Bacterial Diversity and Antimicrobial Resistance of Microorganisms Isolated from Teat Cup Liners in Dairy Farms in Shandong Province, China.

Global milk consumption exceeds 800 million tons a year and is still growing. Milk quality and its products are critical to human health. A teat cup makes direct contact with the cow's teats during milking and its cleanliness is very important for the quality of raw milk. In this study, the microorganism from post-milking teat cup liners were collected from six dairy farms in Shandong Province of China, the bacterial species were identified using microbial mass spectrometry, the minimum inhibitory concentrations of the isolated strains against ten antimicrobial agents were determined using the broth microdilution method, and the antimicrobial resistance genes were detected by PCR. The results indicated that the most frequently isolated bacteria in this study were Bacillus licheniformis (39/276, 14.13%), followed by Bacillus pumilus (20/276, 7.25%), Bacillus cereus (17/276, 6.16%), and Bacillus subtili (16/276, 5.80%). The isolates exhibited the highest average resistance to lincomycin (87.37%), followed by sulfadiazine (61.05%) and streptomycin (42.63%); the highest detection rate of resistance genes was Sul1 (55.43%), followed by ant(4') (51.09%), tet(M) (25.36%), blaKPC (3.62%) and qnrS (3.62%). These findings imply the necessity for enhanced measures in disinfecting cow udders and milking equipment, highlighting the persistently challenging issue of antimicrobial resistance in Shandong Province.

In vitro and in vivo evaluation of antibacterial activity and mechanism of luteolin from Humulus scandens against Escherichia coli from chicken.

Resistance of Escherichia coli (E.coli) to antibiotics has steadily increased over time; hence, there is an urgent need to develop safer alternatives to antibiotics. The present study aimed to evaluate the effect of luteolin (Lut) on E. coli from chicken. The bioactive compound Lut from Humulus scandens was selected by network pharmacology and molecular docking analyses. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) were used to observe the effects of Lut on the morphology and structure of E. coli cells. The data-independent acquisition (DIA) method was used to analyze protein expression level of E. coli before and after Lut treatment. The in vivo evaluation of the antibacterial, anti-inflammatory, and oxidative effects of Lut on E.coli was conducted using E.coli isolated strains infected the SPF chicken model. The network pharmacology analysis revealed 19 distinctive bioactive compounds such as Lut and ß-sitosterol in H. scandens; furthermore, 30 core targets were selected from H. scandens. The KEGG enrichment analysis showed that the PI3K-Akt, TNF, MAPK, IL-17, JAK-STAT, and HIF-1 pathways were related from H. scandens. Based on the results of the network pharmacology analysis, Lut was subjected to screening by molecular docking analysis to determine its antibacterial effect on E. coli and the associated mechanism of action. The minimum inhibitory concentration (MIC) of Lut against E. coli standard strains was 500 µg/mL. SEM, TEM, and CLSM results indicated that Lut damaged the cell wall and cell membrane of E. coli strains and destroyed the cell structure, leading to cell death.The expression level of membrane structure, Phenylalanine metabolism and some other metabolic pathways in E.coli changed after treatment with Lut (P < 0.05). In vivo experiments in the SPF chicken model showed that Lut treatment alleviated the decline in the growth performance of chickens (P < 0.05), prevented pathological changes in the correspond ding organs and suppressed the inflammatory response induced by E. coli infection (P < 0.05), improved the immunity and antioxidant capacity of chickens (P < 0.05), and protected them against infection with E. coli strains. To summarize, Lut from H. scandens can inhibit E. coli growth by damaging the cell membrane structureand affecting the expression level of some metabolic proteins. In vivo experiments also showed that Lut can significantly reduce the damage caused by E. coli isolates on SPF chickens, improve their antioxidant capacity and immunity, and reduce inflammatory responses following E. coli infection.

Role of Rab GTPases in Bacteria Escaping from Vesicle Trafficking of Host Cells.

