Pathogenesis Of Mastitis Research Articles

Succinate exacerbates mastitis in mice via extracellular vesicles derived from the gut microbiota: a potential new mechanism for mastitis

BackgroundA 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.ResultsSuccinate 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.ConclusionsOur 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.Graphical

Comparative analysis of the frequency of the occurrence of mastitis-causing pathogens and assessing their importance in the pathogenesis of mastitis

Comparative analysis of the frequency of the occurrence of mastitis-causing pathogens and assessing their importance in the pathogenesis of mastitis

ANALYSIS OF METABOLIC PATHWAY GENES OF STAPHYLOCOCCUS AUREUS ISOLATED FROM THE MILK OF THE COWS WITH MASTITIS

It is known that the bacteria of the genus Staphylococcus are among the most common bacterial pathogens in the pathogenesis of mastitis. Detailed and comprehensive knowledge of the metabolism of Staphylococcus is necessary to understand its pathogenesis. The aim of the study was to identify the metabolic pathways of S. aureus bacteria isolated from the milk of the cows with mastitis. A total of 88 metabolic pathways were identified for the studied S. aureus. Biosynthesis pathways were strongly dominated in bacteria, the most common of which were biosynthesis of 5-aminoimidazole ribonucleotides, biosynthesis of L-lysine, L-threonine and L-methionine II, biosynthesis of coenzyme A, biosynthesis of L-isoleucine and biosynthesis of uridine monophosphate. A pathway for the synthesis of Nacetylglucosamine has been found in S. aureus bacteria. This pathway may be important for bacterial virulence. The pathway of peptidoglycan III biosynthesis has been discovered. This pathway was previously described for Mycobacteriaceae. In addition, in the cells of S. aureus there have discovered mixed acid fermentation genes, which are more typical for representatives of Enterobacteriaceae. According to the sequencing results, it has been revealed that various folate metabolism pathways are widely represented in S. aureus cells, emphasizing its obvious great importance for this type of bacteria.

Hexadecanamide alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting inflammatory responses and restoring blood-milk barrier integrity.

Subacute ruminal acidosis (SARA) has been demonstrated to promote the development of mastitis, one of the most serious diseases in dairy farming worldwide, but the underlying mechanism is unclear. Using untargeted metabolomics, we found hexadecanamide (HEX) was significantly reduced in rumen fluid and milk from cows with SARA-associated mastitis. Herein, we aimed to assess the protective role of HEX in Staphylococcus aureus (S. aureus)- and SARA-induced mastitis and the underlying mechanism. We showed that HEX ameliorated S. aureus-induced mastitis in mice, which was related to the suppression of mammary inflammatory responses and repair of the blood-milk barrier. In vitro, HEX depressed S. aureus-induced activation of the NF-κB pathway and improved barrier integrity in mouse mammary epithelial cells (MMECs). In detail, HEX activated PPARα, which upregulated SIRT1 and subsequently inhibited NF-κB activation and inflammatory responses. In addition, ruminal microbiota transplantation from SARA cows (S-RMT) caused mastitis and aggravated S. aureus-induced mastitis, while these changes were reversed by HEX. Our findings indicate that HEX effectively attenuates S. aureus- and SARA-induced mastitis by limiting inflammation and repairing barrier integrity, ultimately highlighting the important role of host or microbiota metabolism in the pathogenesis of mastitis and providing a potential strategy for mastitis prevention.

