Comparison of Synthetic Methods and Identification of Several Artificial Antigens of Deoxynivalenol.

The purpose of this experiment was to study the design and modification of hapten molecules and artificial antigen molecules of deoxynivalenol (DON), and to compare the preparation and identification methods of four artificial antigens. According to the characteristics of the molecular structure of DON, four artificial antigen coupling methods were designed-namely, N,N'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), isobutyl chloroformate (IBCF), and N-hydroxysuccinimide (NHS)-to prepare artificial antigens and detection antigens. Through ultraviolet (UV), infrared (IR), and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), along with other physical and chemical identification methods and animal immunisation, the best artificial antigen coupling method was screened. The results showed that the CDI method achieved the best effect among the synthesis methods. The titre of anti-DON polyclonal antibody (pAb) produced by animal immunisation reached 1: (6.4 × 103). The half inhibitory concentration (IC50) was 47.75 ng/mL, the cross-reaction rate with 3-acetyldeoxynivalenol (3-AcDON) was slightly higher at 35.3%, and there was no cross-reaction with other compounds; therefore, four artificial antigens were successfully prepared by using the molecular structure of DON. Through identification, the CDI method was screened as the best artificial antigen synthesis method, with the highest DON pAb titre, the best sensitivity, and the strongest specificity. This will lay a solid antigenic foundation for the preparation of better anti-DON monoclonal antibodies (mAbs) in the future.

Effects of Different Sample Pulverisation Methods on the Extraction of Metabolites from the Fermented Cottonseed Meal Based on UPLC-Q-TOF-MS.

The precondition of studying biological sample is to extract sample metabolites by the best pretreatment methods. There is already limited information about pretreatments of fermented feed metabolites. The study compared the extraction effects of different pulverisation methods used in the sample pretreatment process for the extraction of metabolites from cottonseed meal fermented by Lactobacillus acidophilus based on UPLC-Q-TOF-MS. The extraction effects of three pretreatments (non-pulverisation (WF), pulverisation (F), and high-speed homogenisation methods (YJ)) were compared with the numbers of metabolites and the normalised peak areas of the metabolites. The results showed that the number of metabolites extracted with three pulverisation methods were 1745, 1896, 2132 (ESI+ mode) and 1447, 1675, 2073 (ESI- mode), respectively. The number of variable importance plot (VIP) metabolites and the relative peak areas of metabolites showed that the trend was YJ > F > WF. The extraction effect of high-speed homogenisation method was the best way to extract metabolites from the fermented cottonseed meal. This study built a foundation work for the further research of the fermented feed metabolomics.

Advances in Antibody Preparation Techniques for Immunoassays of Total Aflatoxin in Food.

Aflatoxin (AF) contamination is a major concern in the food and feed industry because of its prevalence and toxicity. Improved aflatoxin detection methods are still needed. Immunoassays are an important method for total aflatoxin (TAF) analysis in food due to its technical advantages such as high specificity, sensitivity, and simplicity, but require high-quality antibodies. Here, we first review the three ways to prepare high-quality antibodies for TAF immunoassay, second, compare the advantages and disadvantages of antigen synthesis methods for B-group and G-group aflatoxins, and third, describe the status of novel genetic engineering antibodies. This review can provide new methods and ideas for the development of TAF immunoassays.

What role does pyroptosis play in microbial infection?

Pyroptosis, a type of programmed cell death mediated by gasdermin, is characterized by the swelling and rupture of cells, release of cellular contents and a strong inflammatory response, which is critical for controlling microbial infection. Pattern recognition receptors recognize the intracellular and extracellular pathogenic microbial components and stimulate the organism's inflammatory response by activating the pyroptosis signaling pathway and releasing interleukin-1ß (IL-1ß), IL-18, and other inflammatory factors to promote pathogen clearance and prevent infection. In the process of continuous evolution, pathogens have developed multiple strategies to modulate the occurrence of pyroptosis and thus enhance their ability to induce disease; that is, the competition between host cells and pathogens controls the occurrence of pyroptosis. Competition can directly affect tissue inflammation outbreaks and even alter cell survival. Studies have shown that various bacterial infections, including Shigella flexneri, Salmonella, Listeria monocytogenes, and Legionella pneumophila, can lead to pyroptosis. Pyroptosis is associated with the occurrence and development of various diseases caused by microbial infection, and the identification of molecules related to the pyroptosis signaling pathway may provide new drug targets for the treatment of related diseases. This study reviews the molecular mechanisms of pyroptosis and the role of pyroptosis in microbial infection-related diseases.

