Characterization of the Hepatic Transcriptome for Divergent Immune-Responding Sheep Following Natural Exposure to Gastrointestinal Nematodes.

Infections with gastrointestinal nematodes (GINs) reduce the economic efficiency of sheep operations and compromise animal welfare. Understanding the host's response to GIN infection can help producers identify animals that are naturally resistant to infection. The objective of this study was to characterize the hepatic transcriptome of sheep that had been naturally exposed to GIN parasites. The hepatic transcriptome was studied using RNA-Sequencing technology in animals characterized as high (n = 5) or medium (n = 6) based on their innate immune acute-phase (AP) response phenotype compared with uninfected controls (n = 4), and with biased antibody-mediated (AbMR, n = 5) or cell-mediated (CMR, n = 5) adaptive immune responsiveness compared to uninfected controls (n = 3). Following the assessment of sheep selected for innate responses, 0, 136, and 167 genes were differentially expressed (DE) between high- and medium-responding animals, high-responding and uninfected control animals, and medium-responding and uninfected control animals, respectively (false discovery rate (FDR) < 0.05, and fold change |FC| > 2). When adaptive immune responses were assessed, 0, 53, and 57 genes were DE between antibody- and cell-biased animals, antibody-biased and uninfected control animals, and cell-biased and uninfected control animals, respectively (FDR < 0.05, |FC| > 2). Functional analyses identified enriched gene ontology (GO) terms and metabolic pathways related to the innate immune response and energy metabolism. Six functional candidate genes were identified for further functional and validation studies to better understand the underlying biological mechanisms of host responses to GINs. These, in turn, can potentially help improve decision making and management practices to increase the overall host immune response to GIN infection.

The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line.

BACKGROUND: Johne's disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne's disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne's disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne's disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 72 h. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved. RESULTS: Immune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and less difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways. CONCLUSIONS: Identifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes.

Identification and characterization of long non-coding RNAs in mammary gland tissues of Chinese Holstein cows.

LncRNAs (Long non-coding RNA) is an RNA molecule with a length of more than 200 bp. LncRNAs can directly act on mRNA, thus affecting the expression of downstream target genes and proteins, and widely participate in many important physiological and pathological regulation processes of the body. In this study, RNA-Seq was performed to detect lncRNAs from mammary gland tissues of three Chinese Holstein cows, including three cows at 7 d before calving and the same three cows at 30 d postpartum (early lactation stage). A total of 1,905 novel lncRNAs were detected, 57.3% of the predicted lncRNAs are ≥ 500 bp and 612 lncRNAs are intronic lncRNAs. The exon number of lncRNAs ranged from 2 to 10. A total of 96 lncRNAs were significantly differentially expressed between two stages, of which 47 were upregulated and 49 were downregulated. Pathway analysis found that target genes were mainly concentrated on the ECM-receptor interaction, Jak-STAT signaling pathway, PI3K-Akt signaling pathway, and TGF-beta signaling pathway. This study revealed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows at non-lactation and early lactation periods, and provided a basis for studying the functions of lncRNAs in Holstein cows during different lactation periods.

The mammary gland of dairy cows is the main place of milk synthesis and secretion, and plays a vital role in the process of milk production. LncRNAs (Long non-coding RNAs) are a class of non-coding RNAs with a length greater than 200 bp and do not encode protein, which can regulate gene expression at the transcriptional, post-transcriptional and chromatin levels, with biological functions such as regulating cell proliferation, differentiation, and apoptosis. Relevant studies in humans and model animals have shown that lncRNAs participate in mammalian mammary gland development and lactation, but there are few studies on lncRNAs regulation of mammary gland development and lactation in dairy cows. Therefore, this study aims to reveal the potential role of lncRNAs in the mammary gland of dairy cows through screening, identification, and functional research of differentially expressed lncRNAs at different periods of mammary gland development (pregnancy and early lactation period). It provides a reference for the follow-up study on the regulatory mechanism of dairy cows' mammary gland health.

