Exploring associations between the teat apex metagenome and Staphylococcus aureus intramammary infections in primiparous cows under organic directives.

The primary objective of this study was to identify associations between the prepartum teat apex microbiome and the presence of Staphylococcus aureus intramammary infections (IMI) in primiparous cows during the first 5 weeks after calving. We performed a case-control study using shotgun metagenomics of the teat apex and culture-based milk data collected longitudinally from 710 primiparous cows on five organic dairy farms. Cases had higher odds of having S. aureus metagenomic DNA on the teat apex prior to parturition compared to controls (OR = 38.9, 95% CI: 14.84-102.21). Differential abundance analysis confirmed this association, with cases having a 23.8 higher log fold change (LFC) in the abundance of S. aureus in their samples compared to controls. Of the most prevalent microorganisms in controls, those associated with a lower risk of post-calving S. aureus IMI included Microbacterium phage Min 1 (OR = 0.37, 95% CI: 0.25-0.53), Corynebacterium efficiens (OR = 0.53, 95% CI: 0.30-0.94), Kocuria polaris (OR = 0.54, 95% CI: 0.35-0.82), Micrococcus terreus (OR = 0.64, 95% CI: 0.44-0.93), and Dietzia alimentaria (OR = 0.45, 95% CI: 0.26-0.75). Genes encoding for Microcin B17 AMPs were the most prevalent on the teat apex of cases and controls (99.7% in both groups). The predicted abundance of genes encoding for Microcin B17 was also higher in cases compared to controls (LFC 0.26). IMPORTANCE: Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem for the dairy industry. The microbiome on the external skin of the teat apex may play a role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 16S approach, which precludes the detection of genomic features such as genes that encode for AMPs. Therefore, further research using a shotgun metagenomic approach is needed to understand what role prepartum teat apex microbiome dynamics play in IMI risk.

Thermographic assessment of mastitis progression in sahiwal cattle: Insights into the patterns in the natural course of infection.

Mastitis is a global concern in the dairy sector, demanding innovative solutions for effective management for quality lifetime milk production. In this study, infrared thermography (IRT) as a non-invasive technology was integrated into routine farm activities for continuous health monitoring of animals. For 30 days, we systematically monitored the udder health status in 40 Sahiwal cows (160 quarters), employing IRT along with the California Mastitis Test (CMT). We also assessed somatic cell count (SCC), microbial identification, and milk quality parameters of representative samples. The thermal imaging data was analyzed, considering both backward propagation from the 0th day to the -10th day and forward propagation from the 0th day to the +10th day. Our findings revealed that on the 0th day, the mean temperatures of the udder surface skin temperature (USST) and teat skin surface temperature (TSST) exhibited differences (p < 0.05) between the quarters affected by sub-clinical mastitis (SCM) and clinical mastitis (CM) in comparison to the healthy quarters, with the highest degree of difference observed. The observed temperature differences between CM and SCM quarters compared to healthy ranged from 1.8 to 3.62 °C and 0.98 to 3.23 °C for USST, and from 1.68 to 3.16 °C and 0.56 to 2.32 °C for TSST, respectively. Furthermore, our observations indicated that both udder and teat quarters responded differently to mastitis. A temperature rise of 1.37 °C in SCM quarters and 1.75 °C in CM quarters was observed between the -10th and -8th day relative to day 0, with the increase being more pronounced in the morning hours. Also, a notable temperature surge occurred during the -2nd and -1st days relative to the 0th day. The log10SCC values and milk quality parameters significantly differed (p < 0.05) between mastitis-affected and healthy samples. In addition, Staphylococcus spp. was identified as the predominant mastitis-causing pathogen in the bacteriological identification conducted in this study. Therefore, IRT efficiently assesses the initiation point of udder infection in Sahiwal cows, aiding in effective udder health management.

N6-methyladenosine-modified lncRNA in Staphylococcus aureus-injured bovine mammary epithelial cells.

