Bovine Cell Line Research Articles

Experimental Senecavirus A Infection of Bovine Cell Lines and Colostrum-Deprived Calves

Senecavirus A (SVA) is a causative agent for vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases of swine including foot-and-mouth disease (FMD). Recently, it was reported that buffalo in Guangdong, China were experiencing clinical symptoms similar to FMD including mouth ulcers and lameness tested positive for SVA. The objective of this study was to determine the susceptibility of cattle (Bos taurus) to SVA infection. Initial in vitro work using the PrimeFlow assay demonstrated that bovine cell lines and peripheral blood mononuclear cells from cattle were susceptible to SVA infection. Subsequently, six colostrum-deprived Holstein calves were challenged with SVA intranasally. No vesicular lesions were observed after challenge. Serum, oral, nasal, and rectal swabs tested for SVA nucleic acid did not support significant viral replication and there was no evidence of seroconversion. Therefore, demonstrating cattle from this study were not susceptible to experimental SVA infection.

Short Communication: effect of sodium butyrate, monensin, and butyric acid on the viability of Eimeria bovis sporozoites and their degree of damage to a bovine epithelial cell line.

This study aimed to determine the viability of sporozoites from Eimeria bovis when exposed to sodium butyrate (SB), monensin (MON), or butyric acid (BA), and to determine the effects of SB on sporozoite invasion of cells in comparison to MON as measured by the damage to a bovine epithelial cell line. To determine viability, isolated sporozoites were suspended in one of four treatments: control (CON) of cell culture medium alone, SB = 0.028 mg/mL suspended in control medium, MON = 0.01 mg/mL suspended in CON, and BA = 0.18 mg/mL suspended in CON. The number of live sporozoites was less for the MON and BA treatments compared to the CON and SB treatments. The number of dead sporozoites was similar regardless of treatment. There was a trend for treatment to affect the percent sporozoite viability. Control, SB and BA treatments were similar, while MON compared to control and SB had decreased percent viability. Results for MON, when compared to BA, were similar for percent viability. Lactate dehydrogenase (LDH) release was used to determine cellular damage to Madin Darby Bovine Kidney (MDBK) cells when exposed to E. bovis sporozoites in vitro. Cells were exposed to similar numbers of sporozoites and treated with: CON, SB = 0.028 mg/mL in control medium, MON = 0.01 mg/mL in control medium. Control LDH result (with sporozoites) was greater than both the SB and MON treatments while the LDH for SB and Mon and cells not exposed to sporozoites were similar. SB and MON were both shown to decrease cellular damage to MDBK cells as determined by decreased LDH release. SB has the potential to act as an anticoccidial alternative to MON.

Generation of a bovine cell line for gene engineering using an HIV-1-based lentiviral vector

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors are indispensable tools for gene engineering in mammalian cells. Conversely, lentiviral vector transduction is severely inhibited in bovine cells. Previous studies demonstrated that this inhibition is caused by the anti-lentiviral host factor tripartite motif containing 5 (TRIM5), which targets incoming HIV-1 virions by interacting with the viral capsid. In this study, we investigated several methods for overcoming the limited applicability of lentiviral vectors in bovine cells. First, we demonstrated that the SPRY domain of bovine TRIM5 is the major determinant of anti-viral activity. Second, we found that mutations that allow the capsid to evade rhesus macaque TRIM5α minimally rescued HIV-1 infectivity in bovine-derived MDBK cells. Third, we found that cyclosporine A, which relieves the inhibition of HIV-1 infection in monkey cells, significantly rescued the impaired HIV-1 infectivity in MDBK cells. Lastly, we successfully generated a bovine cell line lacking intact TRIM5 using the CRISPR/Cas9 technique. This TRIM5 knockout cell line displayed significantly higher susceptibility to an HIV-1-based lentiviral vector. In conclusion, our findings provide a promising gene engineering strategy for bovine cells, thereby contributing to innovations in agriculture and improvements in animal health.

Microfluidic-Based Formulation of Essential Oils-Loaded Chitosan Coated PLGA Particles Enhances Their Bioavailability and Nematocidal Activity.

