Luteolin enhanced antioxidant capability and induced pyroptosis through NF-κB/NLRP3/Caspase-1 in splenic lymphocytes exposure to ammonia.

During feeding process in intensive chicken farms, the prolonged exposure of chickens to elevated level of ammonia leads to substantial economic losses within poultry farming industry. Luteolin (Lut), known as its anti-inflammatory and antioxidant properties, possesses the ability to eliminate free radicals and enhance the activities of antioxidant enzymes, thus rendering it highly esteemed in production. The objective of this study was to examine the effects of Lut on antioxidant and anti-inflammatory responses of chicken splenic lymphocytes exposed to ammonia. In order to achieve this, we have replicated a protective model involving Lut against ammonia exposure in chicken splenic lymphocytes. The findings of the study indicated that Lut mitigated the elevation of lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) induced by ammonia poisoning. Additionally, Lut demonstrated an increase in the expression of antioxidant enzymes, namely superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Furthermore, Lut exhibited a protective effect on cell morphology and ultrastructure following exposure to ammonia. Moreover, Lut exhibited a reduction in the expression of heat shock proteins (HSPs) and inflammatory cytokines, which were found to be highly expressed in splenic lymphocytes after ammonia exposure. Additionally, Lut demonstrated the ability to inhibit the overexpression of pyroptosis-related genes and proteins (NLRP3 and Caspase-1) in splenic lymphocytes following ammonia exposure. Lut exerted an antioxidant effect on lymphocytes, counteracting elevated levels of oxidative stress following exposure to ammonia. Additionally, Lut had the potential to modulate the expression of HSPs, suppressed the inflammatory response subsequent to ammonia exposure, and influenced the expression of NLRP3 and Caspase-1, thereby mitigating pyroptosis induced by ammonia exposure. The exploration of this subject matter can elucidate the protective properties of Lut against NH4Cl-induced damage in chicken splenic lymphocytes, while also offer insights and experimental groundwork for the utilization of natural therapeutics in animal husbandry to prevent and treat ammonia-related conditions.

Screening, Identification, and Probiotic Properties of Bacillus Pumilus From Yak.

The yak has a unique physiological structure suited to life in anoxic and cold environments at high altitudes. The aim of this study was to isolate Bacillus species with good probiotic properties from yak feces. A series of tests were performed on the isolated Bacillus: 16S rRNA identification, antibacterial activity, tolerance to gastroenteric fluid, hydrophobicity, auto-aggregation, antibiotic sensitivity, growth performance, antioxidants, and immune indexes. A safe and harmless Bacillus pumilus DX24 strain with good survival rate, hydrophobicity, auto-aggregation, and antibacterial activity was identified in the yak feces. Feeding mice with Bacillus pumilus DX24 increased their daily weight gain, jejunal villus length, villi/Crypt ratio, blood IgG levels, and jejunum sIgA levels. This study confirmed the probiotic effects of Bacillus pumilus isolated from yak feces and provides the theoretical basis for the clinical application and development of new feed additives.

Enterococcus faecium inhibits NF-κB/NLRP3/IL-1ß signaling pathway and antagonizes Salmonella-mediated inflammatory response.

Aim: This study explored the protective effect of Enterococcus faecium as a probiotic against Salmonella typhimurium infection. Materials & methods: The protective role of E. faecium against tissue damage by S. typhimurium infection and the expression of inflammatory cytokines and tight junction proteins were detected by histological observation, real-time quantitative PCR and immunohistochemical methods. Results: E. faecium demonstrated a regulatory function that affected the expression of Claudin-1 and enhanced tight junctions, suppressed the NF-κB/NLRP3/IL-1ß signaling pathway and reduced the release of IL-6, TNF-α, IFN-γ, TLR4 and MYD88 and inflammatory damage to tissues by S. typhimurium in the duodenum, cecum and colon of mice. Conclusion: E. faecium antagonized S. Typhimurium alleviating inflammatory injury in mice through the NF-κB/NLRP3/IL-1ß signaling pathway.

