Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

FADD promotes type I interferon production to suppress porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) is an epidemic animal infectious disease worldwide, causing huge economic losses to the global swine industry. Fas-associated death domain (FADD) was previously reported to be an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In the current study, we unravel its unidentified role in promoting type I interferon (IFN) production during PRRS virus (PRRSV) infection. We identified that FADD inhibited PRRSV infection via promotion of type I IFN transcription. Overexpression of FADD suppressed the replication of PRRSV, while knockout of FADD increased viral titer and nucleocapsid protein expression. Mechanistically, FADD promoted mitochondrial antiviral signaling protein (MAVS)-mediated production of IFN-ß and some IFN-stimulated genes (ISGs). Furthermore, FADD exerted anti-PRRSV effects in a MAVS-dependent manner and increased the type I IFN signaling during PRRSV infection. This study highlights the importance of FADD in PRRSV replication, which may have implications for the future control of PRRS.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

Preliminary view of the distribution and spread of the plasmid-mediated resistance genes in Glaesserella parasuis.

Introduction. Various plasmid-mediated resistance genes have been reported in Glaesserella parasuis, but little is known about their global distribution features, evolution pattern and spread.Gap Statement. The potential mobilization mechanisms of resistance plasmids in G. parasuis have been poorly explored.Aim. The aim of the study was to investigate the prevalence and diversity of plasmid-mediated resistance genes among G. parasuis isolates, and focus on the analysis of the features of the resistance plasmids from G. parasuis.Method. The plasmids tested were sequenced using the Illumina HiSeq platform in conjunction with PCR and inverted PCR. The susceptibility of the host strains was determined by broth microdilution. The transfer of plasmids tested was conducted by electroporation. The sequence data were compared using bioinformatics tools and the data from our laboratory and the National Center for Biotechnology Information (NCBI) database.Results. Nineteen plasmids were identified from our laboratory and these resistance plasmids were functional and transferable. Moreover, we clustered five types of genetic backbones of plasmids from G. parasuis and revealed the global distribution features of the plasmid-mediated resistance genes.Conclusions. This is the first report of the coexistence of tet(H)-bearing type I plasmid and lnu(C)-bearing type II plasmid in one G. parasuis clinical isolate. In addition, this study provides the first view of the global distribution of plasmid-mediated resistance genes and classifies the plasmids in G. parasuis according to their backbone regions.

Coexpression analysis of lncRNAs and mRNAs identifies potential regulatory long noncoding RNAs involved in the inflammatory effects of lipopolysaccharide on bovine mammary epithelial cells.

BACKGROUND: The infection of bovine mammary glands by pathogenic microorganisms not only causes animal distress but also greatly limits the development of the dairy industry and animal husbandry. A deeper understanding of the host's initial response to infection may increase the accuracy of selecting drug-resistant animals or facilitate the development of new preventive or therapeutic intervention strategies. In addition to their functions of milk synthesis and secretion, bovine mammary epithelial cells (BMECs) play an irreplaceable role in the innate immune response. To better understand this process, the current study identified differentially expressed long noncoding lncRNAs (DE lncRNAs) and mRNAs (DE mRNAs) in BMECs exposed to Escherichia coli lipopolysaccharide (LPS) and further explored the functions and interactions of these lncRNAs and mRNAs. RESULTS: In this study, transcriptome analysis was performed by RNA sequencing (RNA-seq), and the functions of the DE mRNAs and DE lncRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, we constructed a modulation network to gain a deeper understanding of the interactions and roles of these lncRNAs and mRNAs in the context of LPS-induced inflammation. A total of 231 DE lncRNAs and 892 DE mRNAs were identified. Functional enrichment analysis revealed that pathways related to inflammation and the immune response were markedly enriched in the DE genes. In addition, research results have shown that cell death mechanisms, such as necroptosis and pyroptosis, may play key roles in LPS-induced inflammation. CONCLUSIONS: In summary, the current study identified DE lncRNAs and mRNAs and predicted the signaling pathways and biological processes involved in the inflammatory response of BMECs that might become candidate therapeutic and prognostic targets for mastitis. This study also revealed several possible pathogenic mechanisms of mastitis.

