miRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage.

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

Induction of mitophagy via ROS-dependent pathway protects copper-induced hypothalamic nerve cell injury.

Copper (Cu) is one of the essential trace elements in the body, but excessive amounts of Cu harm multiple organs and tissues such as liver, kidneys, testis, ovaries, and brain. However, the mechanism of hypothalamic neurotoxicity induced by Cu is still unknown. This study examined the relationship between reactive oxygen species (ROS) and mitophagy in mouse hypothalamus treated with high Cu. The results demonstrated that high levels of copper sulfate (CuSO4) could cause histopathological and neuronal changes in the mouse hypothalamus, produce a large amount of ROS, induce mitophagy, and lead to an imbalance of mitochondrial fusion/fission. The main manifestations are an increase in the expression levels of LC3-II/LC3-I, p62, DRP1, and FIS1, and a decrease in the expression levels of MFN1 and MFN2. Cu can induce mitophagy also was confirmed by LC3 co-localization with TOMM20 (mitochondrial marker). Next, the effect of oxidative stress on CuSO4-induced mitophagy was demonstrated. The results showed that ROS inhibitor N-acetylcysteine (NAC) diminished CuSO4-induced mitophagy and reversed the disturbance of mitochondrial dynamics. Additionally, a study was carried out to evaluate the role of mitophagy in CuSO4-induced hypothalamic injury. The inhibition of mitophagy using mitophagy inhibitor (Mdivi-1) decreased cell viability and promoted CuSO4-inhibited mitochondrial fusion. The aforementioned results suggested that CuSO4 induced mitophagy via oxidative stress in N38 cells and mouse hypothalamus, and that the activation of mitophagy might generate protective mechanisms by alleviating Cu-induced mitochondrial dynamics disorder. This study provided a novel approach and theoretical basis for studying and preventing Cu neurotoxicity.

Histopathological Changes and Inflammatory Response in Specific Pathogen-Free (SPF) with Porcine Circovirus Type 3 Infection.

Since the first report of PCV3 virus infection in 2016, it has been linked to multisystemic inflammation, reproductive failure, cardiac pathology, and clinical indications resembling porcine dermatitis and nephropathy syndrome (PDNS). However, the pathogenesis and clinical significance of PCV3 is still unclear. In this study, a PCV3 infection model was created using SPF pigs, and histopathology and fluorescence quantitative PCR were utilized to examine PCV3's pathogenicity. Reductions in body weight gain and fever were observed during this study. However, other clinical signs such as Dermatitis and Nephropathy Syndrome were not observed through the study. Viremia was detected in the PCV3-inoculated group from 17 days post-inoculation (p.i.) until the end of the study. Nasal shedding was detected from 21 to 35 dpi and fecal shedding was detected during 25-33 days and 39 days, respectively. Gross lesions and histological evaluation were detected in various tissues and organs, including the lung, heart, kidney, lymph nodes, spleen, liver, small intestine, and testis. The heart, lung, liver, kidney, lymph nodes, and spleen showed pathological changes. The pathological features include swelling, inflammation, cell degeneration, necrosis, and hemorrhage. The lesions are consistent with multisystemic inflammation. Tissue viral load results showed only heart, lung, liver, kidney, lymph nodes, and spleen was positive by qRT-PCR. Moreover, the pro-inflammation cytokines in serum increased a lot in the PCV3-inoculated group compared to the control group, demonstrating that the induced inflammation response may be the cause of tissue damage in PCV3-infection. This study demonstrated that PCV3 can produce mild pathological damage to multiple organs, especially multisystemic inflammatory cell infiltration and prolonged viremia, viral shedding in nasal secretions. This is the first in vivo characterization of PCV3 infection in the SPF piglets model using isolated PCV3 strain, and this is also the first time to show the gross and pathological lesion with all tissue and organs in the PCV3-inoculated group. Our findings might serve as a starting point for more investigation into PCV3's pathogenic mechanism.

The Dysregulation of Inflammatory Pathways Triggered by Copper Exposure.