Enterococcus faecium inhibits NF-κB/NLRP3/Caspase-1 signaling pathway to antagonize enterotoxigenic Escherichia coli-mediated inflammatory response.

Enterotoxigenic Escherichia coli (ETEC) can cause intestinal inflammation and diarrhea in yaks, which has a negative impact on their economic value. In recent years, probiotics have gained increasing attention as a pure, natural, nontoxic, harmless, and residue-free additive. However, the underlying mechanisms by which probiotics safeguard against ETEC are not completely elucidated. This study aimed to investigate the protective effect of Enterococcus faecium (E. faecium) against ETEC infection in mice through oral gavage. Morphological changes were examined through light microscopy. The expressions of inflammatory cytokines (IL-1ß, IL-6, TNF-α, IL-10, NF-κB, and NLRP3), tight junction protein (ZO-1, Claudin-1), and pyroptosis (Caspase-1, Caspase-4, and gasdermin D (GSDMD)) were detected using immunohistochemistry and quantitative real-time PCR. The results indicate that ETEC infection triggers the activation of inflammation-related pathways (NF-κB) and NLRP3 inflammasome, leading to the expression of a large number of inflammatory cytokines. Additionally, the activation of NLRP3 leads to the release of GSDMD activation through Caspase-1, ultimately resulting in inflammatory injury and pyroptosis. Feeding mice E. faecium early resulted in an increase in the expression of tight junction protein, a reduction in inflammatory cytokines, and alleviation of inflammatory injury and pyroptosis in intestinal tissues. Our research indicates that E. faecium has the ability to antagonize ETEC and provide protection to the gastrointestinal mucosa in mice.

The protective effect of Luteolin on chicken spleen lymphocytes from ammonia poisoning through mitochondria and balancing energy metabolism disorders.

Ammonia poses a significant challenge in the contemporary intensive breeding industry, resulting in substantial economic losses. Despite this, there is a dearth of research investigating efficacious strategies to prevent ammonia poisoning in poultry. Consequently, the objective of this study was to investigate the molecular mechanisms through which Luteolin (Lut) safeguards mitochondria and restores equilibrium to energy metabolism disorders, thereby shielding chicken spleen lymphocytes from the detrimental effects of ammonia poisoning. Chicken spleen lymphocytes were categorized into 3 distinct groups: the control group, the ammonia group (with the addition of 1 mmol/L of ammonium chloride), and the Lut group (with the treatment of 0.5 µg/mL of Lut for 12 h followed by the addition of 1 mmol/L of ammonium chloride). These groups were then cultured for a duration of 24 h. To investigate the potential protective effect of Lut on lymphocytes exposed to ammonia, various techniques were employed, including CCK-8 analysis, ultrastructural observation, reagent kit methodology, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). The findings indicate that Lut has the potential to mitigate the morphological damage of mitochondria caused by ammonia poisoning. Additionally, it can counteract the decline in mitochondrial membrane potential, ATP content, and ATPase activities (specifically Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca/Mg2+-ATPase) following exposure to ammonia in lymphocytes. Lut also has the ability to regulate the expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and division (Drp1 and Mff) in spleen lymphocytes after ammonia exposure. This regulation leads to a balanced energy metabolism (HK1, HK2, LDHA, LDHB, PFK, PK, SDHB, and ACO2) and provides protection against ammonia poisoning.

Effects of Probiotic Enterococcus faecium from Yak on the Intestinal Microflora and Metabolomics of Mice with Salmonella Infection.