Comprehensive Analysis of lncRNA and mRNA Expression Profile of Macrophage RAW264.7 Stimulated by Antimicrobial Peptide BSN-37.

BACKGROUND: BSN-37, a novel antimicrobial peptide (AMP) containing 37 amino acid residues isolated from the bovine spleen, has not only antibacterial activity but also immunomodulatory activity. Recent evidence shows that long non-coding RNAs (lncRNAs) play an important role in regulating the activation and function of immune cells. The purpose of this experiment was to investigate the lncRNA and mRNA expression profile of mouse macrophages RAW264.7 stimulated by bovine antimicrobial peptide BSN-37. METHODS: The whole gene expression microarray was used to detect the differentially expressed lncRNA and mRNA between antimicrobial peptide BSN-37 activated RAW264.7 cells and normal RAW264.7 cells. KEGG pathway analysis and GO function annotation analysis of differentially expressed lncRNAs and mRNA were carried out. Eight kinds of lncRNAs and nine kinds of mRNA with large differences were selected for qRT-PCR verification, respectively. RESULTS: In the current study, we found that 1294 lncRNAs and 260 mRNAs were differentially expressed between antibacterial peptide BSN-37 treatment and control groups. Among them, Bcl2l12, Rab44, C1s, Cd101 and other genes were associated with immune responses and were all significantly up-regulated. Mest and Prkcz are related to cell growth, and other genes are related to glucose metabolism and lipid metabolism. In addition, some immune-related terms were also found in the GO and KEGG analyses. At the same time, real-time quantitative PCR was used to verify selected lncRNA and mRNA with differential expression. The results of qRT-PCR verification were consistent with the sequencing results, indicating that our data were reliable. CONCLUSION: This study provides the lncRNA and mRNA expression profiles of RAW264.7 macrophages stimulated by antimicrobial peptide BSN-37 and helps to provide a reference value for subsequent studies on lncRNA regulation of antimicrobial peptide BSN-37 immune function.

Soluble Expression of Antimicrobial Peptide BSN-37 from Escherichia coli by SUMO Fusion Technology.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6 h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

Recombinant Antimicrobial Peptide OaBac5mini Alleviates Inflammation in Pullorum Disease Chicks by Modulating TLR4/MyD88/NF-κB Pathway.

Pullorum disease (PD), caused by Salmonella Pullorum (S. Pullorum), is a serious threat to the poultry industry worldwide. Antimicrobial peptides (AMPs) have drawn extensive attention as new-generation antibiotics because of their broad antimicrobial spectrum, low resistance, and low cytotoxicity. AMP OaBac5mini exhibits strong antibacterial activity against Gram-negative bacteria, but its efficacy and anti-inflammatory effects on chicks with PD remain unclear. The aim of this study was to generate recombinant OaBac5mini via the Escherichia coli (E. coli) recombinant expression system and evaluate its antibacterial effect against S. Pullorum in vitro and in vivo. Real-time cellular analysis (RTCA) results showed that recombinant OaBac5mini exhibited no cytotoxicity on IPEC-J2 and RAW 264.7 cells and significantly alleviated the drop in the cell index of S. Pullorum-infected cells (p < 0.0001). In the chick model of PD, recombinant OaBac5mini significantly attenuated the increase in organ indexes (heart, liver, spleen, and kidney) and bacterial loads (liver and spleen) induced by S. Pullorum. Histopathology examination showed that recombinant OaBac5mini ameliorated histopathological changes and inflammation in chicks with PD, including impaired epithelium of duodenal villi, infiltration of pseudoacidophilic granulocytes in the cecum and bursa of Fabricius, congested blood clots and increased macrophages in the liver, and increased lymphoid nodule and B lymphocytes in the spleen. Western blot and quantitative real-time PCR (qRT-PCR) results indicated that recombinant OaBac5mini alleviated inflammation by modulating innate immunity through the TLR4/MyD88/NF-κB pathway and by suppressing the expression of pro-inflammatory cytokines. These results suggested that recombinant OaBac5mini has good potential as a clinical substitute for antibiotics in PD intervention.

Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli.

Introduction: Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. Methods: In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. Results: The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 µg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 µg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. Conclusion: OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.

