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Background: A high grain diet causes an ecological imbalance in the gut microbiota and serves as an important endogenous trigger of mastitis in dairy cows, but the underlying mechanisms are unclear. Our previous study revealed that subacute rumen acidosis (SARA)-associated mastitis has distinct metabolic profiles in the rumen, especially a significant increase in succinate, but the role of succinate in the pathogenesis of mastitis remains unclear., Results: Succinate treatment exacerbates low-grade endotoxemia-induced mastitis in mice. Specifically, succinate increased the production of gut microbiota-extracellular vehicles (mEVs) containing lipopolysaccharides, which can diffuse across the damaged intestinal barrier into the mammary glands. Administration of mEVs promotes mammary inflammation via activation of the TLR4/NF-κB pathway., Conclusions: Our findings suggest that succinate promotes mastitis through the proliferation of enteric pathogens and mEVs production, suggesting a potential strategy for mastitis intervention on the basis of intestinal metabolic regulation and pathogen inhibition. The role of mEVs in interspecific communication has also been elucidated., Competing Interests: Declarations Ethics approval and consent to participate All animal husbandry and experimental procedures were approved by the Laboratory Animal Management and Committee of Jilin University and complied with the institution’s Laboratory Animal Use and Welfare Guidelines (20210912). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Intramammary infections negatively affect milk quality, animal welfare and productivity in the dairy industry. Somatic cell count (SCC) is the most used screening tool to detect subclinical mastitis caused by intramammary infections. In dairy goats, SCC is greatly influenced by non-infectious factors, which complicates the interpretation. The aim of this research paper was to determine the association between SCC, intramammary infections and non-infectious factors including parity, season, lactation stage, and milk yield in dairy goats. In this longitudinal study, 451 goats from four Norwegian dairy goat herds were sampled for bacteriology and SCC up to nine times during two lactations. Factors like parity, milk yield, and stage of lactation were retrieved from the Norwegian goat recording system., Results: The most prevalent udder pathogen findings were Staphylococcus caprae (6.8%), Staphylococcus warneri (6.3%), and Staphylococcus epidermidis (3.8%), all of which had a mild but significant impact on SCC. Staphylococcus aureus was detected in 3.6% of the udder halves and had a major effect on SCC. Parity, stage of lactation, season, and milk yield significantly influenced SCC., Conclusions: This study highlights that intramammary infections caused by Staphylococcus aureus, along with factors such as increasing parity and the seasonal effects of pasturing, significantly influence the SCC. Understanding these key contributors is essential for improving udder health management and improving milk quality in goat milk production., (© 2024. The Author(s).)

Invasive neonatal infection with Group B Streptococcus (GBS) is a disease of concern that can lead to neurological sequelae. Guidelines for preventing mother-to-child transmission have been introduced to reduce the incidence of early-onset infection, but guidelines for controlling the late-onset form are lacking. Recently, the trans-breastfeeding route of transmission has been highlighted as an example of late-onset infection, but no consensus on how to manage such infections has been reached. In this report, we describe a case of late-onset bacteremia/meningitis in a neonate suspected to have been infected with GBS via breastfeeding. A vaginal culture test of the mother at 35 weeks' gestation was negative for GBS. Since she had symptoms of mastitis, breast milk and nipple cultures were also tested and found to be positive for the strain of GBS identified in the neonate on genetic analysis. Diagnosis of trans-mammary GBS infection is challenging because breastfeeding-related events are difficult to identify. In our case, the diagnosis was based on the mother's history of mastitis, and the patient was treated without escalation to sequelae. When a neonate develops a fever, physicians should consider GBS infection and examine the mother's medical history to facilitate accurate diagnosis, especially if the history includes mastitis. A breast milk culture should be performed if the mother has mastitis, especially in cases of infection in preterm infants and in recurrent cases.

The wide-spreading of multidrug resistance poses a significant threat to human and animal health. Although antimicrobial peptides (AMPs) show great potential application, their instability has severely limited their clinical application. Here, self-assembled AMPs composed of multiple modules based on the principle of associating natural marine peptide N6 with ß-sheet-forming peptide were designed. It is noteworthy that one of the designed peptides, FFN could self-assemble into nanoparticles at 35.46 µM and achieve a dynamic transformation from nanoparticles to nanofibers in the presence of bacteria, resulting in a significant increase in stability in trypsin and tissues by 1.72-57.5 times compared to that of N6. Additionally, FFN exhibits a broad spectrum of antibacterial activity against multidrug-resistant (MDR) gram-positive (G + ) and gram-negative (G - ) bacteria with Minimum inhibitory concentrations (MICs) as low as 2 µM by membrane destruction and complemented by nanofiber capture. In vivo mouse mastitis infection model further confirmed the therapeutic potential and promising biosafety of the self-assembled peptide FFN, which can effectively alleviate mastitis caused by MDR Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and eliminate pathogenic bacteria. In conclusion, the design of peptide-based nanomaterials presents a novel approach for the delivery and clinical translation of AMPs, promoting their application in medicine and animal husbandry., (© 2024. The Author(s).)

Subclinical mastitis is an asymptomatic inflammatory condition that can be difficult to define and diagnose. In the dairy industry, subclinical mastitis is diagnosed by milk somatic cell counts (SCCs) of ≥250,000 cells mL -1 . In this pilot study, we assessed the efficacy of this index to identify human subclinical mastitis by comparing SCC levels with the inflammatory response [interleukin-8 (IL-8) levels] in 37 samples from asymptomatic and 10 clinical mastitis (CM) lactating women. The milk microbiota was determined by 16S rRNA gene sequencing. The SCC of CM samples ranged from 310,000 to 6,600,000 cells mL -1 . However, 14 of 37 (37.8%) asymptomatic samples had high SCC (250,000-460,000 cells mL -1 ), indicating subclinical mastitis. SCC levels significantly ( P < 0.001) and positively correlated with milk IL-8 levels reflecting the escalating inflammatory response across subclinical and clinical mastitis samples. Samples with an SCC of ≥250,000 cells mL -1 showed significant increases in IL-8 responses when compared with milk samples from healthy women. The milk microbiome of CM samples was dominated by streptococcal and staphylococcal species (89.9% combined median relative abundance). In contrast, the combined median streptococcal/staphylococcal relative levels were 75.4% and 66.3% in milks from asymptomatic (subclinical mastitis) and healthy groups, respectively. The Streptococcus genus was increased in samples with an SCC of ≥250,000, although this should be interpreted with caution. Thus, the index of ≥250,000 somatic cells mL -1 could be a reliable indicator of subclinical mastitis in humans and should aid future studies investigating the impact of subclinical mastitis on maternal health, breastfeeding behaviors, infant health, and development., Importance: This pilot study suggests that SCC at a level of (greater than or equal to) 250,000 cells mL -1 , as used in the dairy industry, is a suitable index to identify asymptomatic subclinical mastitis in lactating women since it reflects a significant increase in the inflammatory response compared to milk samples from healthy women. Using this index should aid studies into the short- and long-term consequences of subclinical mastitis for mother and infant., Competing Interests: The authors declare no conflict of interest.