Highly Sensitive Detection of PCV2 Based on Tyramide Signals and GNPL Amplification.

The frequent emergence of secondary infection and immunosuppression after porcine circovirus type 2 (PCV2) infection highlights the need to develop sensitive detection methods. A dual-signal amplification enzyme-linked immunosorbent assay (ELISA) based on a microplate coated with gold nanoparticle layers (GNPL) and tyramide signal amplification (TSA) was established. Results confirmed that the microplates coated with GNPL have a strong binding ability to the antibody without affecting the biological activity of the antibody. The microplates coated with GNPL have strong binding ability to the antibody, and the amplification of the tyramide signal is combined to further improve the sensitivity of PCV2. The PCV2 antibody does not crossreact with other viruses, demonstrating that the method has good specificity. A dual-signal amplification strategy is developed using microplates modified with GNPL and TSA to sensitively detect PCV2.

Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat.

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

Inhibitory Effects of Antimicrobial Peptide JH-3 on Salmonella enterica Serovar Typhimurium Strain CVCC541 Infection-Induced Inflammatory Cytokine Release and Apoptosis in RAW264.7 Cells.

The antibiotic resistance of Salmonella has become increasingly serious due to the increased use of antibiotics, and antimicrobial peptides have been considered as an ideal antibiotic alternative. Salmonella can induce macrophage apoptosis and thus further damage the immune system. The antimicrobial peptide JH-3 has been shown to have a satisfactory anti-Salmonella effect in previous research, but its mechanism of action remains unknown. In this study, the effects of JH-3 on macrophages infected with Salmonella Typhimurium CVCC541 were evaluated at the cellular level. The results showed that JH-3 significantly alleviated the damage to macrophages caused by S. Typhi infection, reduced the release of lactic dehydrogenase (LDH), and killed the bacteria in macrophages. In addition, JH-3 decreased the phosphorylation level of p65 and the expression and secretion of interleukin 2 (IL-2), IL-6, and tumor necrosis factor-α (TNF-α) by inhibiting the activation of the mitogen-activated protein kinase (MAPK) (p38) signaling pathway and alleviating the cellular inflammatory response. From confocal laser scanning microscopy and flow cytometry assays, JH-3 was observed to inhibit the release of cytochrome c in the cytoplasm; the expression of TNF-αR2, caspase-9, and caspase-8; to further weaken caspase-3 activation; and to reduce the S.-Typhi-induced apoptosis of macrophages. In summary, the mechanism by which JH-3 inhibits Salmonella infection was systematically explored at the cellular level, laying the foundation for the development and utilization of JH-3 as a therapeutic alternative to antibiotics.

Methods for the detection and characterization of Streptococcus suis: from conventional bacterial culture methods to immunosensors.

One of the most important zoonotic pathogens worldwide, Streptococcus suis is a swine pathogen that is responsible for meningitis, toxic shock and even death in humans. S. suis infection develops rapidly with nonspecific clinical symptoms in the early stages and a high fatality rate. Recently, much attention has been paid to the high prevalence of S. suis as well as the increasing incidence and its epidemic characteristics. As laboratory-acquired infections of S. suis can occur and it is dangerous to public health security, timely and early diagnosis has become key to controlling S. suis prevalence. Here, the techniques that have been used for the detection, typing and characterization of S. suis are reviewed and the prospects for future detection methods for this bacterium are also discussed.

M protein is sufficient for assembly and release of Peste des petits ruminants virus-like particles.

Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs.

Development of an enzyme-linked immunosorbent assay for detection of clopidol residues in chicken tissues.

BACKGROUND: Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medicine. The aim of this study was to prepare monoclonal antibodies (mAbs) against clopidol (CLOP) and develop an immunoassay for detecting CLOP residues in chicken tissues. After derivation, CLOP hapten was conjugated to carrier proteins to synthesize the artificial antigen, and immunized Balb/C mice were employed to screen mAbs. RESULTS: A sensitive hybridoma named C1G3 was screened out and two indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curves were established. For the traditional two-step assay the linear range was from 0.06 to 98 ng mL(-1) , with half-maximal inhibitory concentration (IC50 ) and limit of detection (LOD) values of 2.76 ng mL(-1) and 0.03 ng mL(-1) respectively, while the rapid one-step icELISA had a working range from 0.08 to 102 ng mL(-1) , with IC50 and LOD values of 3.52 ng mL(-1) and 0.03 ng mL(-1) respectively. It was also indicated that a 10-fold dilution in chicken muscles gave an inhibition curve almost the same as that obtained in phosphate-buffered saline. When applied to spiking tests in chicken samples, the correlation coefficient (R(2) ) between concentrations added and measured was 0.9534. CONCLUSION: The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics.