In this study, poly (lactic-co-glycolic) acid (PLGA) particles were synthesized and coated with chitosan. Three essential oil (EO) components (eugenol, linalool, and geraniol) were entrapped inside these PLGA particles by using the continuous flow-focusing microfluidic method and a partially water-miscible solvent mixture (dichloromethane: acetone mixture (1:10)). Encapsulation of EO components in PLGA particles was confirmed by Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction, with encapsulation efficiencies 95.14%, 79.68%, and 71.34% and loading capacities 8.88%, 8.38%, and 5.65% in particles entrapped with eugenol, linalool, and geraniol, respectively. The EO components’ dissociation from the loaded particles exhibited an initial burst release in the first 8 h followed by a sustained release phase at significantly slower rates from the coated particles, extending beyond 5 days. The EO components encapsulated in chitosan coated particles up to 5 μg/mL were not cytotoxic to bovine gut cell line (FFKD-1-R) and had no adverse effect on cell growth and membrane integrity compared with free EO components or uncoated particles. Chitosan coated PLGA particles loaded with combined EO components (10 µg/mL) significantly inhibited the motility of the larval stage of Haemonchus contortus and Trichostrongylus axei by 76.9%, and completely inhibited the motility of adult worms (p < 0.05). This nematocidal effect was accompanied by considerable cuticular damage in the treated worms, reflecting a synergistic effect of the combined EO components and an additive effect of chitosan. These results show that encapsulation of EO components, with a potent anthelmintic activity, in chitosan coated PLGA particles improve the bioavailability and efficacy of EO components against ovine gastrointestinal nematodes.

PSVIII-20 Patatin-Like Phospholipase Domain-Containing Protein 3 Abundance is Affected by Knockdown of Sterol Regulatory Element-Binding Protein 1c but not of Carbohydrate Response Element Binding Protein

Abstract Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a key lipase involved in fatty liver syndrome onset and recovery in humans and bovine. Regulation of PNPLA3 is mediated by carbohydrate response element binding protein (ChREBP) in rodents and sterol regulatory element-binding protein 1c (SREBP1c) in humans. The objectives of our study were to optimize an siRNA knockdown of ChREBP and SREBP1c and to determine if either transcription factor knockdown would affect PNPLA3 abundance in an immortalized bovine hepatocyte cell line, BFH12. Hepatocytes were cultured in monolayer and at least 80% confluent after 24h when cells were randomly assigned in triplicate to treatment with ChREBP-targeted (siC1, siC2, siC3) or SREBP1c-targeted (siS1, siS2, siS3) siRNA at either 20, 60, or 80µM, or a nonspecific-siRNA control (siNON). Media was replenished after 4h and cells incubated for 20h before harvest in TRIzol for subsequent protein isolation. Protein abundances were determined by Western blot and normalized to total lane protein. Data were analyzed (PROC MIXED, SAS 9.4) with fixed effect of siRNA and concentration. Differences were declared at P≤0.05; tendencies at 0.05

Novel mycoplasma nucleomodulin MbovP475 decreased cell viability by regulating expression of CRYAB and MCF2L2

ABSTRACT Nucleomodulins are secreted bacterial proteins whose molecular targets are located in host cell nuclei. They are gaining attention as critical virulence factors that either modify the epigenetics of host cells or directly regulate host gene expression. Mycoplasma bovis is a major veterinary pathogen that secretes several potential virulence factors. The aim of this study was to determine whether any of their secreted proteins might function as nucleomodulins. After an initial in silico screening, the nuclear localization of the secreted putative lipoprotein MbovP475 of M. bovis was demonstrated in bovine macrophage cell line (BoMac) experimentally infected with M. bovis. Through combined application of ChIP-seq, Electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis, MbovP475 was determined to bind the promoter regions of the cell cycle central regulatory genes CRYAB and MCF2L2. MbovP475 has similar secondary structures with the transcription activator-like effectors (TALEs). Screening of various mutants affecting the potential DNA binding sites indicated that the residues 242NI243 within MbovP475 loop region of the helix-loop-helix domain were essential to its DNA binding activity, thereby contributing to decrease in BoMac cell viability. In conclusion, this is the first report to confirm M. bovis secretes a conserved TALE-like nucleomodulin that binds the promoters of CRYAB and MCF2L2 genes, and subsequently down-regulates their expression and decreases BoMac cell viability. Therefore, this study offers a new understanding of mycoplasma pathogenesis.

The attenuated Mycoplasma bovis strain promotes apoptosis of bovine macrophages by upregulation of CHOP expression.

