Beyond the Coagulation Cascade: Vitamin K and Its Multifaceted Impact on Human and Domesticated Animal Health.

Vitamin K (VK) is an essential micronutrient impacting many systems in the body. This lipid-soluble vitamin is found in various plant and animal products and is absorbed via the lymphatic system. This biomolecule's importance to human health includes but is not limited to its promotion of brain, cardiovascular, bone, and immune functions. These biological properties are also necessary for maintaining domesticated animal health. The synergistic impact of both VK and vitamin D (VD) maximizes these health benefits, specifically for the circulatory and skeletal systems. This manuscript reviews VK's properties, molecular structures, nutrikinetics, mechanisms of action, daily requirements, safety in supplemental form, biomarkers used for its detection, and impacts on various organs. The purpose of synthesizing this information is to evaluate the potential uses of VK for the treatment or prevention of diseases.

Editing of HSF-1 and Na/K-ATPase α1 subunit by CRISPR/Cas9 reduces thermal tolerance of bovine skin fibroblasts to heat shock in vitro.

A follow-up to our previous findings, the present study was planned to evaluate the role of Na/K-ATPase alpha1-subunit (ATP1A1) gene in heat shock tolerance. The primary fibroblast culture was established using ear pinna tissue samples of Sahiwal cattle (Bos indicus). The knockout cell lines of Na/K-ATP1A1 and HSF-1 (heat shock factor-1, as a positive control) genes were developed by CRISPR/Cas9 method and the gene-editing was confirmed by the genomic cleavage detection assay. The two knockout cell lines (ATP1A1 and HSF-1) and wild-type fibroblasts were exposed to heat shock at 42 °C in vitro and different cellular parameters viz., apoptosis, proliferation, mitochondrial membrane potential (ΔΨm), oxidative stress, along with expression pattern of heat-responsive genes were studied. The results showed that in vitro heat shock given to knockout fibroblast cells of both ATP1A1 and HSF-1 genes resulted in decreased cell viability, while increasing the apoptosis rate, membrane depolarization, and ROS levels. However, the overall impact was more in HSF-1 knockout cells as compared to ATP1A1 knockout cells. Taken together, these results indicated that the ATP1A1 gene plays a critical role as HSF-1 under heat stress and helps cells to cope with heat shock.

Expression profile of different classes of proteases in milk derived somatic cells across different lactation stages of indigenous cows (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes - the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

CRISPR-Cas9-mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate-induced inflammation in bovine mammary epithelial cells.

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.

Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9.

BACKGROUND: The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne's disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate. RESULTS: An IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/- IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-α and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative to unedited cells. CONCLUSIONS: The findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.

Identification of Ovine Serum miRNAs Following Bacterial Lipopolysaccharide Challenge.

Host-pathogen interactions are complex and influenced by host genetic and epigenetic modifications. Recently, the significance of microRNAs (miRNAs) in pathogenic infection and the regulation of immune response has been highlighted. However, information on miRNAs' role in the course of inflammation is still very limited in small ruminants. The present study was intended to identify changes in the expression of circulatory miRNAs post-lipopolysaccharide (LPS)-challenge. In this study, young ewes (n = 18) were challenged with Escherichia coli LPS (400 ng/kg i.v.) and blood samples were collected for serum miRNA isolation at two-time points; prior to challenge (T0), and 4 h (T4) post-challenge, reflecting the peak cortisol response. A total of 91 miRNAs were profiled, including 84 miRNAs on a commercial ovine miRNA-PCR array, and seven individual miRNAs. Forty five miRNAs were differentially expressed (DE) with 35 being up-regulated (Fold regulation, FR > 2) and 10 being down-regulated (FR < 1, p < 0.05) at T4. Among the up-regulated miRNAs, 14 were significantly (p < 0.05) induced, including oar-miRs: 369-3p, 495-3p, 376a-3p, 543-3p, 668-3p, 329a-3p, 655-3p, 411a-5p, and 154a-3p, which were located on ovine chromosome 18 forming four miRNA clusters within 10 kb. The elevated miRNAs belonged to different functional classes, playing roles in activating the hypothalamic-pituitary-adrenal axis; increasing cell survival and differentiation; and inducing inflammatory responses and targeted PI3K-Akt and MAPK signaling and chemokine signaling pathways. In summary, these results reveal the dynamic nature of ovine serum miRNAs during LPS-induced stress and highlight the potential role of identified miRNA-clusters on chromosome 18 to understand the regulation of the acute-phase response. Some of these identified circulating miRNAs may also serve as stress biomarkers for livestock in the future.