Genomic Regions Associated with Resistance to Gastrointestinal Nematode Parasites in Sheep-A Review.

Gastrointestinal nematodes (GINs) can be a major constraint and global challenge to the sheep industry. These nematodes infect the small intestine and abomasum of grazing sheep, causing symptoms such as weight loss, diarrhea, hypoproteinemia, and anemia, which can lead to death. The use of anthelmintics to treat infected animals has led to GIN resistance, and excessive use of these drugs has resulted in residue traced in food and the environment. Resistance to GINs can be measured using multiple traits, including fecal egg count (FEC), Faffa Malan Chart scores, hematocrit, packed cell volume, eosinophilia, immunoglobulin (Ig), and dagginess scores. Genetic variation among animals exists, and understanding these differences can help identify genomic regions associated with resistance to GINs in sheep. Genes playing important roles in the immune system were identified in several studies in this review, such as the CFI and MUC15 genes. Results from several studies showed overlapping quantitative trait loci (QTLs) associated with multiple traits measuring resistance to GINs, mainly FEC. The discovery of genomic regions, positional candidate genes, and QTLs associated with resistance to GINs can help increase and accelerate genetic gains in sheep breeding programs and reveal the genetic basis and biological mechanisms underlying this trait.

Novel lncRNA regulatory elements in milk somatic cells of Holstein dairy cows associated with mastitis.

Despite regulatory elements such as long non - coding RNAs representing most of the transcriptome, the functional understanding of long non - coding RNAs in relation to major health conditions including bovine mastitis is limited. This study examined the milk somatic cell transcriptome from udder quarters of 6 Holstein dairy cows to identify differentially expressed long non - coding RNAs using RNA - Sequencing. Ninety - four differentially expressed long non - coding RNAs are identified, 5 of which are previously annotated for gene name and length, 11 are annotated for gene name and 78 are novel, having no gene name or length previously annotated. Significant inflammatory response and regulation of immune response pathways (false discovery rate < 0.05) are associated with the differentially expressed long non - coding RNAs. QTL annotation analysis revealed 31 QTL previously annotated in the genomic regions of the 94 differentially expressed long non - coding RNAs, and the majority are associated with milk traits. This research provides a better understanding of long non - coding RNAs regulatory elements in milk somatic cells, which may enhance current breeding strategies for more adaptable or high mastitis resistant cattle.

Evaluation of Immunomodulatory Effects of Fusarium Mycotoxins Using Bacterial Endotoxin-Stimulated Bovine Epithelial Cells and Macrophages in Co-Culture.

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate animal food and feeds and are capable of inducing a wide range of toxicities. Predictive in vitro models represent valuable substitutes for animal experiments to assess the toxicity of mycotoxins. The complexities of the interactions between epithelial and innate immune cells, vital for upholding barrier integrity and averting infections, remain inadequately understood. In the current study, a co-culture model of bovine epithelial cells (MAC-T) and macrophages (BoMac) was used to investigate the impact of exposure to Fusarium mycotoxins, namely deoxynivalenol (DON), zearalenone (ZEN), enniatin B (ENB), and beauvericin (BEA), on the inflammatory response elicited by the bacterial lipopolysaccharide (LPS) endotoxin. The MAC-T cells and BoMac were seeded on the apical side of a Transwell membrane and in the lower chamber, respectively, and mycotoxin exposure on the apical side of the membrane was carried out with the different mycotoxins (LC20; concentrations that elicited 20% cytotoxicity) for 48 h followed by an LPS immunity challenge for 24 h. The culture supernatants were collected from the basolateral compartment and these samples were submitted for cytokine/chemokine multiplex analysis. RNA-Seq analysis was performed using total RNA extracted from the MAC-T cells to acquire a more detailed insight into their cellular functions. The multiplex analysis indicated that IFN-γ, IL-1α, IL-8, and MCP-1 were significantly induced post-DON treatment when compared to control cells, and levels of IL-1α and IL-8 were enhanced significantly in all mycotoxin-treated groups post-LPS challenge. Analysis of the sequencing results showed that there were 341, 357, and 318 differentially expressed MAC-T cell genes that were up-regulated in the DON, ENB, and BEA groups, respectively. Gene ontology and pathway analysis revealed that these DEGs were significantly enriched in various biological processes and pathways related to inflammation, apoptosis signaling, and Wnt signaling. These results provide a comprehensive analysis of the co-culture cytokine/chemokine production and MAC-T cells' gene expression profiles elicited by Fusarium mycotoxins, which further contributes to the understanding of early endotoxemia post-mycotoxin exposure.