Mycoplasma bovis (M. bovis) is one of the major pathogens in the bovine respiratory disease complex, which includes pneumonia, mastitis, and arthritis and causes a great economic loss in the cattle industry. In China, a live-attenuated vaccine strain M. bovis P150 was obtained by a continuous culture of the wild-type strain M. bovis HB0801 (P1) in vitro for 150 passages. Using the infected bovine macrophage cell line BoMac, this work attempted to investigate the mechanism of P150 attenuation and protective immune response. To begin, we show that M. bovis P150 effectively triggered cytotoxicity and apoptosis in BoMac, although with lower intracellular survival than P1. The transcriptomes of BoMac after infection with M. bovis strains P1 and P150 were sequenced, and bioinformatic analysis identified 233 differentially expressed genes (DEGs), with 185 upregulated and 48 downregulated. Further Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses revealed that the majority of the DEGs were linked to CHOP complex, MAP kinase phosphatase activity and were involved in the IL-17 signaling pathway in immune response, MAPK signaling pathway in signal transduction, and p53 signaling pathway in cell growth and death. Among them, the level of C/EBP homologous protein (CHOP) was significantly upregulated in P150-infected BoMac compared to P1-infected cells at different time points, along with its upstream and downstream genes phosphorylated-PERK, phosphorylated-EIF2α, ATF4, and GADD45A increased in the PERK-dependent ER stress response. The role of CHOP in apoptosis was further verified by M. bovis-induced siCHOP knockdown in BoMac cells. The results showed that CHOP knockdown enhanced P150-induced apoptosis and dramatically increased the M. bovis P1 and P150 intracellular survival, particularly for P150. These data suggest that P150 infection upregulates CHOP expression, which can increase apoptosis and mediate a crosstalk between ER stress and apoptosis during infection, and hence, contribute to high cytotoxicity and low intracellular survival.

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2.

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38β MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38β MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

Transcriptomic Analysis of MDBK Cells Infected with Cytopathic and Non-Cytopathic Strains of Bovine Viral Diarrhea Virus (BVDV).

Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the Pestivirus genus. Infection with BVDV causes a disease with a wide spectrum of clinical symptoms, most often mild, although infections with this virus constitute a serious economic problem all over the world. The virus is characterized by a high genetic variability, while the accumulation of single mutations leads to the formation of its new variants. The aim of this study was to better understand the complicated pathogenesis of this disease at the molecular level via the analysis of the transcriptome of cells infected with this virus. The bovine kidney cell line (MDBK), the cytopathic (cp) reference strain, and two non-cytopathic (ncp) BVD virus field strains were used in transcriptomic studies. The cell transcriptome was tested 24 and 72 h after infection. The results of the microarray analysis revealed changes in the expression levels of numerous genes. Genes with changed expression as a result of infection with the cp strain caused changes in the expression levels of a large number of genes and enriched a number of pathways. Genes with increased expression levels were enriched among other pathways involved in the cell cycle, while genes with reduced expression levels enriched pathways mostly related to metabolism. Genes with increased expression levels as a result of infection with ncp strains enriched a much smaller number of pathways, among them, pathways related to signaling activity 24 h post-infection and serine biosynthetic pathways both 24 and 72 h post-infection. Pathways enriched by genes with reduced expression levels were related to the innate immune response (72 h post-infection) or metabolism (24 and 72 h post-infection). The results of microarray studies can help us to better understand the host’s response to BVDV infection.

The Bovine Hepatic Cell Line BFH12 as a Possible Model for Hepatosteatosis in Dairy Cows.

Hepatosteatosis is a common metabolic disorder of dairy cows, especially during early lactation. Currently, there are a few models of bovine hepatic steatosis available, including primary hepatocytes, liver slices, and animal models. Studies that elucidate the influence of single fatty acids on lipid classes, fatty acid pattern, gene expression, and phenotypic changes are still limited. Hence, we investigated the suitability of the fetal bovine hepatocyte-derived cell line BFH12 as a model for hepatosteatosis. To create a steatotic environment, we treated BFH12 with stearic acid, palmitic acid, or oleic acid in non-toxic doses. Thin-layer chromatography and gas chromatography were used to analyze lipid classes and fatty acid pattern, and qPCR was used to quantify gene expression of relevant target genes. Lipid droplets were visualized with confocal laser scanning microscopy and evaluated for number and size. Treatment with oleic acid increased triglycerides, as well as lipid droplet count per cell and upregulated carnitine palmitoyl transferase 1, which correlates with findings of in vivo models. Oleic acid was largely incorporated into triglycerides, phospholipids, and non-esterified fatty acids. Stearic acid was found mainly in non-esterified fatty acids and triglycerides, whereas palmitic acid was mainly desaturated to palmitoleic acid. All three fatty acids downregulated stearyl-CoA-desaturase 1. In conclusion, BFH12 can acquire a steatotic phenotype by incorporating and accumulating fatty acids. Oleic acid is particularly suitable to produce hepatosteatosis. Therefore, BFH12 may be a useful in vitro model to study bovine hepatosteatosis and its underlying molecular mechanisms.