Most bacteria will use their toxins to interact with the host cell, causing damage to the cell and then escaping from it. When bacteria enter the cell, they will be transported via the endosomal pathway. Rab GTPases are involved in bacterial transport as major components of endosomes that bind to their downstream effector proteins. The bacteria manipulate some Rab GTPases, escape the cell, and get to survive. In this review, we will focus on summarizing the many processes of how bacteria manipulate Rab GTPases to control their escape.

Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG.

Introduction: Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge. Methods: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively. Results: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively. Discussion: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

Cr(VI) induces ferroptosis in DF-1 cells by simultaneously perturbing iron homeostasis of ferritinophagy and mitophagy.

Hexavalent chromium [Cr(VI)] is an environmental pollutant known for its strong oxidizing and carcinogenic effects. However, its potential to induce ferroptosis in poultry remains poorly understood. This study aims to investigate the induction of ferroptosis by Cr(VI) in DF-1 cells and elucidate the underlying mechanisms. DF-1 cells exposed to Cr(VI) showed increased lipid reactive oxygen species and changes in ferroptosis marker genes (decreased expression of GPX4 and increased expression of COX2). Notably, the addition of the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) can reverse this effect. During the cell death process, Cr(VI) induced ferritinophagy, disrupting iron homeostasis and releasing labile iron ions. We predicted by docking that these iron ions would bind to mitochondrial membrane proteins through virtual docking. This binding was validated through colocalization analysis. In addition, Cr(VI) caused mitophagy, which releases additional ferrous ions. Therefore, Cr(VI) can induce the simultaneous release of ferrous ions through these pathways, thereby exacerbating lipid peroxidation and ultimately triggering ferroptosis in DF-1 cells. This study demonstrates that Cr(VI) can induce ferroptosis in DF-1 cells by disrupting intracellular iron homeostasis and providing valuable insights into the toxic effects of Cr(VI) in poultry and potentially other organisms.

Platycodon grandiflorus polysaccharide inhibits the inflammatory response of 3D4/21 cells infected with PCV2.

Porcine circovirus type 2 (PCV2) infection cause multi-systemic inflammation in pigs. Platycodon grandiflorus polysaccharide (PGPSt) has been reported to have the effects of immune regulation and disease resistance. Nevertheless, the role and mechanism of PGPSt in the inflammatory response of 3D4/21 cells induced by PCV2 infection remain unclear. The present study aims to investigate effects of PGPSt on inflammatory response and its possible underlying mechanisms in vitro models. Cells were treated with PCV2 for 36 h to construct a cell inflammation model. The 3D4/21 cell lines were pretreated with or without PGPSt, and the changes of inflammation-related markers and the signaling pathway were detected by CCK-8, ELISA, qPCR and Western blot. The results showed that PGPSt was non-toxic to cells and protected PCV2-infected cells from inflammatory damage. PGPSt could significantly inhibit the high acetylation of histone H3 (AcH3) and histone H4 (AcH4), down-regulate HAT and up-regulate HDAC activity, and reduce the expression of pro-inflammatory enzymes iNOS and COX-2 proteins levels. Then the levels of IL-1ß, IL-6 and TNF-α were significantly inhibited, and the level of IL-10 was promoted. We also observed that PGPSt inhibited the phosphorylation of p65, p38 and Erk1/2, which subsequently inhibited nuclear translocation of NF-κB p65 to express pro-inflammatory factors. In conclusion, PGPSt can reduce the inflammatory response by regulating histone acetylation, reducing the release of inflammatory factors, reducing the expression of pro-inflammatory enzymes, and inhibiting the activation of NF-κB and MAPKs signaling pathways. This suggests that PGPSt had an anti-inflammatory effect on the inflammatory response caused by PCV2 infection, which provided theoretical data support for the research.

Research on Bacterial Diversity and Antibiotic Resistance in the Dairy Farm Environment in a Part of Shandong Province.

