Evaluation of significantly changed chemokine factors of idiopathic granulomatous mastitis in non-puerperal patients.

Idiopathic granulomatous mastitis (IGM), a recurrent inflammation disease of the non-lactating breast, has had an increasing clinical morbidity rate in recent years, and its complicated symptoms and unclear etiology make it challenging to treat. This rare benign inflammatory breast disease, centered on the lobules, represents the most challenging type of non-puerperal mastitis (NPM), also known as non-lactating mastitis. In this study, patients diagnosed with IGM (M, n = 23) were recruited as cases, and patients with benign control breast disease (C, n = 17) were enrolled as controls. Cytokine microarray detection measured and analyzed the differentially expressed cytokine factors between IGM and control patients. Then, we verified the mRNA and protein expression levels of the significantly changed cytokine factors using Q-RT-PCR, ELISA, western blot, and IHC experiments. The cytokine factor expression levels significantly changed compared to the control group. We observed a significant increase between IGM and control patients in cytokine factors expression, such as interleukin-1ß (IL-1ß), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1ß, tumor necrosis factor receptor 2 (TNF RII). Then, we verified the expression of these top five dysregulated factors in both mRNA and protein levels. Our results demonstrated the cytokine map in IGM and indicated that several cytokines, especially chemokines, were associated with and significantly dysregulated in IGM tissues compared to the control group. The chemokine factors involved might be essential in developing and treating IGM. These findings would be helpful for a better understanding of IGM and offer valuable insights for devising novel diagnostic and therapeutic strategies.

Enhanced protein-metabolite correlation analysis: To investigate the association between Staphylococcus aureus mastitis and metabolic immune pathways.

Mastitis is a disease characterized by congestion, swelling, and inflammation of the mammary gland and usually caused by infection with pathogenic microorganisms. Furthermore, the development of mastitis is closely linked to the exogenous pathway of the gastrointestinal tract. However, the regulatory mechanisms governing the gut-metabolism-mammary axis remain incompletely understood. The present study revealed alterations in the gut microbiota of mastitis rats characterized by an increased abundance of the Proteobacteria phylum. Plasma analysis revealed significantly higher levels of L-isoleucine and cholic acid along with 7-ketodeoxycholic acid. Mammary tissue showed elevated levels of arachidonic acid metabolites and norlithocholic acid. Proteomic analysis showed increased levels of IFIH1, Tnfaip8l2, IRGM, and IRF5 in mastitis rats, which suggests that mastitis triggers an inflammatory response and immune stress. Follistatin (Fst) and progesterone receptor (Pgr) were significantly downregulated, raising the risk of breast cancer. Extracellular matrix (ECM) receptors and focal adhesion signaling pathways were downregulated, while blood-milk barrier integrity was disrupted. Analysis of protein-metabolic network regulation revealed that necroptosis, protein digestion and absorption, and arachidonic acid metabolism were the principal regulatory pathways involved in the development of mastitis. In short, the onset of mastitis leads to changes in the microbiota and alterations in the metabolic profiles of various biological samples, including colonic contents, plasma, and mammary tissue. Key manifestations include disturbances in bile acid metabolism, amino acid metabolism, and arachidonic acid metabolism. At the same time, the integrity of the blood-milk barrier is compromised while inflammation is promoted, thereby reducing cell adhesion in the mammary glands. These findings contribute to a more comprehensive understanding of the metabolic status of mastitis and provide new insights into its impact on the immune system.

Lactobacillus reuteri inhibits Staphylococcus aureus-induced mastitis by regulating oxytocin releasing and gut microbiota in mice.