Staphylococcus aureus-induced mastitis is a serious disease in dairy bovine, with no currently effective treatment. Antibiotics demonstrate certain therapeutic potency in dairy husbandry; they generate drug-resistant bacteria, thereby harming public health. LncRNAs and m6A have been verified as potential targets in infectious diseases and have powerful regulatory capabilities. However, the biological regulation of lncRNAs with m6A modification in mastitis needs further investigation. This study aims to determine the m6A-modified lncRNAs in bovine mammary epithelial cells and their diversity during S. aureus induction. Heat-inactivated S. aureus was used to develop the cell injury model, and we subsequently found low cell viability and different m6A modification levels. Our analysis of m6A-modified lncRNA profiles through MeRIP-seq revealed significant differences in 140 peaks within 130 lncRNAs when cells were injured by S. aureus. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that these differential m6A-modified lncRNAs were mainly enriched in the WNT pathway, and their functions were associated with amino acid metabolism, lipid translocation, and metalloproteinase activity. Here, we report for the first time lncRNAs with m6A modification in regulating S. aureus infection, revealing potential mechanisms and targets of infectious diseases, such as mastitis, from an epigenetics perspective.

Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge.

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome.

BACKGROUND: Dairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial. METHODS: Four groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030. RESULTS: In healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance. CONCLUSIONS: At T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Assessment of Lactococcus Cremoris preparations for the pre- and post-milking teat disinfection.

Good hygienic milking practices including the disinfection of the cow teat skin before and after milking aimed at preventing the occurrence of new intramammary infections (IMI) in dairy cows. This practice is generally performed using disinfectants, and in the current context of attention to the emergence of resistances, it is of greatest interest to evaluate alternative solutions that can expand treatment options. We assessed the efficacy of a pre-dipping and a post-dipping product based on the bacteriocin-containing culture of Lactococcus cremoris strain FT27, as compared to commercial disinfectants. FT27 was selected for the bactericidal activity in vitro against food pathogens. In the present study, it also revealed a high bactericidal activity against the main mastitis pathogens, most likely related to Nisin A production, according to genomic and proteomic analysis. The lactococcus-based preparations were applied in two commercial dairy farms in Northern Italy. Overall, 298 dairy cows were enrolled, 141 in the treated group (TR), and 157 in the control group (CTR). The cows were sampled at quarter level every two weeks for 3 months. During the trial, all cows showed a good health status. The hygiene level for udder, flanks and legs was generally good (on average < 3 score); the udder hygiene was significantly worse (P < 0.01) in the CTR group. The teat apex hyperkeratosis was overall low (on average < 2 score). We recorded no significant differences between the two experimental groups in the Somatic Cell Count (SCC) values and the bacteriological results. The overall frequency of new IMI was low, ranging 0.6 - 0.5% for S. aureus in the TR or CTR group respectively, to 2.6-4.4% for NASM. Regarding Str. spp., the new IMI accounted for 1.7% or 1.9% in the TR or CTR group, respectively. Notably, the incidence of S. aureus new IMI did not increase during the study, even though neither of the two herds segregated the positive cows. The non-inferiority test showed that the Lactococcus-based pre- and post-dipping products had an efficacy comparable to proven commercial disinfectants in maintaining udder health and preventing new IMI, thus representing a possible alternative to current teat dip products.

Role of LsrR in the regulation of biofilm formation in mammary pathogenic Escherichia coli.

BACKGROUND: Mammary Pathogenic Escherichia coli (MPEC) is an important pathogen that can escape the attack of the host immune system through biofilm formation and proliferate in the mammary gland continuously, resulting in mastitis in cows and causing enormous economic losses. As an effector of AI-2 quorum sensing, LsrR extensively affects the expression levels of hundreds of genes related to multiple biological processes in model E. coli strain. However, the regulatory role of LsrR in MPEC and whether it is involved in pathogenesis has been seldom reported. RESULTS: In this study, the function of LsrR in strain MPEC5, obtained from a milk sample in dairy cows with mastitis, was investigated by performing high-throughput sequencing (RNA-seq) assays. The results revealed that LsrR down-regulated the transcript levels of fimAICDFGH (encoding Type 1 pili), which have been reported to be associated with biofilm formation process. Biofilm assays confirmed that deletion of lsrR resulted in a significant increase in biofilm formation in vitro. In addition, electrophoretic mobility shift assay (EMSA) provided evidence that LsrR protein could directly bind to the promoter regions of fimAICDFGH in a dose-dependent manner. CONCLUSIONS: These results indicate that LsrR protein inhibits the biofilm formation ability of MPEC5 by directly binding to the fimAICDFGH promoter region. This study presents a novel clue for further exploration of the prevention and treatment of MPEC.

