Alteration of Fecal Microbiota, Fecal Metabolites, and Serum Metabolites in Dairy Cows with Pre-Retained Placenta.

Retained placenta (RP) affects lactation and fertility in dairy cows and causes economic losses to the dairy industry. Therefore, screening for early warning of this disease is important. This study used multi omics techniques to reveal the metabolic differences of dairy cows before RP onset and to find potential warning markers. Fecal samples and serum samples of 90 healthy Holstein cows were collected 7 days pre-calving; 10 healthy and 10 RP cows were enrolled according to normal expulsion of fetal membranes after calving. Fecal samples were subjected to 16S rRNA sequencing and untargeted metabolomics analysis, while plasma was analyzed using targeted metabolomics. Pathogenic bacteria levels increased in the intestines of cows with RP compared to those in healthy cows. Lipid metabolites constituted the largest proportion of differential metabolites between feces and plasma. Six potential warning markers for RP in cows were identified, including two fecal microbiomics markers (Oscillospiraceae UCG-005 and Escherichia-Shigella), one fecal untargeted metabolomics marker (N-acetylmuramic acid), and three plasma targeted metabolomics markers (glycylcholic acid-3 sulfate, 7-ketolithocholic acid, and 12-ketolithocholic acid). These biomarkers can predict RP occurrence in the early perinatal period. These results lay a theoretical foundation for early nutritional intervention and pathogenesis research in dairy cows.

Screening for potential warning biomarkers in cows with ketosis based on host-microbiota co-metabolism analysis.

Introduction: The risk of ketosis is assessed by monitoring changes in plasma metabolites and cow behavior during the peripartum period. However, little is known about changes in the fecal bile acid and microbiota of cows before parturition. Therefore, this study clarified the bile acid profile and screened potential warning biomarkers in heifers 7 days before calving. Methods: Ninety healthy cows were tracked in the transition period, and plasma and feces were collected 7 days before calving, on calving day, and 7 days after calving. The cows were divided into ketosis and healthy groups based on the blood ß-hydroxybutyric acid levels from day 7 after calving. The levels of serum biochemical indices were measured at three time points using commercial kits. Ten cows in the ketosis group (KET-7) and 10 healthy cows (HEA-7) were randomly selected 7 days before calving for metabolome and 16S rRNA amplicon sequencing. Results: No significant differences in serum energy-related indices were observed 7 days before calving. The major bile acids in the feces of the KET-7 group were non-conjugated secondary bile acids (UnconSBA). Differential bile acids were primarily derived from UnconSBA. The potential ketosis warning metabolite in feces for 7 days before delivery was isodeoxycholic acid. The abundance of Rikenellaaceae-RC9-gut-group in the KET-7 group increased, whereas the abundance of Oscillospiraceae UCG-010 bacteria significantly decreased. Lactobacillus and Prevotella-9 in feces were potential warning biomarkers for ketosis in dairy cows 7 days before calving. The variation in differential bile acids in the plasma, consistent with the feces, was mainly derived from UnconSBA. Lithocholic acid in the plasma was a potential ketosis warning metabolite 7 days before delivery. Conclusion: Ketotic cows experienced bile acid metabolism disorders 7 days before calving, and the gut microbiota was closely related to bile acid metabolism disorders. Future studies should investigate the relationship between secondary bile acids and the development of ketosis.

Orally Administered Lactobacillus rhamnosus CY12 Alleviates DSS-Induced Colitis in Mice by Restoring the Intestinal Barrier and Inhibiting the TLR4-MyD88-NF-κB Pathway via Intestinal Microbiota Modulation.

Oral ingestion of probiotics is a promising approach to relieving inflammatory disease through regulating the gut microbiota. A newly discovered strain, Lactobacillus rhamnosus CY12 (LCY12), obtained from cattle-yak milk, displayed numerous probiotic properties. These included enhanced viability in low pH and bile environments, adhesion capabilities, and potent antimicrobial effects. The research aimed to explore the beneficial impacts of the novel LCY12 strain on colitis in mice induced by dextran sulfate sodium (DSS) and to elucidate the underlying molecular mechanisms. The results of the study showed that administration of LCY12 effectively helped to reduce the negative effects of DSS-induced body weight loss, disease activity index score, colon length shortening, loss of goblet cells, and overall histopathological scores in the intestines. Simultaneously, LCY12 administration significantly alleviated intestinal inflammation and safeguarded intestinal barrier integrity by enhancing IL-10 levels, while dampening IL-6, IL-1ß, and TNF-α production. Additionally, LCY12 boosted the presence of tight junction proteins. Furthermore, LCY12 hindered the TLR4/MyD88/NF-κB signaling pathway by downregulating TLR4 and MyD88 expression, inactivating phosphorylated IκBα, and preventing translocation of NF-κB p65 from the cytoplasm to the nucleus. The LCY12 also increased specific intestinal microbial communities and short-chain fatty acid (SCFA) production. Altogether, LCY12 oral administration alleviated colitis induced with DSS in mice by improving intestinal barrier function and regulating inflammatory cytokines, SCFA production, and intestinal microbiota.