An approach to rapidly identify the gender of the pigeon by using cross-priming amplification with immune-chromatographic strip.

Most birds are monomorphic species and breeds, which makes it difficult to determine their gender by appearances, especially the pigeon is a farm animal with economic interest in meat production, ornamentals, sports, and experimental animals. Until now, the available methods for determining the gender of pigeons have mainly consisted of endoscopy, laparoscopy, karyotyping, polymerase chain reaction (PCR), and other similar techniques. Nonetheless, these methods have notable limitations, such as high expenses, invasiveness, and time-consuming procedures, which hinder their practicality for efficiently determining the gender of pigeons. Therefore, an easy, accurate, sensitive, on-site, affordable, and applicable rapid identification of the gender of the pigeon is widely needed for the owner of the pigeon. The purpose of this study was to develop and evaluate the efficacy of Cross-priming amplification (CPA) combined with an immune-chromatographic strip (CPA-strip) for gender identification of the pigeon. The methodology was optimized through various experimental trials. Subsequently, ten samples collected from pigeons were subjected to analysis using the optimized CPA-strip assay, and the results indicated that all female samples were accurately detected. In contrast, the blood samples collected from chickens and ducks were negative when tested with the CPA-strip assay. In conclusion, our study demonstrates the successful establishment of an immune-chromatographic CPA-strip assay for the on-site gender determination of pigeons with high accuracy.

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.

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.

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.

Production and characterisation of monoclonal antibodies against 19-Nortestosterone.

OBJECTIVE: To produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. METHODS: Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). RESULTS: Five hybridoma cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG(1) isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7 × 10(9) L/mol. The titers and IC(50) values of purified ascite fluids were in the range of (0.64-2.56) × 10(5) and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, (-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (<0.01%) with other steroids was observed. CONCLUSION: The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.

Effect of main inorganic metal elements in Panax ginseng field soil on the photodegradation of fluazifop-p-butyl.

The widespread use of pesticides contributes to their existence in the environment. The compounds with photocatalytic activity in environmental matrixes play a significant effect on the photodegradation of pesticides. In order to clear the photolysis effects of the main characteristic inorganic metal elements in the of Panax ginseng field soil on fluazifop-p-butyl, a series of tests were carried out. The obtained results indicated that Mn2+ and Sn+ exhibited a significant photosensitization on the ultraviolet photodegradation of fluazifop-p-butyl. Also the high content of VO3- and Mo7O246- in the photolysis system showed a photoquenching on fluazifop-p-butyl, but the low content is a photosensitive effect. However, in the photolysis system, as the concentration of Co2+ and Li+ increases, the photoquenching effect on fluazifop-p-butyl becomes obvious, and no photosensitization at any tested concentration of them.

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites.

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 µg/L in the supernatants and 18.12 to 31.46 µg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, ß-ZEL, α-ZAL, ß-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 µg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 µg/L, and its linear working range was between 1.03 and 288.55 µg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics.