Mastitis is the most frequent disease of cows and has well-recognized detrimental effects on animal wellbeing and dairy farm profitability. With the advent of the postantibiotic era, alternative antibiotic agents, especially probiotics, have received increasing attention in the treatment of mastitis. Based on research showing that Lactobacillus reuteri (L. reuteri) has anti-inflammatory effects, this study explored the protective effects and mechanisms of L. reuteri against mastitis induced by Staphylococcus aureus (S. aureus) in mice. First, mice with S. aureus-induced mastitis were orally administered L. reuteri, and the inflammatory response in the mammary gland was observed. The results showed that L. reuteri significantly inhibited S. aureus-induced mastitis. Moreover, the concentration of oxytocin (OT) and protein expression of oxytocin receptor (OTR) were measured, and inhibition of OTR or vagotomy reversed the protective effect of L. reuteri or its culture supernatant (LCS) on S. aureus-induced mastitis. In addition, in mouse mammary epithelial cells (MMECs), OT inhibited the inflammation induced by S. aureus by inhibiting the protein expression of OTR. It was suggested that L. reuteri protected against S. aureus-induced mastitis by releasing OT. Furthermore, microbiological analysis showed that the composition of the microbiota was altered, and the relative abundance of Lactobacillus was significantly increased in gut and mammary gland after treatment with L. reuteri or LCS. In conclusion, our study found the L. reuteri inhibited the mastitis-induced by S. aureus via promoting the release of OT, and treatment with L. reuteri increased the abundance of Lactobacillus in both gut and mammary gland.

PFKFB3-Meditated Glycolysis via the Reactive Oxygen Species-Hypoxic Inducible Factor 1α Axis Contributes to Inflammation and Proliferation of Staphylococcus aureus in Epithelial Cells.

Mastitis caused by antibiotic-resistant strains of Staphylococcus aureus is a significant concern in the livestock industry due to the economic losses it incurs. Regulating immunometabolism has emerged as a promising approach for preventing bacterial inflammation. To investigate the possibility of alleviating inflammation caused by S aureus infection by regulating host glycolysis, we subjected the murine mammary epithelial cell line (EpH4-Ev) to S aureus challenge. Our study revealed that S aureus can colonize EpH4-Ev cells and promote inflammation through hypoxic inducible factor 1α (HIF1α)-driven glycolysis. Notably, the activation of HIF1α was found to be dependent on the production of reactive oxygen species (ROS). By inhibiting PFKFB3, a key regulator in the host glycolytic pathway, we successfully modulated HIF1α-triggered metabolic reprogramming by reducing ROS production in S aureus-induced mastitis. Our findings suggest that there is a high potential for the development of novel anti-inflammatory therapies that safely inhibit the glycolytic rate-limiting enzyme PFKFB3.

Employment of mastoparan-like peptides to prevent Staphylococcus aureus associated with bovine mastitis.

Bovine mastitis is a frequent infection in lactating cattle, causing great economic losses. Staphylococcus aureus represents the main etiological agent, which causes recurrent and persistent intramammary infections because conventional antibiotics are ineffective against it. Mastoparan-like peptides are multifunctional molecules with broad antimicrobial potential, constituting an attractive alternative. Nevertheless, their toxicity to host cells has hindered their therapeutic application. Previously, our group engineered three mastoparan-L analogs, namely mastoparan-MO, mastoparan-R1, and [I5, R8] MP, to improve cell selectivity and potential. Here, we were interested in comparing the antibacterial efficacy of mastoparan-L and its analogs against bovine mastitis isolates of S. aureus strains, making a correlation with the physicochemical properties and structural arrangement changes promoted by the sequence modifications. As a result, the analog's hemolytic and/or antimicrobial activity was balanced. All the peptides displayed α-helical folding in hydrophobic and membrane-mimetic environments, as determined by circular dichroism. The peptide [I5, R8] MP stood out for its enhanced selectivity and antibacterial features related to mastoparan-L and the other derivatives. Biophysical approaches revealed that [I5, R8] MP rapidly depolarizes the bacterial membrane of S. aureus, causing cell death by subsequent membrane disruption. Our results demonstrated that the [I5, R8] MP peptide could be a starting point for the development of peptide-based drugs for the treatment of bovine mastitis, with the advantage of no residue in milk, which would help reduce the use of classical antibiotics.IMPORTANCEStaphylococcus aureus is a leading cause of mastitis, the world's most important dairy cattle disease. The multidrug resistance and zoonotic potential of S. aureus, besides the likelihood of antibiotic residues in milk, are of critical concern to public and animal health. Antimicrobial peptides offer a novel antimicrobial strategy. Here, we demonstrate that [I5, R8] MP is a potent and selective peptide, which acts on S. aureus by targeting the bacterial membrane. Therefore, understanding the physicochemical determinants and the modes of action of this class of antimicrobials opens novel prospects for peptide development with enhanced activities in the bovine mastitis context.