Sulforaphane prevents LPS-induced inflammation by regulating the Nrf2-mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis.

BACKGROUND: Mastitis not only deteriorates the composition or quality of milk, but also damages the health and productivity of dairy goats. Sulforaphane (SFN) is a phytochemical isothiocyanate compound with various pharmacological effects such as anti-oxidant and anti-inflammatory. However, the effect of SFN on mastitis has yet to be elucidated. This study aimed to explore the anti-oxidant and anti-inflammatory effects and potential molecular mechanisms of SFN in lipopolysaccharide (LPS)-induced primary goat mammary epithelial cells (GMECs) and a mouse model of mastitis. RESULTS: In vitro, SFN downregulated the mRNA expression of inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6), inhibited the protein expression of inflammatory mediators (cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS)) while suppressing nuclear factor kappa-B (NF-κB) activation in LPS-induced GMECs. Additionally, SFN exhibited an antioxidant effect by increasing Nrf2 expression and nuclear translocation, up-regulating antioxidant enzymes expression, and decreasing LPS-induced reactive oxygen species (ROS) production in GMECs. Furthermore, SFN pretreatment promoted the autophagy pathway, which was dependent on the increased Nrf2 level, and contributed significantly to the improved LPS-induced oxidative stress and inflammatory response. In vivo, SFN effectively alleviated histopathological lesions, suppressed the expression of inflammatory factors, enhanced immunohistochemistry staining of Nrf2, and amplified of LC3 puncta LPS-induced mastitis in mice. Mechanically, the in vitro and in vivo study showed that the anti-inflammatory and anti-oxidative stress effects of SFN were mediated by the Nrf2-mediated autophagy pathway in GMECs and a mouse model of mastitis. CONCLUSIONS: These results indicate that the natural compound SFN has a preventive effect on LPS-induced inflammation through by regulating the Nrf2-mediated autophagy pathway in primary goat mammary epithelial cells and a mouse model of mastitis, which may improve prevention strategies for mastitis in dairy goats.

Association between Tryptophan Metabolism and Inflammatory Biomarkers in Dairy Cows with Ketosis.

Dairy cows with ketosis have high circulating beta-hydroxybutyric acid (BHBA) concentrations alongside which inflammation is concomitantly developed. Tryptophan (Trp) is an essential amino acid that participates in the regulation of the inflammatory response. However, the association between Trp metabolism and inflammation in dairy cows with ketosis remains unclear. Therefore, blood samples from healthy (n = 10) and ketotic (n = 10) primiparous dairy cows were collected at the calving date and the day of ketosis diagnosis (7 days in milk (7 DIM)). Serum levels of non-esterified fatty acids (NEFA), BHBA, haptoglobin (HP), serum amyloid A (SAA), lipopolysaccharide, and cortisol were analyzed. Tryptophan and its metabolites were quantified using liquid chromatography-tandem mass spectrometry. At 7 DIM, the concentrations of NEFA, BHBA, HP, and SAA were higher and the levels of Trp, kynurenine (KYN), indoleacetic acid, indole-3-lactic acid, and 3-indoxyl sulfate were lower in the dairy cows with ketosis compared with those in the healthy cows. However, the KYN/Trp and melatonin/Trp ratios increased in the cows with ketosis. At the calving date, the serum lipopolysaccharide levels did not differ between the healthy and ketotic cows, whereas the levels of NEFA, HP, and cortisol increased in the ketotic cows. Correlation analysis showed that Trp deficiency and elevated Trp metabolism in the dairy cows occurred during ketosis. Overall, our results suggest that abnormal Trp metabolism may contribute to the pathogenesis of ketosis.