Copper (Cu) is an essential micronutrient for both human and animals. However, excessive intake of copper will cause damage to organs and cells. Inflammation is a biological response that can be induced by various factors such as pathogens, damaged cells, and toxic compounds. Dysregulation of inflammatory responses are closely related to many chronic diseases. Recently, Cu toxicological and inflammatory effects have been investigated in various animal models and cells. In this review, we summarized the known effect of Cu on inflammatory responses and sum up the molecular mechanism of Cu-regulated inflammation. Excessive Cu exposure can modulate a huge number of cytokines in both directions, increase and/or decrease through a variety of molecular and cellular signaling pathways including nuclear factor kappa-B (NF-κB) pathway, mitogen-activated protein kinase (MAPKs) pathway, JAK-STAT (Janus Kinase- signal transducer and activator of transcription) pathway, and NOD-like receptor protein 3 (NLRP3) inflammasome. Underlying the molecular mechanism of Cu-regulated inflammation could help further understanding copper toxicology and copper-associated diseases.

Effects of different dietary protein levels on intestinal aquaporins in weaned piglets.

This study was conducted to investigate the relationship between changes in intestinal aquaporins (AQPs) in piglets fed diets with different protein levels and nutritional diarrhoea in piglets. Briefly, 96 weaned piglets were randomly divided into four groups fed diets with crude protein (CP) levels of 18%, 20%, 22% and 24%. The small intestines and colons of the weaned piglets were collected, and several experiments were conducted. In the small intestine, AQP4 protein expression was higher in weaned piglets fed the higher-CP diets (22% and 24% CP) than in those fed the 20% CP diet except at 72 h (p < 0.01). At 72 h, the AQP4 protein expression in the small intestine was lower in the 18% group than in the other three groups (p < 0.01). Under 20% CP feeding, AQP2, AQP4 and AQP9 protein expression in the colons of piglets peaked at certain time points. The AQP2 and AQP4 mRNA levels in the colon and the AQP4 and AQP4 mRNA levels in the distal colon were approximately consistent with the protein expression levels. However, the AQP9 mRNA content in the colon was highest in the 18% group, and the AQP2 mRNA content in the distal colon was significantly higher in the 24% group than in the 20% group. AQP2 and AQP4 were expressed mainly around columnar cells in the upper part of the smooth colonic intestinal villi, and AQP9 was expressed mainly on columnar cells and goblet cells in the colonic mucosa. In conclusion, 20% CP is beneficial to the normal expression of AQP4 in the small intestine, AQP2, AQP4 and AQP9 in the colon of weaned piglets, which in turn maintains the balance of intestinal water absorption and secretion in piglets.

Mfn2 is responsible for inhibition of the RIG-I/IRF7 pathway and activation of NLRP3 inflammasome in Seneca Valley virus-infected PK-15 cells to promote viral replication.

Seneca Valley virus (SVV), a non-enveloped positive single-stranded virus can cause vesicular disease in swine. However, the mechanisms by which SVV activates an innate immune response remain unknown. Mitofusin-2 (MFN2), a mitochondria-shaping protein regulating mitochondrial fusion and fission, plays a crucial role in innate immune responses. But, the roles of Mfn2 in SVV infection have not been elucidated. Here, we show that SVV inhibited Mfn2 expression and NLRP3 inflammasome, activating RIG-I/IRF7 signaling pathway to increase IFN-λ3 expression. Overexpression of Mfn2 inhibited RIG-I/IRF7 signaling pathway, thus decreasing IFN-λ3 expression and promoting SVV replication. Interestingly, overexpression of Mfn2 also activated NLRP3 inflammasome but did not inhibit SVV proliferation. That may mean the RIG-I/IRF7 signaling pathway plays a more important role in SVV proliferation in PK-15 cells. This study could provide important insights into the modulation of host metabolism during SVV infection and provide a strong theoretical basis for a better understanding of the pathogenic mechanism and immune activation mechanism of SVV.

Effects of dietary protein level on small intestinal morphology, occludin protein, and bacterial diversity in weaned piglets.

