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:Bovine mastitis, predominantly caused by pathogenic Staphylococcus aureus, is a major impediment to milk production. Although its prevalence varies from cows to buffaloes, it lacks relevant markers that mark the phases and progression of the disease. Thus, the focus of this study was to identify key proteins that mark the transition from subclinical to clinical states in a species specific manner. Whey proteins isolated from healthy and S. aureus infected milk from both Holstein Friesian cow and Murrah buffalo were characterized by label free quantification using mass spectrometry. Collectively, 662 proteins were identified with atleast two peptides, of which 51 and 80 proteins were found significant by ANOVA (p value < 0.05) with increasing or decreasing trends in the disease progression in HF and Mu, respectively. Linear change in expression of proteins like haptoglobin and fibronectin in HF and spermadhesin and osteopontin in Mu correlated with the stage of the disease and progression in a species specific manner. Similarly, high mobility group proteins, angiogenin, cofilin-1 that were previously reported in oxidative stress were overexpressed whilemembers of ubiquitin family which remove aggregated proteins were downregulated during the progression of infection. Subsequently, selected differentially expressed proteins (e.g. osteopontin, CAP-1 and fibrinogen α) were validated by Western blot analysis. Results of this study provide deeper insights on whey proteome dynamics and a signature pattern indicative of disease progression.
Project description:Staphylococci are major pathogens in humans and animals and emerging antibiotic-resistant strains have further increased the importance of this health issue. The existence of a genetic basis of host response to bacterial infections has been widely documented but the underlying mechanisms and genes are still largely unknown. Previously, two divergent lines of sheep selected on their milk somatic cell count called high and low SCS lines, have been showed to be respectively more and less susceptible to intra mammary infections (IMI). Transcriptional profiling of milk somatic cells (MSC) of high and low SCS sheep infected successively by S. epidermidis and S. aureus was performed to provide enhanced knowledge about the genetic basis of differential host response to IMI with Staphylococci. Gene expression in MSC of high and low SCS sheep collected 12h post-challenge was performed on a 15K gene ovine oligoarray (Agilent). MSC were mainly neutrophils. The high number of differentially expressed genes between the two bacterial strains indicated, among others, increased number of T-cells in MSC after S. aureus, compared to S. epidermidis challenge. Differential regulation of 366 genes between resistant and susceptible animals was largely associated with immune and inflammatory response (including pathogen recognition TLR2 pathway and cell migration), signal transduction, cell proliferation and apoptosis. Close biological connection between most of differentially expressed genes into Ingenuity Pathway Analysis networks further indicated consistency between the genes that were differentially-expressed between resistant and susceptible animals. Gene profiling in high and low SCS sheep provided strong candidates for biological pathway and gene underlying genetically determined resistance and susceptibility towards Staphylococci infections opening new fields for further investigation. Keywords: Staphylococcus epidermidis, Staphylococcus aureus, milk somatic cells, mammalian, transcriptome, immunity, mastitis 22 samples in a two-colour dye-swap experimental design
Project description:Liquid chromatography-mass spectrometry (LC/MS) based label free quantitative proteomics analysis was applied for identification of differentially expressed proteins among the whey samples isolated from, 1) milk from cows with no history of mastitis, negative for S. aureus and somatic cell count (SCC) >2×105 cells\ml and 2) milk samples from sub-clinical mastitis., i.e., positive for S. aureus and SCC >2×105 cells\ml.