Neutrophil Extracellular Traps Mediate Bovine Endometrial Epithelial Cell Pyroptosis in Dairy Cows with Endometritis.

Neutrophils are involved in the development of endometritis, but it remains unknown how neutrophils induce inflammation and tissue damage. Neutrophil extracellular traps (NETs) clear invading pathogens during infection but induce pyroptosis, leading to inflammation and tissue damage. Thus, our objective was to investigate whether NETs participate in bovine endometrial epithelial cell (BEEC) pyroptosis during endometritis. To confirm this, NETs and caspase-1/4; apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC); nod-like receptor protein-3 (NLRP3); and gasdermin D N-terminal (GSDMD-N), TNF-a, IL-1ß, IL-6, and IL-18 in endometrial tissue were detected. Pathological section and vaginal discharge smears were performed to visually determine endometritis in the uterus. BEECs were stimulated with NETs to induce pyroptosis, which was treated with DNase I against pyroptosis. Caspase-1/4, ASC, NLRP3, GSDMD-N, TNF-a, IL-1ß, IL-6, and IL-18 in BEECs were analyzed in endometrial tissue. The results showed that NET formation, as well as pyroptosis-related proteins and proinflammatory, cytokines were elevated in the endometrial tissue of cows with endometritis. Pathological sections and vaginal discharge smears showed increased neutrophils and plasma cells in the uterus, as well as tissue congestion. In BEECs, NETs increased the level of pyroptosis-related proteins and proinflammatory cytokines and were diminished by DNase I. In summary NETs participate BEEC pyroptosis during endometritis in dairy cows.

Altered Fecal Microbiome and Correlations of the Metabolome with Plasma Metabolites in Dairy Cows with Left Displaced Abomasum.

Left displaced abomasum (LDA) in postpartum dairy cows contributes to significant economic losses. Dairy cows with LDA undergo excessive lipid mobilization and insulin resistance. Although gut dysbiosis is implicated, little is known about the role of the gut microbiota in the abnormal metabolic processes of LDA. To investigate the functional links among microbiota, metabolites, and disease phenotypes in LDA, we performed 16S rDNA gene amplicon sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of fecal samples from cows with LDA (n = 10) and healthy cows (n = 10). Plasma marker profiling was synchronously analyzed. In the LDA event, gut microbiota composition and fecal metabolome were shifted in circulation with an amino acid pool deficit in dairy cows. Compared with the healthy cows, salicylic acid derived from microbiota catabolism was decreased in the LDA cows, which negatively correlated with Akkermansia, Prevotella, non-esterified fatty acid (NEFA), and ß-hydroxybutyric acid (BHBA) levels. Conversely, fecal taurolithocholic acid levels were increased in cows with LDA. Based on integrated analysis with the plasma metabolome, eight genera and eight metabolites were associated with LDA. Of note, the increases in Akkermansia and Oscillospira abundances were negatively correlated with the decreases in 4-pyridoxic acid and cytidine levels, and positively correlated with the increases in NEFA and BHBA levels in amino acid deficit, indicating pyridoxal metabolism-associated gut dysbiosis and lipolysis. Changes in branched-chain amino acids implicated novel host-microbial metabolic pathways involving lipolysis and insulin resistance in cows with LDA. Overall, these results suggest an interplay between host and gut microbes contributing to LDA pathogenesis. IMPORTANCE LDA is a major contributor to economic losses in the dairy industry worldwide; however, the mechanisms associated with the metabolic changes in LDA remain unclear. Most previous studies have focused on the rumen microbiota in terms of understanding the contributors to the productivity and health of dairy cows; this study further sheds light on the relevance of the lower gut microbiota and its associated metabolites in mediating the development of LDA. This study is the first to characterize the correlation between gut microbes and metabolic phenotypes in dairy cows with LDA by leveraging multi-omics data, highlighting that the gut microbe may be involved in the regulation of lipolysis and insulin resistance by modulating the amino acid composition. Moreover, this study provides new markers for further research to understand the pathogenesis of the disease as well as to develop effective treatment and prevention strategies.

Cangpu Oral Liquid as a Possible Alternative to Antibiotics for the Control of Undifferentiated Calf Diarrhea.

