Proteomic Analyses of Mammary Glands Provide Insight into the Immunity and Metabolism Pathways Associated with Clinical Mastitis in Meat Sheep.
ABSTRACT: Clinical mastitis is still an intractable problem for sheep breeding. The natural immunologic mechanisms of the mammary gland against infections are not yet understood. For a better understanding of the disease-associated proteins during clinical mastitis in meat sheep, we performed two-dimensional electrophoresis (2-DE)-based comparative proteomic analyses of mammary tissues, including from healthy mammary tissues (HMTs) and from mammary tissues with clinical mastitis (CMMTs). The 2-DE results showed that a total of 10 up-regulated and 16 down-regulated proteins were identified in CMMTs when compared to HMTs. Of these, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses revealed that most proteins were associated with immune responses or metabolisms. The results of qRT-PCR and Western blot for randomly selected four differentially expressed proteins (DEPs) including superoxide dismutase [Mn] (SOD2), annexin A2 (ANAX2), keratin 10 (KRT10) and endoplasmic reticulum resident protein 29 (ERP29) showed that their expression trends were consistent with 2-DE results except ANXA2 mRNA levels. This is an initial report describing the 2-DE-based proteomics study of the meat sheep mammary gland with clinical mastitis caused by natural infection, which provides additional insight into the immune and metabolic mechanisms during sheep mastitis.
Project description:Clinical mastitis in sheep has gravely restrained production performance for a long time. Knowledge of mechanisms of its pathogenesis and resistance in meat sheep mammary gland with clinical mastitis are not yet understood, especially for clinical mastitis caused by natural infection. In this work, RNA-sequencing was firstly used to screen the differentially expressed genes (DEGs) in clinical mastitic mammary tissues (CMMTs) when compared with healthy mammary tissues (HMTs) from meat sheep flocks. We identified 420 DEGs including 316 upregulated and 104 downregulated genes in CMMTs. Gene ontology annotation revealed these DEGs were mainly engaged in immune response and inflammation response. Pathway enrichment showed they were primarily enriched in pathways relevant to inflammation, immune response and metabolism. Alternative splicing analysis showed most common differential splicing genes in CMMTs and HMTs were implicated in immune response. Immunostaining for three immune response-related proteins encoded by DEGs were mainly observed in mammary epithelium from both CMMTs and HMTs, and their positive signals were more intensive in CMMTs than those in HMTs. These findings provide experimental basis and reference for further researching the molecular genetic mechanisms, particularly immune defence mechanisms, of sheep mammary gland during clinical mastitis.
Project description:Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis. The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.
Project description:Caprine arthritis encephalitis virus (CAEV) is a lentivirus that infects both goats and sheep and is closely related to maedi-visna virus that infects sheep; collectively, these viruses are known as small ruminant lentiviruses (SRLV). Infection of goats and sheep with SRLV typically results in discrete inflammatory diseases which include arthritis, mastitis, pneumonia or encephalomyelitis. SRLV-infected animals concurrently demonstrating lentivirus-associated lesions in tissues of lung, mammary gland, joint synovium and the central nervous system are either very rare or have not been reported. Here we describe a novel CAEV promoter isolated from a sheep with multisystemic lentivirus-associated inflammatory disease including interstitial pneumonia, mastitis, polyarthritis and leukomyelitis. A single, novel SRLV promoter was cloned and sequenced from five different anatomical locations (brain stem, spinal cord, lung, mammary gland and carpal joint synovium), all of which demonstrated lesions characteristic of lentivirus associated inflammation. This SRLV promoter isolate was found to be closely related to CAEV promoters isolated from goats in northern California and other parts of the world. The promoter was denoted CAEV-ovine-MS (multisystemic disease); the stability of the transcription factor binding sites within the U3 promoter sequence are discussed.
Project description:The objective of the study was the investigation of the behaviour of cathelicidin-1 in the milk after experimental infection with two prominent bacterial pathogens (experiment 1: Mannheimia haemolytica, experiment 2: M. haemolytica and Staphylococcus chromogenes) as a potential early indicator for diagnosis of mastitis in sheep. In two experiments, after bacterial inoculation into the udder of ewes, bacteriological and cytological examinations of milk samples as well as proteomics examinations [two-dimensional gel electrophoresis analysis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis] were performed sequentially. Cathelicidin-1 was detected and spot densities obtained from PDQuest v.8.0 were recorded. Associations were calculated between cell content and spot densities as well as between presence of mastitis in a mammary gland at a given time-point and detection of cathelicidin-1 in the respective milk sample. All inoculated mammary glands developed mastitis, confirmed by the consistent bacterial isolation from mammary secretion and increased leucocyte content therein. Spot density of cathelicidin-1 in samples from inoculated glands increased 3 h post-inoculation; spot density of cathelicidin-1 in samples from inoculated glands was higher than in samples from uninoculated controls. There was clear evidence of correlation between cell content and cathelicidin-1 spot densities in milk samples. There was significant association between presence of mastitis in the mammary gland and detection of cathelicidin-1 in the respective milk sample; overall accuracy was 0.818-this was significantly greater during the first 24 h post-challenge (0.903) than after the first day (0.704). In conclusion, detection of cathelicidin-1 in milk was significantly associated with presence of mastitis in ewes. The associations were stronger during the first 24 h post-infection than after the first day. Cathelicidin-1 has the advantage that it can be a non-specific biomarker, as simply a "positive" / "negative" assessment would be sufficient.