Evaluation of the efficacy of the antimicrobial peptide HJH-3 in chickens infected with Salmonella Pullorum.

As a promising substitute for antibiotics, increasing attention has been given to the clinical application of antimicrobial peptides (AMPs). In this study, the mode of action of the HJH-3 against Salmonella Pullorum was investigated. The structure and properties of HJH-3 were examined in silico, and minimum inhibitory concentrations (MICs) were determined to evaluate its antimicrobial spectrum. The time-kill kinetics of HJH-3 was determined. The hemolytic activity of HJH-3 was determined by measuring the hemoglobin ultraviolet absorption value, and the cytotoxicity was determined using a CCK-8 kit. The protective effect of HJH-3 on chickens infected with S. Pullorum was evaluated in vivo. The results demonstrated that HJH-3 exhibited strong antibacterial activity against Gram-negative pathogens at MIC values of 1.5625-25 µg/mL and against Gram-positive pathogens at MIC values of 25-50 µg/mL. HJH-3 also showed activity against the Candida albicans (100 µg/mL) and Bacillus subtilis (6.25-12.5 µg/mL). HJH-3 at 100 µg/mL completely killed S. Pullorum after co-incubation for 6 h. Likewise, the hemolysis rate of CRBCs treated with 100 µg/mL HJH-3 (7.31%) was lower than that of CRBCs treated with 100 µg/mL pexiganan (40.43%). Although the hemolysis rate of CRBCs treated with 400 µg/mL HJH-3 was increased to 13.37%, it was much lower than that of 400 µg/mL pexiganan (57.27%). In regards to cytotoxicity, HJH-3 had almost no-effect on the CEF proliferation, pexiganan decreased CEFs proliferation from 56.93 to 31.00% when increasing the concentration from 50 to 200 µg/mL. In a chicken infection model, the results showed that the antibiotic prevention and HJH-3 prevention groups exhibited the best treatment effect, with the chickens being protected from the lethal dose of S. Pullorum, a decreased number of bacteria in the blood and spleen, and less pathological changes in intestinal segments. The prevention of infection by HJH-3 was similar to that by Ampicillin; the effect of treatment after infection was lower than that of treatment before infection, and the survival rate of infected chicks treated with HJH-3 was 70%, which was still higher than that of the infected chickens. These results suggest that HJH-3 has good clinical application potential and can be used as a substitute for antibiotics for the prevention and treatment of S. Pullorum infection.

Evaluation of the mutant selection window of danofloxacin against Actinobacillus pleuropneumoniae in an in vitro dynamic model.

Introduction: The rapid emergence and widespread spread of multidrug-resistant bacteria is a serious threat to the health of humans and animals. The pharmacokinetic/pharmacodynamic (PK/PD) integration model based on mutant selection window (MSW) theory is an important method to optimize the dosage regimen to prevent the emergence and spread of drug-resistant bacteria. Actinobacillus pleuropneumoniae (AP) is a pathogen that can cause pleuropneumonia in pigs. Methods: We employed an in vitro dynamic infection model (DIM) to study the prevention of drug-resistant mutations of danofloxacin against AP. A peristaltic pump was applied to establish an in vitro DIM to simulate the PK of danofloxacin in plasma, and to study the MSW of danofloxacin against AP. A peristaltic-pump in vitro infection model was established to simulate dynamic changes in the danofloxacin concentration in pig plasma. PK and PD data were obtained. Then, the relationship between PK/PD parameters and antibacterial activity was analyzed by the sigmoid Emax model. Results and discussion: The area under the curve during 24 h/ the minimum concentration that inhibits colony formation by 99% (AUC24h/MIC99) had the best-fitting relationship with antibacterial activity. The AUC24h/MIC99 values for a bacteriostatic effect, bactericidal effect, and eradication effect were 2.68, 33.67, and 71.58 h, respectively. We hope these results can provide valuable guidance when using danofloxacin to treat AP infection.

Antimicrobial Peptide MPX with Broad-Spectrum Bactericidal Activity Promotes Proper Abscess Formation and Relieves Skin Inflammation.