Previous studies have demonstrated that gut microbiota dysbiosis promotes the development of mastitis. The interaction of the vagus nerve and gut microbiota endows host homeostasis and regulates disease development, but whether the vagus nerve participates in the pathogenesis of mastitis is unclear. Here, vagotomized mice exhibit disruption of the blood-milk barrier and mammary gland inflammation. Notably, mastitis and barrier damage caused by vagotomy are dependent on the gut microbiota, as evidenced by antibiotic treatment and fecal microbiota transplantation. Vagotomy significantly alters the gut microbial composition and tryptophan metabolism and reduces the 5-hydroxyindole acetic acid (5-HIAA) level. Supplementation with 5-HIAA alleviates vagotomy-induced mastitis, which is associated with the activation of the aryl hydrocarbon receptor (AhR) and subsequent inhibition of the NF-κB pathway. Collectively, our findings indicate the important role of the vagus-mediated gut-mammary axis in the pathogenesis of mastitis and imply a potential strategy for the treatment of mastitis by targeting the vagus-gut microbiota interaction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Aerococcus viridans ( A. viridans ) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log 10 in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log 10) in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans ., Importance: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 μg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans . Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans ., Competing Interests: The authors declare no conflict of interest.

Innovative approaches in nanotechnology provide a potentially promising alternative to untreatable cases of mastitis caused by genus Prototheca spp. algae infections. Drying of the teats of the affected animals or culling are typically the outcomes of mastitis in dairy cattle caused by these pathogens. A major issue in both veterinary medicine and animal breeding is the Prototheca species' widespread resistance to the current methods of managing infections and the available drugs, including antibiotics. Commercial antifungal preparations are also ineffective. Nanotechnology, an emerging discipline, has the potential to create an effective alternative treatment for protothecal mastitis . The aim of the paper is to combine the literature data on the use of nanotechnology in the control of mastitis , taking into account data on combating mastitis caused by Prototheca spp. infections. The databases employed were PubMed, Google Scholar, and Scopus, focusing on literature from the last 20 years to ensure relevance and currency. Studies conducted in vitro have demonstrated that nanomaterials have significant biocidal activity against mastitis infections of different etiologies. Analyzed research papers show that (NPs), such as AgNPs, CuNPs, AuNPs, etc., may not negatively impact various cell lines and may be effective agents in reducing the pathogens' viability. However, it is also critical to assess the risks involved in using nanomaterials.

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

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 β-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., Competing Interests: Declaration of Competing Interest The authors declare no competing conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mastitis is an inflammatory disease of the mammary gland with a high incidence in lactating animals, significantly impacting their health and breastfeeding. Moreover, mastitis adversely affects milk quality and yield, resulting in substantial economic losses for the dairy farming industry. Forsythiaside A (FTA), a phenylethanol glycoside analog extracted from Forsythia, exhibits notable anti-inflammatory and antioxidant properties. However, its protective effects and specific mechanisms against mastitis remain unclear. In this study, a lipopolysaccharide (LPS)-induced mouse mastitis model was used to investigate the protective effect of FTA on LPS-induced mastitis and its potential mechanism using histological assays, Western blot, qRT-PCR, FITC-albumin permeability test, 16s rRNA gene sequencing analysis and non-targeted metabolomics assays to investigate the protective effect of FTA on LPS-induced mastitis model and its potential mechanism. The results demonstrated that FTA significantly mitigated LPS-induced mouse mastitis by reducing inflammation and apoptosis levels, modulating the PI3K/AKT/mTOR signaling pathways, inducing autophagy, and enhancing antioxidant capacity and the expression of tight junction proteins. Furthermore, FTA increased the abundance of beneficial microbiota while decreasing the levels of harmful microbiota in mice, thus counteracting the gut microbiota disruption induced by LPS stimulation. Intestinal metabolomics analysis revealed that FTA primarily regulated LPS-induced metabolite alterations through key metabolic pathways, such as tryptophan metabolism. This study confirms the anti-inflammatory and antioxidant effects of FTA on mouse mastitis, which are associated with key metabolic pathways, including the restoration of gut microbiota balance and the regulation of tryptophan metabolism. These findings provide a novel foundation for the treatment and prevention of mammalian mastitis using FTA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Beta-glucan (β-glucan) is a polysaccharide containing β-glycosidic bonds that is an important structure part of different yeast cells., Aim: The purpose of the study is to characterize β-glucan obtained from Candida albicans (C. albicans) isolated from caprine mastitis ., Methods: The β-glucan was extracted by using utilizing an Alkaline-acidic extraction technique. The dry weight of extracted β-glucan was 7.47/150 g with 4.98%., Results: The findings demonstrated that the extracted β-glucan had similarity in the primary peak 5.78 of liquid samples using the method of high-performance liquid chromatography when compared to the standard form of β-glucan. However, scanning electron microscopy studies revealed that the standard of β-glucan was distinct in morphology but similar to β-glucan isolated from C. albicans in terms of particle sizes in the range of 1.60-2.65 m and the lack of cell wall traces. The findings of an investigation using energy-dispersive X-ray fluorescence spectroscopy (EDS/EDX) of extracted and standard β-glucan, showed the principal elements discovered were carbon (C), oxygen (O), and nitrogen (N). Aluminum (Al), silicon (Si), nickel (Ni), and gold (Au) were also present, but in less amounts., Conclusion: The extracted β-glucan displayed a high degree of similarity and purity to the standard β-glucan, according to the findings of Fourier transform infrared spectroscopy (FT-IR) research., Competing Interests: All authors have no conflict of interest to disclose.

A young woman experienced pain and swelling in a non-lactating breast. The culture test result showed an unusual microbe, which is increasingly prevalent in Norway and internationally.

Background: Mastitis in goats is unquestionably a grave concern, with far-reaching implications for both animal well-being and productivity, while also presenting a potential threat to public health., Aim: The study aimed to compare culture methods and multiplex PCR (m-PCR) in the detection of the most three common mastitis-causing pathogens ( Staphylococcus aureus , Escherichia coli , and Streptococcus spp.) and investigate the gene expression, single nucleotide polymorphisms (SNPs), serum concentrations of immunological and antioxidant indicators linked to mastitis in Shami goats., Methods: One hundred Shami do (50 Shami goats with clinical mastitis and 50 normal goats taken as control group). The culture methods and m-PCR analysis were used to find the bacteria in the milk samples. Blood samples were obtained to assess some hemato-biochemical parameters, detect SNPs, and determine the expression of certain immunological and antioxidant indicators in the genes., Results: The culture method detected the pathogens causing mastitis in 90% of the milk samples, but m-PCR detected them in 100% of the milk samples. SNPs linked to mastitis resistance/susceptibility in examined does were detected through DNA sequencing of immunological and antioxidant indicators. The magnitude of gene expression varied significantly between the resistant and mastitis-affected groups. Significant ( P ˂ 0.05) elevations were noticed in WBCs count, mainly neutsrophils count, serum levels of BHB, NEFA, triglycerides, LDL-C, AST, ALT, ALP, creatinine, total protein, globulin, Ca, K, GPx, MDA, acute phase proteins, and cytokines in mastitis affected does as compared to control. While RBCs count, PCV%, lymphocytes count, serum concentration of glucose, cholesterol, HDL-C, albumin, Na, Cl, P, GSH, SOD, and catalase significantly ( P ˂ 0.05) diminished in mastitis affected does compared to healthy ones. APPs and pro-inflammatory cytokines scored high sensitivities and NPVs but TNF-α and serum amyloid A (SAA) had the highest percentages of increase., Conclusion: The study confirmed that m-PCR is the most sensitive method for bacteria identification ( S. aureus , E. coli, and Strept. spp.) while SNPs in antioxidant and immunological genes may be important genetic indicators for mastitis risk or resistance in Shami does. To establish an effective management plan and forecast the most sensitive risk time for illness onset, gene expression profiles of the tested genes may also be employed as proxy biomarkers. TNF-α and SAA may be precious indicators for the detection of caprine mastitis., Competing Interests: The authors declare that they have no conflict of interest.