Assessing Larval Zebrafish Survival and Gene Expression Following Sodium Butyrate Exposure and Subsequent Lethal Bacterial Lipopolysaccharide (LPS) Endotoxin Challenge.

As aquaculture production continues to grow, producers are looking for more sustainable methods to promote growth and increase fish health and survival. Butyrate is a short-chain fatty acid (SCFA) with considerable benefits to gut health, and in recent years, butyrate has been commonly used as an alternative to antimicrobials in livestock production. In this study, we aimed to assess the protective effects of sodium butyrate (NaB) on larval zebrafish subjected to a lethal Pseudomonas aeruginosa lipopolysaccharide (LPS) endotoxin challenge and to elucidate potential protective mechanisms of action. Larval zebrafish were pre-treated with 0, 3000, or 6000 µM NaB for 24 h at 72 h post-fertilization (hpf), then immune challenged for 24 h with 60 µg/mL of LPS at 96 hpf. Our results demonstrate that larval zebrafish pre-treated with 6000 µM of NaB prior to lethal LPS challenge experienced significantly increased survival by 40%, and this same level of NaB significantly down-regulated the expression of pro-inflammatory Tumor Necrosis Factor α (TNF-alpha). Findings from this study are consistent with the beneficial effects of NaB on other vertebrate species and support the potential use of NaB in aquaculture.

N-Acetylcysteine and Its Immunomodulatory Properties in Humans and Domesticated Animals.

N-acetylcysteine (NAC), an acetylated derivative of the amino acid L-cysteine, has been widely used as a mucolytic agent and antidote for acetaminophen overdose since the 1960s and the 1980s, respectively. NAC possesses antioxidant, cytoprotective, anti-inflammatory, antimicrobial, and mucolytic properties, making it a promising therapeutic agent for a wide range of diseases in both humans and domesticated animals. Oxidative stress and inflammation play a major role in the onset and progression of all these diseases. NAC's primary role is to replenish glutathione (GSH) stores, the master antioxidant in all tissues; however, it can also reduce levels of pro-inflammatory tumor necrosis factor-alpha (TNF-∝) and interleukins (IL-6 and IL-1ß), inhibit the formation of microbial biofilms and destroy biofilms, and break down disulfide bonds between mucin molecules. Many experimental studies have been conducted on the use of NAC to address a wide range of pathological conditions; however, its effectiveness in clinical trials remains limited and studies often have conflicting results. The purpose of this review is to provide a concise overview of promising NAC usages for the treatment of different human and domestic animal disorders.

Identification and Classification of Long Non-Coding RNAs in the Mammary Gland of the Holstein Cow.