Bacteria have developed antibiotic resistance during the large-scale use of antibiotics, and multidrug-resistant strains are common. The development of new antibiotics or antibiotic substitutes has become an important challenge for humankind. MPX is a 14 amino acid peptide belonging to the MP antimicrobial peptide family. In this study, the antibacterial spectrum of the antimicrobial peptide MPX was first tested. The antimicrobial peptide MPX was tested for antimicrobial activity against the gram-positive bacterium S. aureus ATCC 25923, the gram-negative bacteria E. coli ATCC 25922 and Salmonella enterica serovar Typhimurium CVCC541, and the fungus Candida albicans ATCC 90029. The results showed that MPX had good antibacterial activity against the above four strains, especially against E. coli, for which the MIC was as low as 15.625 µg/mL. The study on the bactericidal mechanism of the antimicrobial peptide revealed that MPX can destroy the integrity of the cell membrane, increase membrane permeability, and change the electromotive force of the membrane, thereby allowing the contents to leak out and mediating bacterial death. A mouse acute infection model was used to evaluate the therapeutic effect of MPX after acute infection of subcutaneous tissue by S. aureus. The study showed that MPX could promote tissue repair in S. aureus infection and alleviate lung damage caused by S. aureus. In addition, skin H&E staining showed that MPX treatment facilitated the formation of appropriate abscesses at the subcutaneous infection site and facilitated the clearance of bacteria by the skin immune system. The above results show that MPX has good antibacterial activity and broad-spectrum antibacterial potential and can effectively prevent the invasion of subcutaneous tissue by S. aureus, providing new ideas and directions for the immunotherapy of bacterial infections.

NOX2 oxidase inhibitor GSK2795039 possess antiviral activity against H1N1 influenza A virus in vitro and vivo.

The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. The previous research has shown that NOX2 involved in IAV replication, but the detailed mechanism has not been reported. In the present study we investigated the roles of NOX2 in host inflammatory response and IAV replication using a novel inhibitor GSK2795039. The drug significantly reduced H1N1 virus induced NOX2 activity and ROS release in human lung epithelial cells. The results of time course experiments suggested that GSK2795039 inhibited an early post-entry step of viral infection. Concomitantly, there was a decreased expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-ß and interleukin (IL)-6) in NOX2 suppressed cells. In vivo, compared with control groups, suppression of NOX2 improved the survival rate of mice infected with H1N1 virus (42.9% in GSK2795039 treated mice versus >0% of control mice) and viral burden also decreased in the GSK2795039 treated group. Thus, our data demonstrated a critical role for NOX2 in the establishment of H1N1 infection and subsequent inflammatory reactions, which suggest that GSK2795039 may be a potential therapeutic drug for IAV infection.

Effects of the Antimicrobial Peptide Mastoparan X on the Performance, Permeability and Microbiota Populations of Broiler Chickens.

Restrictions on antibiotics are driving the search for alternative feed additives to promote gastrointestinal health and development in broiler chicken production. Proteins including antimicrobial peptides can potentially be applied as alternatives to antibiotics and are one of the most promising alternatives. We investigated whether the addition of MPX to the diet affects the production performance, immune function and the intestinal flora of the caecal contents of broiler chickens. One hundred one-day-old chickens were randomly divided into two groups: control (basal diet) and MPX (20 mg/kg) added to the basal diet. The results indicated that dietary supplementation with MPX improved the performance and immune organ index, decreased the feed conversion ratio, increased the villus length, maintained the normal intestinal morphology and reduced the IL-6 and LITNF mRNA expression levels of inflammation-related genes. In addition, MPX increased the mRNA expression of the digestive enzymes FABP2 and SLC2A5/GLUT5 and the tight junction proteins ZO-1, Claudin-1, Occludin, JAM-2 and MUC2, maintained the intestinal permeability and regulated the intestinal morphology. Moreover, MPX increased the CAT, HMOX1 and SOD1 mRNA expression levels of the antioxidant genes. Furthermore, a 16S rRNA microflora analysis indicated that the abundance of Lactobacillus and Lactococcus in the cecum was increased after addition of MPX at 14 d and 28 d. This study explored the feasibility of using antimicrobial peptides as novel feed additives for broiler chickens and provides a theoretical basis for their application in livestock.

Effect of Gan Cao (Glycyrrhiza uralensis Fisch) polysaccharide on growth performance, immune function, and gut microflora of broiler chickens.