Characterizing binding sites of heat responsive microRNAs and their expression pattern in heat stressed PBMCs of native cattle, exotic cattle and riverine buffaloes.

It is generally believed that due to evolutionary differences and adaptation to tropical conditions, Indian native cattle has superior heat tolerant ability than Bos taurus cattle. In the present study, 3'-UTR of two most important heat responsive genes i.e., heat shock protein 70.1 (HSP70.1) and heat shock factor- 1 (HSF-1) were sequence characterized in different breeds of Indian native cattle to identify the variations and miRNA binding sites. In addition, the impact of heat stress was assessed in a total of 57 PBMCs samples of native Sahiwal cows (Bos indicus), exotic Holstein cows (Bos taurus) and Murrah buffaloes (Bubalus bubalis) using various cellular parameters like cell viability, cytotoxicity and apoptosis. Further, expression profile of 12 heat responsive miRNAs were also evaluated in unstressed and stressed PBMCs to understand post transcriptional changes in native cows, exotic cows and Murrah buffaloes. The sequence data showed 3'-UTR of HSP70.1 gene of Indian cattle to be exactly similar to Bos taurus with no miRNA binding site. Whereas, sequencing of 3'-UTR of HSF-1 gene revealed 3 SNPs at positions G1762T; C1811T and C1983T with 7 well conserved miRNA binding sites. The impact of heat stress on various cellular parameters in terms of cell viability, cytotoxicity and apoptosis was highest in PBMCs of Holstein cows followed by Murrah buffaloes and Sahiwal cows. Further, in contrast to Holstein Frisian cows and Murrah buffaloes, the expression pattern of 12 heat responsive miRNAs, in heat stressed PBMCs of Sahiwal cows were quite distinct. There was a significant (p < 0.05) induction in expression of most of the miRNAs after heat stress in PBMCs of Sahiwal cows followed by a rapid decline. The distinct cellular response and pattern of miRNA expression across cattle types and buffaloes might be influencing their PBMCs tolerance level to heat stress.

Evaluation of Milk Colostrum Derived Lactoferrin of Sahiwal (Bos indicus) and Karan Fries (Cross-Bred) Cows for Its Anti-Cancerous Potential.

Lactoferrin (Lf) is an iron-binding glycoprotein protein known to have immune-modulatory role and recently, its anticancerous effect against different cancer cell types was emphasized. In the present investigation, a comparative evaluation of anticancer potential of colostrum-derived lactoferrin from Indian native zebu cow (Sahiwal, SAC), crossbred (Karan Fries, KFC) and commercially available (C-Lf) lactoferrin from exotic cow using cellular models was made. A protocol was standardized successfully to purify Lf protein from colostrum of both breeds using HPLC and purity was confirmed by LC-MS. A standardized dose of 750 µg/mL Lf was used to treat two cell types MDA-MB-231 and MCF-7 with Lf from three different sources; SAC-Lf, KFC-Lf and C-Lf for 48 h and 72 h. Different cellular parameters including cytotoxicity, viability, apoptosis and cell proliferation were determined. Comparatively, Lf from commercial source (C-Lf) had maximum effect in both cell types followed by SAC-Lf and KFC-Lf. Further, transcriptional changes in genes associated with apoptosis (Bax and Bcl-2), tumor progression (p53, p21, CD44 and NF-κß) and survival (survivin) were evaluated in Lf treatment. The overall results strongly emphasized to the fact that Lf purified from cow colostrum has the capacity to inhibit the in vitro growth of cancerous cell lines albeit to a varied extent.

Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism.

Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three-dimensional (3D) environment as important signals are lost when cells are cultured in two-dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1-casein (CSN1S1), alpha S2-casein (CSN1S2), beta-casein (CSN2), kappa-casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol-3-phosphate acyltransferase (GPAM), fatty acid-binding protein 3 (FABP3), and stearoyl-CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT-PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome-like structures were formed by day 5, whereas polarized acinus-like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10-day-old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism-related genes. Therefore, matrix-based 3D culture of MEC that recapitulate the structural and functional context of normal tissues could provide a better in vitro model to understand the mammary gland functioning of buffaloes.

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum modulates immunoglobulin levels and cytokines expression in whey proteins sensitised mice.

BACKGROUND: Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. This study investigated whether orally administrated probiotics could suppress sensitisation in whey proteins (WP)-induced allergy mouse model. Two types of probiotic Dahi were prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3. Mice were fed with probiotic Dahi (La-Dahi and LaBb-Dahi) from 7 days before sensitisation with WP, respectively, in addition to milk protein-free basal diet, and control group received no supplements. RESULTS: Feeding of probiotic Dahi suppressed the elevation of whey proteins-specific IgE and IgG response of WP-sensitised mice. In addition, sIgA levels were significantly (P < 0.001) increased in intestinal fluid collected from mice fed with La-Dahi. Production of T helper (Th)-1 cell-specific cytokines, i.e. interferon-γ (IFN-γ), interleukin (IL)-12, and IL-10 increased, while Th2-specific cytokines, i.e. IL-4 decreased in the supernatant of cultured splenocytes collected from mice fed with probiotic Dahi as compared to the other groups. Moreover, the splenic mRNA levels of IFN-γ, interleukin-10 were found to be significantly increased, while that of IL-4 decreased significantly in La-Dahi groups, as compared to control groups. CONCLUSION: Results of the present study indicate that probiotic Dahi skewed Th2-specific immune response towards Th1-specific response and suppressed IgE in serum. Collectively, this study shows the potential use of probiotics intervention in reducing the allergic response to whey proteins in mice. © 2015 Society of Chemical Industry.

Dietary supplementation of Butyrivibrio fibrisolvens alters fatty acids of milk and rumen fluid in lactating goats.

BACKGROUND: Conjugated linoleic acid (CLA) isomers have high health amelioration potential and hence it is of great interest to increase the CLA content in dairy products. The present study was conducted to investigate the effect of administration of high CLA producing Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk and rumen fluid in lactating goats. Four groups (n = 5) of lactating goats were assigned the following treatments: Control (C) (basal diet); T1 (basal diet + linoleic acid source), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 10(9) CFU head(-1)) and T3 (basal diet + linoleic acid source + suspension of Butyrivibrio fibrisolvens In-1, 10(9) CFU head(-1)). RESULTS: Rumen liquor and milk samples were collected on days 0, 15, 30, 60 and 90 of the experiment and linoleic isomerase enzyme (LA-I) activity and fatty acid profiles were elucidated. Major effects of treatments were seen on day 30 of the experiment. Total CLA content of rumen fluid increased (P < 0.05) by 218.72, 182.26 and 304% whereas total saturated fatty acid (SFA) content was lowered (P < 0.05) by 6.1, 4.44 and 9.55% in T1, T2 and T3, respectively, as compared to control. Vaccenic acid in groups T2 and T3 increased (P < 0.05) by 66.67% and 105.7% as compared to control. In milk, total CLA increased by 2.03, 1.61 and 0.61 folds in T3, T2 and T1, respectively. Total monounsaturated fatty acid and polyunsaturated fatty acid content increased (P < 0.05) in group T3 by 14.15 and 37.44%, respectively. CONCLUSION: Results of the present study indicated that administration of B. fibrisolvens In-1 along with a linoleic acid (LA) source is a useful strategy to alter the biohydrogenation pattern in the rumen that subsequently decreased SFA content while increased CLA and unsaturated fatty acids in ruminant's milk.

