Monovalent, bivalent and biparatopic nanobodies targeting S1 protein of porcine epidemic diarrhea virus efficiently neutralized the virus infectivity.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets, which has brought huge economic losses to the pork industry worldwide since its first discovery in the early 1970s in Europe. Passive immunization with neutralizing antibodies against PEDV is an effective prevention measure. To date, there are no effective therapeutic drugs to treat the PEDV infection. RESULTS: We conducted a screening of specific nanobodies against the S1 protein from a phage display library obtained from immunized alpacas. Through competitive binding to antigenic epitopes, we selected instead of chose nanobodies with high affinity and constructed a multivalent tandem. These nanobodies were shown to inhibit PEDV infectivity by the neutralization assay. The antiviral capacity of nanobody was found to display a dose-dependent pattern, as demonstrated by IFA, TCID50, and qRT-PCR analyses. Notably, biparatopic nanobody SF-B exhibited superior antiviral activity. Nanobodies exhibited low cytotoxicity and high stability even under harsh temperature and pH conditions, demonstrating their potential practical applicability to animals. CONCLUSIONS: Nanobodies exhibit remarkable biological properties and antiviral effects, rendering them a promising candidate for the development of anti-PEDV drugs.

Saikosaponin B2, Punicalin, and Punicalagin in Vitro Block Cellular Entry of Feline Herpesvirus-1.

In the realm of clinical practice, nucleoside analogs are the prevailing antiviral drugs employed to combat feline herpesvirus-1 (FHV-1) infections. However, these drugs, initially formulated for herpes simplex virus (HSV) infections, operate through a singular mechanism and are susceptible to the emergence of drug resistance. These challenges underscore the imperative to innovate and develop alternative antiviral medications featuring unique mechanisms of action, such as viral entry inhibitors. This research endeavors to address this pressing need. Utilizing Bio-layer interferometry (BLI), we meticulously screened drugs to identify natural compounds exhibiting high binding affinity for the herpesvirus functional protein envelope glycoprotein B (gB). The selected drugs underwent a rigorous assessment to gauge their antiviral activity against feline herpesvirus-1 (FHV-1) and to elucidate their mode of action. Our findings unequivocally demonstrated that Saikosaponin B2, Punicalin, and Punicalagin displayed robust antiviral efficacy against FHV-1 at concentrations devoid of cytotoxicity. Specifically, these compounds, Saikosaponin B2, Punicalin, and Punicalagin, are effective in exerting their antiviral effects in the early stages of viral infection without compromising the integrity of the viral particle. Considering the potency and efficacy exhibited by Saikosaponin B2, Punicalin, and Punicalagin in impeding the early entry of FHV-1, it is foreseeable that their chemical structures will be further explored and developed as promising antiviral agents against FHV-1 infection.

Exploring PLGA-OH-CATH30 Microspheres for Oral Therapy of Escherichia coli-Induced Enteritis.

Antibiotic therapy effectively addresses Escherichia coli-induced enteric diseases, but its excessive utilization results in microbial imbalance and heightened resistance. This study evaluates the therapeutic efficacy of orally administered poly (lactic-co-glycolic acid) (PLGA)-loaded antimicrobial peptide OH-CATH30 microspheres in murine bacterial enteritis. Mice were categorized into the healthy control group (CG), untreated model group (MG), OH-CATH30 treatment group (OC), PLGA-OH-CATH30 treatment group (POC), and gentamicin sulfate treatment group (GS). Except for the control group, all other experimental groups underwent Escherichia coli-induced enteritis, followed by a 5-day treatment period. The evaluation encompassed clinical symptoms, intestinal morphology, blood parameters, inflammatory response, and gut microbiota. PLGA-OH-CATH30 microspheres significantly alleviated weight loss and intestinal damage while also reducing the infection-induced increase in spleen index. Furthermore, these microspheres normalized white blood cell count and neutrophil ratio, suppressed inflammatory factors (IL-1ß, IL-6, and TNF-α), and elevated the anti-inflammatory factor IL-10. Analysis of 16S rRNA sequencing results demonstrated that microsphere treatment increased the abundance of beneficial bacteria, including Phocaeicola vulgatus, in the intestinal tract while concurrently decreasing the abundance of pathogenic bacteria, such as Escherichia. In conclusion, PLGA-OH-CATH30 microspheres have the potential to ameliorate intestinal damage and modulate the intestinal microbiota, making them a promising alternative to antibiotics for treating enteric diseases induced by Escherichia coli.