Salmonella spp. are pathogenic bacteria that cause diarrhea, abortion, and death in yak and severely harm livestock breeding. Therefore, it is vital to identify a probiotic that effectively antagonizes Salmonella. To the best of our knowledge, few prior studies have investigated the efficacy of Enterococcus faecium against Salmonella. Here, we evaluated the enteroprotective mechanism of E. faecium in a mouse Salmonella infection model using hematoxylin-eosin (H&E) staining, quantitative real-time polymerase chain reaction (Q-PCR) technology, microbial diversity sequencing, and metabonomics. Enterococcus faecium inhibited the proinflammatory cytokines IL-1ß, IL-6, TNF-α, and IFN-γ and promoted the anti-inflammatory cytokine IL-10. The Firmicutes/Bacteroidota (F/B) ratio and the abundances of Firmicutes and Akkermansia were significantly higher in the E. faecium than in the Salmonella group. Metabonomics and microbial diversity sequencing disclosed five different metabolites with variable importance in the projection (VIP) > 3 that were characteristic of both the Salmonella and E. faecium groups. Combined omics revealed that Lactobacillus and Bacteroides were negatively and positively correlated, respectively, with cholic acid, while Desulfovibrio was positively correlated with lipids in both the control and Salmonella groups. Desulfovibrio was also positively correlated with lipids in both the Salmonella and E. faecium groups. Enterococcus faecium antagonizes Salmonella by normalizing the abundance of the intestinal microorganisms and modulating their metabolic pathways. Hence, it may efficaciously protect the host intestine against Salmonella infection.

The orf virus 129 protein can inhibit immune responses by interacting with host complement C1q binding protein in goat turbinate bone cells.

OBJECTIVE: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1ß and IFN-γ. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and co-immunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1ß and IFN-γ. C1QBP overexpression induced IFN-γ production and reduced IL-6 and IL-1ß production. Conversely, C1QBP knockdown induced IL-1ß production and reduced IFN-γ and IL-1ß production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1ß, and IFN-γ induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.

Metabolomic Analysis of Multiple Biological Specimens (Feces, Serum, and Urine) by 1H-NMR Spectroscopy from Dairy Cows with Clinical Mastitis.

Due to huge economic losses to the dairy industry worldwide, mastitis can be considered as one of the most common diseases in dairy cows. This work aimed to study this disease by comparing multiple biological specimens (feces, serum, and urine) from individuals with or without clinical mastitis. This was performed by a single analytical platform, namely 1H-NMR, through a multi-matrix strategy. Thanks to the high reproducibility of 1H-NMR, we could characterize 120 molecules across dairy cow feces, serum, and urine. Among them, 23 molecules were in common across the three biofluids. By integrating the results of multi-matrix metabolomics, several pathways pertaining to energy metabolism and amino acid metabolism appeared to be affected by clinical mastitis. The present work wished to deepen the understanding of dairy cow mastitis in its clinical form. Simultaneous analysis of metabolome changes across several key biofluids could facilitate knowledge discovery and the reliable identification of potential biomarkers, which could be, in turn, used to shed light on the early diagnosis of dairy cow mastitis in its subclinical form.

Extraction of Polysaccharides from Root of Pseudostellaria heterophylla (Miq.) Pax. and the Effects of Ultrasound Treatment on Its Properties and Antioxidant and Immune Activities.

The hydrophilic polysaccharides (PS) were isolated and purified from the tuberous roots of Pseudostellaria heterophylla. The extraction process of PS from Pesudostellariae radix was optimized by single-factor experiments and orthogonal design. The extract was purified by DEAE cellulose column to obtain the pure polysaccharide PHP. Then PHP was treated with different intensities of sonication to study the effect of sonication on PHP's characteristics and its biological activity in vitro and in vivo. The results of this study revealed that ultrasound treatment did not significantly change the properties of PHP. Further, with the increase of ultrasound intensity, PHP enhanced the proliferation and phagocytosis of macrophage RAW264.7. Meanwhile, it could also significantly improve the body's antioxidant activity and immune function. The results of this study demonstrated that PHP has the potential as a food additive with enhanced antioxidant and immune functions, and its biological activities could be enhanced by sonication.

First detection and molecular characteristics of bopivirus from goats in China.