Due to the physiological characteristics of piglets, the morphological structure and function of the small intestinal mucosa change after weaning, which easily leads to diarrhea in piglets. The aim of this study was to investigate effects of crude protein (CP) levels on small intestinal morphology, occludin protein expression, and intestinal bacteria diversity in weaned piglets. Ninety-six weaned piglets (25 days of age) were randomly divided into four groups and fed diets containing 18%, 20%, 22%, and 24% protein. At 6, 24, 48, 72, and 96 h, changes in mucosal morphological structure, occludin mRNA, and protein expression and in the localization of occludin in jejunal and ileal tissues were evaluated. At 6, 24, and 72 h, changes in bacterial diversity and number of the ileal and colonic contents were analyzed. Results showed that structures of the jejunum and the ileum of piglets in the 20% CP group were intact. The expression of occludin mRNA and protein in the small intestine of piglets in the 20% CP group were significantly higher than those in the other groups. As the CP level increased, the number of pathogens, such as Clostridium difficile and Escherichia coli, in the intestine increased, while the number of beneficial bacteria, such as Lactobacillus, Bifidobacterium, and Roseburia, decreased. It is concluded that maintaining the CP level at 20% is beneficial to maintaining the small intestinal mucosal barrier and its absorption function, reducing the occurrence of diarrhea, and facilitating the growth and development of piglets.

Antiviral Effect of Selenomethionine on Porcine Deltacoronavirus in Pig Kidney Epithelial Cells.

Porcine deltacoronavirus (PDCoV) is an emerging porcine intestinal coronavirus in recent years, which mainly causes different degrees of vomiting and diarrhea in piglets and has caused great harm to the swine husbandry worldwide since its report. Selenium is an essential trace element for organisms and has been demonstrated to have antiviral effects. In this study, pig kidney epithelial (LLC-PK) cells were used to study the antiviral activity of selenomethionine (Se-Met) (2, 4, 8, and 16 µM) against PDCoV by detecting the replication of the virus, the expression of the mitochondrial antiviral signal protein (MAVS) protein, and the phosphorylation of interferon regulatory factor-3 (IRF-3), IFN-α, and IFN-ß, and the changes in glutathione content, glutathione peroxidase, superoxide dismutase activity, and hydrogen peroxide content in the cells. The results showed that Se-Met at higher than physiological concentrations (16 µM) could significantly inhibit the replication of PDCoV in LLC-PK cells and enhance the expression of MAVS protein and the phosphorylation of IRF-3. In addition, Se-Met also improved the intracellular production of IFNα/ß and antioxidant capacity with increasing doses. These data suggest that the availability of selenium through selenomethionine supports the antiviral response in porcine kidney cells, and the specific mechanism is attributed to the improved cellular antioxidant capacity and activation of the MAVS pathway by Se-Met.

Effect of Selenium on Brain Injury in Chickens with Subacute Arsenic Poisoning.

The aim of this study was to investigate the effects of different doses of selenium (Se) on oxidative damage and neurotransmitter-related parameters in arsenic (As)-induced broiler brain tissue damage. Two hundred 1-day-old avian broilers were randomly divided into five groups and fed the following diets: control group (As 0.1 mg/kg + Se 0.2 mg/kg), As group (As 3 mg/kg + Se 0.2 mg/kg), low-Se group (As 3 mg/kg + Se 5 mg/kg), medium-Se group (As 3 mg/kg + Se 10 mg/kg), and high-Se group (As 3 mg/kg + Se 15 mg/kg). Glutathione (GSH), glutathione peroxidase (GSH-PX), nitric oxide (NO), nitric oxide synthase (NOS) activity, glutamate (Glu) concentration, glutamine synthetase (GS) activity, acetylcholinesterase (TchE) activity, and the apoptosis rate of brain cells were measured. The results showed that 3 mg/kg dietary As could induce oxidative damage and neurotransmitter disorder of brain tissue, increase the apoptosis rate of brain cells and cause damage to brain tissue, decrease activities of GSH and GSH-PX, decrease the contents of NO, decrease the activities of iNOS and tNOS, increase contents of Glu, and decrease activities of Gs and TchE. Compared with the As group, the Se addition of the low-Se and medium-Se groups protected against As-induced oxidative damage, neurotransmitter disorders, and the apoptosis rate of brain cells, with the addition of 10 mg/kg Se having the best effect. However, 15 mg/kg Se not only did not produce a protective effect against As damage but actually caused similar or severe damage.

Effects of Selenium on the Immunotoxicity of Subacute Arsenic Poisoning in Chickens.