The mammary glands, responsible for milk secretion, are regulated at a local level by various hormones, growth factors, non-coding RNAs, and other elements. Recent research has discovered the presence of lncRNAs in these glands, with suggestions that they may be essential for the maintenance and function of mammary glands. Besides directly controlling the gene and protein expression, lncRNAs are believed to play a significant part in numerous physiological and pathological processes. This study focused on examining the mammary gland tissues of Chinese Holstein cows, to identify and categorize long non-coding RNAs (lncRNAs). The research intended to distinguish lncRNAs in the mammary tissues of Holstein cows and contrast them between lactation and non-lactation periods. In this study, mammary gland tissues were sampled from three Holstein cows in early lactation (n = 3, 30 days postpartum) and non-lactation (n = 3, 315 days postpartum) on a large dairy farm in Jiangsu province. Mammary tissue samples were collected during early lactation and again during non-lactation. In total, we detected 1905 lncRNAs, with 57.3% being 500 bp and 612 intronic lncRNAs. The exon count for lncRNAs varied from 2 to 10. It was observed that 96 lncRNA expressions markedly differed between the two stages, with 83 genes being upregulated and 53 downregulated. Enrichment analysis results revealed that Gene Ontology (GO) analysis was primarily abundant in cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that target genes were predominantly abundant in metabolic pathways, fatty acid biosynthesis, the immune system, and glycosphingolipid biosynthesis. This study analyzed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows during both lactation and non-lactation stages, forming a foundation for further investigation into the functional roles of lncRNAs in Holstein cows throughout lactation.

Remediation Strategies for Mycotoxins in Animal Feed.

Mycotoxins occur widely in various animal feedstuffs, with more than 500 mycotoxins identified so far [...].

Dietary protein, lipid and insect meal on growth, plasma biochemistry and hepatic immune expression of lake whitefish (Coregonus clupeaformis).

Studies are lacking that investigate the dietary nutrient requirements of lake whitefish (Coregonus clupeaformis), a newly farmed fish species in Ontario, Canada. Dietary levels of protein and lipid must be optimized to ensure high growth performance for the commercial success of this species. Additionally, the inclusion of insect meal in the diet may improve growth and immune response. The objective of this study was to evaluate the effects of dietary protein:lipid ratios and insect meal as a feed additive on the growth performance and hepatic immune function of juvenile lake whitefish (301 ± 10 g). A 16-week (112 day) trial was performed with five diets including a commercial control diet (BCC), and four experimental diets with high or low levels of protein (54 and 48%, respectively) and lipid (18 and 12%, respectively). The high protein dietary groups contained 5% of full-fat black soldier fly larvae (Hermetia illucens). Fish weights, viscera, liver, and blood were collected for further analysis. Specific growth rate, thermal growth coefficient and weight gain were significantly higher in fish fed with the BCC and high protein high lipid (HPHL) diets. However, viscerosomatic index was found to be significantly higher in fish fed the BCC diet, thus HPHL is more optimal for non-visceral weight gain. Higher levels of plasma phosphorus, aspartate aminotransferase and potassium indicated poor growth and stress in fish fed low lipid diets. Relative expression of HSP70, involved in cellular repair, was significantly downregulated in fish fed high lipid diets, and no effects were found on the expression of innate immune and oxidative stress genes. Also, IL8 (CXCL8) and catalase were upregulated (non-significant) in fish fed the HPHL diet with the largest weight gain. No effects of insects were found on growth, plasma biochemistry or gene expression, which suggests 5% dietary inclusion was too low. Overall, we recommend a HPHL diet for the cultivation of lake whitefish based on improved growth performance, low viscera weight, improved plasma biochemistry and downregulation of cellular repair genes.

Impact of heat stress on dairy cattle and selection strategies for thermotolerance: a review.

Climate change is a problem that causes many environmental issues that impact the productivity of livestock species. One of the major issues associated with climate change is an increase of the frequency of hot days and heat waves, which increases the risk of heat stress for livestock species. Dairy cattle have been identified as being susceptible to heat stress due to their high metabolic heat load. Studies have shown heat stress impacts several biological processes that can result in large economic consequences. When heat stress occurs, dairy cattle employ several physiological and cellular mechanisms in order to dissipate heat and protect cells from damage. These mechanisms require an increase and diversion in energy toward protection and away from other biological processes. Therefore, in turn heat stress in dairy cattle can lead numerous issues including reductions in milk production and reproduction as well as increased risk for disease and mortality. This indicates a need to select dairy cattle that would be thermotolerant. Various selection strategies to confer thermotolerance have been discussed in the literature, including selecting for reduced milk production, crossbreeding with thermotolerant breeds, selecting based on physiological traits and most recently selecting for enhanced immune response. This review discusses the various issues associated with heat stress in dairy cattle and the pros and cons to the various selection strategies that have been proposed to select for thermotolerance in dairy cattle.