Mastitis is a common and serious bacterial infection of the mammary gland. Saikosaponin A (SSA) is a triterpenoid saponin isolated from Bupleurum falcatum that has the ability to treat various diseases. However, little is known about the role of SSA in achieving mastitis remission. Here, we found that SSA alleviated Staphylococcus aureus (S. aureus)-induced mastitis by attenuating inflammation and maintaining blood-milk barrier integrity. Furthermore, S. aureus activated nuclear factor kappa B (NF-κB) pathway by upregulated p-p65 and p-IκB. S. aureus also induced ferroptosis in mammary gland in mice, mainly characterized by excessive iron accumulation, mitochondrial morphological changes and impaired antioxidant production. However, S. aureus-induced NF-κB activation and ferroptosis were prevented by SSA. Moreover, SAA could upregulate the expression of SIRT1, Nrf2, HO-1 and GPX4. And the inhibitory effects of SAA on inflammation and ferroptosis were reversed by SIRT1 inhibitor EX-527. In conclusion, SAA protected S. aureus-induced mastitis through suppressing inflammation and ferroptosis by activating SIRT1/Nrf2 pathway., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.

The increase in bacterial resistance to antibiotics is a global problem for public health. In our previous works, it was shown that the application of AgNPs in cow mastitis treatment increased S. aureus and S . dysgalactiae susceptibility to 31 antibiotics due to a decrease in the bacterial efflux effect. The aim of the present work was to shed light on whether the change in adhesive and anti-lysozyme activities caused by AgNPs also contribute to the restoration of bacterial susceptibility to antibiotics. In vivo sampling was performed before and after cow mastitis treatments with antibiotics or AgNPs. The isolates were identified, and the adhesive and anti-lysozyme activities were assessed. These data were compared with the results obtained for in vitro pre-treatment of reference bacteria with AgNPs or antibiotics. The present study revealed that bacterial treatments in vitro and in vivo with AgNPs: (1) decrease the bacterial ability to adhere to cells to start an infection and (2) decrease bacterial anti-lysozyme activity, thereby enhancing the activity of lysozyme, a natural "antibiotic" present in living organisms. The obtained data contribute to the perspective of the future application of AgNPs for recovering the activity of antibiotics rapidly disappearing from the market.

Although emerging evidence shows that gut microbiota-mediated metabolic changes regulate intestinal pathogen invasions, little is known about whether and how gut microbiota-mediated metabolites affect pathogen infection in the distal organs. In this study, untargeted metabolomics was performed to identify the metabolic changes in a subacute ruminal acidosis (SARA)-associated mastitis model, a mastitis model with increased susceptibility to Staphylococcus aureus (S. aureus). The results showed that cows with SARA had reduced cholic acid (CA) and deoxycholic acid (DCA) levels compared to healthy cows. Treatment of mice with DCA, but not CA, alleviated S. aureus-induced mastitis by improving inflammation and the blood-milk barrier integrity in mice. DCA inhibited the activation of NF-κB and NLRP3 signatures caused by S. aureus in the mouse mammary epithelial cells, which was involved in the activation of TGR5. DCA-mediated TGR5 activation inhibited the NF-κB and NLRP3 pathways and mastitis caused by S. aureus via activating cAMP and PKA. Moreover, gut-dysbiotic mice had impaired TGR5 activation and aggravated S. aureus-induced mastitis, while restoring TGR5 activation by spore-forming bacteria reversed these changes. Furthermore, supplementation of mice with secondary bile acids producer Clostridium scindens also activated TGR5 and alleviated S. aureus-induced mastitis in mice. These results suggest that impaired secondary bile acid production by gut dysbiosis facilitates the development of S. aureus-induced mastitis and highlight a potential strategy for the intervention of distal infection by regulating gut microbial metabolism., (© 2023. The Author(s).)

Cefquinome is a new generation cephalosporin that is effective in the treatment of mastitis in animals. In this study, we evaluated the associations between the specific pharmacokinetics and pharmacodynamics (PK/PD) of cefquinome and its antibacterial activity against Streptococcus agalactiae in a mouse model of mastitis. After a single intramammary dose of cefquinome (30, 60, 120, and 240 μg/mammary gland), the concentration of cefquinome in plasma was analysed by liquid chromatography with tandem mass spectrometry (HPLC/MS-MS). The PK parameters were calculated using a one-compartment first-order absorption model. Antibacterial activity was defined as the maximum change in the S. agalactiae population after each dose. An inhibitory sigmoid Emax model was used to evaluate the relationships between the PK/PD index values and antibacterial effects. The duration for which the concentration of the antibiotic (%T) remained above the minimum inhibitory concentration (MIC) was defined as the optimal PK/PD index for assessing antibacterial activity. The values of %T > MIC to reach 0.5-log10CFU/MG, 1-log10 CFU/MG and 2-log10 CFU/MG reductions were 31, 47, and 81%, respectively. When the PK/PD index %T > MIC of cefquinome was >81% in vivo, the density of the Streptococcus agalactiae was reduced by 2-log10. These findings provide a valuable understanding to optimise the dose regimens of cefquinome in the treatment of S. agalactiae infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Escherichia coli ( E. coli ) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio ( Z. morio ) hemolymph against mastitis pathogens. In this study, we established E. coli -induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli -induced mastitis in vivo and in vitro. In mice with E. coli , Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4 + T/CD8 + T, and increased the production of IL-2 triggered by E. coli . Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli -induced mastitis. In E. coli -stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli -induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli -induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli -induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