This study aimed to determine the effects of selenium on the immune toxicity of subacute arsenic poisoning in chickens. Two hundred 8-day-old broilers were randomly divided into 5 groups: the control group (0.1 mg/kg As + 0.2 mg/kg Se), the As group (3 mg/kg As + 0.2 mg/kg Se), As + Se group I (3 mg/kg As + 5 mg/kg Se), As + Se group II (3 mg/kg As + 10 mg/kg Se), and As + Se group III (3 mg/kg As + 15 mg/kg Se). The conclusions were drawn based on the following measurements: 3.0 mg/kg added to feed led to a decrease in the growth performance of the broilers, reduced the level and conversion rate of ANAE, reduced the blood protein content of the broilers but had no effect on the albumin/globulin ratio, and had an inhibitory effect on erythrocyte immunity. Selenium-added of 5 and 10 mg/kg in daily feed leads to increased growth performance, increases the positive rate and conversion rate of ANAE, increases the hemoglobin content of broilers, and promotes erythrocyte immunity, which indicates that the selenium-added reduces the toxic effects of arsenic; 3.0 mg/kg arsenic with 15 mg/kg selenium had the most severe toxic effects. Fifteen milligrams per kilogram of selenium added in daily feed increases the toxicity of arsenic to broilers. The dose of 10 mg/kg selenium showed the best inhibitory effect on subacute arsenic poisoning in the broilers.

Effects of Selenium on Arsenic-Induced Liver Lesions in Broilers.

The aim of the present study was to investigate the abilities of selenium to counteract the toxic damage of arsenic (As). Two hundred 1-day-old healthy male broilers were randomly divided into five groups and fed the following diets: control group (0.1 mg/kg As + 0.2 mg/kg Se), As group (3 mg/kg As + 0.2 mg/kg Se), As + Se group I (3 mg/kg As + 5 mg/kg Se), As + Se group II (3 mg/kg As + 10 mg/kg Se), and As + Se group III (3 mg/kg As + 15 mg/kg Se), respectively. The relative weight of the liver, hepatic protein content, GSH-Px levels, SOD activities, NO contents, iNOS and tNOS activities, and increased malondialdehyde contents, ALT and AST activities, and the apoptotic hepatocytes were analyzed. Adding 3 mg/kg arsenic to the diet caused the growth and development of chicken liver to be blocked, resulting in decrease of protein contents in liver tissue, decrease of SOD and GSH-Px activities, increase of MDA contents, decrease of NO contents, decrease of iNOS and TNOs activities, increase of ALT and AST activities, increase of apoptosis rates of liver cells. Compared to the 3-mg/kg arsenic group, adding 5 mg/kg and 10 mg/kg selenium, respectively, could repair the liver growth retardation and steatosis caused by arsenic, increase the protein contents in liver tissue, increase the activities of SOD and GSH-Px, reduce the contents of MDA, increase the contents of NO, enhance the activities of iNOS and TNOs, reduce the activities of ALT and AST, and reduce the rates of apoptosis of liver cells, in which the best effects are to add 10 mg/kg selenium. While 15 mg/kg of sodium selenite may induce progression of As-induced hepatic lesions, the results indicated that 5 and 10 mg/kg of sodium selenite supplied in the diet, through mechanisms of oxidative stress and apoptosis regulation, may ameliorate As-induced hepatic lesions in a dose-dependent manner.

High Prevalence of Antimicrobial Resistance and Integron Gene Cassettes in Multi-Drug-Resistant Klebsiella pneumoniae Isolates From Captive Giant Pandas (Ailuropoda melanoleuca).