Impact of Mycobacterium avium subsp. paratuberculosis infection on bovine IL10RA knockout mammary epithelial (MAC-T) cells.

Mycobacterium avium subsp. Paratuberculosis (MAP) is an intracellular pathogen that causes Johne's disease (JD) in cattle and other ruminants. IL10RA encodes the alpha chain of the IL-10 receptor that binds the cytokine IL-10, and is one of the candidate genes that have been found to be associated with JD infection status. In this study, a previously developed IL10RA knockout (IL10RAKO) bovine mammary epithelial (MAC-T) cell line and wild-type (WT) MAC-T cells were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of IL10RA. Cytokine and chemokine concentrations in culture supernatants were measured by multiplexing immunoassay. Total RNA was extracted from the MAC-T cells, and qPCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of TNF-α, IL-6, CXCL8, CXCL10, CCL2, and CCL3 were significantly induced in WT MAC-T cells and IL-10 was significantly inhibited post-MAP infection. However, IL10RAKO MAC-T cells had greater secretion of TNF-α, IL-6, IFN-γ, CCL3, CCL4, CXCL8, and CXCL10, and lower secretion of VEGF-α. Moreover, the expression of inflammatory genes (TNF-α, IL-1α, IL-6) was also more significantly induced in IL10RAKO cells than in WT MAC-T cells post-MAP-infection, and unlike the WT cells, anti-inflammatory cytokines IL-10 and SOCS3 and chemokines CCL2 were not significantly induced. In addition, the expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP-infection; however, there was no significant induction of these miRNAs in the IL10RAKO cells, which suggests IL10 receptor is somehow involved in regulating the miRNA response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in interleukin signaling, and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of IL10RA in the regulation of innate immune response to MAP.

Identifying hepatic genes regulating the ovine response to gastrointestinal nematodes using RNA-Sequencing.

Gastrointestinal nematode (GIN) infections are considered the most important disease of grazing sheep and due to increasing anthelmintic resistance, chemical control alone is inadequate. Resistance to Gastrointestinal nematode infection is a heritable trait, and through natural selection many sheep breeds have higher resistance. Studying the transcriptome from GIN-exposed and GIN-unexposed sheep using RNA-Sequencing technology can provide measurements of transcript levels associated with the host response to Gastrointestinal nematode infection, and these transcripts may harbor genetic markers that can be used in selective breeding programs to enhance disease resistance. The objective of this study was to compare liver transcriptomes of sheep naturally exposed to Gastrointestinal nematode s, with either high or low parasite burdens, to GIN-unexposed control sheep in order to identify key regulator genes and biological processes associated with Gastrointestinal nematode infection. Differential gene expression analysis revealed no significant differentially expressed genes (DEG) between sheep with a high or low parasite burden (p-value ≤0.01; False Discovery Rate (FDR) ≤ 0.05; and Fold-Change (FC) of > ±2). However, when compared to the control group, low parasite burden sheep showed 146 differentially expressed genes (64 upregulated and 82 downregulated in the low parasite burden group relative to the control), and high parasite burden sheep showed 159 differentially expressed genes (57 upregulated and 102 downregulated in the low parasite burden group relative to the control) (p-value ≤0.01; FDR ≤0.05; and FC of > ±2). Among these two lists of significant differentially expressed genes, 86 differentially expressed genes (34 upregulated, 52 downregulated in the parasited group relative to the control) were found in common between the two parasite burden groups compared to the control (GIN-unexposed sheep). Functional analysis of these significant 86 differentially expressed genes found upregulated genes involved in immune response and downregulated genes involved in lipid metabolism. Results of this study offer insight into the liver transcriptome during natural Gastrointestinal nematode exposure that helps provide a better understanding of the key regulator genes involved in Gastrointestinal nematode infection in sheep.