Prevalence and characterization of community-associated Staphylococcus aureus isolates from human mastitis in Beijing, China.

OBJECTIVES: Staphylococcus aureus (S. aureus) spreads worldwide and occurrence of mastitis caused by it holds significant implications for public health. We aim to reveal the molecular typing, antibiotic resistance and virulence gene profile of S. aureus causing mastitis through investigation. METHODS: A total of 200 isolates of S. aureus were collected from outpatients infected with mastitis in a hospital in Beijing from 2020.7 to 2021.7. The molecular characteristics were analyzed by MLST and spa typing, virulence genes were screened by PCR, antibiotic susceptible test was performed by VITEK® 2 Compact system and phylogenetic analysis was performed by MEGA11 and iTOL. RESULTS: Nineteen sequence types (STs) belonging to 9 clone complexes (CCs) were identified. ST22 was the most dominant clone (77.0%, 154/200). MRSA accounted for 19.0% (38/200) and 89.5% (34/38) of MRSA isolates belonged to CC22 and CC59. The isolates had relatively low levels of antibiotic resistance, with the exception of ß-lactams and macrolides with resistance rates above 50.0%. The carrying rate of pvl in the ST22-MRSA strains were 84.2% and the detection rates of seb and pvl in the MRSA isolates were significantly higher than those in the MSSA isolates, while the hlg, fnbA and sdrD showed opposite results. Whole genome sequenced specimens of MRSA strains X4 and B5 show the same evolutionary origin as ST22 EMRSA-15 (HE681097), which is popular in Europe. CONCLUSIONS: The method based on molecular epidemiology is an important tool for tracking the spread of S. aureus infections. We need to be alert to the major MRSA clones CC22 and CC59 in the region and be vigilant to the possible pandemic and spread of ST22 EMRSA-15.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Genetic basis of antibiotic resistance in bovine mastitis and its possible implications for human and ecological health.

Bovine mastitis is a mammary gland inflammation that can occur due to infectious pathogens, Staphylococcus aureus and Escherichia coli, which are, respectively, the most prevalent Gram-positive and Gram-negative bacteria associated with this disease. Currently, antibiotic treatment has become more complicated due to the presence of resistant pathogens. This review, therefore, aims to identify the most common resistance genes reported for these strains in the last four years. During the review, it was noted that blaZ, blaSHV, blaTEM, and blaampC are the most reported genes for S. aureus and E. coli, associated with drug inactivation, mainly ß-lactamases. They are characterized by generating bacterial resistance to ß-lactam antibiotics, the most common treatment in animal and human bacterial treatments (penicillins and cephalosporins, among others). Genes associated with efflux systems were also present in the two strains and included norA, tetA, tetC, and tetK, which generate resistance to macrolide and tetracycline antibiotics. Additionally, the effects of spreading resistance between animals and humans through direct contact (such as consumption of contaminated milk) or indirect contact (through environmental contamination) has been deeply discussed, emphasizing the importance of having adequate sanitation and antibiotic control and administration protocols.

Presentation and Management of Granulomatous Mastitis in the United States: Results of an American Society of Breast Surgeons Registry Study.