Background: Breast abscess is developed on the basis of acute mastitis, which will cause damage to the physical and mental health of lactating women and is an important factor affecting the rate of breastfeeding. This study examined the risk factors for mastitis to develop into breast abscess, and analyzed the distribution of pathogenic bacteria, bacterial resistance, and treatment outcome., Methods: The medical records of 316 cases of mastitis and 219 cases of breast abscess were retrospectively collected. We analyzed the bacterial distribution of mastitis and breast abscess, and compared the differences of bacterial drug resistance. Univariate analysis and binary logistic regression were used to analyze the following aspects: age, primiparity or not, history of breast surgery, body temperature, puerperium or not, onset time, located in the nipple/areolar complexe area or not, history of massage by non-professionals, staphylococcus aureus/methicillin-resistant staphylococcus aureus (MRSA) infection or not, diabetes and white blood cell count., Results: Of the 535 patients, 203 (37.9%) were positive for staphylococcus aureus. There were 133 (65.5%) cases of methicillin-sensitive staphylococcus aureus (MSSA) and 70 (34.5%) cases of MRSA. Concerning bacterial drug resistance, a statistical analysis showed that MSSA had high resistance rate to penicillin (96.2%), ampicillin (91%), clindamycin (42.9%) and erythromycin (45.9%). MRSA had a high resistance rate to penicillin (100%), ampicillin (98.6%), oxacillin (95.7%), erythromycin (81.4%), clindamycin (80%), and amoxicillin (31.7%). Risk factors for progression of mastitis to breast abscess include a body temperature<38.5°C, a postpartum time ≥ 42 days, an onset time ≥ 2 days, lesions in the nipple/areolar complex area, a history of massage by non-medical staff and bacterial cultures for milk or pus that test positive for staphylococcus aureus or MRSA (P < 0.001)., Conclusions: The most common pathogenic bacteria of mastitis and breast abscess is staphylococcus aureus. There are many risk factors for mastitis to develop into breast abscess. We should take effective measures for its risk factors and select sensitive antibiotics according to the results of bacterial culture to reduce the formation of breast abscess., Competing Interests: The authors have declared that no competing interests exist.

The lactation capacity of dairy cows is critical to the productivity of the animals. Mastitis is a disease that directly affects the lactation capacity of cows. Staphylococcus aureus (S. aureus) is one of the most important pathogens that causes mastitis in dairy cows. The anti-inflammatory effect of Salvia miltiorrhiza polysaccharides (SMPs) has been demonstrated in mice and chickens. However, the effectiveness of SMPs in preventing and treating mastitis is unclear. Therefore, the purpose of this study was to explore the protective effect and mechanism of SMPs on mastitis caused by S. aureus. S. aureus was used to induce mastitis in rats, and three doses of SMPs (87.5, 175, 350 mg/kg, BW/d) were administered as treatments. The bacterial load, histopathology, and myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGase) activities of mammary glands were observed and measured. Cytokines, including interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor α (TNF-α), were examined by qRT-PCR and ELISA. Key proteins in the NF-κB and MAPK signaling pathways were analyzed by Western blotting. The results showed that SMP supplementation could significantly reduce the colonization of S. aureus and the recruitment of inflammatory cells in mammary glands. S. aureus-induced gene transcription and protein expression of IL-1β, IL-6, and TNF-α were significantly suppressed in mammary glands. In addition, the increase in NF-κB and MAPK protein phosphorylation was inhibited by SMPs. These results revealed that supplementation with SMPs protected the mammary gland of rats against damage caused by S. aureus and alleviated the inflammatory response. This study provides a certain experimental basis for the treatment of S. aureus-induced mastitis with SMPs in the future., (© 2022. The Author(s).)

The symptoms of mastitis caused by Staphylococcus aureus ( S. aureus ) in dairy cows are not obvious and difficult to identify, resulting in major economic losses. N 6 -Methyladenosine (m 6 A) modification has been reported to be closely associated with the occurrence of many diseases. However, only a few reports have described the role of m 6 A modification in S. aureus -induced mastitis. In this study, after 24 h of treatment with inactivated S. aureus , MAC-T cells (an immortalized bovine mammary epithelial cell line) showed increased expression levels of the inflammatory factors IL-1 β , IL-6, TNF- α , and reactive oxygen species. We found that the mRNA levels of METLL3, METLL14, WTAP, and ALKBH5 were also upregulated. Methylated RNA immunoprecipitation sequencing analysis revealed that 133 genes were m 6 A hypermethylated, and 711 genes were m 6 A hypomethylated. Biological functional analysis revealed that the differential m 6 A methylated genes were mainly related to oxidative stress, lipid metabolism, inflammatory response, and so on. In the present study, we also identified 62 genes with significant changes in m 6 A modification and mRNA expression levels. These findings elucidated the m 6 A modification spectrum induced by S. aureus in MAC-T cells and provide the basis for subsequent m 6 A research on mastitis., Competing Interests: There is no conflict of interest., (Copyright © 2022 Ting Li et al.)

Lactational mastitis is an excellent target to study possible interactions between HMOs, immune factors and milk microbiota due to the infectious and inflammatory nature of this condition. In this work, microbiological, immunological and HMO profiles of milk samples from women with (MW) or without (HW) mastitis were compared. Secretor status in women (based on HMO profile) was not associated to mastitis. DFLNH, LNFP II and LSTb concentrations in milk were higher in samples from HW than from MW among Secretor women. Milk from HW was characterized by a low bacterial load (dominated by Staphylococcus epidermidis and streptococci), high prevalence of IL10 and IL13, and low sialylated HMO concentration. In contrast, high levels of staphylococci, streptococci, IFNγ and IL12 characterized milk from MW. A comparison between subacute (SAM) and acute (AM) mastitis cases revealed differences related to the etiological agent (S. epidermidis in SAM; Staphylococcus aureus in AM), milk immunological profile (high content of IL10 and IL13 in SAM and IL2 in AM) and milk HMOs profile (high content of 3FL in SAM and of LNT, LNnT, and LSTc in AM). These results suggest that microbiological, immunological and HMOs profiles of milk are related to mammary health of women., (© 2022. The Author(s).)

Staphylococcus aureus is a vital bovine mastitis pathogen causing huge economic losses to the dairy industry worldwide. In our previous studies, leukotoxin ED (LukED) was detected in most S. aureus strains isolated from bovine mastitis. Here, four single-chain fragment variables (scFvs) (ZL8 and ZL42 targeting LukE, ZL22 and ZL23 targeting LukD) were obtained using purified LukE and LukD proteins as the antigens after five rounds of bio-panning. The complementarity-determining region 3 (CDR3) of the VH domain of these scFvs exhibited significant diversities. In vitro , the scFvs significantly decreased LukED-induced cell killing by inhibiting the binding of LukED to chemokine receptors (CCR5 and CXCR2) and reduced the death rates of bovine neutrophils and MAC-T cells caused by LukED and S. aureus ( p < 0.05). In an S. aureus -induced mouse mastitis model, histopathology and MPO results revealed that scFvs ameliorated the histopathological damages and reduced the infiltration of inflammatory cells ( p < 0.05). The ELISA and qPCR assays showed that scFvs reduced the transcription and expression levels of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-18 ( p < 0.05). The overall results demonstrated the protective anti-inflammatory effect of scFvs in vitro and in vivo, enlightening the potential role of scFvs in the prevention and treatment of S. aureus -induced mastitis.

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus . RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus . A major differential response was the activation of innate immune response by E. coli , but not by S. aureus . Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus . Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli . Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus -stimulated inflammatory response. We also show that, unlike E. coli , S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.