Antibiotics are essential in the prevention of calf diarrhea epidemics. As more antibiotics become ineffective due to drug-resistant bacteria, attention must be directed toward alternative treatments for calf diarrhea. Natural antibiotic alternatives, such as Chinese herbal medicine, have become a research hotspot in the clinical treatment of diseases such as calf diarrhea due to their characteristics of fewer side effects, low cost, little residue, and no drug resistance. The Cangpu Oral Liquid (CP) was modified from a traditional herbal formula that had been widely used in ancient China to treat gastrointestinal diseases in animals. In order to evaluate the treatment effect of CP on neonatal calf diarrhea, a randomized controlled field trial was performed. Two hundred and forty-six diarrheal Holstein calves of 2-15 days old were selected and randomly divided into two treatment groups receiving either apramycin or CP. 101 out of 123 calves recovered from diarrhea in the CP group, whereas 77 out of 123 calves showed recovery after antibiotic therapy. There were no differences in initial weight between both groups, while the final weight was significantly different (P = 0.892, P = 0.025, respectively). The mean average daily gain (ADG) of calves (211.45 gram/day) in the CP group was significantly higher compared to the antibiotic group (164.56 gram/day) (P = 0.001). The CP group also showed a shorter recovery time from diarrhea (3.90 days vs. 6.62 days, P = 0.001). The current results indicate that the CP has a beneficial clinical effect on the treatment of diarrhea in neonatal calves and is an effective alternative treatment option.

Inhibition of Neutrophil Extracellular Traps Formation by Cl-Amidine Alleviates Lipopolysaccharide-Induced Endometritis and Uterine Tissue Damage.

Endometritis is a common disease that affects the production in dairy cows and leads to severe losses in the dairy industry. Neutrophil extracellular traps (NETs) formation promotes pathogenic invasions of the lumen of the tissue, leading to inflammatory diseases such as mastitis, pancreatitis, and septic infection. However, research that could show the relationship between NETs and endometritis is scarce. Cl-amidine has been shown to ameliorate the disease squealing and clinical manifestation in various disease models. In this study, we investigated the role of NETs in LPS-triggered endometritis in rats and evaluated the therapeutic efficiency of Cl-amidine. An LPS-induced endometritis model in rats was established and found that the formation of NETs can be detected in the rat's uterine tissues in vivo. In addition, Cl-amidine treatment can inhibit NETs construction in LPS-induced endometritis in rats. Myeloperoxidase (MPO) activity assay indicated that Cl-amidine treatment remarkably alleviated the inflammatory cell infiltrations and attenuated the damage to the uterine tissue. The Western blot results indicated that Cl-amidine decreased the expression of citrullinated Histone H3 (Cit-H3) and high-mobility group box 1 protein (HMGB1) protein in LPS-induced rat endometritis. The ELISA test indicated that Cl-amidine treatment significantly inhibited the expression of the pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. The NETs were determined by Quant-iTTMPicoGreen dsDNA kit®, which indicated that Cl-amidine significantly inhibited the NETs in rat serum. All results showed that Cl-amidine effectively reduced the expression of Cit-H3 and HMGB1 proteins by inhibiting the formation of NETs, thereby attenuating the inflammatory response to LPS-induced endometritis in rats. Hence, Cl-amidine could be a potential candidate for the treatment of endometritis.

MicroRNA miR-24-3p Mediates the Negative Regulation of Lipopolysaccharide-Induced Endometrial Inflammatory Response by Targeting TNF Receptor-Associated Factor 6 (TRAF6).

PURPOSE: Endometritis is a female reproductive disease that affects the cattle industries development and microRNAs (miRNAs) play a pivotal role and critical regulators of the innate immune response in varieties of diseases. The present study intends to investigate the regulatory role of miR-24-3p in the innate immune response involved in endometritis and evaluate its therapeutic potential. METHODS: Whole mice uteri and bovine endometrial epithelial cells (BEECs) were separately stimulated with LPS. The BEECs were also transfected with miR-24-3p mimic and negative control; siTRAF6 and siNC; pcDNA3.1 empty and pcDNA3.1(+)TRAF6 separately with LPS stimulation. The expression levels of miR-24-3p and TRAF6 were measured via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. LPS-induced inflammatory response assessed by inflammatory cytokines secretion and expression via ELISA and qRT-PCR. Bioinformatics analysis and luciferase reporter assay validated the interaction between miR-24-3p and TRAF6. The activation of the NF-ĸB/MAPK pathway and p65 phosphorylation was investigated by Western blot and immunofluorescence assay, respectively. RESULTS: The expression of miR-24-3p was decreased, and TRAF6 was elevated with an increased level of pro-inflammatory cytokines in LPS-treated BEECs and mice uterus. The overexpression of miR-24-3p suppressed LPS-induced secretion of inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) and deactivation of NF-ĸB/MAPK pathways. The downregulation of TRAF6 inhibited LPS-induced inflammatory response in BEECs. TRAF6 is validated as a target of miR-24-3p, and miR-24-3p reversed the overexpression of cloned TRAF6 on inflammation response in BEECs. CONCLUSION: Our findings demonstrate that the overexpression of miR-24-3p attenuates endometrial inflammation and the expression of pro-inflammatory mediators via suppressing TRAF6. Therefore, modulating the pathogenesis of endometritis and possibly, a therapeutic potential against endometritis.

