proteins produced by these strains. Genotypic characterisation found that strain MOK124 (CC151) encoded more toxins, including the ruminant-specific leukocidin LukMF, compared to strain MOK023 (CC97). In addition, MOK124 secreted more toxins in vitro, compared to MOK023. Immunoproteomic analysis was performed using sera from cows infected with either MOK023 or MOK124. One-dimensional serum blotting revealed that cows infected with MOK023 predominantly generated a humoral response against high molecular weight proteins while cows infected with MOK124 primarily generated a humoral response against low molecular weight proteins. Two-dimensional serum blotting demonstrated that antibodies produced by an MOK023 infected cow could cross react with some of the extracellular proteins produced by MOK124 and vice versa. Mass spectrometry analysis of immunoreactive proteins identified common candidate immunogens produced by both strains, including α-hemolysin and ß-hemolysin. In addition, strain-specific candidate immunogens were also identified. This study demonstrates that genes encoding important S. aureus secreted virulence factors, the production of cognate gene products, and the humoral immune response to infection is, to an extent, strain-dependent. However, the identification of some common candidate immunogens suggests that there are proteins that can be exploited for further vaccine or diagnostic research that targets S. aureus strains from a variety of lineages.

Label-free quantitative proteomic analysis of M. longissimus dorsi from cattle during dietary restriction and subsequent compensatory growth.

Compensatory growth (CG) is a naturally occurring physiological process whereby an animal has the ability to undergo enhanced growth following a period of restricted feeding. This studies objective was to identify key proteins involved in the expression of CG. Forty Holstein Friesian bulls were equally assigned to one of four groups. R1 and R2 groups were subjected to restricted feed allowance for 125 days (Period 1). A1 and A2 animals had ad libitum access to feed in Period 1. Following Period 1, all animals from R1 and A1 were slaughtered. Remaining animals (R2 and A2) were slaughtered following ad libitum access to feed for successive 55 days (Period 2). M. longissimus dorsi samples were collected at slaughter from all animals. Proteins were isolated from samples and subjected to label-free mass spectrometry proteomic quantification. Proteins which were differentially abundant during CG (n = 39) were involved in cellular binding processes, oxidative phosphorylation and mitochondrial function. There was also evidence for up regulation of three pathways involved in nucleotide biosynthesis. Genetic variants in or regulating genes pertaining to proteins identified in this study may hold potential for use as DNA based biomarkers for genomic selection of animals with a greater ability to undergo CG.

Alterations in hepatic miRNA expression during negative energy balance in postpartum dairy cattle.

BACKGROUND: Negative energy balance (NEB), an altered metabolic state, occurs in early postpartum dairy cattle when energy demands to support lactation exceed energy intake. During NEB the liver undergoes oxidative stress and increased breakdown of fatty acids accompanied by changes in gene expression. It is now known that micro RNAs (miRNA) can have a role in mediating such alterations in gene expression through repression or degradation of target mRNAs. miRNA expression is known to be altered by metabolism and environmental factors and miRNAs are implicated in expression modulation of metabolism related genes. RESULTS: miRNA expression was profiled in the liver of moderate yielding dairy cattle under severe NEB (SNEB) and mild NEB (MNEB) using the Affymetrix Gene Chip miRNA_2.0 array with 679 probe sets for Bos-taurus miRNAs. Ten miRNAs were found to be differentially expressed using the 'samr' statistical package (delta = 0.6) at a q-value FDR of < 12%. Five miRNAs including miR-17-5p, miR-31, miR-140, miR-1281 and miR-2885 were validated using RT-qPCR, to be up-regulated under SNEB. Liver diseases associated with these miRNAs include non-alcoholic fatty liver (NAFLD) and hepatocellular carcinoma (HCC). miR-140 and miR-17-5p are known to show differential expression under oxidative stress. A total of 32 down-regulated putative target genes were also identified among 418 differentially expressed hepatic genes previously reported for the same animal model. Among these, GPR37 (G protein-coupled receptor 37), HEYL (hairy/enhancer-of-split related with YRPW motif-like), DNJA1, CD14 (Cluster of differentiation 14) and GNS (glucosamine (N-acetyl)-6-sulfatase) are known to be associated with hepatic metabolic disorders. In addition miR-140 and miR-2885 have binding sites on the most down-regulated of these genes, FADS2 (Fatty acid desaturase 2) which encodes an enzyme critical in lipid biosynthesis. Furthermore, HNF3-gamma (Hepatocyte nuclear factor 3-gamma), a hepatic transcription factor (TF) that is involved in IGF-1 expression regulation and maintenance of glucose homeostasis is a putative target of miR-31. CONCLUSIONS: This study shows that SNEB affects liver miRNA expression and these miRNAs have putative targets in hepatic genes down-regulated under this condition. This study highlights the potential role of miRNAs in transcription regulation of hepatic gene expression during SNEB in dairy cattle.