PSVI-7 Snorna Dysregulated Expression and Potential Roles in Bovine Sub-Clinical Mastitis Due to Staphylococcus Chromogenes

Abstract Staphylococcus chromogenes (SC), a frequent non-aureus staphylococcus on Canadian herds causes both clinical and subclinical forms of mastitis and contributes to large economic losses on Canadian farms. However, little research attention has been directed to uncovering the molecular mechanisms underlying SC mastitis pathogenesis, including the roles of small nucleolar RNAs (snoRNAs). SnoRNAs function to regulate host ribosomal RNAs (rRNAs) biogenesis and processing, and are known to have regulatory functions in several human diseases, but no study has examined their regulatory functions in bovine mastitis. To understand the regulatory roles of snoRNAs in cows with SC subclinical mastitis, the snoRNA transcriptome of milk somatic cells from three SC infected cows [positive for SC only, have high somatic cell counts (SCC ≥350,000 cells/mL) for ≥ 3 months consecutively, only one positive udder sampled], and four healthy cows [low SCC (< 100,000 cells/mL) for ≥3 months consecutively, compositive sample of all quarters] was characterized by sequencing and bioinformatics analysis. Among 232 known snoRNAs found in the samples, 33 were differentially expressed (DE; 14 downregulated and 19 upregulated; FDR < 0.05). Out of these, SnoRA3/snoRA45 (upregulated) belongs to H/ACA snoRNA box type while SNORD14 (downregulated) belongs to C/D box snoRNA box type. According to RNAsnoop analysis, SnoRA3/snoRA45 interacted with 18S-1667 rRNA [minimum free energy (MFE) = -36Kcal/Mol] while SNORD14 interacted with 28S-3926 rRNA (MFE = -15.40Kcal/Mol). The observed strong MFE suggests that SnoRA3/snoRA45 and SNORD14 might potentially participate in rRNA modifications which could affect important functional sites of the ribosome with implications on the pathogenesis of bovine SC mastitis. SnoRA3/snoRA45 have been reported to guide site specific pseudouridylation of rRNA by upregulating the ribosomal pathway as well as have important roles in cell cycle progression in human. SNORD14 is expressed from the intron of its host gene RPS13, which is a ribosomal protein involved in protein biosynthesis. Also, SNORD14 has been shown to be potentially involved in intracellular protein transport and positive regulation of MAPK cascade. Our results suggest that the DE snoRNAs could potentially regulate the host rRNA biogenesis and processing in response to mastitis due to SC. To the best of our knowledge, this is the first study to report dysregulated snoRNA expression and potential involvement in bovine SC subclinical mastitis.

Mammary Epithelial Cell-Derived Exosomal miR-221-3p Regulates Macrophage Polarization by Targeting Igf2bp2 during Mastitis.

Mastitis affects the milk quality and yield and is the most expensive disease in dairy cows. Elucidation of the pathogenesis of mastitis is of great importance for disease control. As a medium of intercellular communication, exosomes play key roles in various inflammatory diseases by regulating macrophage polarization. However, the molecular factors in exosomes that mediate the intercellular communication between mammary epithelial cells and macrophages during mastitis remain to be further explored. In this study, we isolated and identified mammary epithelial cell-derived exosomes from a lipopolysaccharide (LPS)/lipoteichoic acid (LTA)-induced mastitis cell model, and we demonstrated that exosomes from LPS/LTA-stimulated mammary epithelial cells promote M1-type macrophage polarization in vivo and in vitro. Based on the results of high-throughput sequencing, we constructed a differential miRNA (microRNA) expression profile of exosomes and demonstrated that miR-221-3p was highly expressed. Furthermore, in vivo and in vitro experiments, combined with coculture experiments and fluorescence tracing, showed that high miR-221-3p expression promoted M1-type macrophage polarization, demonstrating the transcellular role of miR-221-3p. Mechanistically, dual luciferase reporter gene assays and rescue assays showed that miR-221-3p regulated macrophage polarization by targeting Igf2bp2. The results of this study will deepen our understanding of the pathogenesis of mastitis, and the molecular regulatory axis that was established in this study is expected to be a target for mastitis treatment.