Multi-drug-resistant Klebsiella pneumoniae (MDR K. pneumonia) is increasingly being reported with corresponding increase in morbidity and mortality all over the world. However, limited information is available concerning MDR K. pneumonia in giant pandas. The objective of this study was to grasp the drug resistance profile of MDR K. pneumonia isolated from giant pandas. A total of 182 K. pneumoniae isolates were collected from fresh feces of 94 captive giant pandas of different ages and sex and separated by season. We performed a standard disk diffusion antimicrobial susceptibility test with the isolates and further evaluated the antibiotic resistance genes (ARGs) of multi-drug-resistant strains by high-throughput quantitative PCR. In addition, we then analyzed mobile genetic elements (MGEs), integron gene cassettes, and the multi-locus sequence typing of multi-drug-resistant strains by PCR. Antimicrobial susceptibility testing results demonstrated that a total of 30 (16.5%) K. pneumoniae isolates showed multiple drug resistances. The thirty MDR K. pneumonia isolates were mainly resistant to amoxicillin (100.0%), doxycycline (86.7%), chloramphenicol (60.0%), compound trimethoprim (60.0%) and trimethoprim (56.7%). Fifty different types of antibiotic resistance genes were found, which included a total of 671 antibiotic resistance genes, in the 30 multi-drug-resistant isolates. The top ten resistance genes were: vanTC-02, aacC, blaCTX-M-04, blaSHV-01, blaSHV-02, ampC-04, blaOXY, tetD, blaTEM and tetA-02. Thirteen mobile genetic elements were detected, of which IS26 (96.67%) and intI1 (96.67%) had the highest frequency. The thirty MDR K. pneumonia isolates were negative for the traA, traF, tnsA, IS1133, ISpa7, ISkpn6, intI2 and intI3 genes. Moreover, a further investigation of integrons revealed that two types of specific gene cassettes (dfrA12 + orfF + aadA2 and dfrA12 + orfF) were identified in class 1 integrons. Multi-locus sequence typing results showed that 22 STs in the thirty MDR K. pneumonia isolates were identified, the main type was ST37 (5/30). Our results illustrate that effective surveillance and strict biosecurity strategies should be taken to prevent the spread of multi-drug-resistant bacteria, and monitor the emergence of mobile genetic elements and integrons.

Effects of deoxynivalenol on mitochondrial dynamics and autophagy in pig spleen lymphocytes.

Deoxynivalenol (DON) is a type of cytotoxic mycotoxin that targets the mitochondria in cells. However, effects of DON on mitochondrial dynamics have not yet been reported. In this study, we investigated the effects of DON on mitochondrial dynamics and mitochondrial autophagy. Spleen lymphocytes of primary pigs were treated with different concentrations of DON. 24 h later, the cells were collected and the indexes were measured as follow: contents of ROS and MDA; activities of SDH and SOD; total antioxidant capacity; expression of MFN1, MFN2 and OPA1; the mRNA expression of the genes encoding mitochondrial autophagy proteins LC3 and P62, as well as mitochondrial respiratory chain complex activities. The results showed that, compared with the control group, the contents of ROS and MDA in the DON groups increased, while the activities of T-AOC, SDH and SOD decreased in a dose-dependent manner. With increasing DON concentrations, the expression levels of MFN1/2, OPA1 and mitochondrial respiratory chain complex activities decreased, while LC3, P62 increased. We suspect that DON causes oxidative damage, which in turn leads to down-regulation of MFN1, MFN2, OPA1 and up-regulation of LC3 and P62 mRNA, thereby inhibiting mitochondrial fusion and promoting mitochondrial autophagy.

Effects of antibacterial peptides on rumen fermentation function and rumen microorganisms in goats.

Although many studies have confirmed that antimicrobial peptides (AMPs: PBD-mI and LUC-n) can be used as feed additives, there are few reports of their use in ruminants. The present study aimed to investigate the impact of AMPs on ameliorating rumen fermentation function and rumen microorganisms in goats. Eighteen 4-month-old Chuanzhong black goats were used in a 60-day experiment (6 goats per group). Group I was used as the control and was fed a basal diet, the group II were fed the basal diet supplemented with 2 g of AMPs [per goat/day] and group III were fed the basal diet supplemented 3 g of AMPs [per goat/day], respectively. Rumen fluid samples were collected at 0, 20 and 60 days. Bacterial 16S rRNA genes and ciliate protozoal 18S rRNA genes were amplified by PCR from DNA extracted from rumen samples. The amplicons were sequenced by Illumina MiSeq. Rumen fermentation parameters and digestive enzyme activities were also examined. Our results showed that dietary supplementation with AMPs increased the levels of the bacterial genera Fibrobacter, Anaerovibrio and Succiniclasticum and also increased the ciliates genus Ophryoscolex, but reduced the levels of the bacterial genera Selenomonas, Succinivibrio and Treponema, and the ciliate genera Polyplastron, Entodinium, Enoploplastron and Isotricha. Supplementation with AMPs increased the activities of xylanase, pectinase and lipase in the rumen, and also increased the concentrations of acetic acid, propionic acid and total volatile fatty acids. These changes were associated with improved growth performance in the goats. The results revealed that the goats fed AMPs showed improved rumen microbiota structures, altered ruminal fermentation, and improved efficiency regarding the utilization of feed; thereby indicating that AMPs can improve growth performance. AMPs are therefore suitable as feed additives in juvenile goats.