Project description:BACKGROUND: Proteomics and bioinformatics may help us better understand the biological adaptations occurring during bovine mastitis. This systems approach also could help identify biomarkers for monitoring clinical and subclinical mastitis. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to screen potential proteins associated with mastitis at late infectious stage. RESULTS: Healthy and mastitic cows' mammary gland tissues were analyzed using iTRAQ combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Bioinformatics analyses of differentially expressed proteins were performed by means of Gene Ontology, metabolic pathways, transcriptional regulation networks using Blast2GO software, the Dynamic Impact Approach and Ingenuity Pathway Analysis. At a false discovery rate of 5%, a total of 768 proteins were identified from 6,499 peptides, which were matched with 15,879 spectra. Compared with healthy mammary gland tissue, 36 proteins were significantly up-regulated (>1.5-fold) while 19 were significantly down-regulated (<0.67-fold) in response to mastitis due to natural infections with Staphylococci aureus. Up-regulation of collagen, type I, alpha 1 (COL1A1) and inter-alpha (Globulin) inhibitor H4 (ITIH4) in the mastitis-infected tissue was confirmed by Western blotting and Immunohistochemistry. CONCLUSION: This paper is the first to show the protein expression in the late response to a mastitic pathogen, thus, revealing mechanisms associated with host tissue damage. The bioinformatics analyses highlighted the effects of mastitis on proteins such as collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan 2. Our findings provide additional clues for further studies of candidate genes for mastitis susceptibility. The up-regulated expression of COL1A1 and ITIH4 in the mastitic mammary gland may be associated with tissue damage and repair during late stages of infection.
Project description:Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. The objective of this study was to establish a rat model of mastitis induced by S. aureus infection and to explore changes in the proteomes of mammary tissue in different udder states, providing a better understanding of the host immune response to S. aureus mastitis. On day 3 post-partum, 6 rats were randomly divided into two groups (n = 3), with either 100 ?L of PBS (blank group) or a S. aureus suspension containing 2×107 CFU·mL-1 (challenge group) infused into the mammary gland duct. After 24 h of infection, the rats were sacrificed, and mammary gland tissue was collected. Tandem mass tag (TMT)-based technology was applied to compare the proteomes of healthy and mastitic mammary tissues. Compared with the control group, the challenge group had 555 proteins with significant differences in expression, of which 428 were significantly upregulated (FC>1.2 and p<0.05) and 127 were downregulated (FC>0.83 and p<0.05 or p<0.01). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that upregulated differentially significant expressed proteins (DSEPs) were associated with mainly immune responses, including integrin alpha M, inter-?-trypsin inhibitor heavy chain 4, and alpha-2-macroglobulin. This study is the first in which a rat model of S. aureus-induced mastitis was used to explore the proteins related to mastitis in dairy cows by TMT technology, providing a model for replication of dairy cow S. aureus-induced mastitis experiments.
Project description:Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3'-untranslated regions (UTR) (52 bp+167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. +1283 C>T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3'-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold (p<0.05) in mastitis-infected mammary tissues (n = 5) compared with the healthy mammary gland mammary tissues (n = 5). Except for bta-miR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold (p<0.05), whereas miR-2318 was downregulated in the mastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation.