Integrated analysis of inflammatory mRNAs, miRNAs, and lncRNAs elucidates the molecular interactome behind bovine mastitis

Mastitis is known as intramammary inflammation, which has a multifactorial complex phenotype. However, the underlying molecular pathogenesis of mastitis remains poorly understood. In this study, we utilized a combination of RNA-seq and miRNA-seq techniques, along with computational systems biology approaches, to gain a deeper understanding of the molecular interactome involved in mastitis. We retrieved and processed one hundred transcriptomic libraries, consisting of 50 RNA-seq and 50 matched miRNA-seq data, obtained from milk-isolated monocytes of Holstein–Friesian cows, both infected with Streptococcus uberis and non-infected controls. Using the weighted gene co-expression network analysis (WGCNA) approach, we constructed co-expressed RNA-seq-based and miRNA-seq-based modules separately. Module-trait relationship analysis was then performed on the RNA-seq-based modules to identify highly-correlated modules associated with clinical traits of mastitis. Functional enrichment analysis was conducted to understand the functional behavior of these modules. Additionally, we assigned the RNA-seq-based modules to the miRNA-seq-based modules and constructed an integrated regulatory network based on the modules of interest. To enhance the reliability of our findings, we conducted further analyses, including hub RNA detection, protein–protein interaction (PPI) network construction, screening of hub-hub RNAs, and target prediction analysis on the detected modules. We identified a total of 17 RNA-seq-based modules and 3 miRNA-seq-based modules. Among the significant highly-correlated RNA-seq-based modules, six modules showed strong associations with clinical characteristics of mastitis. Functional enrichment analysis revealed that the turquoise module was directly related to inflammation persistence and mastitis development. Furthermore, module assignment analysis demonstrated that the blue miRNA-seq-based module post-transcriptionally regulates the turquoise RNA-seq-based module. We also identified a set of different RNAs, including hub-hub genes, hub-hub TFs (transcription factors), hub-hub lncRNAs (long non-coding RNAs), and hub miRNAs within the modules of interest, indicating their central role in the molecular interactome underlying the pathogenic mechanisms of S. uberis infection. This study provides a comprehensive insight into the molecular crosstalk between immunoregulatory mRNAs, miRNAs, and lncRNAs during S. uberis infection. These findings offer valuable directions for the development of molecular diagnosis and biological therapies for mastitis.

Teat-apex colonizer Bacillus from healthy cows antagonizes mastitis-causing Staphylococcus aureus biofilms

Staphylococcus aureus is the most frequent causal agent of bovine mastitis, which is largely responsible for milk production losses worldwide. The pathogen's ability to form stable biofilms facilitates intramammary colonization and may explain disease persistence. This virulence factor is also highly influential in the development of chronic intramammary infections refractory to antimicrobial therapy, which is why novel therapies that can tackle multiple targets are necessary. Since udder microbiota have important implications in mastitis pathogenesis, they offer opportunities to develop alternative prophylactic and therapeutic strategies. Here, we observed that a Bacillus strain from the teat apex of lactating cows was associated to reduce colonization by S. aureus. The strain, identified as Bacillus sp. H21, was able to antagonize in-formation or mature S. aureus biofilms associated to intramammary infections without affecting cell viability. When exploring the metabolite responsible for this activity, we found that a widespread class of Bacillus exopolysaccharide, levan, eliminated the pathogenic biofilm under evaluated conditions. Moreover, levan had no cytotoxic effects on bovine cellular lines at the biologically active concentration range, which demonstrates its potential for pathogen control. Our results indicate that commensal Bacillus may counteract S. aureus-induced mastitis, and could therefore be used in novel biotechnological strategies to prevent and/or treat this disease.