Antimicrobials are extensively utilized in dairy farms to prevent and control diseases in cattle. However, their use contributes to the emergence of antimicrobial-resistant bacteria (ARB) and antimicrobial-resistant genes (ARG), and these can be transmitted to the environment. Regular monitoring of antimicrobial resistance (AMR) is crucial for implementing effective mitigation strategies. This research aimed to assess the environmental microbial species present on dairy farms in Shandong Province and characterize the antimicrobial resistance profiles of the isolates. Five dairy farms located in Shandong Province were selected, representing the prevalent large-scale farming patterns in the area. Sampling took place from April to June 2022, with a total of 223 isolates collected from various environmental locations within each farm (bedding, sports field, and milking parlor). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was employed to identify the species of the clinical isolates. The main pathogens isolated were Aerococcus viridans (5.38%, n = 12), Corynebacterium xerosis (4.93%, n = 11), and Acinetobacter lwoffii (4.03%, n = 9). Among the bacterial isolates, resistance to lincomycin was highest at 91%, and 88% were resistant to sulfadiazine. Antimicrobial resistance genes were detected in only a small proportion of the isolates, the most common of which was sul1. These findings highlight the necessity for careful evaluation of antimicrobial usage in maintaining their effectiveness in human medicine. Understanding the microbial species present and their antimicrobial resistance profiles aids in focusing efforts toward sustainable antimicrobial use and safeguarding human health.

Role of PERK-Mediated Endoplasmic Reticulum Stress in Ferroptosis Caused by Hexavalent Chromium in Chicken Hepatocytes.

This study aimed to investigate whether Cr(VI) can induce ferroptosis in chicken hepatocytes and determine the role of PERK-mediated endoplasmic reticulum stress (ERS). First, a model of Cr(VI) poisoning was established by exposing chicken hepatocytes to Cr(VI). The levels of ferroptosis-related proteins, meanwhile, GSH, SOD, MDA, and lipid ROS, were measured. Furthermore, the expression of GRP78 and PERK proteins was examined. Changes in ERS and ferroptosis were evaluated by silencing the PERK gene. Results showed that Cr(VI) led to the accumulation of lipid ROS, decreased expression of GPX4 and HSP27, increased expression of COX2, and induced ferroptosis in chicken hepatocytes. Exposure to Cr(VI) increased the protein expression of GRP78 and PERK, and silencing of PERK worsened Cr(VI)-induced ferroptosis. In conclusion, Cr(VI) can induce ferroptosis in chicken hepatocytes, and PERK plays an important role as a negative regulator.

The surface morphology of Atractylodes macrocephala polysaccharide and its inhibitory effect on PCV2 replication.

BACKGROUND: Porcine infection with Porcine circovirus type 2 (PCV2) causes immunosuppression, which is easy to cause concurrent or secondary infection, making the disease complicated and difficult to treat, and causing huge economic losses to the pig industry. Total polysaccharide from the rhizoma of Atractylodes macrocephala Koidz. (PAMK) is outstanding in enhancing non-specific immunity and cellular immunity, and effectively improving the body's disease resistance, indicating its potential role in antiviral immunotherapy. RESULTS: PAMK had the characteristics of compact, polyporous and agglomerated morphology, but does not have triple helix conformation. PCV2 infection led to the increase in LC3-II, degradation of p62 and the increase of viral Cap protein expression and viral copy number. PAMK treatment significantly alleviated PCV2-induced autophagy and inhibited PCV2 replication. Moreover, PAMK treatment significantly attenuated the increase of PINK1 protein expression and the decrease of TOMM20 protein expression caused by PCV2 infection, alleviated Parkin recruitment from cytoplasm to mitochondria and intracellular reactive oxygen species accumulation, restored mitochondrial membrane charge, alleviated viral Cap protein expression. CONCLUSION: PAMK alleviates PCV2-induced mitophagy to suppress PCV2 replication by inhibiting the Pink 1/Parkin pathway. These findings may provide new insights into the prevention and treatment of PCV2. © 2023 Society of Chemical Industry.