A metavirome analysis was performed and detected bopivirus in the diarrhoeal fecal samples of goats in China. A total of 136 fecal samples were collected from yeanlings between the dates of June 2021 and January 2022 in Sichuan province, China. Moreover, "Bopivirus B" strains were detected by a specific RT-PCR targeting the 3D gene of the virus. The results showed that the overall detection rate of "Bopivirus B" was 19.12% (26/136). Additionally, there was a higher detection rate (24.05%, 19/79) in the fecal samples collected from yeanlings with diarrhea compared to those from asymptomatic animals (12.28%, 7/57). In these samples, no other common diarrhea-causing pathogens were detected except for three enteric viruses, namely caprine enterovirus, caprine kobuvirus and caprine hunnivirus (with detection rates of 13.97, 13.97, and 8.82%, respectively). Subsequently, full-length VP4, VP2, VP3, and VP1 genes from "Bopivirus B"-positive samples were amplified, cloned, sequenced, and analyzed. The phylogenetic analysis performed on the VP1 genes revealed that the identified bopivirus belonged to genotype B1 (seven strains) and B2 (three strains) and presented a high genetic diversity. Furthermore, a complete genome sequence of a "Bopivirus B" strain (SWUN/B1/2022) was obtained using PCR from fecal sample of a diarrhoeal yeanling. The complete genome was 7,309 nucleotides in length with a standard picornavirus genome organization, and shares 93.10% and 91.10% nucleotide similarity with bopivirus B1 genotype strain ovine/TB14/2010-HUN and bopivirus B2 genotype strain goat/AGK16/2020-HUN, respectively. According to the species classification criteria put forward by the International Committee on Taxonomy of Viruses and VP1 genotype, the strain SWUN/B1/2022 belongs to the bopivirus B1. This strain has unique amino acid substitutions in the VP4, VP2, VP3, and VP1 genes. Moreover, genomic recombination analysis revealed that this strain may be a minor parental strain of bopivirus B1 ovine/TB14/2010-HUN. Evolutionary analysis based on the 2C and 3CD genes revealed that the new bopivirus B1 strain SWUN/B1/2022 presents a unique evolutionary pattern. This study provided evidence to suggest that "Bopivirus B" is circulating with substantial genetic diversity in goats in China at present, and the mixed infection of "Bopivirus B" with other enteric viruses should be considered to be a composite factor in the occurrence of viral diarrhea in goats.

HABP4 overexpression promotes apoptosis in goat turbinate bone cells.

Hyaluronic acid-binding protein (HABP4) plays important roles in regulating cell cycle and apoptosis. However, its functions in regulating cell apoptosis remain unclear. To reveal the effects of HABP4 on cell proliferation, cell cycle and apoptosis, the HABP4 sequence was cloned, and we investigated the gain and loss functions of HABP4 in goat turbinate bone cells. Our results showed that a 1,496-bp HABP4 sequence was cloned successfully. The interference effect of siRNA1 on HABP4 was the strongest, reducing its mRNA expression level by 83%, decreasing the cells in the G0/G1 and S phases of the cell cycle and inhibiting cell growth and apoptosis. The overexpression of HABP4 produced contrasting results. Furthermore, an HABP4 knockdown caused the up-regulated expression of genes associated with apoptosis, including Bcl-2 and BCL2L11, but the down-regulation of Caspase3, Caspase7, Bax, PARP1, SOCS2 and P53 mRNA levels. Additionally, HABP4 overexpression significantly up-regulated the expression levels of Bax, Caspase3, Caspase7, BCL2L11, P53, SOCS2 and PARP1. However, the expression of Bcl-2 was down-regulated. These data provide an important foundation for further in-depth studies of HABP4 functions.

Development and validation of an insulated isothermal PCR assay for the rapid detection of Mannheimia haemolytica.