Sialic acid exacerbates gut dysbiosis-associated mastitis through the microbiota-gut-mammary axis by fueling gut microbiota disruption

BackgroundMastitis is one of the most severe diseases in humans and animals, especially on dairy farms. Mounting evidence indicates that gastrointestinal dysbiosis caused by induction of subacute ruminal acidosis (SARA) by high-grain diet consumption and low in dietary fiber is associated with mastitis initiation and development, however, the underlying mechanism remains unknown.ResultsIn the present study, we found that cows with SARA-associated mastitis have altered metabolic profiles in the rumen, with increased sialic acids level in particular. Consumption of sialic acid (SA) in antibiotic-treated mice, but not healthy mice, induced marked mastitis. SA treatment of antibiotic-treated mice also induced mucosal and systemic inflammatory responses, as evidenced by increased colon and liver injuries and several inflammatory markers. In addition, gut dysbiosis caused by antibiotic impaired gut barrier integrity, which was aggravated by SA treatment. SA potentiated serum LPS level caused by antibiotic treatment, leading to increased activation of the TLR4-NF-κB/NLRP3 pathways in the mammary gland and colon. Moreover, SA facilitated gut dysbiosis caused by antibiotic, and especially enhanced Enterobacteriaceae and Akkermansiaceae, which correlated with mastitis parameters. Fecal microbiota transplantation from SA-antibiotic-treated mice mimicked mastitis in recipient mice. In vitro experiments showed that SA prompted Escherichia coli growth and virulence gene expression, leading to higher proinflammatory cytokine production in macrophages. Targeting the inhibition of Enterobacteriaceae by sodium tungstate or treating with the commensal Lactobacillus reuteri alleviated SA-facilitated mastitis. In addition, SARA cows had distinct ruminal microbial structure by the enrichment of SA-utilizing opportunistic pathogenic Moraxellaceae and the depletion of SA-utilizing commensal Prevotellaceae. Treating mice with the specific sialidase inhibitor zanamivir reduced SA production and Moraxellaceae abundance, and improved mastitis in mice caused by ruminal microbiota transplantation from cows with SARA-associated mastitis.ConclusionsThis study, for the first time, indicates that SA aggravates gut dysbiosis-induced mastitis by promoting gut microbiota disturbance and is regulated by commensal bacteria, indicating the important role of the microbiota-gut-mammary axis in mastitis pathogenesis and suggesting a potential strategy for mastitis intervention based on gut metabolism regulation.-WPXBcu1emhBxPP-wo4QqAVideo

Fecal microbiome transplant from patients with lactation mastitis promotes mastitis in conventional lactating mice.

Lactation mastitis seriously severely affects the health of lactating females and their infants, yet the underlying causes of clinical lactation mastitis remain unclear. In this study, we used microbiota-humanized mice as a model to investigate the role of gut microbiota in lactation mastitis. We compared the fecal microbiota of lactation mastitis patients and healthy individuals and conducted fecal microbiota transplantation (FMT) experiments in an antibiotic-pretreated mouse model to test whether gut microbes contribute to human lactation mastitis. Our results showed that gut microbiota diversity was reduced and dysbiosis was present in lactating mastitis patients. FMT from lactation mastitis patients (M-FMT), but not from healthy individuals (H-FMT), to antibiotic-treated mice resulted in lactation mastitis. The inflammation in mice caused by gut microbiota from lactating mastitis patients appears to be pervasive, as hepatocytes from mice that received feces from lactating mastitis patients showed marked swelling. In addition, serum pro-inflammatory factors, including IL-4, IL-17, MPO, IL-6, IL-1β, and TNF-α, were significantly increased in the M-FMT group. The Firmicutes/Bacteroidetes ratio (F/B), a biomarker of gut dysbiosis, was significantly increased in the M-FMT group. At the phylum level, Actinobacteria were significantly increased, and Verrucomicrobia were significantly decreased in the M-FMT group. At the genus level, Ruminococcus and Faecalibacterium were significantly reduced, while Parabacteroides were significantly increased in the feces of both patients with lactation mastitis and M-FMT mice. Moreover, our study revealed an "amplification effect" on microbiota differences and mastitis disease following human-to-mouse FMT. Collectively, our findings demonstrate that the gut microbiota in lactating mastitis patients is dysbiotic and contributes to the pathogenesis of mastitis.