BACKGROUND: Granulomatous mastitis (GM) is a benign, chronic, inflammatory disease lacking clear treatment guidelines. The purpose of this American Society of Breast Surgeons (ASBrS) prospective, multisite registry was to characterize the presentation of GM and identify treatment strategies associated with symptom resolution and optimal cosmesis. METHODS: ASBrS members entered data into a registry on patient demographics, treatment, symptoms, and cosmesis over a 1-year period. Initial symptoms were graded as mild, moderate, or severe. The Chi-square test and logistic regression were used to identify factors related to symptom improvement and cosmesis. RESULTS: Overall, 112 patients with a mean age of 36 years were included. More patients were Hispanic (49.1%) and from the Southwest (41.1%), and management included observation (4.5%), medical (70.5%), surgical (5.4%), or combination treatment (19.6%). Immunosuppression was used in 83 patients (74.1%), including 43 patients who received intralesional steroid injections. Patients with severe symptoms were more likely to undergo surgical intervention compared with those with mild or moderate symptoms (21.4% vs. 0% and 7.5%, respectively; p = 0.004). Within 1 year, 85 patients (75.9%) experienced symptom improvement and/or resolution at a median of 3 months. Receipt of immunosuppressive therapy was predictive of improvement or resolution at 1 month (odds ratio 4.22; p = 0.045). One-year physician-assessed cosmesis was excellent or good for 20/35 patients (57.1%) and was not associated with type of treatment or symptom severity. CONCLUSION: Although GM can have a protracted course, the majority of patients in this registry resolved within 1 year, with good cosmetic result. Treatment with immunosuppression appears to be most beneficial, and a symptom-based algorithm may be helpful to guide treatment.

The inhibition effect of caffeic acid on NOX/ROS-dependent macrophages M1-like polarization contributes to relieve the LPS-induced mice mastitis.

The mammary gland is an adipose tissue containing not only adipocytes but also epithelial, endothelial, and immune cells. Epithelial cells and macrophages, as the integral components of the immune system, are on the front line of defense against infection. Our preliminary work proved that caffeic acid (CA) can effectively inhibit the inflammatory cascade of bovine mammary epithelial cells (BMEC) induced by lipopolysaccharide (LPS) and maintain cellular integrity and viability. Here, we investigated the therapeutic effect of CA on LPS-induced mice mastitis and explored its regulatory mechanism on macrophage inflammatory response induced by LPS in vitro. Firstly, the mice mastitis model was established by intramammary injection with 10 µg LPS, after which different CA doses (5, 10, 15 mg/kg) were administered. Then, the pathological section, myeloperoxidase (MPO) activity, proinflammatory factors and chemokines releasement, and redox state of mammary tissues were assessed, confirming CA's effectiveness on mice mastitis. In vitro, we validated the therapeutic relevance of CA in relieving LPS-induced RAW264.7 inflammatory and oxidative stress responses. Moreover, we further provided evidence that CA significantly reduced LPS-induced reactive oxygen species (ROS) generation via NADPH oxidase (NOX), which improved the imbalance relationship between nuclear factor kappa-B (NF-κB) and NF-E2 p45-related factor 2 (Nrf2) and led to a marked weakening of M1 polarization. The NOX-ROS signal inhibited by CA weakened the oxidative burst and neutrophil chemotaxis of macrophages, thus alleviating the immune cascade in mammary gland tissue and reducing the LPS-induced inflammatory damage. Collectively, CA would be a potential candidate or antibacterial synergist for curbing mastitis.

Comparative analysis of Streptococcus agalactiae serotypes Ia and II isolates from China and Pakistan in a murine model: A focus on pathogenesis and immune response.

Bovine mastitis, caused by Streptococcus agalactiae (Group B Streptococcus; GBS), poses significant economic challenges to the global dairy industry. Mouse models serves as valuable tools for assessing GBS-induced infections as an alternative to large animals. This study aimed to investigate the LD50 dose, organ bacterial load, and quantification of peritoneal leukocyte populations for GBS serotypes Ia and II isolates from China and Pakistan. Additionally, we measured indicators such as lactoferrin, albumin, and myeloperoxidase (MPO) activity. Pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-2) and anti-inflammatory cytokines (IL-10 and TGF-ß) in serum and tissue samples were evaluated using ELISA and qPCR, respectively. BALB/c mice (4 mice per group) received individual intraperitoneal injections of 100 µl containing specific bacterial inoculum concentrations (ranging from 105 to 109 CFU per mouse) of Chinese and Pakistani GBS isolates (serotypes Ia and II). Control groups received 100 µL of sterile PBS. Results revealed that the LD50 bacterial dose causing 50 % mortality in mice was 107 CFU. The highest bacterial load in all experimental groups was quantified in the peritoneum, followed by blood, mammary gland, liver, spleen, lungs, and brain. The most significant bacterial dissemination was observed in mice inoculated with Pakistani serotype Ia at 24 h, with a subsequent notable decline in bacterial counts at day 3. Notably, infection with Pakistani serotype Ia showed a trend of increased total leukocyte counts, significantly higher than Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II. A substantial influx of neutrophils and lymphocytes was observed in response to all tested serotypes, with Pakistani serotype Ia inducing a significantly higher influx compared to other groups (Pakistani serotype II, Chinese serotype Ia, and Chinese serotype II). Furthermore, TNF-α, IL-1ß, IL-2, and IL-6 expressions were significantly increased in mice one day after infection with the Pakistani serotype Ia. Compared to mice infected with the Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II, those infected with the Pakistani serotype Ia isolate exhibited the highest production of IL-10 and TGF-ß, along with significantly increased concentrations of lactoferrin, albumin, and MPO. These findings suggest that the persistence and severity of infection caused by the Pakistani serotype Ia may be linked to its ability to spread to deeper tissues. This study enhances our understanding of the clinical characteristics of bovine mastitis caused by S. agalactiae in China and Pakistan.