Virulence gene profiles: alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China.

BACKGROUND: Staphylococcus aureus, a common cause of bovine mastitis, is known for its ability to acquire to antimicrobial resistance and to secrete numerous virulence factors that can exacerbate inflammation. In addition, alpha-hemolysin has an important role in S. aureus infections, diversity of the hla gene (that produces alpha-hmolysin) in S. aureus isolated from bovine mastitis has not been well characterized. The objective was, therefore, to determine diversity of virulence genes, hla gene sequences, and clonal profiles of S. aureus from bovine mastitis in Chinese dairy herds, and to evaluate inter-relationships. RESULTS: The antimicrobials resistance varies from as low as 1.9% (2/103) for CTX to as high as 76.7% (79/103) for penicilin in the 103 isolates and 46 (44.7%) S. aureus were determined as multi-resistant isolates with diverse resistance patterns. Thirty-eight virulence gene patterns (with variable frequencies) were identified in the 103 isolates and correlated with MLST types, indicating a great diversity. Although the hla gene also had great diversity (14 genotypes), Hla peptides were relatively more conserved. With 7 clonal complexes identified from 24 spa types and 7 MLST types. Regarding the letter, ST 97 was the dominant type in S. aureus from bovine mastitis in China. Furthermore, based on phylogenetic analysis, there was a distinct evolutionary relationship between the hla gene and MLST. CONCLUSION: Multi-resistant S. aureus occurred in bovine mastitis with diverse resistance patterns. The diversity of virulence gene profiles, especially the hla gene and, their relationship with molecular types were reported for the first time in S. aureus from bovine mastitis, which will be useful for future studies on immunogenicity and vaccine development. In addition, based on the distinct evolutionary relationship between the hla gene and MLST types, we inferred that the hla gene has potential role for molecular typing of S. aureus.

Combined effects of deoxynivalenol and zearalenone on oxidative injury and apoptosis in porcine splenic lymphocytes in vitro.

Deoxynivalenol (DON) and zearalenone (ZEA) are the two most common mycotoxins in animal feed. In this study, we examined oxidative injury and apoptosis of porcine splenic lymphocytes induced by DON or ZEA and their combination in vitro. Based on IC50 values, porcine splenic lymphocytes were treated with 0.06, 0.3, 1.5, and 7.5µg/mL DON, 0.08, 0.4, 2, and 10µg/mL ZEA, or both DON and ZEA at 0.06 and 0.08µg/mL, 0.3 and 0.4µg/mL, and 1.5 and 2µg/mL, respectively. After 48h of DON and/or ZEA exposure, the cells were analyzed for antioxidant functions, apoptosis, and mRNA and protein expression of apoptosis-related genes p53, Bcl-2, Bax, caspase-3, and caspase-8 to determine their apoptosis and oxidative damage effects and mechanisms. The results showed that, compared with the control group, SOD, CAT, GPx, GSH, and Bcl-2 mRNA and protein expression levels were significantly reduced in exposed groups (P<0.05 or P<0.01). Furthermore, MDA contents, apoptosis rates, and p53, Bax, caspase-3 and caspase-8 protein and mRNA expression levels were increased significantly (P<0.01). The effects of DON and ZEA were dose dependent and synergistic in combination. These data suggest that DON and ZEA induce oxidative damage and apoptosis of porcine splenic lymphocytes.

Prototheca zopfii isolated from bovine mastitis induced oxidative stress and apoptosis in bovine mammary epithelial cells.