A fiber-enriched diet alleviates Staphylococcus aureus-induced mastitis by activating the HDAC3-mediated antimicrobial program in macrophages via butyrate production in mice.

Mounting evidence suggests that the gut microbiota plays an important role in the pathogenesis of mastitis, an important disease affecting the health of lactating women and the development of the dairy industry. However, the effect of the regulation of the gut microbiota by dietary components on mastitis development remains unknown. In this study, we found that a fiber-enriched diet alleviated Staphylococcus aureus (S. au)-induced mastitis in mice, which was dependent on the gut microbiota as depletion of the gut microbiota by antibiotics abolished this protective effect. Likewise, fecal microbiota transplantation (FMT) from high-inulin (HI)-treated mice (HIF) to recipient mice improved S. au-induced mastitis in mice. Consumption of an HI diet and HIF increased fecal short-chain fatty acid (SCFA) levels compared with the control group. Moreover, treatment with SCFAs, especially butyrate, alleviated S. au-induced mastitis in mice. Mechanistically, consumption of an HI diet enhanced the host antimicrobial program in macrophages through inhibiting histone deacetylase 3 by the production of butyrate. Collectively, our results suggest that modulation of the gut microbiota and its metabolism by dietary components is a potential strategy for mastitis intervention and serve as a basis for other infectious diseases.

Detection of ICAA and ICAD genes for biofilm production in Staphylococcus aureus isolates from goat mastitis

Staphylococcus aureus is the most important bacterial pathogen associated with mastitis in dairy animals including goats. The production of biofilm by S. aureus is an important virulence factor in the pathogenesis of mastitis. In the study, 70 milk samples were collected from goats affected with clinical mastitis. After preliminary identification, 29 S. aureus isolates were confirmed through 23S rRNA ribotyping. The confirmed S. aureus isolates were further screened for biofilm formation by Congo red agar method and icaA and icaD genes based polymerase chain reaction. On Congo red agar, 25 (86.20%) isolates produced biofilm while 26 (89.65%) and 25 (86.20%) isolates possessed icaA and, icaD genes, respectively. In conclusion, a high prevalence of biofilm producing S. aureus was identified from goat mastitis and possessed adhesion genes.

Subcutaneous Streptococcus dysgalactiae GAPDH vaccine in mice induces a proficient innate immune response.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of Streptococcus dysgalactiae, coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor. This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections. Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with S. dysgalactiae in vivo. They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods. Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against S. dysgalactiae was investigated. In vitro experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB. GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.

Immunopathological Responses to the Bovine Mastitis Associated with Staphylococcus Species Infection

Bovine mastitis is a disease that concerns animals' welfare and increases the economic production losses. Bacterial agents such as Staphylococcus species are the main causative agent of bovine mastitis. This bacterial agent expresses some inflammatory cytokines that might enhance the cell-mediated, which may promote the pathogenesis of mastitis. The objective of the current study was to investigate the bovine innate immune response circulating levels of pro-inflammatory and anti-inflammatory cytokines. A total of 10 mL of milk specimens were collected randomly from 100 clinically mastitic cows, and another 20 clinically healthy cows were considered as a control group for the California Mastitis test. The microbiological cultures of milk specimens were performed. The interleukins (ILs)that involved IL-4, IL-6, and IL-10 were detected using the ELISA test for the evaluation of the pro-inflammatory bovine mastitis pathophysiology. The results of this study showed that Staphylococcus aureus detection was in 31.2% of mastitic milk and 8.7% of non-mastitic milk specimens; and the coagulase-negative Staphylococcus was detected in 14.8% and 18.7% in the mastitic and non-mastitic milk specimens, respectively. The IL-6 level was shown significantly higher (P<0.05)in the specimens of mastitic milk (194±12.8 pg/mL) compared to the non-mastitic milk (31±2.9 pg/mL). In conclusion, the elevated level of expression of IL-6 cytokine in the milk of cows with mastitis suggested that IL-6 might be used as a potentially suitable biomarker for early bovine mastitis diagnosis