MicroRNA Bta-miR-24-3p Suppressed Galectin-9 Expression through TLR4/NF-ĸB Signaling Pathway in LPS-Stimulated Bovine Endometrial Epithelial Cells.

Endometritis is a major infectious disease affecting dairy development. MicroRNAs are recognized as critical regulators of the innate immune response. However, the role and mechanism of Bta-miR-24-3p in the development of endometritis are still unclear. This study aimed to investigate the effect of Bta-miR-24-3p on the inflammatory response triggered by lipopolysaccharide (LPS) and to clarify the possible mechanism. LPS-treated bovine endometrial epithelial cells (BEECs) were cultured to investigate the role of Bta-miR-24-3p. The expression levels of Bta-miR-24-3p were downregulated, and galectin-9 (LGALS9) were measured by quantitative real-time polymerase chain reaction. The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured by using enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Activation of nuclear factor-κB (NF-κB) and TLR4 pathway was assessed by Western blot. The interaction between Bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay. LPS-induction in BEECs with Bta-miR-24-3p was overexpressed leads inhibition of pro-inflammatory cytokines, LGALS9 expression, and TLR4/NF-ĸB pathway deactivation. Knockdown of LGALS9 inhibited the LPS-induced inflammatory response in BEECs. LGALS9 was validated as a target of Bta-miR-24-3p. Cloned overexpression of LGALS9 failed to alter the effect of Bta-miR-24-3p on the inflammatory response in BEECs. Overall, Bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9. The immunotherapeutic stabilisation of Bta-miR-24-3p could give a therapeutic option for endometritis and other disorders commonly associated with endometritis, suggesting a novel avenue for endometritis treatment.

Expression Pattern of Cathelicidins in Dairy Cows During Endometritis and Role of Bovine Endometrial Epithelial Cells in Production of Cathelicidins.

Endometritis is a common bacterial disease of dairy cows. Cathelicidins are host-defense peptides that play important roles in clearance of bacteria. However, the expression pattern of these peptides during endometritis is still unclear. We hypothesize that the levels of bovine cathelicidins increased during endometritis. This study was to investigate the changes of bovine cathelicidins during endometritis. Forty-four post-partum cows (28-35 days after calving) involved in this study were grouped according to the character of vaginal discharge (VD) into three groups. These were (1) cows with clear fluid (n = 8, healthy cows group, N); (2) cows with VD containing <50% off-white mucopurulent material (n = 20, moderate endometritis cows, M); (3) cows with VD containing > 50% yellow or white purulent material (n = 16, severe endometritis cows, S). The blood, VD, and endometrial biopsies samples were collected from each cow to assess the levels of cathelicidin 1-7. Furthermore, bovine endometrial epithelial cells (BEECs) were stimulated with different concentration of Escherichia coli (2 × 106 and 2 × 107 CFU/mL) to detect the cellular source of cathelicidins. Quantitative real-time PCR (RT-qPCR) was used to detect the relative mRNA expression of cathelicidins, and enzyme-linked immune sorbent assay (ELISA) method were used to measure the protein levels. The mRNA and protein levels of cathelicidin 1-7 significantly increased during bovine endometritis (both moderate and severe endometritis), while samples from severe cases showed lower levels of cathelicidins compared to moderate cases. BEECs can express cathelicidin 1-7, and E. coli triggered the release of these proteins. High concentration of E. coli decreased the mRNA and protein levels of cathelicidins. Taken together, our results supported that cathelicidins are released as host defense molecules against the bacteria during bovine endometritis, and BEECs play an active role in expression and production of cathelicidins.