Glycyrrhiza uralensis Fisch, also called Gan Cao, is a commonly prescribed herb in traditional Chinese medicine. Gan Cao is associated with immune-modulation and antitumor potential though its mechanism of action is not well-known. To explore the effects of different dietary levels of Gan Cao polysaccharide (GCP) on broilers, a total of 400 Avian broiler chickens were randomly divided into 4 groups with 10 replicates of 10 broilers each. The broilers in the control group were fed a basal diet, while those in the experimental groups were fed the basal diet supplemented with 0.5%, 1.0%, and 1.5% GCP, respectively, for 42 d. The results showed a significant increase in the growth performance in the GCP groups. The antibody titer of NDV and the phagocytosis index was higher in the birds with GCP treatment than in the control group, with the 1% GCP addition displaying the highest titer. The Lactobacillus and Bifidobacteria count in the cecum content of the birds in the 1% GCP group was higher compared to the other groups. In conclusion, dietary supplementation with GCP had a substantial impact on the growth performance, immune response, and microflora population in the cecum of the birds, especially at a level of 1% addition.

Effect of Weaning at 21 Days of Age on the Content of Bile Acids in Chyme of Cecum.

This experiment was conducted to investigate the effects of weaning at 21 days of age on cecal chyme bile acids (BAs) in piglets. According to a 2 × 3 factorial design, the main factors were lactation and weaning, and the other factor was 22, 24, and 28 days of age, respectively. Piglets were randomly divided into two groups of eighteen piglets each and six piglets were selected for slaughter at 22, 24, and 28 days of age, respectively, to determine the content of different types of Bas in the intestinal lumen of the cecum. Results: (1) There was a significant interaction between weaning and age on intestinal primary Bas hyocholic acid (HCA) and chenodeoxycholic acid (CDCA) (p < 0.05), and weaning significantly increased the content of primary BAs in piglets' intestines, which showed a trend of decreasing and then increasing with the increase in piglets' age. (2) There was a significant interaction between weaning and age on intestinal secondary BAs deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) (p < 0.05). DCA and LCA in piglets' intestines tended to decrease with increasing age, while UDCA showed a trend of decreasing and then increasing with increasing piglets' age; weaning significantly increased the content of secondary BAs in piglets' intestines. (3) There was a significant interaction between weaning and age on intestinal glycine chenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA), but not on taurohyocholic acid (THCA), taurohyodeoxycholic acid (THDCA), and taurineursodeoxycholic acid (TUDCA) (p > 0.05). Weaning significantly increased the contents of GCDCA, TCDCA, TLCA, THDCA, and TUDCA in the intestinal tract (p < 0.05), while THCA content was not significant. In conclusion, weaning can increase the BAs content in the cecum of piglets, and there is an interaction between group and weaning age on BAs content.

Combined Antibody Tagged HRP Gold Nanoparticle Probe for Effective PCV2 Screening in Pig Farms.

Introduction: Porcine circovirus type 2 (PCV2) causes immune repression and intercurrent infections in pigs, resulting in a huge economic loss to the pig breeding industry. Additionally, the spread of PCV2 in pig farms can pollute the living environment of the residents in the farm's vicinity, which increases the rate of infections. Therefore, rapid and sensitive detection methods are needed for disease prevention and timely environmental cleaning. Methods: This research describes a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that utilizes gold nanoparticles (AuNPs) in a functional, specific antibody labeled probe for the detection of PCV2. Due to their high specific surface area and histocompatibility, AuNPs were used as carriers of HRP labeled anti-PCV2 antibodies to amplify the detection signal. Results: Compared to conventional sandwich ELISA procedures, this method resulted in higher sensitivity (51-fold) and a shorter assay time with a limit of detection of 195 TCID50/mL. The cross-reactivity assay demonstrated that this assay was PCV2 specific. Conclusion: The amplified Ab (HRP) labeled AuNPs probe provides a sensitive analytical approach for the determination of the traces of the PCV2 antigen in early diagnosis.

Antimicrobial peptide MPX attenuates LPS-induced inflammatory response and blood-testis barrier dysfunction in Sertoli cells.