Genome of Bovine Viral Diarrhea Virus (BVDV) Contaminating a Continuous LFBK-αVβ6 Cell Line.

ABSTRACTHere, we report the genome of bovine viral diarrhea virus 1 (BVDV-1) contaminating a continuous fetal bovine kidney cell line. The cell line (LFBK-αVβ6) is used for the rapid isolation and serotyping of foot-and-mouth disease virus (FMDV). The sequence contains the full polyprotein-coding sequence and partial untranslated regions (UTRs).

Diurnal variations of triglyceride accumulation in mouse and bovine adipocyte-derived cell lines.

Several studies have suggested a strong interaction between the circadian clock and lipid metabolism in mammals. The circadian clock is driven by endogenous cyclic gene expression patterns, commonly referred to as clock genes, and transcription-translation negative feedback loops. Clock genes regulate the transcription of some lipid metabolism-related genes; however, the relationship between the circadian clock and triglyceride (TG) accumulation at the cellular level remains unclear. Here, we evaluated rhythms of intracellular TG accumulation levels as well as the expression of clock genes and lipid metabolism-related genes for 54 h in mouse and bovine adipose-derived cell cultures. To the best of our knowledge, this study represents the first report demonstrating that TG accumulation exhibits diurnal variations, with the pattern differing among cell types. Furthermore, we found that expression of clock genes and corresponding lipid metabolism-related genes exhibited circadian rhythms. Our results suggest that the cellular clock regulates lipid metabolism-related genes to relate circadian rhythms of TG accumulation in each cell type. We anticipate that the amount of fat stored depends on the timing of the supply of glucose-the precursor of fat. The findings of this study will contribute to the advancement of chrono-nutrition.

Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells.

Immortalized cell lines have been used in a wide range of applications in research on immune disorders and cellular metabolic regulation due to the stability and uniformity of their cellular characteristics. At present, the investigation into molecular functions and signaling pathways within bovine cells remains largely limited by the lack of immortalized model cells. Current methods for immortalizing bovine cells are mainly restricted to the ectopic expression of human telomerase reverse transcriptase (hTERT) through transient transfection or virus-mediated delivery, which have defects in efficiency and reliability. In this study, we identified bovine TERT (bTERT) as a novel potent biofactor for immortalizing bovine cells with great advantages over hTERT, and established an efficient and easily manipulated strategy for the immortalization of bovine primary cells. Through the homology-mediated end-joining-based insertion of bTERT at the ROSA26 locus, we successfully generated immortalized bovine fetal fibroblast cell lines with stable characteristics. The observed limitation of this strategy in immortalizing bovine bone marrow-derived macrophages was attributed to the post-translational modification of bTERT, causing inhibited nuclear localization and depressed activity of bTERT in this terminally differentiated cell. In summary, we constructed an innovative method to achieve the high-quality immortalization of bovine primary cells, thereby expanding the prospects for the future application of immortalized bovine model cell lines.

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance.

Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Delivery Capacity and Anticancer Ability of the Berberine-Loaded Gold Nanoparticles to Promote the Apoptosis Effect in Breast Cancer.