Anti-Inflammatory and Antibacterial Potential of Qicao Rukang Powder in Bovine Subclinical Mastitis.

BACKGROUND: Subclinical mastitis is one of the most common reproductive diseases in dairy cows. Qicao Rukang powder is a Chinese herbal compound mixture developed to treat subclinical mastitis in dairy cows by clearing heat, tonifying qi, and improving blood and milk circulation. The study aimed to determine the anti-inflammatory and antimicrobial efficacy of Qicao Rukang powder in treating subclinical mastitis in dairy cows at the manufacturer's recommended dose. METHODS: Forty (40) Holstein dairy cows with milk somatic cell count (SCC) ≥ 500,000 cellml-1 were randomly assigned to treatment (n = 20) and control (n = 20) groups. Cows in the treatment group were administered with 150 grams of Qicao Rukang powder orally for five days, while the control group received no treatment. The authors analyzed the milk SCC, milk composition, bacteriological cure rate of the drug, blood serum levels of interleukins (IL-6, IL-1ß, and IL-8), tumor necrosis factor (TNF-α), and interferon gamma (INF-γ) quantified by using ELISA kits on day 0 and day 6. RESULTS: SCC of the treated group reduced very significantly (P < 0.001) compared with the control group. Milk fat, protein, and total solids increased significantly (P < 0.05) after treatment, whereas lactose and milk urea nitrogen levels showed a nonsubstantial rise. The bacteriological cure percentage of Qicao Rukang powder therapy was 77.8% for Aeromonas spp. (14 of 18), 75% for Pseudomonas spp. (6 of 8), and 100% for Acinetobacter spp. and Enterococcus spp. giving 81.8% cured for all isolates (27 of 33). Only 26.7% (8 of 30) of untreated cows recovered spontaneously. Analysis of IL-1ß, IL-6, and INF-γ in the blood serum of the treated group revealed a significant decrease (P < 0.01) with nonsignificant rises in TNF-α and IL-8 levels. CONCLUSIONS: This research demonstrates that Qicao Rukang powder has potent antibacterial and anti-inflammatory actions, supporting its use as an alternative to conventional treatment for subclinical dairy cow mastitis. However, further investigations will be required to explain the role of the active ingredients and the mechanisms involved in the pharmacological activities of the Qicao Rukang powder.

Antimicrobial Resistance and Virulence Factor of Streptococcus dysgalactiae Isolated from Clinical Bovine Mastitis Cases in Northwest China.

OBJECTIVE: Streptococcus dysgalactiae is a major pathogen in bovine mastitis. The purpose of this study was to survey the prevalence, antimicrobial resistance, as well as the spread of resistance and virulence-associated gene of S. dysgalactiae. METHODS: A total of 60 S. dysgalactiae strains were obtained from 830 milk samples from Holstein cows with clinical mastitis. Antimicrobial resistance was examined by the disk diffusion method. Antimicrobial resistance and virulence genes were investigated by PCR, agarose gel electrophoresis and 16S rRNA gene sequencing. RESULTS: All isolates were resistant to tetracycline and showed a high level of resistance to aminoglycoside antibiotics, where 81.67% of the strains were multi-resistant to these ten sorts of antibiotics. In addition, the most prevalent resistance gene in S. dysgalactiae was aphA-1 (98.33%), followed by blaTEM (96.67%), ermB (83.3%), aadA1/aadA2 (78.33%) and tetL (73.33%). Totally, seven virulence genes with 25 combination patterns were detected in these isolates, and each isolates harbored at least one virulence gene. 21.67% of the isolates carried three or more virulence genes, while one strain with seven virulence-related genes and belonged to cfb+lmb+eno+napr+bca+scpB+cyl. CONCLUSION: These findings indicate that S. dysgalactiae isolated from clinical bovine mastitis cases in Northwest China show a variety of molecular ecology and are highly resistant to antibiotics commonly used in dairy farms. This research will help investigators better understand the pathophysiology S. dysgalactiae in bovine mastitis and choose the appropriate antibiotics to treat mastitis.

Increasing of matrix metalloproteinase 3 in bovine endometritis.