The immune enhancing effect of antimicrobial peptide LLv on broilers chickens.

To evaluate the effect and its mechanism of heat-resistant antimicrobial peptide LLv on broilers, three hundred 1-day-old healthy AA+ female broilers were allocated into 5 groups with 6 replicates in each group and 10 birds in each replicate. Birds were given a basal diet, an antibiotic diet (10.2 mg/kg chlortetracycline hydrochloride), and the basal diet supplemented with 10, 50, and 100 mg/kg LLv for 42 d, respectively. Compared with the group which birds were fed an antibiotic-free basal diet (control group), supplementing 100 mg/kg LLv increased 21-day IgA, IgM, IL-4, AIV-Ab, IFN-γ levels and 42-day IgA, IgM, IL-4, AIV-Ab levels and reduced 42-day IL-1 levels in serum (P < 0.05). Compared with antibiotic group, the 10 and 50 mg/kg LLv decreased 42-day IgM levels in serum (P < 0.05). The 100 mg/kg LLv increased 21-day AIV-Ab levels and 42-day IL-4, AIV-Ab levels and reduced 42-day IL-1 levels in serum (P < 0.05). Compared with control group, the 100 mg/kg LLv increased the expression rate of sIgA secretory cells and sIgA content in jejunal mucosa at 21 d and 42 d (P < 0.05), which did not differ from antibiotic group (P > 0.05). Compared with antibiotic group, the 10 mg/kg LLv reduced 21-day sIgA content and the 50 mg/kg LLv reduced 42-d the expression rate of sIgA secretory cells in jejunal mucosa (P < 0.05). Compared with control group, the 100 mg/kg LLv increased the expression of TCR, IL-15, CD28, BAFF, CD86, CD83, MHC-II, and CD40 genes in jejunal mucosa at 21 d and 42 d (P < 0.05). Compared with antibiotic group, the 100 mg/kg LLv increased the expression of 21-day BAFF, CD40, MHC-II, CD83 genes and the expression of 42-day BAFF, TCR, IL-15, CD40, CD83 genes in jejunal mucosa (P < 0.05). The results showed that the addition of LLv to the ration had a promotional effect on the immune function of broiler chickens.

High expression of antimicrobial peptides cathelicidin-BF in Pichia pastoris and verification of its activity.

Antibacterial peptides are endogenous polypeptides produced by multicellular organisms to protect the host against pathogenic microbes, they show broad spectrum antimicrobial activities against various microorganisms and possess low propensity for developing resistance. The purpose of this study is to develop recombinant antibacterial peptide cathelicidin-BF by genetic engineering and protein engineering technology, and study its antibacterial activity in vitro and in vivo, so as to provide reference for the production and application of recombinant antibacterial peptide cathelicidin-BF. In this study, on account of Pichia pastoris eukaryotic expression system, we expressed and prepared antibacterial peptide cathelicidin-BF. Then, the minimum inhibitory concentration of antibacterial peptide cathelicidin-BF and the comparison with the antibacterial activity of antibiotics were determined through the antibacterial experiment in vitro. Chickens as infection model were used to verify the antibacterial peptide activity in vivo. The results show that the bacteriostatic ability of antibacterial peptide cathelicidin-BF is similar to that of antibiotics in certain concentration, and can reach the treatment level of antibiotics. Although the mode of administration of antibacterial peptide is still limited, this study can provide reference for the future research of antibacterial peptide.