Simple SummaryIn this research, we aimed to evaluate the biological effects of physically gold nanoparticle-collagen nanocarrier incorporated with alkaloid berberine (Au-Col-BB) on non-transformed bovine aortic endothelial cells (BAEC) and Her-2 breast cancer cell lines through in vitro and in vivo assessments. Au-Col-BB showed better cytotoxicity, as well as significantly induced cell apoptosis in Her-2 cancer cells compared with normal cells (non-transformed BAEC). Further, Au-Col-BB also demonstrated better anti-cancer capacity for inhibiting cell growth in Her-2 tumor-bearing mice. In brief, we confirmed that the Au-Col-BB nanocarrier could be a potential nanodrug for increasing the efficiency of specific therapeutic effects in breast cancer disease.Gold nanoparticles (AuNPs) were fabricated with biocompatible collagen (Col) and then conjugated with berberine (BB), denoted as Au-Col-BB, to investigate the endocytic mechanisms in Her-2 breast cancer cell line and in bovine aortic endothelial cells (BAEC). Owing to the superior biocompatibility, tunable physicochemical properties, and potential functionalization with biomolecules, AuNPs have been well studied as carriers of biomolecules for diseases and cancer therapeutics. Composites of AuNPs with biopolymer, such as fibronectin or Col, have been revealed to increase cell proliferation, migration, and differentiation. BB is a natural compound with impressive health benefits, such as lowering blood sugar and reducing weight. In addition, BB can inhibit cell proliferation by modulating cell cycle progress and autophagy, and induce cell apoptosis in vivo and in vitro. In the current research, BB was conjugated on the Col-AuNP composite (“Au-Col”). The UV-Visible spectroscopy and infrared spectroscopy confirmed the conjugation of BB on Au-Col. The particle size of the Au-Col-BB conjugate was about 227 nm, determined by dynamic light scattering. Furthermore, Au-Col-BB was less cytotoxic to BAEC vs. Her-2 cell line in terms of MTT assay and cell cycle behavior. Au-Col-BB, compared to Au-Col, showed greater cell uptake capacity and potential cellular transportation by BAEC and Her-2 using the fluorescence-conjugated Au-Col-BB. In addition, the clathrin-mediated endocytosis and cell autophagy seemed to be the favorite endocytic mechanism for the internalization of Au-Col-BB by BAEC and Her-2. Au-Col-BB significantly inhibited cell migration in Her-2, but not in BAEC. Moreover, apoptotic cascade proteins, such as Bax and p21, were expressed in Her-2 after the treatment of Au-Col-BB. The tumor suppression was examined in a model of xenograft mice treated with Au-Col-BB nanovehicles. Results demonstrated that the tumor weight was remarkably reduced by the treatment of Au-Col-BB. Altogether, the promising findings of Au-Col-BB nanocarrier on Her-2 breast cancer cell line suggest that Au-Col-BB may be a good candidate of anticancer drug for the treatment of human breast cancer.

Establishment of a bovine rumen epithelial cell line.

Rumen epithelium plays an essential role in absorption, transport, and metabolism of short-chain fatty acids, the main products of rumen fermentation, and in preventing microbes and other potentially harmful rumen contents from entering the systemic circulation. The objective of this study was to generate an immortal rumen epithelial cell line that can be used as a convenient model of rumen epithelial cells in vitro. We isolated primary rumen epithelial cells from a steer through trypsin digestion and transduced them with lentiviruses expressing the Simian Virus (SV) 40 T antigen. We cloned the transduced cells by limiting dilution. Western blotting analysis confirmed the expression of the SV40 T antigen in two single-cell clones. Cells from one clone, named bovine rumen epithelial clone 1 (BREC1), displayed a flat and squamous morphology in culture. RNA sequencing revealed that BREC1 cells expressed many markers of epithelial cells, including keratins, the epidermal growth factor receptor, and the short-chain fatty acid transporters monocarboxylic acid transporter (MCT) 1 (MCT-1) and MCT-4. RNA sequencing revealed that BREC1 cells expressed key enzymes such as 3-hydroxymethyl-3-methylglutaryl-CoA lyase and 3-hydroxy-3-methylglutaryl-CoA synthase 1 involved in ketogenesis, a unique function of rumen epithelial cells. RNA sequencing also revealed the expression of genes encoding tight junctions, desmosomes, anchoring junctions, and polarized plasma membranes, structures typical of epithelial cells, in BREC1 cells. Cell proliferation assays indicated that BREC1 cells were similar to primary rumen epithelial cells in response to insulin-like growth factor 1, insulin, and butyrate. In conclusion, BREC1 is not only a convenient but an appropriate model for studying the factors and mechanisms that control proliferation, apoptosis, differentiation, nutrient transport, metabolism, and barrier function in rumen epithelium.

Antibacterial Activity of Ikarugamycin against Intracellular Staphylococcus aureus in Bovine Mammary Epithelial Cells In Vitro Infection Model.

Simple SummaryAntibiotics are widely used for the treatment and control of bovine mastitis. However, the treatment has only been partially effective, as the cure percentage only ranging from 10–30%. Infection by Staphylococcus aureus (S. aureus) is particularly difficult to treat due to the bacteria’s ability to enter and resides inside the host cells. Most antibiotics are ineffective against intracellular bacterial due to the poor penetration into host cells to achieve optimal intracellular bactericidal bioavailability levels. There is therefore, an increasing need to evaluate candidate active substances and develop novel antibiotics effective against intracellular persistence infection. In this study, we examine the potential antibacterial properties of ikarugamycin compound as an alternative drug candidate to be explored for treating persistent bovine mastitis caused by intracellular S. aureus using bovine mammary cell line as an in vitro infection model. We also assessed the potential cytotoxicity effect of ikarugamycin in the infection model. We found that, the ikarugamycin possessed intracellular killing activity against S. aureus within the mammary epithelial cell. This finding highlights the potential application of ikarugamycin as a novel antimicrobial for the treatment of S. aureus mastitis.Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 μg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.