The rumen microbiota contributed to the development of mastitis induced by subclinical ketosis.

BACKGROUND: Mastitis is a serious disease which affects animal husbandry, particularly in cow breeding. The etiology of mastitis is complex and its pathological mechanism is not yet fully understood. Our previous research in clinical investigation has revealed that subclinical ketosis can increase the number of somatic cell counts (SCC) in milk, although the underlying mechanism remains unclear. Recent studies have further confirmed the significant role of mastitis. RESULTS: In this study, we aimed to examine the SCC, rumen microbiota, and metabolites in the milkmen of cows with subclinical ketosis. Additionally, we conducted a rumen microbiota transplant into mice to investigate the potential association between rumen microbiota disturbance and mastitis induced by subclinical ketosis in dairy cows. The study has found that cows with subclinical ketosis have a higher SCC in their milk compared to healthy cows. Additionally, there were significant differences in the rumen microbiota and the level of volatile fatty acid (VFA) between cows with subclinical ketosis and healthy cows. Moreover, transplanting the rumen microbiota from subclinical ketosis and mastitis cows into mice can induce mammary inflammation and liver function damage than transplanting the rumen flora from healthy dairy cows. CONCLUSIONS: In addition to the infection of mammary gland by pathogenic microorganisms, there is also an endogenous therapeutic pathway mediated by rumen microbiota. Targeted rumen microbiota modulation may be an effective way to prevent and control mastitis in dairy cows.

Dietary anti-inflammatory and anti-bacterial medicinal plants and its compounds in bovine mastitis associated impact on human life.

Bovine mastitis (BM) is the most common bacterial mediated inflammatory disease in the dairy cattle that causes huge economic loss to the dairy industry due to decreased milk quality and quantity. Milk is the essential food in the human diet, and rich in crucial nutrients that helps in lowering the risk of diseases like hypertension, cardiovascular diseases and type 2 diabetes. The main causative agents of the disease include various gram negative, and positive bacteria, along with other risk factors such as udder shape, age, genetic, and environmental factors also contributes much for the disease. Currently, antibiotics, immunotherapy, probiotics, dry cow, and lactation therapy are commonly recommended for BM. However, these treatments can only decrease the rise of new cases but can't eliminate the causative agents, and they also exhibit several limitations. Hence, there is an urgent need of a potential source that can generate a typical and ideal treatment to overcome the limitations and eliminate the pathogens. Among the various sources, medicinal plants and its derived products always play a significant role in drug discovery against several diseases. In addition, they are also known for its low toxicity and minimum resistance features. Therefore, plants and its compounds that possess anti-inflammatory and anti-bacterial properties can serve better in bovine mastitis. In addition, the plants that are serving as a food source and possessing pharmacological properties can act even better in bovine mastitis. Hence, in this evidence-based study, we particularly review the dietary medicinal plants and derived products that are proven for anti-inflammatory and anti-bacterial effects. Moreover, the role of each dietary plant and its compounds along with possible role in the management of bovine mastitis are delineated. In this way, this article serves as a standalone source for the researchers working in this area to help in the management of BM.