Involvement of adhesins (EcpD, FdeC, FimH) expressed in mammary pathogenic Escherichia coli on adhesion to bovine mammary epithelial cells.

Mammary pathogenic Escherichia coli (MPEC) causes mastitis, which results in substantial economic losses to the dairy industry. A high percentage of Escherichia coli isolated from cows with clinical mastitis harbor adhesin genes, such as fimH. However, it is unclear whether these adhesins are important in the adhesion of MPEC to bovine mammary epithelial cells (BMECs). Therefore, we investigated the effect of adhesins (EcpD, FdeC, and FimH) in MPEC on adherence to the bovine mammary epithelium using cultured BMECs. For this purpose, we used wild-type MPEC as well as single- and double-mutants of fimH, ecpD, and fdeC, and performed adhesion assays with BMECs. First, BMECs were cultured in the presence of lactogenic hormones to induce milk component production and tight junction formation. The bacterial count of the wild-type strain that adhered to the BMECs increased in a dose-dependent manner. In deletion mutant strains, the ΔfimH strain showed lower adhesion (P < 0.05), whereas the adhesion ratio of the ΔecpD and ΔfdeC strains was not statistically different compared with that of the wild-type strain (P > 0.05). Additionally, the fimH/fdeC double-deletion mutants showed the lowest adhesion to BMECs. In conclusion, FimH is crucial in the adhesion of MPEC to BMECs. Overall, our work identifies FimH or FimH/FdeC as interesting targets for future drugs or vaccines to improve the treatment, prevention or chronicity of mastitis induced by MPEC.

Variations in milk, udder skin, and fecal microbiota and their relationships with blood metabolites and milk composition in dairy cows.

This study examined the milk, udder skin, feces, and bedding microbiota in a dairy farm. Blood metabolites concentration and milk composition were also determined to examine their relationship with variations in the microbiota. Samples were collected from 10 healthy cows during the summers of 2018 and 2020. Milk protein, fat, and solid-not-fat contents were higher, and blood urea nitrogen and nonesterified fatty acid levels were lower in the 2020 samples. Principal coordinate analysis demonstrated that milk, udder skin, and fecal microbiota were separate groups. Year-to-year differences were distinct for milk and udder skin microbiota; however, the fecal microbiota of the 2018 and 2020 samples were similar. The bedding microbiota grouped with the udder skin microbiota of the 2018 samples. Although nonpathogens found as prevalent taxa in udder skin microbiota were likely to be found as abundant taxa in milk microbiota, selection and elimination occurred during transmission. Network analysis suggested that bacterial taxa of milk, udder skin, and fecal microbiota were unrelated to blood metabolites and milk composition, regardless of pathogens or nonpathogens.

An in-depth investigation of the microbiota and its virulence factors associated with severe udder cleft dermatitis lesions.