Evaluation of light-emitting diode colors and intensities on slaughter performance, meat quality and serum antioxidant capacity in caged broilers.

OBJECTIVE: This study was to evaluate the interaction of three different light-emitting diode (LED) light colors (white, green, and blue) and three intensities (5, 10, and 15 lx) on slaughter performance, meat quality and serum antioxidant capacity of broilers raised in three-layer cages. METHODS: A total of 648 (8-days-old) male broiler chicks (Cobb-500) were randomly assigned in 3×3 factorially arranged treatments: three light colors (specifically, white, blue, and green) and three light intensities (namely, 5, 10, and 15 lx) for 35 days. Each treatment consisted of 6 replicates of 12 chicks. The test lasted for 35 days. RESULTS: The semi-eviscerated weight percentage (SEWP) in 5 lx white was higher than that in 15 lx (p<0.01). The eviscerated weight percentage (EWP) (p<0.05) and water-loss percentage (WLP) (p<0.01) decreased in 10 lx white light than those in green light. Under blue light, the content of hypoxanthine (Hx) in muscle was lower than that under white and green light (p<0.01). The content of malondialdehyde (MDA) in 15 lx blue light was higher than that in 10 lx green light (p<0.05). Light color had an extremely significant effect on thigh muscle percentage, WLP, Hx, and crude protein content (p<0.01). Light intensity had a significant effect on SEWP (p<0.05), EWP (p<0.05), lightness (L*) value (p<0.05), WLP (p<0.01), and the contents of superoxide dismutase (p<0.05), MDA (p<0.01), glutathione peroxidase (p<0.01). CONCLUSION: Using white LED light with 10 lx light intensity can significantly improve the chicken quality of caged Cobb broilers, improve the content of inosine acid in chicken breast and enhance the antioxidant capacity of the body. We suggest that the broiler farm can use 10 lx white LED light source for lighting in 8 to 42 days.

Knockdown of Adra2a Increases Secretion of Growth Factors and Wound Healing Ability in Diabetic Adipose-Derived Stem Cells.

Studies showed that compared to normal adipose-derived stem cells (ASCs), ASCs from type 2 diabetic (T2D) mice were less effective in treating diabetic cutaneous wounds. However, the mechanisms remain unclear. Our transcriptomic profiling comparison showed that the expression of α2A-adrenergic receptor (Adra2a) was significantly increased in ASCs from T2D mice (T2D ASCs). Therefore, the purpose of this study was to investigate whether the elevated Adra2a is involved in the diminished wound-healing capabilities of T2D ASCs. RNA-seq was used to compare the transcriptomic profiles of T2D and normal ASCs. The differential genes were verified by real-time RT-qPCR. Clonidine was used to active Adra2a, and lentivirus-mediated RNAi was used to knockdown Adra2a. The secretion and expression of growth factors were detected by enzyme-linked immunosorbent assay (ELISA) and real-time RT-qPCR, respectively. The cAMP and PKA activity were also detected. Wound healing abilities of various ASCs were assessed in T2D mouse excisional wound models. The results showed Adra2a agonist clonidine decreased the expression and secretion of growth factors, cAMP content, and activity of PKA in ASCs, while Adra2a knockdown T2D ASCs showed the opposite effects. Adra2a knockdown T2D ASCs also showed increased wound-healing capabilities compared to untreated T2D ASCs. Altogether, T2D increased Adra2a expression, which may subsequently decrease the expression and secretion of growth factors and eventually diminish the wound-healing capabilities of T2D ASCs. Adra2a knockdown can restore the secretion of growth factors in T2D ASCs and then accelerate the wound healing, which may provide a new possibility in the treatment of diabetic wounds.

Drinking Water Supplemented with Acidifiers Improves the Growth Performance of Weaned Pigs and Potentially Regulates Antioxidant Capacity, Immunity, and Gastrointestinal Microbiota Diversity.