The Immunomodulatory Effects of Selenium: A Journey from the Environment to the Human Immune System.

Selenium (Se) is an essential nutrient that has gained attention for its impact on the human immune system. The purpose of this review is to explore Se's immunomodulatory properties and to make up-to-date information available so novel therapeutic applications may emerge. People acquire Se through dietary ingestion, supplementation, or nanoparticle applications. These forms of Se can beneficially modulate the immune system by enhancing antioxidant activity, optimizing the innate immune response, improving the adaptive immune response, and promoting healthy gut microbiota. Because of these many actions, Se supplementation can help prevent and treat pathogenic diseases, autoimmune diseases, and cancers. This review will discuss Se as a key micronutrient with versatile applications that supports disease management due to its beneficial immunomodulatory effects. Further research is warranted to determine safe dosing guidelines to avoid toxicity and refine the application of Se in medical treatments.

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.

Exploring the ameliorative potential of probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum on dextran sodium sulphate induced colitis in mice.

Conventional medical therapies for ulcerative colitis (UC) are still limited due to the adverse side effects like dose-dependent diarrhoea and insufficient potency to keep in remission for long-term periods. So, new alternatives that provide more effective and safe therapies for ulcerative colitis are constantly being sought. In the present study, probiotic LaBb Dahi was selected for investigation of its therapeutic effect on DSS-induced colitis model in mice. LaBb Dahi was prepared by co-culturing Dahi culture of Lactococci along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 in buffalo milk. Four groups of mice (12 each) were fed for 17 d with buffalo milk (normal control), buffalo milk plus DSS (Colitis control), Dahi plus DSS, and LaBb Dahi plus DSS, respectively, with basal diet. The disease activity scores, weight loss, organ weight, colon length, myeloperoxidase (MPO) and ß-glucoronidase activity was assessed, and the histopathological picture of the colon of mice was studied. All colitis control mice evidenced significant increase in MPO, ß-glucoronidase activity and showed high disease activity scores along with histological damage to colonic tissue. Feeding with LaBb Dahi offered significant reduction in MPO activity, ß-glucoronidase activity and improved disease activity scores. We found significant decline in length of colon, organ weight and body weight in colitis induced controls which were improved significantly by feeding LaBb Dahi. The present study suggests that LaBb Dahi can be used as a potential nutraceutical intervention to combat UC related changes and may offer effective adjunctive treatment for management of UC.

Effect of thermal processing of cow and buffalo milk on the allergenic response to caseins and whey proteins in mice.

BACKGROUND: Heat treatment is the most common method for reducing pathogen load, but it remains controversial in reducing the incidence of hyperimmune reactions. The aim of this study was to compare the allergenicity of caseins (CSN) and whey proteins (WP) of thermally processed cow and buffalo milk in a mouse model. Swiss albino mice were sensitised by intraperitoneal injections (administered in three doses at weekly intervals) of CSN or WP from cow or buffalo milk for the evaluation of humoral response and splenocyte stimulation index. RESULTS: After 3 weeks of intraperitoneal stimulation of mice with milk proteins, the sterilised milk protein group displayed significantly lowered (P ≤ 0.05) serum IgG and IgE levels, while considerably increased cow milk protein-specific responses (IgE) were shown by proteins of pasteurised milk compared with those of raw milk. The stimulation index of splenocytes induced by CSN or WP of boiled and sterilised milk was also lower (P ≤ 0.05) than that of raw milk of both cow and buffalo. CONCLUSION: The experiment showed that boiling and sterilisation of cow and buffalo milk clearly affect the allergenicity by decreasing the humoral and cell-mediated responses in mice. All results indicated that CSN and WP of sterilised milk are less allergenic than those of raw milk in mice.

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.

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.

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.

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.

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.