Role of Toll-Like Receptor 4 in Mycobacterium avium subsp. paratuberculosis Infection of Bovine Mammary Epithelial (MAC-T) Cells In Vitro.

Toll-like receptor 4 (TLR4) encodes an innate immune cell pattern-recognition receptor implicated in the recognition of Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in ruminants. Polymorphisms in TLR4 have been associated with susceptibility to MAP infection. In this study, a previously developed TLR4 knockout (TLR4KO) bovine mammary epithelial (MAC-T) cell line and wild-type MAC-T cells (WT) were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of TLR4. Cytokines/chemokines production in culture supernatants was measured by multiplexing immunoassay. Total RNA was extracted from the remaining MAC-T cells, and quantitative PCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), CXCL8, CXCL10, CCL4, and CCL3 were significantly induced in WT MAC-T cells during MAP infection. However, TLR4KO MAC-T cells had greater secretion of CCL3, IL-6, vascular endothelial growth factor (VEGF-α), and TNF-α and decreased secretion of CXCL10 and CCL2. Moreover, the expression of inflammatory genes was induced in TLR4KO cells. The expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP infection; however, there was no significant induction of these miRNAs in TLR4KO cells, which suggests they are involved in regulating the innate immune response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in TLR and interleukin signaling and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of TLR4 in the regulation of innate immune response to MAP. IMPORTANCE Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent for paratuberculosis or Johne's disease (JD) in ruminants, a disease clinically very similar to Crohn's disease in humans. Polymorphisms in the bovine Toll-like receptor genes (TLR1, TLR2, and TLR4) have been shown to affect MAP recognition and host innate immune response and have been associated with increased susceptibility of cattle to paratuberculosis. Our results demonstrated that knocking out the TLR4 gene in bovine MAC-T cells enhanced inflammation in response to MAP. These findings show divergent roles for TLR4 in Escherichia coli lipopolysaccharide and mycobacterial infections, and this may have important consequences for the treatment of these inflammatory diseases and for genetic selection to improve disease resistance. It advances our understanding of the role of TLR4 in the context of MAP infection.

Ferroptosis is involved in deoxynivalenol-induced intestinal damage in pigs.

BACKGROUND: Deoxynivalenol (DON) is a widespread issue for feed and food safety, leading to animal and human health risks. The objective of this study was to determine whether ferroptosis is involved in DON-induced intestinal injury in piglets. Three groups of 21-day-old male weanling piglets (n = 7/group) were fed a control diet, or diet adding 1.0 or 3.0 mg DON/kg. At week 4, serum and small intestines were collected to assay for biochemistry, histology, redox status and ferroptosis-related genes expression. In addition, the involvement of ferroptosis and the role of FTL gene in DON-induced cell death were further verified in the IPEC-J2 cells. RESULTS: Compared to the control, dietary supplementation of DON at 1.0 and 3.0 mg/kg induced different degrees of damage in the duodenum, jejunum and ileum, and increased (P < 0.05) serum lipopolysaccharide concentration by 46.2%-51.4%. Dietary DON supplementation at 1.0 and (or) 3.0 mg/kg increased (P < 0.05) concentrations of malondialdehyde (17.4%-86.5%) and protein carbonyl by 33.1%-92.3% in the duodenum, jejunum and ileum. In addition, dietary supplemented with DON upregulated (P < 0.05) ferroptotic gene (DMT1) and anti-ferroptotic genes (FTL and FTH1), while downregulated (P < 0.05) anti-ferroptotic genes (FPN, FSP1 and CISD1) in the duodenum of the porcine. Furthermore, the in vitro study has demonstrated that deferiprone, a potent ferroptotic inhibitor, mitigated (P < 0.05) DON-induced cytotoxicity in porcine small intestinal IPEC-J2 cells. Additionally, deferiprone prevented or alleviated (P < 0.05) the dysregulation of ferroptosis-related genes (ACSL4 and FTL) by DON in IPEC-J2 cells. Moreover, specific siRNA knockdown FTL gene expression compromised the DON-induced cell death in IPEC-J2 cells. CONCLUSIONS: In conclusion, this study revealed that ferroptosis is involved in DON-induced intestinal damage in porcine, and sheds a new light on the toxicity of DON to piglets.