This study is designed to discuss the antimicrobial resistance, virulence determinants and biofilm formation capacity of Enterococcus spp. isolated from milk of sheep and goat with subclinical mastitis in Qena, Egypt. The obtained isolates were identified by the VITEK2 system and 16S rDNA sequencing as E. faecalis, E. faecium, E. casseliflavus and E. hirae. Overall, E. faecalis and E. faecium were the dominant species recovered from mastitic milk samples. The antimicrobial susceptibility test evidenced multidrug resistance of the isolates against the following antimicrobials: oxacillin (89.2.%), followed by vancomycin (75.7%) and linezolid (70.3%). Also, most of these isolates (73%) could form biofilms. For example, 18.9% of Enterococcus strains formed strong biofilm, whereas 32.4% of isolates formed moderate biofilm and 21.6% of isolates formed weak biofilm. The most prevalent resistance genes found in our isolates were blaZ (54%), vanA (40%), ermB (51.4%), tetM (13.5%) and optrA (10.8%). Moreover, asa1 (37.8%), cylA (42.3%), gelE (78.4%), esp (32.4%), EF3314(48.6%) and ace (75.5%) were the most common virulence genes. A significant correlation was found between biofilm formation, multidrug resistance and virulence genes of the isolates. This study highlights several aspects of virulence and harmfulness of Enterococcus strains isolated from subclinical mastitic milk, which necessitates continuous inspection and monitoring of dairy animals., Competing Interests: The authors have declared that no competing interests exist.

Cow mastitis, which significantly lowers milk quality, is mainly caused by pathogenic bacteria such as E. coli . Previous studies have suggested that lactic acid bacteria can have antagonistic effects on pathogenic bacteria that cause mastitis. In the current study, we evaluated the in vitro and in vivo alleviative effects of L. plantarum KLDS 1.0344 in mastitis treatment. In vitro antibacterial experiments were performed using bovine mammary epithelial cell (bMEC), followed by in vivo studies involving mastitis mouse models. In vitro results indicate that lactic acid was the primary substance inhibiting the E. coli pathogen. Meanwhile, treatment with L. plantarum KLDS 1.0344 can reduce cytokines' mRNA expression levels in the inflammatory response of bMEC induced by LPS. In vivo , the use of this strain reduced the secretion of inflammatory factors IL-6, IL-1β, and TNF-α, and decreased the activity of myeloperoxidase (MPO), and inhibited the secretion of p-p65 and p-IκBα. These results indicate that L. plantarum KLDS 1.0344 pretreatment can reduce the expression of inflammatory factors by inhibiting the activation of NF-κB signaling pathway, thus exerting prevent the occurrence of inflammation in vivo . Our findings show that L. plantarum KLDS 1.0344 has excellent properties as an alternative to antibiotics and can be developed into lactic acid bacteria preparation to prevent mastitis disease., Competing Interests: Author QX is employed by Heilongjiang Feihe Dairy Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Wang, Guo, Xie, Evivie, Song, Li and Huo.)

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake ( znuABC ) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The Δ znuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated Δ kpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the Δ kpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

Staphylococcus aureus is one of the main bacterial agents responsible for cases of mastitis in ruminants, playing an important role in the persistence and chronicity of diseases treated with antimicrobials. Using the multilocus sequence typing technique, network approaches and study of the population diversity of microorganisms, we performed analyzes of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM). The most strains of ES-GPM were categorically different phylogenetically from the others and could be divided into two lineages: one with a majority belonging to ES-GPM and the other to varied strains. These two lineages were separated by 27 nuclear polymorphisms. The 43 strains comprised 22 clonal complexes (CCs), of which the ES-GPM strains were present in CC133, CC5 and a new complex formed by the sequence type 4966. The genetic diversity of some alleles showed be greater diversity and polymorphism than others, such as of the aroE and yqiL genes less than glpF gene. In addition, the sequences ES-GPM to the arc gene and glpF alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM. Therefore, this study identified genetic polymorphisms characteristic of S. aureus isolated from milk of goats diagnosed with persistent mastitis after the failed treatment with the antibiotic enrofloxacin. This study may help in the future to identify and discriminate this agent in cases of mastitis, and with that, the most appropriate antibiotic treatment can be performed in advance of the appearance of persistent mastitis caused by the agent, reducing the chances of premature culling and animal suffering., (© 2021. The Author(s).)

Subclinical mastitis is one of the major problems affecting dairy animals' productivity and is classified based on milk somatic cell counts (SCC). Previous data showed that marine-derived Bacillus amyloliquefaciens - 9 (GB-9) improved the immunity and the nonspecific immune defense system of the body. In this study, the potential role of GB-9 in improving subclinical mastitis was assessed with Radix Tetrastigmae (RT) as a positive control in subclinical mastitis Saanen dairy goats. The current data showed that GB-9 and RT significantly reduced the SCC in dairy goats. After being fed with GB-9 or RT, the decreased concentrations of malondialdehyde, IgA, IgM, IL-2, IL-4, and IL-6 were observed. The amplicon sequencing analysis of fecal samples revealed that GB-9 significantly altered the bacterial community. Bacteroides and Phascolarctobacterium were the major genera that respond to GB-9 feeding. The correlation analysis using weighted gene co-expression network analysis showed a MePink module was most associated with the serum concentrations of immunoglobulin and interleukin. The MePink module contained 89 OTUs. The feeding of GB-9 in decreasing the SCC was associated with the altered abundance of Bacteroides , which was correlated with the concentrations of immunoglobulins and chemokines. Collectively, the current data suggested that marine-derived GB-9 could be a helpful probiotic to control subclinical mastitis.

Objective: A crosssectional study was conducted between September 2015 and August 2016 in the district of Afar Regional State, Northeastern Ethiopia, to characterize the most prevalent bacterial pathogens and identify the associated risk factors of camel subclinical mastitis. California mastitis test (CMT) was used as a screening test, and standard bacteriological methods were carried out for isolation and identification of the pathogens., Results: Among the total 96 lactating camels examined, 25 were found positive with the overall prevalence of 26%, with 25% and 1% subclinical and clinical mastitis cases, respectively. Totally, 384 quarters of udder were examined; of these, 10 of them were blind while the rest 374 were nonblind teats. The quarter level prevalence of subclinical mastitis was 8.9%. The analysis showed that statistically significant difference ( P < 0.05) of tick infestation and subclinical mastitis. Additionally, among the bacteriologically tested 34 CMT positive milk samples, all of them showed growth on nutrient and blood agar plate. Out of these culture isolates, the major bacterial pathogens identified were Staphylococcus aureus (8.7%), Staphylococcus hyicus (6.52%) , Staphylococcus intermedius (6.52), Coagulase-negative staphylococci (19.57%), Bacillus (19.57%), Escherichia coli (6.52%), and Pasteurella multocida (6.52%) species. Therefore, appropriate control measures and awareness creation to the community should be practiced., Competing Interests: All authors declare that there is no competing interest., (Copyright © 2021 Atnaf Alebie et al.)

