Project description:Mastitis, the inflammation of the mammary gland, is one of the most prevalent diseases in dairy farming worldwide. Unfortunately, the disease is most often present in a subclinical type with no clear symptoms. The sooner the infection is detected, the less opportunities for the disease to progress and the more treatment options remain available. Milk microRNA (miRNA) encapsulated in extracellular vesicles (EV) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EV analysis regarding sampling time-point or natural infections. In order to estimate the reliability of EV measurements in raw bovine milk, we first evaluated the changes in EV size, concentration and miRNA cargo during three consecutive days. Then, we compared milk EV differences from natural infected quarters with high somatic cell count (SCC) with their healthy adjacent quarters with low SCC and quarters from uninfected udders. We found that milk EV miRNA cargo is very stable along three days and that infected quarters do not induce relevant changes in milk EV of adjacent healthy quarters, making them suitable controls. We observed cow-individual changes in immunoregulatory miRNA in quarters with chronic subclinical mastitis, pointing towards infection-specific alterations. Finally, we proposed bta-miR-223 as a potential indicator of subclinical mastitis prognosis in raw milk.
Project description:Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are a novel class of bioactive food compounds. Milk produced by cows with subclinical mastitis threatens animals healthy and milk safety. However, little is known about the differentially expressed miRNA in milk-derived EVs related to subclinical mastitis. This study profiled miRNAs in milk-derived EVs from healthy cows and cows with subclinical mastitis. The potential targets for differentially expressed (DE) miRNAs were predicted. Milk-derived EVs were isolated from healthy cows (n = 7, the control group) and cows with subclinical (n = 7, the SM group). Two hundred ninety miRNAs (221 known and 69 novel ones) were identified. The top 20 miRNAs were commonly abundant (> 0.1% of the total read counts) in Healthy and SM groups, were regarded as abundant bovine milk-derived EVs miRNAs. MiR-21-5p was the most highly expressed known miRNA. Target genes of the top 20 abundant miRNAs were significantly enriched in Ras signaling pathway. The bta-miR-21-5p, bta-miR-30a-5p and miR-6-1096 were differentially expressed. For DE miRNAs, there was no significantly enriched pathways were found in the KEGG enrichment analysis. The linkage between the validated target genes and diseases suggested that we pay particular attention to exosome miRNAs from mastitic milk in milk safety.
Project description:Bovine mastitis, the infection of the mammary gland which leads to great health and economic challenges for dairy farmers is accompanied by dramatic changes in the milk proteome. In this study of naturally occurring mastitis not only have the changes in the milk proteome been quantified in subclinical and clinical mastitis but simultaneous changes in the serum proteome have also been characterised and quantified. Milk and serum samples from healthy dairy cows (n=12) were compared to those of cows with subclinical (n=10) and clinical mastitis (n=112) using TMT label-based proteomic approach. The study included the milk and serum samples taken from thirty-two dairy cows ( kept on private farms located in Croatia. All cows were checked by physical examination. Somatic cells count (SCC) and mastitis test in milk samples were performed. According to the results, cows were assigned into three groups: Group I (control, n=10) consisted of healthy cows with SCC below 400,000 cells/ml on the monthly check-up and a negative mastitis test and without any clinical sign of mastitis. Group II (subclinical mastitis, n=12) comprised cows without clinical signs of mastitis but with SCC above 400,000 cells/ml on the monthly basis and a positive mastitis test at the time of sampling. Group III (clinical mastitis, n=10) consisted of cows with clinical signs of mastitis which include changes in milk appearance (flakes and clots in milk), different stages of udder inflammation (hyperemia, edema, pain, udder enlargement and elevated udder temperature) and disturbance of general health (depression, relaxed cold ears, dehydration, elevated body temperature, increased heart and respiratory rate, decreased ruminal contraction and decreased appetite). Blood samples were taken from v. coccygea and centrifuged at 3000 g for 15 min after clotting for two hours at room temperature. Serum samples were stored at -80°C until analysis. Milk samples were taken aseptically before the morning milking. First few streams were discarded. Teat ends were disinfected with cotton swabs soaked with 70% ethanol. Samples were taken into sterile tubes and transported to laboratory on ice within a few hours.
Project description:Bovine mastitis causes changes in the serum exosomal miRNAs expression. Serum samples from healthy dairy cows (n = 7) were compared to those of cows with subclinical (n = 7 ) using small RAN sequencing. Three hundred fifty-five miRNAs (341 known and 14 novel ones) were identified. There were 42 miRNAs up-regulated in serum-derived EVs from cows with subclinical mastitis, including bta-miR-1246, bta-miR-2431-3p, bta-miR-126-3p, bta-miR-29a, etc. The MAPK signaling pathway was the most affected pathway by clinical mastitis. Thus, miRNA alterations in mastitis serum-derived EVs support the potential regulator role of specific miRNAs as exosomal cargo in clinical mastitis physiology.