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function.

High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants. Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland. The bovine udder plays a pivotal role in maintaining milk yield and composition, thus, human health. However, toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied. In this study, the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol (DON), enniatin B (ENB) and beauvericin (BEA) on bovine mammary gland homeostasis. Results indicated that exposure to DON, ENB and BEA for 48 h significantly decreased cell viability in a concentration-dependent manner (P < 0.001). Exposure to DON at 0.39 µmol/L and BEA at 2.5 µmol/L for 48 h also decreased paracellular flux of FITC-40 kDa dextran (P < 0.05), whereas none of the mycotoxins affected transepithelial electrical resistance after 48 h exposure. The qPCR was performed for assessment of expression of gene coding tight junction (TJ) proteins, toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48 h of exposure. DON, ENB and BEA significantly upregulated the TJ protein zonula occludens-1, whereas markedly downregulated claudin 3 (P < 0.05). Exposure to DON at 1.35 µmol/L for 4 h significantly increased expression of occludin (P < 0.01). DON, ENB and BEA significant downregulated TLR4 (P < 0.05). In contrast, ENB markedly increased expression of cytokines interleukin-6 (IL-6) (P < 0.001), tumor necrosis factor α (TNF-a) (P < 0.05) and transforming growth factor-ß (TGF-ß) (P < 0.01). BEA significantly upregulated IL- 6 (P < 0.001) and TGF-ß (P = 0.01), but downregulated TNF-α (P < 0.001). These results suggest that DON, ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.

Alpha-class glutathione S-transferases involved in the detoxification of aflatoxin B1 in ducklings.

The objective of this study was to identify the key glutathione S-transferase (GST) isozymes involved in the detoxification of Aflatoxin B1 (AFB1) in ducks' primary hepatocytes. The full-length cDNA encoding the 10 GST isozymes (GST, GST3, GSTM3, MGST1, MGST2, MGST3, GSTK1, GSTT1, GSTO1 and GSTZ1) were isolated/synthesized from ducks' liver and cloned into the pcDNA3.1(+) vector. The results showed that pcDNA3.1(+)-GSTs plasmids were successfully transferred into the ducks' primary hepatocytes and the mRNA of the 10 GST isozymes were overexpressed by 1.9-3274.7 times. Compared to the control, 75 µg/L (IC30) or 150 µg/L (IC50) AFB1 treatment reduced the cell viability by 30.0-50.0% and increased the LDH activity by 19.8-58.2% in the ducks' primary hepatocytes. Notably, the AFB1-induced changes in cell viability and LDH activity were mitigated by overexpression of GST and GST3. Compared to the cells treated with AFB1, exo-AFB1-8,9-epoxide (AFBO)-GSH, as the major detoxified product of AFB1, was increased in the cells overexpression of GST and GST3. Moreover, the sequences, phylogenetic and domain analysis revealed that the GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4. In conclusion, this study found that the ducks' GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4, which were involved in the detoxification of AFB1 in ducks' primary hepatocytes.

Expression Profile of miRNA from High, Middle, and Low Stress-Responding Sheep during Bacterial Endotoxin Challenge.