Mastitis is the economically most important disease of dairy cows. This study used PacBio single-molecule real-time sequencing technology to sequence the full-length 16S rRNAs from 27 milk samples (18 from mastitis and nine from healthy cows; the cows were at different stages of lactation). We observed that healthy or late stage milk microbiota had significantly higher microbial diversity and richness. The community composition of the microbiota of different groups also varied greatly. The healthy cow milk microbiota was predominantly comprised of Lactococcus lactis , Acinetobacter johnsonii , and Bacteroides dorei , while the milk from mastitis cows was predominantly comprised of Bacillus cereus . The prevalence of L. lactis and B. cereus in the milk samples was confirmed by digital droplets PCR. Differences in the milk microbiota diversity and composition could suggest an important role for some these microbes in protecting the host from mastitis while others associated with mastitis. The results of our research serve as useful references for designing strategies to prevent and treat mastitis.

Streptococcus uberis ( S. uberis ) is an important pathogen causing mastitis, which causes continuous inflammation and dysfunction of mammary glands and leads to enormous economic losses. Most research on infection continues to be microbial metabolism-centric, and many overlook the fact that pathogens require energy from host. Mouse is a common animal model for studying bovine mastitis. In this perspective, we uncover metabolic reprogramming during host immune responses is associated with infection-driven inflammation, particularly when caused by intracellular bacteria. Taurine, a metabolic regulator, has been shown to effectively ameliorate metabolic diseases. We evaluated the role of taurine in the metabolic regulation of S. uberis -induced mastitis. Metabolic profiling indicates that S. uberis exposure triggers inflammation and metabolic dysfunction of mammary glands and mammary epithelial cells (the main functional cells in mammary glands). Challenge with S. uberis upregulates glycolysis and oxidative phosphorylation in MECs. Pretreatment with taurine restores metabolic homeostasis, reverses metabolic dysfunction by decrease of lipid, amino acid and especially energy disturbance in the infectious context, and alleviates excessive inflammatory responses. These outcomes depend on taurine-mediated activation of the AMPK-mTOR pathway, which inhibits the over activation of inflammatory responses and alleviates cellular damage. Thus, metabolic homeostasis is essential for reducing inflammation. Metabolic modulation can be used as a prophylactic strategy against mastitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lan, Wan, Xu, Wang, Fu, Zhou, Lin, Han, Luo, Miao and Yin.)

Staphylococcus aureus is the most common mastitis-related pathogen in dairy cattle, goats, and sheep worldwide. However, the population structure and genomic characteristics of mastitis-associated S. aureus in small ruminants are limited. Furthermore, the genotypic and phenotypic characteristics involved in the pathogenicity of S . aureus have been thoroughly defined, yet their association with the severity of mastitis is not fully established. Here, we performed genotyping by pulsed-field gel electrophoresis (PFGE) and spa analyses to assess the genetic diversity and relatedness of 162 S. aureus strains recovered from clinical mastitis (CM) and subclinical mastitis (SCM) cases from goats, sheep, and bovines. PFGE analysis revealed 108 distinguishable pulsotypes and 3 main clusters that comprised isolates from the three host species, while according to spa typing, 32 different spa types were identified. Genotypic analysis revealed a spreading of genetically related or indistinguishable S. aureus strains among ovine, caprine, and bovine farms of distant geographical regions. In total, 28 different staphylococcal enterotoxin (SE) gene profiles were observed, revealing a diverse range of SE genes among isolates. By evaluating the antimicrobial resistance, we found low phenotypic antimicrobial resistance among all ruminant isolates. We also performed multiple correspondence analysis, which indicated that the presence of the sec gene, biofilm production, and high autoaggregation ability are associated with CM cases.

Background: Staphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB&#95;SauM&#95;SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms., Results: SDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 10 9  CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ., Conclusions: Our results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.

Background: The incidence of mastitis has increased, and this disease can lead to long antibiotic courses and complications. Here, we aimed to identify the factors associated with antibiotic duration and recurrence of complicated mastitis., Materials and Methods: This retrospective cohort study was conducted in a tertiary hospital in Taiwan. All hospitalized patients diagnosed with mastitis (ICD-9 code 611.0) from Jan. 1, 2012, to Dec. 31, 2016, were enrolled. Patient characteristics and clinical data were obtained from the medical charts. Recurrence was defined as mastitis within the first year after the discontinuation of antibiotics for at least 7 days., Results: In total, 214 females with a median age of 37 years old (IQR 33-45) were enrolled. A total of 148 patients (69.2%) underwent debridement, and 122 (57.0%) underwent biopsy. Histopathological examinations revealed granulation tissue in 44.6% (62/139) of the patients. Positive cultures were obtained in 65.9% (141/214) of the patients. Coagulase-negative staphylococci (64/141, 45.4%) was the most common pathogen, followed by Corynebacterium species (42/141, 29.8%). The median hospitalization length and antibiotic course were 7 (IQR 4-13) and 37 days (IQR 22-77), respectively. Three patients died of breast cancer during treatment. The recurrence rate was 18.5% (39/211). Younger age, corynebacterial infection, and pregnancy were associated with longer treatment durations (P < 0.001, 0.003, <0.001). Corynebacterial infection was associated with a 2.16-fold (95% CI: 1.11-4.20) increase in recurrence after adjusting for age., Conclusion: Corynebacterial infection is associated with longer treatment courses and an increased recurrence rate of complicated mastitis. Therefore, specific treatments should be considered., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

Sub-acute mastitis (SAM) is a prevalent disease among lactating women, being one of the main reasons for early weaning. Although the etiology and diagnosis of acute mastitis (AM) is well established, little is known about the underlying mechanisms causing SAM. We collected human milk samples from healthy and SAM-suffering mothers, during the course of mastitis and after symptoms disappeared. Total (DNA-based) and active (RNA-based) microbiota were analysed by 16S rRNA gene sequencing and qPCR. Furthermore, mammary epithelial cell lines were exposed to milk pellets, and levels of the pro-inflammatory interleukin IL8 were measured. Bacterial load was significantly higher in the mastitis samples and decreased after clinical symptoms disappeared. Bacterial diversity was lower in SAM milk samples, and differences in bacterial composition and activity were also found. Contrary to AM, the same bacterial species were found in samples from healthy and SAM mothers, although at different proportions, indicating a dysbiotic ecological shift. Finally, mammary epithelial cell exposure to SAM milk pellets showed an over-production of IL8. Our work therefore supports that SAM has a bacterial origin, with increased bacterial loads, reduced diversity and altered composition, which partly recovered after treatment, suggesting a polymicrobial and variable etiology.