Understanding mastitis: Microbiome, control strategies, and prevalence - A comprehensive review.

Mastitis significantly affects the udder tissue in dairy cattle, leading to inflammation, discomfort, and a decline in both milk yield and quality. The condition can be attributed to an array of microbial agents that access the mammary gland through multiple pathways. The ramifications of this ailment are not merely confined to animal welfare but extend to the financial viability of the livestock industry. This review offers a historical lens on mastitis, tracing its documentation back to 1851, and examines its global distribution with a focus on regional differences in prevalence and antimicrobial resistance (AMR) patterns. Specific microbial genes and communities implicated in both mastitis and AMR are explored, including Staphylococcus aureus, Streptococcus agalactiae,Streptococcus dysagalactiae, Streptococcus uberis Escherichia coli, Klebsiella pneumoniae, Mycoplasma bovis, Corynebacterium bovis, among others. These microorganisms have evolved diverse strategies to elude host immune responses and neutralize commonly administered antibiotics, complicating management efforts. The review aims a comprehensive overview of the current knowledge and research gaps on mastitis and AMR, and to highlight the need for a One Health approach to address this global health issue. Such an approach entails multi-disciplinary cooperation to foster judicious antibiotic use, enhance preventive measures against mastitis, and bolster surveillance and monitoring of AMR in pathogens responsible for mastitis.

Lycopene promoted M2 macrophage polarization via inhibition of NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to Escherichia coli infection in J744A.1 cells.

Escherichia coli (E. coli) can induce severe clinical bovine mastitis, which is to blame for large losses experienced by dairy farms. Macrophage polarization into various states is in response to pathogen infections. Lycopene, a naturally occurring hydrocarbon carotenoid, relieved inflammation by controlling M1/M2 status of macrophages. Thus, we wanted to explore the effect of lycopene on polarization states of macrophages in E. coli-induced mastitis. Macrophages were cultivated with lycopene for 24, before E. coli inoculation for 6 h. Lycopene (0.5 µmol/L) significantly enhanced cell viabilities and significantly reduced lactic dehydrogenase (LDH) levels in macrophages, whereas 2 and 3 µmol/L lycopene significantly enhanced LDH activities. Lycopene treatment significantly reduced the increase in LDH release, iNOS, CD86, TNF-α, IL-1ß and phosphatase and tensin homolog (PTEN) expressions in E. coli group. 0.5 µmol/L lycopene significantly increased E. coli-induced downregulation of CD206, arginase I (ARG1), indoleamine 2,3-dioxygenase (IDO), chitinase 3-like 3 (YM1), PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, jumonji domain-containing protein-3 (JMJD3) and interferon regulatory factor 4 (IRF4) levels. Moreover, Ginkgolic acid C17:1 (a specific PTEN inhibitor), 740YPDGFR (a specific PI3K activator), SC79 (a specific AKT activator) or CHPG sodium salt (a specific NF-κB activator) significantly decreased CD206, AGR1, IDO and YM1 expressions in lycopene and E. coli-treated macrophages. Therefore, lycopene increased M2 macrophages via inhibiting NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to E. coli infection in macrophages. These results contribute to revealing the pathogenesis of E. coli-caused bovine mastitis, providing the new angle of the prevention and management of mastitis.

Using registry data to identify individual dairy cows with abnormal patterns in routinely recorded somatic cell counts.

Data from the Danish milk recording system routinely enter the Danish Cattle Database, including somatic cell counts (SCC) for individual animals. Elevated SCC can signal intramammary inflammation, suggesting subclinical mastitis. Detecting mastitis is pivotal to limit severity, prevent pathogen spread, and target treatment or culling. This study aimed to differentiate normal and abnormal SCC patterns using recorded registry data. We used registry data from 2010 to 2020 for dairy cows in herds with 11 annual milk recordings. To create consistency across herds, we used data from 13,996 unique animals and eight different herds, selected based on the amount of data available, only selecting Holstein animals and conventional herds. We fitted log10-transformed SCC to days in milk (DIM) using the Wilmink and Wood's curve functions, originally developed for milk yield over the lactation. We used Nonlinear Least Square and Nonlinear Mixed Effect models to fit the log10-transformed SCC observations to DIM at animal level. Using mean squared residuals (MSR), we found a consistently better fit using a Wood's style function. Detection of MSR outliers in the model fitting process was used to identify animals with log10(SCC) curves deviating from the expected "normal" curve for that same animal. With this study, we propose a method to identify single animals with SCC patterns that indicate abnormalities, such as mastitis, based on registry data. This method could potentially lead to a registry data-based detection of mastitis cases in larger dairy herds.