Animals respond to stress by activating a wide array of physiological and behavioral responses that are collectively referred to as the stress response. MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in the regulation of homeostasis. There are many reports demonstrating examples of stress-induced miRNA expression profiles. The aim of this study was to determine the circulatory miRNA profile of variable stress-responding lambs (n = 112) categorized based on their cortisol levels as high (HSR, 336.2 ± 27.9 nmol/L), middle (MSR, 147.3 ±9.5 nmol/L), and low (LSR, 32.1 ± 10.4 nmol/L) stress responders post-LPS challenge (400 ng/kg iv). Blood was collected from the jugular vein at 0 (T0) and 4 h (T4) post-LPS challenge, and miRNAs were isolated from four animals from each group. An array of 84 miRNAs and 6 individual miRNAs were evaluated using qPCR. Among 90 miRNAs, there were 48 differentially expressed (DE) miRNAs (log fold change (FC) > 2 < log FC) in the HSR group, 46 in the MSR group, and 49 in the LSR group compared with T0 (control) samples. In the HSR group, three miRNAs, miR-485-5p, miR-1193-5p, and miR-3957-5p were significantly (p < 0.05) upregulated, while seven miRNAs, miR-376b-3p, miR-376c-3p, miR-411b-5p, miR-376a-3p, miR-376b-3p, miR-376c-3p, and miR-381-3p, were downregulated (p < 0.05) as compared to the LSR and MSR groups. Functional analysis of DE miRNAs revealed their roles in Ras and MAPK signaling, cytokine signaling, the adaptive immune system, and transcription pathways in the HSR phenotype, implicating a hyper-induced acute-phase response. In contrast, in the LSR group, enriched pathways included glucagon signaling metabolic regulation, the transportation of amino acids and ions, and the integration of energy metabolism. Taken together, these results indicate variation in the acute-phase response to an immune stress challenge, and these miRNAs are implicated in regulating responses within cortisol-based phenotypes.

Comparative Assessment of Different Yeast Cell Wall-Based Mycotoxin Adsorbents Using a Model- and Bioassay-Based In Vitro Approach.

Frequently reported occurrences of deoxynivalenol (DON), beauvericin (BEA), and, to a lesser extent, ochratoxin A (OTA) and citrinin (CIT) in ruminant feed or feedstuff could represent a significant concern regarding feed safety, animal health, and productivity. Inclusion of yeast cell wall-based mycotoxin adsorbents in animal feeds has been a common strategy to mitigate adverse effects of mycotoxins. In the present study, an in vitro approach combining adsorption isotherm models and bioassays was designed to assess the efficacy of yeast cell wall (YCW), yeast cell wall extract (YCWE), and a postbiotic yeast cell wall-based blend (PYCW) products at the inclusion rate of 0.5% (w/v) (ratio of adsorbent mass to buffer solution volume). The Hill's adsorption isotherm model was found to best describe the adsorption processes of DON, BEA, and CIT. Calculated binding potential for YCW and YCWE using the Hill's model exhibited the same ranking for mycotoxin adsorption, indicating that BEA had the highest adsorption rate, followed by DON and CIT, which was the least adsorbed. PYCW had the highest binding potential for BEA compared with YCW and YCWE. In contrast, the Freundlich isotherm model presented a good fit for OTA adsorption by all adsorbents and CIT adsorption by PYCW. Results indicated that YCW was the most efficacious for sequestering OTA, whereas YCWE was the least efficacious. PYCW showed greater efficacy at adsorbing OTA than CIT. All adsorbents exhibited high adsorption efficacy for BEA, with an overall percentage average of bound mycotoxin exceeding 60%, whereas moderate efficacies for the other mycotoxins were observed (up to 37%). Differences in adsorbent efficacy of each adsorbent significantly varied according to experimental concentrations tested for each given mycotoxin (p < 0.05). The cell viability results from the bioassay using a bovine mammary epithelial cell line (MAC-T) indicated that all tested adsorbents could potentially mitigate mycotoxin-related damage to bovine mammary epithelium. Results from our studies suggested that all tested adsorbents had the capacity to adsorb selected mycotoxins in vitro, which could support their use to mitigate their effects in vivo.