Staphylococcus aureus , an important cause of mastitis in mammals, is becoming increasingly problematic due to the development of resistance to conventional antibiotics. The ability of S. aureus to invade host cells is key to its propensity to evade immune defense and antibiotics. This study focuses on the functions of cathelicidins, small cationic peptides secreted by epithelial cells and leukocytes, in the pathogenesis of S. aureus mastitis in mice. We determined that endogenous murine cathelicidin (CRAMP; Camp ) was important in controlling S. aureus infection, as cathelicidin knockout mice ( Camp -/- ) intramammarily challenged with S. aureus had higher bacterial burdens and more severe mastitis than did wild-type mice. The exogenous administration of both a synthetic human cathelicidin (LL-37) and a synthetic murine cathelicidin (CRAMP) (8 μM) reduced the invasion of S. aureus into the murine mammary epithelium. Additionally, this exogenous LL-37 was internalized into cultured mammary epithelial cells and impaired S. aureus growth in vitro We conclude that cathelicidins may be potential therapeutic agents against mastitis; both endogenous and exogenous cathelicidins conferred protection against S. aureus infection by reducing bacterial internalization and potentially by directly killing this pathogen., (Copyright © 2020 American Society for Microbiology.)

Mastitis is usually caused by a variety of pathogenic bacteria that include both Gram-positive and Gram-negative bacteria. Lipopolysaccharide (LPS) is the pathogen-associated molecular pattern (PAMP) of Gram-negative bacteria, and peptidoglycan (PGN) and lipoteichoic acid (LTA) are those of Gram-positive bacteria. The effects of LPS, PGN and/or LTA on inflammatory response and lactation in bovine mammary epithelial cells (BMECs) are well studied, but the epigenetic mechanisms of their effects received less attention. Furthermore, since the three PAMPs are often simultaneously present in the udder of cows with mastitis, it has implications in practice to study their additive effects. The results show that co-stimulation of bovine mammary epithelial cells with PGN, LTA, and LPS induced a higher number of differentially expressed genes (DEGs) and greater expressions of inflammatory factors including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6. In addition, co-stimulation further increased DNA hypomethylation compared with sole LPS stimulation. Co-stimulation greatly decreased casein expression but did not further decrease histone acetylation levels and affect the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC), compared with sole LPS stimulation. Collectively, this study demonstrated that PGN, LTA, and LPS had an additive effect on inducing transcriptome changes and inflammatory responses in BMECs, probably through inducing a greater decrease in DNA methylation. Co-stimulation with PGN, LTA, and LPS decreased casein expression to a greater degree, but it might not be linked to histone acetylation and HAT and HDAC activity.

Background: Mastitis is a disease of economic concern that affects dairy industry worldwide. This study aimed to investigate and identify possible etiologies encountered in an episode of acute gangrenous mastitis in lactating she-camels in Al Dhafra region, Abu Dhabi Emirate, United Arab Emirates (UAE). Beside the routine clinical examination, conventional bacteriological methods were used to isolate and identify possible aerobic/anaerobic bacterial or fungal pathogens from cultured milk samples collected from the mastitic she-camels. Moreover, quantitative real-time polymerase chain reaction (qPCR) was used for the detection of Mycoplasma agalactiae and Mycoplasma bovis strains, and the 16S rRNA gene was sequenced to confirm the isolation. The isolates were also tested for their susceptibility to antimicrobials., Results: Acute gangrenous mastitis is reported in the dromedary camel herd with about 80% morbidity rate among lactating she-camels exhibited acute, painful hard swelling of affected teat, quarter or entire udder. About 41.7% of the infected animals were stamped out for culling due to complete or partial amputation of udder quarters. Streptococcus agalactiae was the sole isolated organism (6 isolates). The antimicrobial susceptibility testing revealed that, the Streptococcus agalactiae isolates were sensitive to both penicillin and ampicillin. Comparison of the 16S rRNA gene sequencing results by BLASTN confirmed the presence of Streptococcus agalactiae with high confidence (100% identity). Phylogenetic analysis indicated clustering of one isolate (CMAUAE accession number; MN267805.1) with Streptococcus agalactiae that infects multi-hosts including humans, while strains (CMBUAE to CMFUAE with accession numbers; MN267806.1 to MN267810.1 respectively) clustered with Streptococcus agalactiae that infects humans. No Mycoplasma spp was detected by qPCR analysis., Conclusions: In the present study, the Streptococcus agalactiae was found to be the main cause of acute gangrenous mastitis in dromedary camels in UAE. More research should be done to investigate other possible causes of clinical or subclinical mastitis in dromedary camels in UAE.

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: Staphylococcus aureus is one of the main causative agents of mastitis in small ruminants. Antimicrobial use is the major treatment, but there are many flaws linked to resistance, tolerance or persistence. This study aimed to verify changes in resistance, virulence and clonal profiles of S. aureus isolated from persistent mastitis goat milk before and after enrofloxacin treatment., Results: MIC increased to at least one antimicrobial in S. aureus isolates after enrofloxacin treatment compared to before. The most detected resistance genes before and after treatment were tetK, tetM, and blaZ, with more resistance genes detected after enrofloxacin treatment (p < 0.05). Occasional variations in efflux system gene detection were observed before and after treatment. Nine virulence genes (hla, fnbA, fnbB, eta, etb, sea, sec, seh, and sej) were detected at both times, and between these, the hla and eta genes were detected more in isolates after treatment. All isolates of S. aureus belonged to the same sequence type (ST) 133, except for two S. aureus isolates prior to enrofloxacin treatment which were classified as ST5 and the other as a new one, ST4966. Isolates of S. aureus 4, 8, and 100 from before and after treatment had identical pulse types, while others obtained from other animals before and after treatment were classified into distinct pulse types., Conclusion: There were occasional changes in the studied profiles of S. aureus isolated before and after treatment of animals with enrofloxacin, which may have contributed to the permanence of bacteria in the mammary gland, even when using traditional treatment, resulting in persistent mastitis.

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 μL 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-α-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., Competing Interests: The authors have declared that no competing interests exist.