Bovine protothecal mastitis results in considerable economic losses worldwide. However, Prototheca zopfii induced morphological alterations and oxidative stress in bovine mammary epithelial cells (bMECs) is not comprehensively studied yet. Therefore, the aim of this current study was to investigate the P. zopfii induced pathomorphological changes, oxidative stress and apoptosis in bMECs. Oxidative stress was assessed by evaluating catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) contents and lactate dehydrogenase (LDH) activity, while ROS generation and apoptosis was measured by confocal laser scanning microscopy. The results revealed that infection of P. zopfii genotype II (GTII) significantly changed bMECs morphology, increased apoptotic rate and MDA contents at 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control group, in time-dependent manner. LDH activity and ROS generation was also increased (p < 0.01) at 12 h and 24 h. However, SOD and CAT contents in bMECs infected with GTII were decreased (p < 0.05) at 12 h, while GPx (p < 0.01), SOD (p < 0.05) and CAT (p < 0.01) levels were reduced at 24 h. In case of GTI, only CAT and GPx activities were significantly decreased when the duration prolonged to 24 h but lesser than GTII. This suggested that GTII has more devastating pathogenic effects in bMECs, and the findings of this study concluded that GTII induced apoptosis and oxidative stress in bMECs via the imbalance of oxidant and antioxidant defenses as well as the production of intracellular ROS.

Cloning, Expression, and Immunogenicity of Fimbrial-F17A Subunit Vaccine against Escherichia coli Isolated from Bovine Mastitis.

There is a need to identify and select new promising immunodominant antigens that have the ability to provide protective immunity against E. coli causing bovine mastitis. Recently we showed that f17a was found to be the most prevalent and crucial virulent factor among the pathogenic E. coli isolated from bovine mastitis. Here, in this report, the recombinant F17A based subunit vaccine adjuvant with MF59 was tested for immunogenicity against E. coli in a murine model. The vaccinated mice did not show any abnormal behavioral changes and histopathological lesions after vaccination. The specific antibody level against F17A was significantly higher in MF59-adjuvant-group, and also lasted for longer duration with a significant (P < 0.01) production level of IgG1 and IgG2a. Moreover, we noted higher survival rate in mice injected with F17A-MF59-adjuvant group after challenging with the clinical E. coli strain. Our findings of bacterial clearance test revealed that elimination rate from liver, spleen, and kidney in MF59-adjuvant-group was significantly higher than the control group. Finally, the proportion of CD4+T cells was increased, while CD8+ was decreased in MF59-adjuvant group. In conclusion, the current study reveals the capability of F17A-MF59 as a potential vaccine candidate against pathogenic E. coli causing mastitis in dairy animals.

Effects of deoxynivalenol on calcium homeostasis of concanavalin A--Stimulated splenic lymphocytes of chickens in vitro.

In this study, the in vitro effects of the treatment of concanavalin A (Con A)--stimulated splenic lymphocytes with DON were examined. Splenic lymphocytes isolated from chickens were stimulated with 12.5 µg/mL Con A and exposed to deoxynivalenol (DON) (0-50 µg/mL) for 48 h. The intracellular calcium concentration ([Ca(2+)]i), pH, calmodulin (CaM) mRNA levels, and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities were detected. With the DON exposure concentrations increased, the [Ca(2+)]i and CaM mRNA levels gradually increased in a dose-dependent manner, and all the evaluated conconcentrations affected ATPase activity to the same extent. There were significant differences (P<0.05 or P<0.01) between the treatment groups and the control group. These results indicate that an imbalance in calcium homeostasis and intracellular acidification are components of DON cytotoxicity in chicken lymphocytes.

Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen species-mediated mitochondrial pathway.

We investigated the immunotoxicity and cytotoxicity of deoxynivalenol (DON), a mycotoxin, and the mechanism by which it induces apoptosis. Chicken splenic lymphocytes treated with 0-50µg/mL DON for 48h inhibited growth of splenic lymphocytes in a dose-dependent manner, as revealed by the Cell Counting Kit-8 (CCK-8) bioassay. Annexin V-fluorescein isothiocyanate staining indicated that the number of apoptotic and necrotic cells were significantly higher compared with the control (P<0.01). DON treatment induced ROS accumulation, resulting in reduced mitochondrial transmembrane potential, as detected by flow cytometry and 2',7'-dichlorofluorescein acetate and rhodamine 123 labeling, respectively. Enzyme linked immunosorbent assays revealed that the concentrations of p53, Bax, Bak-1, and Caspase-3 increased with increasing DON concentration (P<0.05 or P<0.01), whereas the concentrations of Bcl-2 decreased (P<0.01) compared with the control. These data suggest that DON induces apoptosis in splenic lymphocytes via a ROS-mediated mitochondrial pathway.