Udder cleft dermatitis (UCD) is a skin condition affecting the anterior parts of the udder in dairy cattle. In the present study, we aimed to shed light on the microbiota in severe UCD lesions versus healthy udder skin by putting forward a taxonomic and functional profile based on a virulence factor analysis. Through shotgun metagenomic sequencing, we found a high proportion of bacteria in addition to a low abundance of archaea. A distinct clustering of healthy udder skin versus UCD lesion samples was shown by applying principal component analysis and (sparse) partial least squares analysis on the metagenomic data. Proteobacteria, Bacillota, and Actinomycetota were among the most abundant phyla in healthy udder skin samples. In UCD samples, Bacteroidota was the most abundant phylum. At genus level, Bifidobacterium spp. had the highest relative abundance in healthy skin samples, whereas Porphyromonas spp. and Corynebacterium spp. had the highest relative abundance in UCD samples. In the differential abundance analysis, Porphyromonas spp. and Bacteroides spp. were significantly differentially abundant in UCD samples, whereas Bifidobacterium spp., Staphylococcus sp. AntiMn-1, and Staphylococcus equorum were more commonly found in healthy samples. Moreover, the abundance of several treponeme phylotypes was significantly higher in lesion samples. The streptococcal cysteine protease speB was among the most abundant virulence factors present in severe UCD lesions, while a plethora of virulence factors such as the antitoxin relB were downregulated, possibly contributing to creating the ideal wound climate for the dysbiotic community. Network analysis showed healthy lesion samples had a large network ofpositive, correlations between the abundances of beneficial species such as Aerococcus urinaeequi and Bifidobacterium angulatum, indicating that the healthy skin microbiome forms an active protective bacterial network, which is disrupted in case of UCD. In UCD samples, a smaller microbial network mainly consisting of positive correlations between the abundances of Bacteroides fragilis and anaerobic Bacteroidota was exposed. Moreover, a high correlation between the taxonomic data and virulence factors was revealed, concurrently with 2 separate networks of microbes and virulence factors. One network, matching with the taxonomic findings in the healthy udder skin samples, showcased a community of harmless or beneficial bacteria, such as Bifidobacterium spp. and Butyrivibrio proteoclasticus, associated with hcnB, hcnC, relB, glyoxalase, and cupin 2. The other network, corresponding to UCD samples, consisted of pathogenic or facultative pathogenic and mainly anaerobic bacteria such as Treponema spp., Mycoplasmopsis spp., and bovine gammaherpesvirus 4, that correlated with virulence factors SpvB, fhaB, and haemagglutination activity domain-associated factor. Our results point toward a dysbiotic community with a notable decrease in diversity and evenness, with a loss of normal skin inhabitants and innocuous or useful species making way for predominantly anaerobic, facultative pathogens. The shift in the abundance of virulence factors such as fhaB and SpvB could play a role in the manifestation of a local micro-environment favorable to the microbiome associated with udder skin lesions. Lastly, the presence of specific networks between microbial species, and between microbes and virulence factors was shown.

The anti-hyperplasia effect of polysaccharide from Prunella vulgaris L. on mammary gland hyperplasia in rats through regulation of the AKT-FOXO3a signaling pathway and intestinal flora.

BACKGROUND: Prunella vulgaris L., a medicinal and edible homologous plant, is often used to treat conditions such as breast hyperplasia, thyroid enlargement and lymphatic tuberculosis. Research has demonstrated that it is particularly effective in the treatment of mammary gland hyperplasia (MGH). However, the material basis and mechanism of its efficacy are still unclear. RESULTS: Our results showed that in rats with MGH, polysaccharide from Prunella vulgaris L. (PVP) led to a reduction in the levels of estradiol, prolactin and malondialdehyde, while simultaneously increasing the concentrations of progesterone (P), superoxide dismutase (SOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) in the serum. In addition, results obtained from 16S rRNA sequencing demonstrated that PVP had the capacity to increase the richness and diversity of the intestinal microbiota in MGH rats, as well as modify the structure of the microbiota. Correlation analysis revealed that the levels of P, SOD, MnSOD and CAT were positively associated with Allobaculum, Romboutsia, Faecalibaculum and Clostridium, while negatively correlated with Turicibacter. CONCLUSIONS: The mechanism of PVP in treating MGH might be through inhibiting the phosphorylation of the AKT-FOXO3a signaling pathway and then activating the expression of downstream antioxidant enzymes, such as MnSOD and CAT. At the same time, PVP could restore intestinal flora homeostasis in rats with MGH by regulating the flora changes of Allobaculum, Romboutsia, Clostridium and Faecalibaculum, thereby reducing oxidative stress in rats with MGH. © 2024 Society of Chemical Industry.

Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus.

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log2 fold change [|log2FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis. IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

Streptococcus uberis induced expressions of pro-inflammatory IL-6, TNF-α, and IFN-γ in bovine mammary epithelial cells associated with inhibited autophagy and autophagy flux formation.