Matrix metalloproteinase 3 (MMP3), a key member of the MMPs family, is involved in the modulation of endometrial inflammation and innate immunity. However, the role of MMP3 in bovine endometritis remains unknown. To investigate the role of MMP3 in bovine endometritis, endometrial MMP3 expression were determined in uterine biopsies from twenty 40-60 days postpartum dairy cows, which six were healthy cows and fourteen were endometritic cows. Moreover, MMP3 expression were also detected at different intensity of inflammatory response, which was induced by graded concentrations (0, 1, 5, 10 µg/ml) of LPS in bovine endometrial epithelial cells (BEECs) in vitro. RT-qPCR was used to test the mRNA levels of MMP3 in tissues or cells. Western blot was conducted to measure protein levels, and enzyme-linked immunosorbent assay (ELISA) was used for TNF-α and IL-1ß in cell supernatant. Results showed that MMP3 mRNA and protein levels significantly increased and positive correlative with severity of endometritis in vivo. Likewise, MMP3 expression also positive correlative with intensity of LPS inflammatory response in BEECs in vitro. These results indicate that increasing of MMP3 directly correlates with bovine endometritis, and its increasing may contribute to progression of bovine endometritis.

LPS Mediates Bovine Endometrial Epithelial Cell Pyroptosis Directly Through Both NLRP3 Classical and Non-Classical Inflammasome Pathways.

As a highly inflammatory form of programmed cell death, pyroptosis is triggered by pro-inflammatory signals and associated with inflammation. It is characterized by cell swelling and large bubbles emerging from the plasma membrane, which release cytokines during inflammation. Compared with other types of cell death, pyroptosis has a distinct morphology and mechanism and involves special inflammasome cascade pathways. However, the inflammasome mechanism through which endometrial epithelial cell pyroptosis occurs in LPS-mediated inflammation remains unclear. We confirmed that there was an increased mRNA and protein expression of the IL-6, TNF-α, IL-1ß, IL-18 cytokines, the inflammasome molecules NLRP3, CASPASE-1, CASPASE-4, and GSDMD in LPS-induced primary bovine endometrial epithelial cells (BEECs) in an in vitro established inflammatory model using ELISA, real-time PCR (RT-PCR), vector construction and transfection, and Western blotting. Scanning electron microscopy and lactate dehydrogenase (LDH) activity assays revealed induced cell membrane rupture, which is the main characteristic of pyroptosis. In conclusion, the cytolytic substrate GSDMD's cleavage by caspase-1 or caspase-4 through the NLRP3 classical and non-classical inflammasome pathways, GSDMD N-terminus bind to the plasma membrane to form pores and release IL -18, IL-1ß cause cell death during LPS induced BEECs inflammation.

Using vaginal discharge score (VDS) grading system to evaluate the effect of clinical endometritis on reproductive performance of dairy cows in China.

Clinical endometritis (CE) is a major cause in affecting the reproductive performance of dairy cows. The objectives of this study were to ascertain the prevalence of CE and to evaluate the effect of CE on reproductive performance in dairy cows using vaginal discharge score (VDS) grading system. 803 dairy cows were examined by vaginoscope with 4-point VDS at 26 ± 3 days in milk (DIM) and classified into six groups: non-endometritis with VDS 0 (control; CON), endometritis with VDS 1 (MEM), non-treated endometritis with VDS 2 (NTME), treated endometritis with VDS 2 (TME), non-treated endometritis with VDS 3 (NTPE), and treated endometritis with VDS 3 (TPE). Cows in TME and TPE groups were treated with 200 mL of 50% dextrose solution by intrauterine infusion. The prevalence of CE was 33% at 26 ± 3 DIM. Binary logistic regression analysis revealed cows in MEM, NTME and NTPE groups had a less likelihood of first artificial insemination (AI) pregnancy than those in CON group (P < 0.05). Kaplan-Meier survival curves for days open were statistically different (P = 0.004). In Cox regression model, cows in NTME and NTPE groups had a reduced pregnancy rate than those in CON group (P < 0.05). The hazard of pregnancy in NTME group was lower than that in TME group (P = 0.044). Similarly, it was lower for the hazard of pregnancy in NTPE group than in TPE group (P = 0.048). Cows in MEM, NTME, and NTPE groups required more services per pregnancy than those in CON group (P < 0.05). In conclusion, CE examined by the VDS grading system impaired reproductive performance, and mild endometritis with VDS 1 should be treated in the early postpartum period to ameliorate fertility in dairy herds.

Modulation of Bovine Endometrial Cell Receptors and Signaling Pathways as a Nanotherapeutic Exploration against Dairy Cow Postpartum Endometritis.