Antimicrobial and antibiofilm effect of cannabinoids from Cannabis sativa against methicillin-resistant Staphylococcus aureus (MRSA) causing bovine mastitis.

Antimicrobial resistance (AMR) poses a serious threat to human, animal, and plant health on a global scale. Search and elimination techniques should be used to effectively counter the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. With only a few novel drugs in clinical development, the quest for plant-based alternatives to prevent the spread of antibiotic resistance among bacteria has accelerated. Treatment of MRSA infections is challenging owing to rapidly emerging resistance mechanisms coupled with their protective biofilms. In the present research, we examined the antibacterial properties of ten plant-derived ethanolic leaf extracts. The most effective ethanolic leaf extract against MRSA in decreasing order of zone of inhibition, Cannabis sativa L. > Syzygium cumini > Murraya koenigii > Eucalyptus sp. > while Aloe barbadensis, Azadirachta indica, had very little impact. Mangifera indica, Curcuma longa, Tinospora cordifolia, and Carica papaya did not exhibit inhibitory effects against MRSA; hence, Cannabis was selected for further experimental study. The minimal inhibitory concentration (MIC) of Cannabis sativa L. extract was 0.25 mg ml-1 with 86% mortality. At a sub-MIC dosage of 0.125 mg ml-1, the biofilm formation was reduced by 71%. The two major cannabinoids detected were cannabidiol and delta-9-tetrahydrocannabinol (Δ9-THC), which were majorly attributed to substantial inhibitory action against MRSA. The time-kill kinetics demonstrated a bactericidal action at 4 MIC over an 8-20-h time window with a 90% reduction in growth rate. The results from SEM, and light microscopy Giemsa staining revealed a reduction in cells in the treated group with increased AKP activity, indicating bacterial cell membrane breakdown. These findings suggested cannabinoids may be a promising alternative to antibiotic therapy for bovine biofilm-associated MRSA.

The Effect of Local Steroid Administration on Idiopathic Granulomatous Mastitis: A Systematic Review andMeta-Analysis.

INTRODUCTION: Idiopathic granulomatous mastitis (IGM) is a rare, benign inflammatory breast disease. Corticosteroids and surgery are the primary treatment options, and a growing number of publications have shown the effectiveness of local steroid administration (intralesional injection and topical corticosteroids). However, less is known about the specific details and effects of this treatment approach. The purpose of this meta-analysis was to summarize the details and evaluate the efficacy of local steroid administration for IGM. METHODS: The PubMed, Embase, Cochrane Library, and SinoMed databases were systematically searched from inception to July 2023 to identify relevant randomized controlled trials. The quality of the included studies was assessed, and meta-analysis and subgroup analysis were conducted to obtain the pooled effect sizes of the outcomes of interest. RESULTS: Eight trials comprising 613 patients were included. Local steroid administration included intralesional injection and topical steroid ointment, and control groups were mainly given systemic therapy (oral steroid) and surgical treatment. The meta-analysis showed that local steroid administration had a significant effect on the response rate (risk ratio [RR] = 1.35, 95% CI = [1.14-1.59], P = 0.0004). The incidence of side effects was also lower than that of systemic treatment (RR = 0.24, 95% CI = [0.13-0.43], P＜0.0001). There was no difference in the recurrence rate (RR = 0.8, 95% CI = [1.42-1.51], P = 0.48). CONCLUSIONS: Local steroid administration can increase the RR and decrease the incidence of side effects for IGM patients. There is no significant difference in the recurrence rate between the local steroid administration group and the control group. Further studies are needed to identify the effect in different stages and among pregnant women.

Soluble CD14 produced by bovine mammary epithelial cells modulates their response to full length LPS.

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

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.