We developed a rapid insulated isothermal PCR (iiPCR) assay for on-site detection of Mannheimia haemolytica using a primer and probe set targeting the superoxide dismutase (sodA) gene. Our iiPCR assay detected M. haemolytica clinical isolates successfully and produced negative results on other bovine or ovine respiratory pathogens, including Histophilus somni, Bibersteinia trehalosi, Trueperella pyogenes, Streptococcus suis, and Mycoplasma spp., indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected as few as 21 copies of genomic DNA and 17.2 cfu/mL of bacterial culture, which was 10 and 100 times more sensitive than conventional PCR, respectively. Our iiPCR assay can be performed on a portable device in a total of 58 min and may be a useful tool for the detection of M. haemolytica in bovine and ovine respiratory disease in the field.

Complete Genome Sequence of a Parabacteroides distasonis Strain (CavFT hAR46) Isolated from a Gut Wall-Cavitating Microlesion in a Patient with Severe Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that Parabacteroides distasonis could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.

A real-time PCR for detection and quantification of Mycoplasma ovipneumoniae.

A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 10(2) to 2.2 × 10(7) genomes.

Selection of reference genes for quantitative real-time PCR analysis in chicken embryo fibroblasts infected with avian leukosis virus subgroup J.

BACKGROUND: The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J). FINDINGS: The expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF. CONCLUSIONS: The RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

An experimental study of the pathogenicity of a duck hepatitis A virus genotype C isolate in specific pathogen free ducklings.

Duck hepatitis A virus genotype C (DHAV-C), recognized recently, is one of the pathogens causing fatal duck viral hepatitis in ducklings, especially in Asia. To demonstrate the pathogenesis of the DHAV-C isolate, 3-day-old specific pathogen free ducklings were inoculated subcutaneously with a DHAV-C isolate and the clinical signs were observed. Virus distribution, histological and apoptotic morphological changes of various tissues were examined at different times post inoculation. The serial, characteristic changes included haemorrhage and swelling of the liver. Apoptotic cells and virus antigen staining were found in all of the tissues examined. Where more virus antigen staining was detected, there were more severe histopathological and apoptotic changes. The amount of virus antigen and the histological and apoptotic morphological changes agreed with each other and became increasingly severe with length of time after infection. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages and monocytes in immune organs such as the bursa of Fabricius, thymus and spleen, and in liver, kidney and cerebral cells. Necrosis was also observed within 72 h post inoculation in all organs examined, except the cerebrum, and was characterized by cell swelling and collapsed plasma membrane. These results suggest that the recent outbreak of disease caused by DHAV-C virus is pantropic, causing apoptosis and necrosis of different organs. The apoptosis and necrosis caused by the DHAV-C field strain in this study is associated with pathogenesis and DHAV-C-induced lesions.

Genome sequence of Mycoplasma ovipneumoniae strain SC01.

Mycoplasma ovipneumoniae is associated with chronic nonprogressive pneumonia in both sheep and goats. Studies concerning its molecular pathogenesis, genetic analysis, and vaccine development have been hindered due to limited genomic information. Here, we announce the first complete genome sequence of this organism.

Characteristics of the molecular diversity of the outer membrane protein A gene of Haemophilus parasuis.

The molecular diversity of the gene encoding the outer membrane protein A (OmpA) of Haemophilus parasuis has been unclear. In this study, the structural characteristics, sequence types, and genetic diversity of ompA were investigated in 15 H. parasuis reference strains of different serovars and 20 field isolates. Three nucleotide lengths of the complete open reading frame (ORF) of ompA were found: 1098 base pairs (bp), 1104 bp, and 1110 bp. The OmpA contained 4 hypervariable domains, mainly encoding the 4 putative surface-exposed loops, which makes it a potential molecular marker for genotyping. Western blot analysis showed that the recombinant OmpAs of serovars 4 and 5 could cross-react with antiserum to all 15 serovars. Hence, although ompA of H. parasuis exhibited high variation among serovars, this variation did not seem to affect the strong antigenic characteristics of OmpA.

Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV.

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID(50) of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the ß-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.