Gut dysbiosis induces the development of mastitis through a reduction in host anti-inflammatory enzyme activity by endotoxemia

BackgroundMounting experimental evidence has shown that the gut microbiota plays a significant role in the pathogenesis of mastitis, and clinical investigations have found that the occurrence of mastitis is correlated with ruminal dysbiosis. However, the underlying mechanism by which the ruminal microbiota participates in the development of mastitis remains unknown.ResultsIn the present study, we found that cows with clinical mastitis had marked systemic inflammation, which was associated with significant ruminal dysbiosis, especially enriched Proteobacteria in the rumen. Ruminal microbiota transplantation from mastitis cows (M-RMT) to mice induced mastitis symptoms in recipient mice along with increased mammary proinflammatory signature activation of the TLR4-cGAS-STING-NF-κB/NLRP3 pathways. M-RMT also induced mucosal inflammation and impaired intestinal barrier integrity, leading to increased endotoxemia and systemic inflammation. Moreover, we showed that M-RMT mirrored ruminal microbiota disruption in the gut of recipient mice, as evidenced by enriched Proteobacteria and similar bacterial functions, which were correlated with most proinflammatory parameters and serum lipopolysaccharide (LPS) levels in mice. Recurrent low-grade LPS treatment mirrored gut dysbiosis-induced endotoxemia and caused severe mastitis in mice. Furthermore, we found that gut dysbiosis-derived LPS reduced host alkaline phosphatase activity by activating neuraminidase (Neu), which facilitates low-grade LPS exposure and E. coli-induced mastitis in mice. Conversely, treatment with calf intestinal alkaline phosphatase or the Neu inhibitor zanamivir alleviated low-grade LPS exposure and E. coli-induced mastitis in mice.ConclusionsOur results suggest that ruminal dysbiosis-derived low-grade endotoxemia can cause mastitis and aggravate pathogen-induced mastitis by impairing host anti-inflammatory enzymes, which implies that regulating the ruminal or gut microbiota to prevent low-grade systemic inflammation is a potential strategy for mastitis intervention.F8E2W2ZseYxr6TzxhA5jTLVideo

Bta-miR-223 Targeting the RHOB Gene in Dairy Cows Attenuates LPS-Induced Inflammatory Responses in Mammary Epithelial Cells.

Bovine mammary epithelial cells (bMECs) are part of the first line of defense against pathogens. In recent studies, bta-miR-223 has been reported to activate congenital and innate immunity against inflammatory damage during the pathogenesis of mastitis in dairy cows. The purpose of this study was to identify the regulatory mechanism of bta-miR-223 and its downstream target genes in inflammatory bMECs. A double luciferase reporter gene assay demonstrated that ras homolog family member B (RHOB) was the target gene of bta-miR-223. To further elucidate the role of bta-miR-223 in congenital immune responses, bta-miR-223 mimics (mimic/inhibitor) were transfected into bMECs stimulated with lipopolysaccharide (LPS), which activates the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect secreted inflammatory factors. Results showed that bta-miR-223 expression during inflammation in bMECs reduced the secretion of inflammatory factors by targeting RHOB and deactivation of NF-κB gene activity. Silencing RHOB inhibited LPS-induced inflammatory response in bMECs. Overall, bta-miR-223 attenuated LPS-induced inflammatory response, and acted as a negative feedback regulator via targeting RHOB, providing a novel avenue for mastitis treatment.

Characterization of peripheral white blood cells transcriptome to unravel the regulatory signatures of bovine subclinical mastitis resistance.