The in vitro antiviral activity of Lacticaseibacillus casei MCJ protein-based metabolites on bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) is a ubiquitous immunosuppressive etiological agent which is economically important for a wide host range in the livestock industry. Lactobacillus spp. has widely been using in the field of management and treatment of gastro-enteric disease for both humans and animals. The ability of Lacticaseibacillus casei MCJ protein-based metabolized to suppress BVDV infection in Madin–Darby Bovine Kidney cell line was demonstrated in this study. The protein-based metabolites were extracted from the cultured L. casei to obtain the safest and beneficial form of the probiotic bacteria. It is revealed that LPM have no cytotoxic effect and the cell viability remain more than 80% even after the cells are treated with 3000 µg/mL of LPM. The results of the plaque formation assay showed that LPM can reduce the viral infection rate. To know the mechanism of LPM for anti-BVDV activity, MDBK cells were exposed to LPM before, after and co-incubation of virus infection. The co-treatment of LPM with BVDV revealed the best results. The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective candidate for the prevention and control of BVDV infection in an animal.

Isolation of Bovine leukemia virus from cows with persistent lymphocytosis in Iraq

This is the first study to report on the isolation of bovine leukemia virus (BLV) from peripheral blood mononuclear cells of two cross bred cows in Iraq. The cattle were seropositive by ELISA when selected while being surveyed for the detection of BLV. Among six cows, two were cases of persistent lymphocytosis (PL). Cytopathology was characterized by the formation of multinucleated giant cells (syncytia) and cytoplasmic vacuoles. Moreover, the viruses produced clear plaques on the monolayer of the primary fetal calf kidney (FCK) cells. Inhibition of plaque formation by BLV-antisera suggested a diagnosis of BLV, which was further confirmed by PCR. Cells infected with the isolates were positive to a monoclonal antibody against the viral gp51 trans-membrane glycoprotein by immunocytochemistry. Both isolates replicated and induced cytopathic effects in bovine and human cell line cultures. Phylogenetic analysis based on partial gp51 env gene sequences revealed that Iraqi strain highly homogenous with Turkey strain (100%) and had 1% distance value with other world strains. In conclusion, this present study found that BLV-infected cattle with PL can be a source for viral isolation, and the cytopathological features of the virus infection are arranged and differ depending on the cell type. This is the first study to report on the isolation of the EBL virus in Iraq, and it provides the basis for further studies about a BLV Iraqi strain that can help control this disease.

Discovering the Protective Effects of Resveratrol on Aflatoxin B1-Induced Toxicity: A Whole Transcriptomic Study in a Bovine Hepatocyte Cell Line.

Aflatoxin B1 (AFB1) is a natural feed and food contaminant classified as a group I carcinogen for humans. In the dairy industry, AFB1 and its derivative, AFM1, are of concern for the related economic losses and their possible presence in milk and dairy food products. Among its toxic effects, AFB1 can cause oxidative stress. Thus, dietary supplementation with natural antioxidants has been considered among the strategies to mitigate AFB1 presence and its toxicity. Here, the protective role of resveratrol (R) has been investigated in a foetal bovine hepatocyte cell line (BFH12) exposed to AFB1, by measuring cytotoxicity, transcriptional changes (RNA sequencing), and targeted post-transcriptional modifications (lipid peroxidation, NQO1 and CYP3A enzymatic activity). Resveratrol reversed the AFB1-dependent cytotoxicity. As for gene expression, when administered alone, R induced neglectable changes in BFH12 cells. Conversely, when comparing AFB1-exposed cells with those co-incubated with R+AFB1, greater transcriptional variations were observed (i.e., 840 DEGs). Functional analyses revealed that several significant genes were involved in lipid biosynthesis, response to external stimulus, drug metabolism, and inflammatory response. As for NQO1 and CYP3A activities and lipid peroxidation, R significantly reverted variations induced by AFB1, mostly corroborating and/or completing transcriptional data. Outcomes of the present study provide new knowledge about key molecular mechanisms involved in R antioxidant-mediated protection against AFB1 toxicity.