This study evaluated the potential effects of adding acidifiers to the drinking water on the growth performance, complete blood count, antioxidant indicators, and diversity of gastrointestinal microbiota for weaned pigs. A total of 400 weaned pigs were randomly divided into four treatments. Pigs were fed the same basal diet and given either water (no acidifier was added, control) or water plus blends of different formulas of acidifiers (acidifier A1, A2, or A3) for 35 days. On d 18 and 35 of the experimental period, 64 pigs (four pigs per pen) were randomly selected to collect blood for a CBC test (n = 128) and an antioxidant indicators test (n = 128); 24 pigs (six pigs per group) were randomly selected to collect fresh feces (n = 48) from the rectum for 16S rRNA gene sequencing. Compared to the control, supplementing the drinking water with acidifiers improved the growth performance and survival rate of weaned pigs. Acidifier groups also increased serum catalase (CAT) and total antioxidant capacity (T-AOC) activities, while also displaying a decreased malondialdehyde (MDA) concentration compared to the control. The relative abundance of Firmicutes in the acidifier A1 group was greater than that in the control group (p < 0.05) on d 35; the relative abundance of Lactobacillus in the acidifier A1 group was greater than that in the control group (p < 0.05) on d 18 and 35. The microbial species Subdoligranulum or Ruminococcaceae_UCG-005 had significantly positive correlations with ADG and ADFI or with serum antioxidant indicators, respectively. These findings suggest that supplementing the drinking water with an acidifier has a potential as an antioxidant, which was reflected in the improvement of growth performance, immunity, antioxidant capacity, and intestinal flora.

Interactions between color and intensity of LED light on growth performance, serum biochemical profile, immune response variable, and nutrient apparent utilization in broiler chicken.

This study investigated the effects of light-emitting diode (LED) color and intensity of broilers. One-day-old Cobb-500 broilers (n = 648) were fed nine groups with six replicates; three light colors (white, blue, & green) and intensities (for 1 to 7 days, viz., 20, 40, and 60 lx; for 8 to 42 days, viz., 5, 10, and 15 lx) were applied. Test lasted for 42 days. Results indicated that compared with blue light, 60-lx white light for 1 to 7 days increased the average daily gain (ADG) and average daily feed intake (ADFI) of broilers (p < 0.01). In the 10-lx light groups, the levels of interleukin-2 (IL-2) and the concentrations of albumin (ALB) (p < 0.05) increased. Moreover, the nutrient apparent utilization for ether extract (EE) under 10-lx green light was higher than that under 15-lx blue light (p < 0.01). The interaction effects of light intensity and light color had an extremely significant influence on the ADG for 1 to 7 days, IL-2 level, ALB content, and EE apparent utilization rate (p < 0.01) and had a significant influence on the ADFI and F/G for 1 to 7 days (p < 0.05). The production performance of broilers reared in three-layer cage could be improved by using 60-lx white LED light for 1 to 7 days and 5- to 10-lx green LED light or 10-lx white LED light for 8 to 42 days.

Baicalin protects LPS-induced blood-brain barrier damage and activates Nrf2-mediated antioxidant stress pathway.

The integrity of the BBB is closely related to brain microvascular endothelial cells and TJs, and its dysfunction can lead to stroke, multiple sclerosis, extracranial injury and neurodegenerative diseases. Baicalin is one of the main bioactive extracts from Scutellaria Baicalensis Georgi, which has anti-inflammatory and anti-oxidation pharmacological functions. Preventive protection with baicalin for seven consecutive days can significantly improve the appearance of cell apoptosis and Fluorescein sodium infiltration in the brain tissue of BALB/C mice. In addition, baicalin can inhibit the production of pro-inflammatory cytokines induced by LPS in mice and bEnd.3 cells, including IL-1ß and TNF-α. At the same time, LPS caused a decrease in tight junction proteins in the blood-brain barrier, but baicalin can alleviate the damage of the blood-brain barrier by up-regulating Claudin-5 and ZO-1 protein expression. In addition, the results showed that baicalin reduced the production of ROS and MDA in bEnd.3 cells and promoted the production of SOD, and up-regulated the expression of Nrf2, HO-1 and NQO1. The mechanism of this change was mediated by activating the Nrf2 signaling pathway. All in all, Baicalin protected LPS-induced blood-brain barrier damage and activateed Nrf2-mediated antioxidant stress pathway.