Mastitis is an important constraint to milk production in pastoralist camel (Camelus dromedarius) herds in Kenya. The objective of this study was to investigate the prevalence, risk factors, and bacterial panorama of subclinical mastitis (SCM) in pastoralist camel herds in Isiolo County, Kenya. Furthermore, antimicrobial susceptibility in udder pathogens was studied. A cross-sectional sample of 206 camels from 20 milking herds was screened using the California Mastitis Test (CMT), and quarter milk was subjected to bacterial culturing. Isolates were confirmed using MALDI-TOF mass spectrometry analysis, and antimicrobial susceptibility was determined using the broth microdilution method. Interviews focusing on herd management were conducted with camel owners. Subclinical mastitis, defined as a CMT score ≥ 3 (scale 1 to 5) and absence of clinical symptoms in the udder, were present in all visited herds. On the individual level, 46% of the camels had at least 1 quarter affected with SCM, and on the quarter level the prevalence was 26%. Intramammary infections (IMI) were common; out of 798 quarter milk samples, 33% yielded conclusive bacterial growth. The sensitivity and specificity of CMT for correctly identifying quarters with IMI were 82% and 92%, respectively. The most prevalent pathogen was Streptococcus agalactiae (72% of IMI-positive quarters), followed by non-aureus staphylococci (19%) and Staphylococcus aureus (13%). Antimicrobial susceptibility testing revealed that only a low proportion (4.9%) of Strep. agalactiae isolates was sensitive to tetracycline. For Staph. aureus, 59.1% of isolates exhibited sensitivity to penicillin. Skin lesions on the teats or udder were a risk factor for SCM. Increased age, parity, and stage of lactation were associated with increased risk of both SCM and IMI. Older camels with a blind teat or a previous history of mastitis were more likely to be infected with Strep. agalactiae. Hygiene routines for milking were largely absent in the observed herds, and knowledge of adequate milk handling was limited. The poor udder health is likely to depend on multiple factors, most prominently the within-herd maintenance of contagious udder pathogens, in combination with difficult sanitary conditions and lack of awareness among camel keepers. This study showed that in pastoralist camel herds around Isiolo town, SCM and IMI specifically caused by Strep. agalactiae are common udder health problems and are associated with increasing age, parity, and stage of lactation, and skin lesions on the teats and udder. Resistance to tetracycline in Strep. agalactiae was common. Control strategies specifically targeting SCM and adapted to pastorally managed camel herds need to be developed to reduce disease, combat antimicrobial resistance, and improve the livelihoods of pastoralists., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ) are the most common pathogens of mastitis, and S. aureus generally causes subclinical mastitis which is more persistent and resistant to treatment. Peptidoglycan (PGN) and lipoteichoic acid (LTA) are cell wall components of S. aureus . Although the roles of PGN and LTA in causing inflammation are well studied, the epigenetic mechanisms of the effects of PGN and LTA on the inflammation and lactation remain poorly understood. This study characterized the gene expression profiling by RNA sequencing and investigated DNA methylation and histone acetylation in relation to inflammation and lactation in the immortalized bovine mammary epithelial cell line (MAC-T). The cells were cultured for 24 h with neither PGN nor LTA (CON), PGN (30 μg/mL), LTA (30 μg/mL), and PGN (30 μg/mL) + LTA (30 μg/mL), respectively. The number of differentially expressed genes (DEGs) and the expression of proinflammatory factors including interleukin (IL)-1β, IL-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6 of the treatments increased in the following order: CON < PGN < LTA < PGN + LTA, and the DEGs mainly enriched on the cytokine-cytokine receptor interaction and chemokine signaling pathway. LTA and PGN + LTA induced hypomethylation of global DNA by suppressing DNA methyltransferase (DNMT) activity. PGN and LTA, alone or combined, decreased the mRNA expression of casein genes ( CSN1S1, CSN2, and CSN3 ) and the expression of two caseins (CSN2 and CSN3), and reduced histone H3 acetylation by suppressing histone acetyltransferase (HAT) activity and promoting histone deacetylase (HDAC) activity. Collectively, this study revealed that PGN and LTA induced inflammation probably due to decreasing DNA methylation through regulating DNMT activity, and decreased lactation possibly through reducing histone H3 acetylation by regulating HAT and HDAC activity in bovine mammary epithelial cells.

In dairy cattle, mastitis is a disease of the mammary gland caused by pathogens such as bacteria, viruses, fungi, and algae. Mastitis causes economic losses to dairy farms as well as public health concerns. The reproductive efficiency of commercial dairy herds has important implications for the economic success of dairy operations and is strongly associated with the health status of cows. Mastitis has previously been linked with decreased fertility of dairy cows, but the effect of specific pathogens on the severity of fertility reduction is still unclear. In this study, cows diagnosed with mastitis caused by major pathogens (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Klebsiella spp., Mycoplasma spp., and environmental Streptococcus) needed more artificial inseminations (AI) than did cows with mastitis caused by minor pathogens (coagulase-negative Staphylococcus and Corynebacterium spp.) and healthy cows. Cows diagnosed with mastitis, independent of what pathogen was causing mastitis, had more days open compared with nonmastitic cows. The percentage of cows that successfully established pregnancy at first AI was greater for the control group than for the major pathogens group but not significantly different from the minor pathogens group. Pregnancy loss was lower in the control group than in the major pathogens group; however, there was no difference compared with the minor pathogen group. Mastitis caused by gram-negative bacteria decreased the percentage of pregnancy per first AI and increased days open and pregnancy loss compared with the control group. Cows with mastitis caused by gram-positive bacteria also had increased days open compared with control cows. This study shows that different mastitis-causing bacteria can affect the fertility of cows differently. Mastitis events caused by major pathogens and gram-negative bacteria were associated with the greatest decrease in reproductive efficiency., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the effect of intramammary infusions of natural composition GLP 810 with immunomodulating properties on the local nonspecific cellular and humoral immune response in cows with subclinical mastitis. The composition GLP 810 consists of lactic acid, lysozyme, glycopeptides, and 0.9% solution of NaCl. The following parameters were studied: (1) leukocyte differential distribution in milk, (2) expression of cytokines in milk leukocytes, (3) antibacterial activity, and (4) milk quality. Nineteen mammary glands in five lactating cows were infused with 10 mL of GLP 810, and nineteen other glands from five control cows were treated with 10 mL 0.9% NaCl. The results showed that after intramammary administration of the composition GLP 810 three times with 48 h intervals, the following effects on leukocyte populations in milk were observed: (1) an increase in the number of polymorphonuclear leukocytes and lymphocytes and (2) a decrease in the number of macrophages. A reduction in the number of pathogenic bacteria was also detected. The analyses of tumour necrosis factor-alpha, interleukin-10, and beta-defensin-2 revealed that the production of the aforementioned cytokines significantly increased, whereas no significant effects on interleukin-1 and caspase-6 expression in milk leukocytes were recorded. However, there were significant differences between mammary glands with high and low milk somatic cell count. The results suggest that the composition GLP 810 has an immunomodulatory effect on mammary glands and it could be used for improving the immune response in cows with subclinical mastitis during lactation., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Gundega Gulbe et al.)

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Staphylococcus xylosus (S. xylosus) is one of the emerging pathogens causing bovine mastitis with high rate of isolation in most of the reported clinical and field cases. To verify the role of glutamine synthetase (GS) in the pathogenesis of S. xylosus, we evaluated the virulence level of the wild-type strain and its glnA mutant strain in biofilm assays in vitro and murine infection model in vivo. From the results, it was observed that the glnA mutant strain was attenuated and could reduce tissue damage. 1-Hydroxyanthraquinone (1-HAQ) is a kind of anthraquinones, it exhibited a significant inhibitory effect on the growth of S. xylosus and biofilm formation in vitro and provided anti-inflammatory effects in vivo. In addition, the rate at which it inhibits the biofilm, inflammatory factors, and CFU of wild-type strains were significantly higher than that of the mutant strains, indicating that 1-hAQ might have pharmacological effects against S. xylosus through the regulation of GS protein. The effect of 1-hAQ on GS was further confirmed by the down-regulation of glnA expression, reduced GS activity, Gln content and the results of molecular docking. Taken together, these findings suggest that 1-hAQ facilitated a significant attenuation of S. xylosus pathogenicity by regulating the GS protein: a vital virulence factor. Therefore, it can be inferred that 1-hAQ may serve as a potential source of organic compound for the development of novel alternative drugs in mitigating the menace of bovine mastitis., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)