Project description:Early detection of bovine subclinical mastitis may improve treatment strategies and reduce the use of antibiotics. Herein, individual milk samples from Holstein cows affected by subclinical mastitis induced by S. agalactiae and Prototheca spp. were analyzed by untargeted and targeted mass spectrometry approaches to assess changes in their peptidome profiles and identify new potential biomarkers of the pathological condition. Results showed a higher amount of peptides in milk positive at the bacteriological examination when compared with the negative control. However, the different pathogens seemed not to trigger specific effects on milk peptidome. The peptides that best distinguish positive from negative samples are mainly derived from the most abundant milk proteins, especially from β- and αs1-casein, but also include the antimicrobial peptide casecidin 17. These results provide new insights into the physiopathology of mastitis. Upon further validation, the panel of potential discriminant peptides could help to the development of new diagnostic and therapeutic tools.
Project description:Previous studies have investigated the peptidomic changes occurring in cow milk during mastitis; however, these focused mainly on clinical mastitis, either spontaneous (Mansor et al., 2013) or induced by experimental infection (Thomas et al., 2016). Mansor and coworkers were the first to use mass spectrometry to demonstrate that several peptides found increased in milk from cows with clinical S. aureus or E. coli mastitis were mainly derived from aS1- and b-casein. In that study, 48 peptides were significantly different between the milks of healthy and mastitic cows. Non-mastitic samples were confirmed to be non-mastitic by having SCC <100,000 cells/mL (Mansor et al., 2013). Thomas and coworkers expanded the peptidomic repertoire in a study evaluating the kinetics of experimental S. uberis infection, and found signature peptides with potential as mastitis markers (Thomas et al., 2016). Only one study evaluated the milk peptidome in subclinical mastitis (Guerrero et al., 2015) demonstrating that even subclinical infections can cause significant increases in the total number of released peptides when compared to uninfected milk. However, neither the IMI agents nor the somatic cell counts were reported. With the aim of understanding high abundance protein and peptidomic changes due to subclinical CNS mastitis, to identify signature peptides with potential for subclinical mastitis detection, and to compare the proteomic and peptidomic findings with those reported in clinical mastitis, we investigated the influence of CNS IMI on high abundance milk proteins by SDS-PAGE and densitometric analysis, followed by a detailed characterization of the milk peptidome by means of high-performance liquid chromatography/tandem mass spectrometry and bioinformatic analysis.
Project description:Mastitis is defined as inflammation of the mammary gland and one of the most serious concerns with regards to milk production and animal health in dairy industry. Indeed, mastitis have marked influence on the milk yield, milk constituents, milk quality and nourishment of the neonates. We performed a comparative proteome analysis of the bovine milk obtained from healthy, sub-clinical and clinical mastitis from Indian indigenous cattle Karan Fries.
Project description:Mastitis is a very costly and common disease in the dairy industry. The study of the transcriptome from healthy and mastitic milk somatic cell samples using RNA-Sequencing technology (RNA-Seq) can provide measurements of transcript levels associated with the immune response to the infection. The objective of this study was to characterize the Holstein milk somatic cells transcriptome from 6 cows to determine host response to intramammary infections. RNA-Sequencing was performed on two samples from each cow from two separate quarters, one classified as healthy (n = 6) and one as mastitic (n = 6). In total, 449 genes were differentially expressed between the healthy and mastitic quarters (P-value < 0.01, FDR < 0.05, FC > ±2). Among the differentially expressed genes, the most expressed genes based on Reads Per Kilo base per Million mapped reads (RPKM) in the healthy group were associated with milk components (CSN2 and CSN3), and in the mastitic group they were associated with immunity (B2M and CD74). In-silico functional analysis was performed using the list of 449 differentially expressed genes, which identified 36 significantly enriched metabolic pathways (FDR < 0.01), some of which were associated with the immune system, such as cytokine-cytokine interaction and cell adhesion molecules. Seven functional candidate genes were selected, based on the criteria of being highly expressed and present in significant pathways that are relevant to the inflammatory process (GLYCAM1, B2M, CD74, BoLA DR-Alpha, FCER1G, SDS and NFKBIA). Lastly, we identified the differentially expressed genes that are located in QTL regions previously known to be associated with mastitis, specifically clinical mastitis, somatic cell count and somatic cell score. It was concluded that there are multiple genes within QTL regions that could potentially impact host response to mastitis causing agents, making some cows more susceptible to intramammary infections. The identification of key genes with functional, statistical, biological and positional relevance associated with host defense to infection, will contribute to a better understanding of the underlying genetic architecture associated with mastitis. This in turn will improve the sustainability of agricultural practices, by facilitating the selection of cows with improved host defense leading to increased resistance to mastitis.
Project description:Escherichia coli and Staphylococcus aureus are two common pathogenic microorganisms that cause mastitis in dairy cows. They can cause clinical mastitis and subclinical mastitis. In recent studies, lncRNAs have been found to play an important role in the immune responses triggered by microbial inducers. However, the actions of lncRNAs in bovine mastitis remain unclear. The purpose of this study was to explore the lncRNA profile on mastitis.