Orchitis accounts for a high proportion of male animal reproductive disorders. Hence, it is urgent to identify drugs for the prevention and treatment of orchitis. Antimicrobial peptides (AMPs) are currently recognized as one of the most promising alternatives to antibiotics. However, the protective effects of AMPs on lipopolysaccharide (LPS)-induced orchitis have not been reported. In this study, we developed an LPS-induced orchitis model in which primary bovine Sertoli cells were used as model cells. MPX was indicated to effectively reduce the inflammatory response of Sertoli cells. MPX attenuated the gene expression of the proinflammatory cytokines TNF-α, IL-6 and IL-1ß by suppressing the MAPK pathway, especially the phosphorylation of p38 and ERK. MPX also decreased the oxidative stress response caused by LPS and upregulated Occludin and Claudin-1 expression, thereby maintaining the integrity of the blood-testis barrier. Moreover, we found that MPX inhibited apoptosis in Sertoli cells. In a mouse model, we found that MPX significantly inhibited the disruptive effects of LPS, reducing seminiferous epithelium damage, vacuolations, hyperplasia, and apoptosis in spermatogenic cells and rescuing spermatogenesis. In addition, the expression of inflammatory factors such as IL-1ß, IL-18, IL-6 and TNF-α was decreased after MPX treatment in the mouse testes. MPX had no effect on other organs in mice, indicating its safety. This study was undertaken to investigate how MPX regulates the inflammatory response in Sertoli cells and provide a reference for the clinical prevention and treatment of male animal orchitis.

Intracellular mechanism of antimicrobial peptide HJH-3 against Salmonella pullorum.

To explore the potential intracellular mechanism of the antimicrobial peptide HJH-3 in killing Salmonella, a DNA blocking test and scanning electron microscopy (SEM) were used to determine the ability of the peptide to bind bacterial DNA in vitro. Laser confocal analysis and electron microscopy were used to observe the binding of antimicrobial peptide HJH-3 and Salmonella DNA, and flow cytometry was used to analyze the effect of antimicrobial peptides on cell division in vivo. The results showed that HJH-3 can bind to DNA to block the diffusion and migration of DNA in agarose gel. Laser confocal microscopy revealed that antimicrobial peptide HJH-3 penetrated the bacterial cell membrane and bound with bacterial DNA. Transmission electron microscopy showed that antimicrobial peptide HJH-3 aggregated in the nucleoid of Salmonella cells, and through a channel in the membrane destroyed by the antimicrobial peptide, DNA and other intracellular contents were excreted, and polymerized DNA was fragmented. The results of the flow cytometry analysis confirmed that the death rate of Salmonella increased significantly after exposure to antimicrobial peptide HJH-3 and increased with increasing antimicrobial peptide concentration. These results suggest that AMP HJH-3 may be a candidate antimicrobial agent to treat infectious diseases caused by Salmonella pullorum.

The Antimicrobial Peptide MPX Can Kill Staphylococcus aureus, Reduce Biofilm Formation, and Effectively Treat Bacterial Skin Infections in Mice.

Staphylococcus aureus is a common pathogen that can cause pneumonia and a variety of skin diseases. Skin injuries have a high risk of colonization by S. aureus, which increases morbidity and mortality. Due to the emergence of multidrug-resistant strains, antimicrobial peptides are considered to be among the best alternatives to antibiotics due to their unique mechanism of action and other characteristics. MPX is an antibacterial peptide extracted from wasp venom that has antibacterial activity against a variety of bacteria. This study revealed that MPX has good bactericidal activity against S. aureus and that its minimum inhibitory concentration (MIC) is 0.08 µM. MPX (4×MIC) can kill 99.9% of bacteria within 1 h, and MPX has good stability. The research on the bactericidal mechanism found that MPX could destroy the membrane integrity, increase the membrane permeability, change the membrane electromotive force, and cause cellular content leakage, resulting in bactericidal activity. Results from a mouse scratch model experiment results show that MPX can inhibit colonization by S. aureus, which reduces the wound size, decreases inflammation, and promotes wound healing. This study reports the activity of MPX against S. aureus and its mechanism and reveals the ability of MPX to treat S. aureus infection in mice, laying the foundation for the development of new drugs for bacterial infections.