Effects of Enteromorpha polysaccharide dietary addition on the diversity and relative abundance of ileum flora in laying hens.

This experiment explored the effects of different levels of Enteromorpha polysaccharide dietary addition on the intestinal flora structure in laying hens. A total of 300 Hy-line brown laying hens aged 280 days old were selected according to the principle of equal weight and egg production rate. Group 1 was the blank control group fed with basic diet, Group 2 was the antibiotic control group supplemented with bacitracin zinc (0.005%) and basic diet, and Groups 3-5 were the experimental groups that received 0.1%, 0.2%, and 0.4% Enteromorpha polysaccharides in their diets, respectively. Four replicates per group and 15 repeats per replicate were prepared. The pretrial period was 10 days, and the normal trial period was 42 days. The ileum contents of laying hens were collected aseptically toward the end of the test to detect the diversity and relative abundance of the flora. Results were as follows. (1) Bacterial abundance (ACE and Chao1) and diversity (Simpson and Shannon) indexes were not significantly different between the control and test groups (P > 0.05). (2) Compared with that in group 1, the relative abundance of Firmicutes in groups 4 and 5 significantly increased by 14.13% (P < 0.05) and 13.70% (P < 0.05), respectively. The relative abundance of Bacilli in group 4 was significantly increased by 11.94% (P < 0.05) and 12.86% (P < 0.05) compared with those in groups 1 and 3, respectively. The relative abundance of Lactobacillales in group 4 was significantly increased by 27.02% (P < 0.05) compared with that in group 1. The relative abundance of Lactobacillaceae in group 4 was significantly increased by 22.92% (P < 0.05) and 11.4% (P < 0.05) compared with those in groups 1 and 3, respectively. The relative abundance of Lactobacillus in groups 4 and 5 was increased by 19.75% (P < 0.05) and 18.54% (P < 0.05), respectively. CONCLUSION: The dietary addition of 0.2% Enteromorpha polysaccharides can remarkably increase the relative abundance of Firmicutes phylum, Bacilli class, Lactobacillales order, Lactobacillaceae family, and Lactobacillus genus in the ileum of laying hens. This effect was equivalent to the action of bacitracin zinc and had no substantial influence on the diversity of ileum flora.

mRNA expression in different developmental stages of the chicken bursa of Fabricius.

The bursa of Fabricius, the central humoral immune organ unique to birds, plays an important role in B-lymphocyte differentiation. In order to gain a better understanding of the molecular mechanism of critical biological processes like B-cell immigration, differentiation, and final emigration, the transcriptional changes during embryonic and posthatch development of this organ were investigated. We generated a cDNA library from total RNA isolated from 3 representative developmental stages (embryonic day [ED] 10, posthatch d 2 and d 21). We generated over 70 million high-quality reads from the cDNA library by using deep sequencing. The uniquely mapped sequences of ED 10, d 2 and d 21 were 71087280, 59167491 and 70263675 respectively. All of the differential expressed genes were involved in Vitamin A metabolism, regulation of actin cytoskeleton, Wnt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, Notch signaling pathway, Toll-like receptor signaling pathway. The RNA-seq analysis provides a powerful method for analyzing the transcriptome and investigating the transcriptional changes of different development stages of bursa of Fabricius. The assembled bursa transcriptome provides an essential resource for future investigations about chicken Bursa development.

WITHDRAWN: Development of an RT-PCR-RFLP assay for the detection and differentiation of wild-type and vaccine strains of classical swine fever virus.

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