Autophagy is a highly conserved cellular defensive mechanism that can eliminate bacterial pathogens such as Streptococcus uberis, that causes mastitis in cows. However, S. uberis induced autophagy is still unclear. In this study, we tested if certain inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, critical in mastitis due to S. uberis infection, regulate autophagy activation in bovine mammary epithelial cells (bMECs). Using Western blot and laser scanning confocal microscope in bMECs challenged by S. uberis, showed that the expression of IL-6, TNF-α, IFN-γ oscillated with the expressions of autophagic Atg5, ULK1, PTEN, P62, and LC3ӀӀ/LC3Ӏ. S. uberis infection induced autophagosomes and LC3 puncta in bMECs with upregulation of Atg5, ULK1, PTEN, LC3ӀӀ/LC3Ӏ, and downregulation of P62. The levels of IL-6, TNF-α, and IFN-γ increased during autophagy flux formation to decrease during autophagy induction. Autophagy inhibition increased the expression of IL-6, TNF-α, and IFN-γ and increased S. uberis burden. This study indicates autophagy is induced during S. uberis infection and IL-6, TNF-α, and IFN-γ contribute to autophagy and autophagy flux formation.

Scavenger receptor B1 facilitates the endocytosis of Escherichia coli via TLR4 signaling in mammary gland infection.

SCARB1 belongs to class B of Scavenger receptors (SRs) that are known to be involved in binding and endocytosis of various pathogens. SRs have emerging role in regulating innate immunity and host-pathogen interactions by acting in co-ordination with Toll-like receptors.Query Little is known about the function of SCARB1 in milk-derived mammary epithelial cells (MECs). This study reports the role of SCARB1 in infection and its potential association in TLR4 signaling on bacterial challenge in Goat mammary epithelial cells (GMECs). The novelty in the establishment of MEC culture lies in the method that aims to enhance the viability of the cells with intact characteristics upto a higher passage number. We represent MEC culture to be used as a potential infection model for deeper understanding of animal physiology especially around the mammary gland. On E.coli challenge the expression of SCARB1 was significant in induced GMECs at 6 h. Endoribonuclease-esiRNA based silencing of SCARB1 affects the expression of TLR4 and its pathways i.e. MyD88 and TRIF pathways on infection. Knockdown also affected the endocytosis of E.coli in GMECs demonstrating that E.coli uses SCARB1 function to gain entry in cells. Furthermore, we predict 3 unique protein structures of uncharacterized SCARB1 (Capra hircus) protein. Overall, we highlight SCARB1 as a main participant in host defence and its function in antibacterial advances to check mammary gland infections. Video Abstract.

Priming of the murine mammary gland with Staphylococcus chromogenes IM reduces bacterial growth of Streptococcus uberis: a proof-of-concept study.

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation.

Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction.

Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity.

Bovine mastitis is mainly caused by bacterial infection and is responsible for important economic losses as well as alterations of the health and welfare of animals. The increase in somatic cell count (SCC) in milk during mastitis is mainly due to the influx of neutrophils, which have a crucial role in the elimination of pathogens. For a long time, these first-line defenders have been viewed as microbe killers, with a limited role in the orchestration of the immune response. However, their role is more complex: we recently characterized a bovine neutrophil subset expressing major histocompatibility complex class II (MHC-II) molecules (MHC-IIpos), usually distributed on antigen-presenting cells, as having regulatory capacities in cattle. In this study, our objective was to evaluate the implication of different neutrophils subsets in the mammary gland immunity during clinical and subclinical mastitis. Using flow cytometry, we analyzed the presence of MHC-IIpos neutrophils in blood and in milk during clinical mastitis at different time points of inflammation (n = 10 infected quarters) and during subclinical mastitis, defined as the presence of bacteria and an SCC >150,000 cells/mL (n = 27 infected quarters). Our results show, for the first time, that in blood and milk, neutrophils are a heterogeneous population and encompass at least 2 subsets distinguishable by their expression of MHC-II. In milk without mastitis, we observed higher production of reactive oxygen species and higher phagocytosis capacity of MHC-IIpos neutrophils compared with their MHC-IIneg counterparts, indicating the high bactericidal capacities of MHC-IIpos neutrophils. MHC-IIpos neutrophils are enriched in milk compared with blood during subclinical mastitis but not during clinical mastitis. Moreover, we observed a positive and highly significant correlation between MHC-IIpos neutrophils and T lymphocytes present in milk during subclinical mastitis. Our experiments involved a total of 47 cows (40 Holstein and 7 Normande cows). To conclude, our study opens the way to the discovery of new biomarkers of mastitis inflammation.