Project description:BACKGROUND: Mastitis is still considered to be the most economically important infectious disease in dairy cattle breeding. The immune response in mammary gland tissues could help in developing support strategies to combat this disease. The role of neutrophils and macrophages in the innate response of mammary gland is well known. However, the immune response in mammary gland tissues, including levels of antimicrobial peptide transcripts, has not been well recognized. Moreover, most studies are conducted in vitro, on cell cultures, or on artificially infected animals, with analysis being done within a several dozen hours after infection.The aim of the study was to examine the in vivo transcript levels of beta-defensin and cathelicidins genes in cow mammary gland secretory tissue (parenchyma) with the chronic, recurrent and incurable mammary gland inflammation induced by coagulase-positive or coagulase-negative Staphyloccoci vs. bacteria-free tissue. RESULTS: The mRNA of DEFB1, BNBD4, BNBD5, BNBD10 and LAP genes, but not of TAP gene, were detected in all investigated samples regardless of the animals' age and microbiological status of the mammary gland, but at different levels. The expression of most of the beta-defensin genes was shown to be much higher in tissues derived from udders infected with bacteria (CoPS or CoNS) than from bacteria-free udders, regardless of parity. Cathelicidins (CATH4, CATH5 and CATH6) showed expression patterns contrasting those of ?-defensins, with the highest expression in tissues derived from bacteria-free udders. CONCLUSION: Increased expression of genes encoding ?-defensins in the infected udder confirms their crucial role in the defense of the cow mammary gland against mastitis. On the other hand, the elevated cathelicidin transcripts in non-infected tissues indicate their role in the maintenance of healthy mammary tissues. The expression levels of investigated genes are likely to depend on the duration of the infection and type of bacteria.
Project description:BACKGROUND: Bovine mastitis is one of the most costly and prevalent diseases affecting dairy cows worldwide. In order to develop new strategies to prevent Escherichia coli-induced mastitis, a detailed understanding of the molecular mechanisms underlying the host immune response to an E. coli infection is necessary. To this end, we performed a global gene-expression analysis of mammary gland tissue collected from dairy cows that had been exposed to a controlled E. coli infection. Biopsy samples of healthy and infected utter tissue were collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent the acute phase response (APR) and chronic stage, respectively. Differentially expressed (DE) genes for each stage were analyzed and the DE genes detected at T = 24 h were also compared to data collected from two previous E. coli mastitis studies that were carried out on post mortem tissue. RESULTS: Nine-hundred-eighty-two transcripts were found to be differentially expressed in infected tissue at T = 24 (P < 0.05). Up-regulated transcripts (699) were largely associated with immune response functions, while the down-regulated transcripts (229) were principally involved in fat metabolism. At T = 192 h, all of the up-regulated transcripts were associated with tissue healing processes. Comparison of T = 24 h DE genes detected in the three E. coli mastitis studies revealed 248 were common and mainly involved immune response functions. KEGG pathway analysis indicated that these genes were involved in 12 pathways related to the pro-inflammatory response and APR, but also identified significant representation of two unexpected pathways: natural killer cell-mediated cytotoxicity pathway (KEGG04650) and the Rig-I-like receptor signalling pathway (KEGG04622). CONCLUSIONS: In E. coli-induced mastitis, infected mammary gland tissue was found to significantly up-regulate expression of genes related to the immune response and down-regulate genes related to fat metabolism. Up to 25% of the DE immune response genes common to the three E. coli mastitis studies at T = 24 h were independent of E. coli strain and dose, cow lactation stage and number, tissue collection method and gene analysis method used. Hence, these DE genes likely represent important mediators of the local APR against E. coli in the mammary gland.
Project description:Milk yield is the most important dairy sheep trait and constitutes the key genetic improvement goal via selective breeding. Mastitis is one of the most prevalent diseases, significantly impacting on animal welfare, milk yield and quality, while incurring substantial costs. Our objectives were to determine the feasibility of a concomitant genetic improvement programme for enhanced milk production and resistance to mastitis. Individual records for milk yield, and four mastitis-related traits (milk somatic cell count, California Mastitis Test score, total viable bacterial count in milk and clinical mastitis presence) were collected monthly throughout lactation for 609 ewes of the Chios breed. All ewes were genotyped with a mastitis specific custom-made 960 single nucleotide polymorphism (SNP) array. We performed targeted genomic association studies, (co)variance component estimation and pathway enrichment analysis, and characterised gene expression levels and the extent of allelic expression imbalance. Presence of heritable variation for milk yield was confirmed. There was no significant genetic correlation between milk yield and mastitis traits. Environmental factors appeared to favour both milk production and udder health. There were no overlapping of SNPs associated with mastitis resistance and milk yield in Chios sheep. Furthermore, four distinct Quantitative Trait Loci (QTLs) affecting milk yield were detected on chromosomes 2, 12, 16 and 19, in locations other than those previously identified to affect mastitis resistance. Five genes (DNAJA1, GHR, LYPLA1, NUP35 and OXCT1) located within the QTL regions were highly expressed in both the mammary gland and milk transcriptome, suggesting involvement in milk synthesis and production. Furthermore, the expression of two of these genes (NUP35 and OXCT1) was enriched in immune tissues implying a potentially pleiotropic effect or likely role in milk production during udder infection, which needs to be further elucidated in future studies. In conclusion, the absence of genetic antagonism between milk yield and mastitis resistance suggests that simultaneous genetic improvement of both traits be achievable.