Subclinical bovine mastitis is a pathogenic infection of the breast characterized by a marked decrease in milk production and quality. As it has no obvious clinical symptoms, diagnosis and treatment are challenging. Therefore, searching for biomarkers in cows’ peripheral white blood cells is valuable for preventing and treating subclinical mastitis. Thus, in this study, the transcriptome of peripheral blood from healthy and subclinical mastitis cows was characterized to find the regulatory signatures of bovine subclinical mastitis using RNA-seq. A total of 287 differentially expressed genes (DEGs) and 70 differentially expressed lncRNAs (DELs) were detected, and 37 DELs were documented near known Quantitative Trait Loci (QTL) associated with the mastitis of cows. Bioinformatic analysis indicated that lncRNAs MSTRG25101.2, MSTRG.56327.1, and MSTRG.18968.1, which are adjacent to the SCS QTL and SCC QTL, may be candidate lncRNAs that influence the pathogenesis of mastitis in cows by up-regulating the expression of genes TLR4, NOD2, CXCL8, and OAS2. Moreover, the alternative splicing (AS) pattern of transcriptional sequence differences between healthy cows and subclinical mastitis cows suggested a molecular mechanism of mastitis resistance and susceptibility. A total of 2,212 differential alternative splicing (DAS) events, corresponding to 1,621 unique DAS genes, were identified in both groups and significantly enriched in immune and inflammatory pathways. Of these, 29 DAS genes were subject to regulation by 32 alternative splicing SNPs, showing diverse and specific splicing patterns and events. It is hypothesized that the PIK3C2B and PPRPF8 splice variants associated with AS SNPs (rs42705933 and rs133847062) may be risk factors for susceptibility to bovine subclinical mastitis. Altogether, these key blood markers associated with resistance to subclinical mastitis and SNPs associated with alternative splicing of genes provide the basis for genetic breeding for resistance to subclinical mastitis in cows.

Matrine attenuates bovine mammary epithelial cells inflammatory responses induced by Streptococcus agalactiae through inhibiting NF-κB and MAPK signaling pathways

Streptococcus agalactiae is one of the main pathogens associated with bovine mastitis. The invasion of S. agalactiae in bovine mammary epithelial cells (BMECs) has been implicated as a key event in the pathogenesis of mastitis. Matrine is known for its various pharmacological activities, such as immune response regulation and anti-inflammation. The primary aim of the research was to investigate the preventive effect of matrine on S. agalactiae-induced inflammation in BMECs along with underlying molecular mechanisms. Our data showed matrine at the concentrations of 50–100 μg/mL promoted BMECs proliferation without infection, and decreased cytotoxicity induced by S. agalactiae. Subsequently, BMECs were pre-treated with matrine (50, 75, or 100 μg/mL) for 24 h, followed by the infection with S. agalactiae for an additional 6 h. Pretreatment with matrine followed by S. agalactiae treatment decreased cell apoptosis of BMECs. Also, pretreatment of matrine to BMECs prevented the invasion of S. agalactiae. The mRNA abundances of IL-1β, IL-6, IL-8, and TNF-α were down-regulated in S. agalactiae-infected cells pretreated with matrine. In addition, the greater ratios of protein NF-κB p-p65/p65, p-IκBα/IκBα, p-38/38, and p-ERK/ERK induced by S. agalactiae were attenuated due to matrine treatment. Furthermore, pretreatment of BMECs with matrine impeded the degradation of TAK1 induced by S. agalactiae infection. These results suggest matrine could be a potential modulator in immune response of the mammary gland. In conclusion, matrine prevents cellular damage due to S. agalactiae infection by the modulation of NF-κB and MAPK signaling pathways and pro-inflammatory cytokine production.