Evaluation of test-day milk somatic cell count to predict intramammary infection in late lactation grazing dairy cows.

Use of selective dry cow antimicrobial therapy requires to precisely differentiate cows with an intramammary infection (IMI) from uninfected cows close to drying-off to enable treatment allocation. Milk somatic cell count (SCC) is an indicator of an inflammatory response in the mammary gland and is usually associated with IMI. However, SCC can also be influenced by cow-level variables such as milk yield, lactation number and stage of lactation. In recent years, predictive algorithms have been developed to differentiate cows with IMI from cows without IMI based on SCC data. The objective of this observational study was to explore the association between SCC and subclinical IMI, taking cognizance of cow-level predictors on Irish seasonal spring calving, pasture-based systems. Additionally, the optimal test-day SCC cut-point (maximized sensitivity and specificity) for IMI diagnosis was determined. A total of 2,074 cows, across 21 spring calving dairy herds with an average monthly milk weighted bulk tank SCC of ≤200,000 cells/mL were enrolled in the study. Quarter-level milk sampling was carried out on all cows in late lactation (interquartile range = 240-261 d in milk) for bacteriological culturing. Bacteriological results were used to define cows with IMI, when ≥1 quarter sample resulted in bacterial growth. Cow-level test-day SCC records were provided by the herd owners. The ability of the average, maximum and last test-day SCC to predict infection were compared using receiver operator curves. Predictive logistic regression models tested included parity (primiparous or multiparous), yield at last test-day and a standardized count of high SCC test-days. In total, 18.7% of cows were classified as having an IMI, with first parity cows having a higher proportion of IMI (29.3%) compared with multiparous cows (16.1%). Staphylococcus aureus accounted for the majority of these infections. The last test-day SCC was the best predictor of infection with the highest area under the curve. The inclusions of parity, yield at last test-day, and a standardized count of high SCC test-days as predictors did not significantly improve the ability of last test-day SCC to predict IMI. The cut-point for last test-day SCC which maximized sensitivity and specificity was 64,975 cells/mL. This study indicates that in Irish seasonal pasture-based dairy herds, with low bulk tank SCC control programs, the last test-day SCC (interquartile range days in milk = 221-240) is the best predictor of IMI in late lactation.

Invited review: Selective treatment of clinical mastitis in dairy cattle.

Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.

Investigation of intramammary infections in primiparous cows during early lactation on organic dairy farms.

Previous studies have shown that organically raised dairy cows have an increased prevalence of Staphylococcus aureus compared with conventionally raised dairy cows. However, little information exists about the dynamics of intramammary infection (IMI) in primiparous cows during early lactation on organic dairy farms. The objective of this study was to describe the IMI dynamics of primiparous cows on certified organic farms during early lactation. This longitudinal study enrolled 503 primiparous cows from 5 organic dairy farms from February 2019 to January 2020. Quarter-level milk samples were collected aseptically on a weekly basis during the first 5 wk of lactation. Samples were pooled by cow and time point into composite samples inside a sterilized laminar hood and submitted for microbiological culture. For each of the different microorganisms identified, we estimated the prevalence in each postpartum sample, period prevalence (PP), cumulative incidence, and persistence of IMI. Logistic regression models were used to investigate whether the prevalence of IMI differed by farm or sampling time points and whether IMI persistence differed between detected microorganisms. Our findings revealed a high prevalence of Staphylococcus aureus (PP = 18.9%), non-aureus staphylococci and closely related mammaliicoccal species (PP = 52.1%), and Streptococcus spp. and Streptococcus-like organisms (PP = 32.1%) within the study population. The prevalence of these microorganisms varied significantly between farms. Staphylococcus aureus and Staphylococcus chromogenes exhibited significantly higher IMI persistence compared with other detected bacterial taxa, confirming the divergent epidemiological behavior in terms of IMI chronicity across different microorganisms. This study improves our understanding of the epidemiology of mastitis-causing pathogens in organically raised primiparous cows, which can be used to tailor mastitis control plans for this unique yet growing subpopulation of dairy cows.