In order to control and prevent bovine endometritis, there is a need to understand the molecular pathogenesis of the infectious disease. Bovine endometrium is usually invaded by a massive mobilization of microorganisms, especially bacteria, during postpartum dairy cows. Several reports have implicated the Gram-negative bacteria in the pathogenesis of bovine endometritis, with information dearth on the potentials of Gram-positive bacteria and their endotoxins. The invasive bacteria and their ligands pass through cellular receptors such as TLRs, NLRs, and biomolecular proteins of cells activate the specific receptors, which spontaneously stimulates cellular signaling pathways like MAPK, NF-kB and sequentially triggers upregulation of pro-inflammatory cytokines. The cascade of inflammatory induction involves a dual signaling pathway; the transcription factor NF-κB is released from its inhibitory molecule and can bind to various inflammatory genes promoter. The MAPK pathways are concomitantly activated, leading to specific phosphorylation of the NF-κB. The provision of detailed information on the molecular pathomechanism of bovine endometritis with the interaction between host endometrial cells and invasive bacteria in this review would widen the gap of exploring the potential of receptors and signal transduction pathways in nanotechnology-based drug delivery system. The nanotherapeutic discovery of endometrial cell receptors, signal transduction pathway, and cell biomolecules inhibitors could be developed for strategic inhibition of infectious signals at the various cell receptors and signal transduction levels, interfering on transcription factors activation and pro-inflammatory cytokines and genes expression, which may significantly protect endometrium against postpartum microbial invasion.

Decreasing of S100A4 in bovine endometritis in vivo and in vitro.

Endometritis is a prevalent reproductive disease in dairy cows, and is a superficial inflammation of the endometrium. S100 calcium-binding protein A4 (S100A4) is suggested to be implicated in the progression of inflammation. However, to our knowledge, no study has reported the changes of S100A4 during bovine endometritis. The objective of this study was to investigate S100A4 gene expression and protein levels in the uterus with endometritis in dairy cows. Vaginal mucus samples were collected for diagnosis of the severity degree of endometritis and the detection of S100A4 protein content. Blood samples and endometrial biopsies were collected and divided into the control (CN), mild endometrtis (M), and severe endometritis (S) groups according to the characteristics of the vaginal mucus type. The isolated bovine endometrial epithelial cells (BEECs) were challenged with E. coli (2 × 106 CFU/mL, 2 × 107 CFU/mL) or lipopolysaccharide (LPS, 3 and 10 µg/mL) as an inflammatory model. RT-qPCR was used to detect the gene expression levels of S100A4 and cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF-α), in tissues or cells. Enzyme-linked immunosorbent assay (ELISA) was used for S100A4 protein level detection in tissues, cells, cell supernatant, vaginal mucus, and serum samples. The results showed that S100A4 gene and protein levels decreased in bovine endometrium with endometritis and in E. coli- or LPS-stimulated BEECs. We failed to detect S100A4 in the cell supernatant, vaginal mucus, and serum samples. This study suggested that S100A4 is a pathogenesis-related protein of endometritis, and decreased expression of S100A4 may pave the way for the development of endometritis in dairy cows.

Serum Metabolomics Revealed the Differential Metabolic Pathway in Calves with Severe Clinical Diarrhea Symptoms.

The complex etiology, higher morbidity and mortality, poor prognosis, and expensive cost of calf diarrhea have made it a catastrophic disease in the dairy industry. This study aims to assess the biomarkers in calves with diarrhea and to predict the biomarkers related to the pathway. As subjects, nine calves with diarrhea and nine healthy calves were enrolled, according to strict enrollment criteria. The serum metabolites were detected by a liquid chromatographic tandem mass spectrometry (LC-MS/MS), and then analyzed by online multivariate statistical analysis software to further screen the biomarkers. In addition, the biomarkers involved in the metabolic pathways of calves with diarrhea and healthy calves were analyzed. In the serum of calves with diarrhea, nine biomarkers were found to which several biomarkers exhibited a certain relation. Moreover, these biomarkers were involved in important metabolic pathways, including protein digestion and absorption, ABC transporters, aminoacyl-tRNA biosynthesis, mineral absorption, and fatty acid biosynthesis. All these findings suggested that the imbalance of these markers was closely related to the occurrence and development of calf diarrhea. The targeted regulation of metabolic pathways involved in these biomarkers may facilitate the diagnosis, treatment, and discussion of the mechanism of calf diarrhea.