BACKGROUND: This study aimed to explore the zearalenone (ZEN) immunogen synthesis method, immunogenicity, and antibody characteristics and to lay a foundation for the establishment of immunoassay methods for ZEN single residue and ZEN and its analogs total residue. METHODS: Based on the molecular structure and active sites of ZEN, oxime active ester (OAE), condensation mixed anhydride (CMA), formaldehyde (FA), and 1,4-butanediol diglycidyl ether method (BDE) were designed and used for immunogen (ZEN-BSA) synthesis. The immunogens were identified by infrared (IR) and ultraviolet (UV) spectra and gel electrophoresis (SDS-PAGE) and were then used to immunize Balb/c mice to prepare ZEN polyclonal antibody (ZEN pAb). The titers and sensitivity of the ZEN pAb were determined by indirect noncompetitive ELISA (inELISA) and indirect competitive ELISA (icELISA), respectively, and its specificity was assessed by the cross-reaction test (CR). RESULTS: ZEN-BSA was successfully synthesized, and the molecular binding ratios of ZEN to BSA were 17.2 : 1 (OAE), 14.6 : 1 (CMA), 9.7 : 1 (FA), and 8.3 : 1 (BDE), respectively. The highest inELISA titers of ZEN pAb of each group were 1 : (6.4 × 103) (OAE), 1 : (3.2 × 103) (CMA), 1 : (1.6 × 103) (FA), and 1 : (1.6 × 103) (BDE), respectively. The 50% inhibition concentrations (IC50) for ZEN by icELISA of each group were 11.67 µg/L (OAE), 16.29 µg/L (CMA), 20.92 µg/L (FA) and 24.36 µg/L (BDE), respectively. ZEN pAb from the mice immunized with ZEN-BSA (OAE) and ZEN-BSA (CMA) had class broad specificity to ZEN and its analogs. The CRs of ZEN pAb with α-ZAL, ß-ZAL, α-ZOL, ß-ZOL, and ZON were 36.53%, 16.98%, 64.33%, 20.16%, and 10.66%, respectively. ZEN pAb from the mice immunized with ZEN-BSA (FA) and ZEN-BSA (BDE) had high specificity for ZEN. The CRs of ZEN pAb with its analogs were all less than 1.0%. CONCLUSION: This study demonstrated that the preparation of the class broad-specificity antibodies of ZEN and its analogs can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the OAE method, while the preparation of highly specific antibodies can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the FA method. These findings lay the material and technical foundation for immunoassay of ZEN single residue and ZEN and its analogs total residue.

The Antimicrobial Peptide Mastoparan X Protects Against Enterohemorrhagic Escherichia coli O157:H7 Infection, Inhibits Inflammation, and Enhances the Intestinal Epithelial Barrier.

Escherichia coli can cause intestinal diseases in humans and livestock, destroy the intestinal barrier, exacerbate systemic inflammation, and seriously threaten human health and animal husbandry development. The aim of this study was to investigate whether the antimicrobial peptide mastoparan X (MPX) was effective against E. coli infection. BALB/c mice infected with E. coli by intraperitoneal injection, which represents a sepsis model. In this study, MPX exhibited no toxicity in IPEC-J2 cells and notably suppressed the levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO), and lactate dehydrogenase (LDH) released by E. coli. In addition, MPX improved the expression of ZO-1, occludin, and claudin and enhanced the wound healing of IPEC-J2 cells. The therapeutic effect of MPX was evaluated in a murine model, revealing that it protected mice from lethal E. coli infection. Furthermore, MPX increased the length of villi and reduced the infiltration of inflammatory cells into the jejunum. SEM and TEM analyses showed that MPX effectively ameliorated the jejunum damage caused by E. coli and increased the number and length of microvilli. In addition, MPX decreased the expression of IL-2, IL-6, TNF-α, p-p38, and p-p65 in the jejunum and colon. Moreover, MPX increased the expression of ZO-1, occludin, and MUC2 in the jejunum and colon, improved the function of the intestinal barrier, and promoted the absorption of nutrients. This study suggests that MPX is an effective therapeutic agent for E. coli infection and other intestinal diseases, laying the foundation for the development of new drugs for bacterial infections.

Cloning and Identification of PK15 Cells for Enhanced Replication of Classical Swine Fever Virus.

INTRODUCTION: Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs, leading to economic losses around the world. Attenuated live vaccines with CSFV antigens have played an important role in the prevention and control of the disease. Porcine kidney 15 (PK15) cells have been widely used for the propagation of CSFV, but this cell line is not efficient or homogeneously susceptible to viral infection. MATERIAL AND METHODS: To achieve a homogeneous PK15 cell line which enabled high titre replication of CSFV, we used the limiting dilution cell cloning method. RESULTS: We developed two cell clones, PK15-1A6 and PK15-3B1, which respectively have high- and low-permissive phenotypes to CSFV infection. The PK15-1A6, PK15-3B1, and PK15 parent cells showed different characteristics in cell proliferation rate, susceptibility to CSFV infection, and CSFV production. The mean virus titres per millilitre reflected by TCID50 values in PK15-1A6, PK15-3B1, and PK15 parent cells were 106.85, 103.63, and 104.74, respectively. CONCLUSION: The PK15-1A6 cell clone is more permissive to CSFV infection than the PK15 parent cells. The screened high-permissive cells will be useful for CSFV propagation and vaccine development in vitro, and facilitate research on the pathogenicity of CSFV.