Induction of mastitis by cow-to-mouse fecal and milk microbiota transplantation causes microbiome dysbiosis and genomic functional perturbation in mice

BackgroundMastitis pathogenesis involves a wide range of opportunistic and apparently resident microorganims including bacteria, viruses and archaea. In dairy animals, microbes reside in the host, interact with environment and evade the host immune system, providing a potential for host-tropism to favor mastitis pathogenesis. To understand the host-tropism phenomena of bovine-tropic mastitis microbiomes, we developed a cow-to-mouse mastitis model.MethodsA cow-to-mouse mastitis model was established by fecal microbiota transplantation (FMT) and milk microbiota transplantation (MMT) to pregnant mice to assess microbiome dysbiosis and genomic functional perturbations through shotgun whole metagenome sequencing (WMS) along with histopathological changes in mice mammary gland and colon tissues.ResultsThe cow-to-mouse FMT and MMT from clinical mastitis (CM) cows induced mastitis syndromes in mice as evidenced by histopathological changes in mammary gland and colon tissues. The WMS of 24 samples including six milk (CM = 3, healthy; H = 3), six fecal (CM = 4, H = 2) samples from cows, and six fecal (CM = 4, H = 2) and six mammary tissue (CM = 3, H = 3) samples from mice generating 517.14 million reads (average: 21.55 million reads/sample) mapped to 2191 bacterial, 94 viral and 54 archaeal genomes. The Kruskal–Wallis test revealed significant differences (p = 0.009) in diversity, composition, and relative abundances in microbiomes between CM- and H-metagenomes. These differences in microbiome composition were mostly represented by Pseudomonas aeruginosa, Lactobacillus crispatus, Klebsiella oxytoca, Enterococcus faecalis, Pantoea dispersa in CM-cows (feces and milk), and Muribaculum spp., Duncaniella spp., Muribaculum intestinale, Bifidobacterium animalis, Escherichia coli, Staphylococcus aureus, Massilia oculi, Ralstonia pickettii in CM-mice (feces and mammary tissues). Different species of Clostridia, Bacteroida, Actinobacteria, Flavobacteriia and Betaproteobacteria had a strong co-occurrence and positive correlation as the indicator species of murine mastitis. However, both CM cows and mice shared few mastitis-associated microbial taxa (1.14%) and functional pathways regardless of conservation of mastitis syndromes, indicating the higher discrepancy in mastitis-associated microbiomes among lactating mammals.ConclusionsWe successfully induced mastitis by FMT and MMT that resulted in microbiome dysbiosis and genomic functional perturbations in mice. This study induced mastitis in a mouse model through FMT and MMT, which might be useful for further studies- focused on pathogen(s) involved in mastitis, their cross-talk among themselves and the host.Graphical

Integrated Bacteria-Fungi Diversity Analysis Reveals the Gut Microbial Changes in Buffalo With Mastitis.

The gut microbial community is closely related to mastitis, but studies regarding the influences of mastitis on gut microbiota in buffalo remain scarce. Herein, we characterized the differences in gut bacterial and fungal communities between mastitis-affected and healthy buffalos. Interestingly, although mastitis had no effect on gut bacterial and fungal diversities in the buffalos, some bacterial and fungal taxa were significantly altered. Bacterial and fungal taxonomic analysis showed that the preponderant bacterial phyla (Firmicutes and Bacteroidetes) and fungal phyla (Ascomycota and Basidiomycota) in buffalo were the same regardless of health status. At the level of genus, the changes in some gut bacterial and fungal abundances between both groups were gradually observed. Compared with healthy buffalos, the proportions of 3 bacterial genera (uncultured_bacterium_f_Muribaculaceae, Eubacterium_nodatum_group, and Lachnoclostridium_10) and 1 fungal genus (Pichia) in the mastitis-affected buffalo were significantly increased, whereas 4 bacterial genera (Ruminococcus_2, Candidatus_Stoquefichus, Turicibacter, and Cellulosilyticum) and 4 fungal genera (Cladosporium, Thermothelomyces, Ganoderma and Aspergillus) were significantly decreased. Taken together, this research revealed that there was significant difference in the compositions of the gut microbial community between the healthy and mastitis-affected buffalos. To our knowledge, this is the first insight into the characteristics of the gut microbiota in buffalos with mastitis, which is beneficial to understand the gut microbial information of buffalo in different health states and elucidate the pathogenesis of mastitis from the gut microbial perspective.