Isolation and identification of the broad-spectrum high-efficiency phage vB_SalP_LDW16 and its therapeutic application in chickens.

BACKGROUND: Salmonella infection in livestock and poultry causes salmonellosis, and is mainly treated using antibiotics. However, the misuse use of antibiotics often triggers the emergence of multi-drug-resistant Salmonella strains. Currently, Salmonella phages is safe and effective against Salmonella, serving as the best drug of choice. This study involved 16 Salmonella bacteriophages separated and purified from the sewage and the feces of the broiler farm. A phage, vB_SalP_LDW16, was selected based on the phage host range test. The phage vB_SalP_LDW16 was characterized by the double-layer plate method and transmission electron microscopy. Furthermore, the clinical therapeutic effect of phage vB_SalP_LDW16 was verified by using the pathogenic Salmonella Enteritidis in the SPF chicken model. RESULTS: The phage vB_SalP_LDW16 with a wide host range was identified to the family Siphoviridae and the order Caudoviridae, possess a double-stranded DNA and can lyse 88% (22/25) of Salmonella strains stored in the laboratory. Analysis of the biological characteristics, in addition, revealed the optimal multiplicity of infection (MOI) of vB_SalP_LDW16 to be 0.01 and the phage titer to be up to 3 × 1014 PFU/mL. Meanwhile, the phage vB_SalP_LDW16 was found to have some temperature tolerance, while the titer decreases rapidly above 60 ℃, and a wide pH (i.e., 5-12) range as well as relative stability in pH tolerance. The latent period of phage was 10 min, the burst period was 60 min, and the burst size was 110 PFU/cell. Furthermore, gastric juice was also found to highly influence the activity of the phage. The clinical treatment experiments showed that phage vB_SalP_LDW16 was able to significantly reduce the bacterial load in the blood through phage treatment, thereby improving the pathological changes in the intestinal, liver, and heart damage, and promoting the growth and development of the chicken. CONCLUSIONS: The phage vB_SalP_LDW16 is a highly lytic phage with a wide host range, which can be potentially used for preventing and treating chicken salmonellosis, as an alternative or complementary antibiotic treatment in livestock farming.

Nitric oxide mediates apoptosis and mitochondrial dysfunction and plays a role in growth hormone deficiency by nivalenol in GH3 cells.

Nivalenol (NIV), a type B trichothecenes commonly found in cereal crops, can cause growth impairment in animals. However, limited information about its mechanisms is available. Trichothecenes have been characterized as an inhibitor of protein synthesis and induce apoptosis in cells. Oxidative stress is considered an underlying mechanism. However, whether NIV can induce oxidative stress and apoptosis in rat pituitary cells line GH3 is unclear. The present study showed that NIV significantly reduced the viability of cells and caused oxidative stress in GH3 cells. Further experiments showed that nitric oxide (NO), but not ROS, mediated NIV-induced oxidative stress. Additionally, NIV induced caspase-dependent apoptosis, decrease in mitochondrial membrane potential and mitochondrial ultrastructural changes. However, NIV-induced caspase activation, mitochondrial damage and apoptosis were partially alleviated by Z-VAD-FMK or NO scavenger hemoglobin. Finally, NIV changed the expression of growth-associated genes and pro-inflammatory cytokines. NIV also reduced the GH secretion in GH3 cells, which was reversed by hemoglobin. Taken together, these results suggested that NIV induced apoptosis in caspase-dependent mitochondrial pathway in GH3 cells, which might be an underlying mechanism of NIV-induced GH deficiency. Importantly, NO played a critical role in the induction of oxidative stress, apoptosis and GH deficiency in NIV-treated GH3 cells.

Nitric oxide (NO)-mediated mitochondrial damage plays a critical role in T-2 toxin-induced apoptosis and growth hormone deficiency in rat anterior pituitary GH3 cells.

T-2 toxin, a major compound of trichothecenes, induces cell apoptosis and growth hormone (GH) deficiency and causes considerable growth retardation in animals and human cells. However, the mechanism underlying its growth suppression still remains unclear. Recent studies have suggested that ROS induced cell apoptosis and animal feed intake reduction, but there are limited reports on the role of RNS in T-2 toxin-mediated mitochondrial damage, cell apoptosis and growth retardation. Herein, T-2 toxin-induced GH3 cell damage and apoptosis were tested by MTT assay, LDH leakage and flow cytometry, respectively. Intracellular NO and antioxidant enzyme activity, ΔΨm, morphometric changes of mitochondria, the caspase pathway, and inflammatory factors were investigated. Free radical scavengers NAC, SOD and NO scavenger haemoglobin were used to explore the role of oxidative stress and the relationship between NO production and caspase pathway. The results clearly revealed that T-2 toxin caused significant increases in NO generation, cell apoptosis, GH deficiency, increased iNOS activity, upregulation of inflammatory factors and caspase pathway, decreases in ΔΨm and morphosis damage. These data suggest that mitochondria are a primary target of T-2 toxin-induced NO, and NO is a key mediator of T-2 toxin-induced cell apoptosis and GH deficiency via the mitochondria-dependent pathway in cells.

Metabolites and JAK/STAT pathway were involved in the liver and spleen damage in male Wistar rats fed with mequindox.

Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides antibacterial agent and growth promoter in animal husbandry. This study was to investigate whether reactive oxygen species (ROS), the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, suppressors of cytokine signaling (SOCS) and inflammatory cytokines were involved in toxicities of MEQ. Our data demonstrated that high dose of MEQ (275 mg/kg) apparently led to tissue impairment combined with imbalance of redox in liver. In liver and spleen samples, hydroxylation metabolites and desoxymequindox were detected, directly confirming the potential link of N→O group reduction metabolism with its organ toxicity. Moreover, up-regulation of JAK/STAT, SOCS family, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were also observed in the high-dose group. Meanwhile, significant changes of oxidative stress indices in liver were observed in the high-dose group. As for NADPH subunit, the mRNA levels of many subunits were significantly up-regulated at low doses but down-regulated in a dose-dependent manner in liver and spleen, suggesting an involvement of NADPH in MEQ metabolism and ROS generation. In conclusion, we reported the dose-dependent long-term toxicity as well as the discussion of the potential mechanism and pathways of MEQ, which raised further awareness of its toxicity following with the dose change.

Interactions of NADPH oxidase, renin-angiotensin-aldosterone system and reactive oxygen species in mequindox-mediated aldosterone secretion in Wistar rats.

High doses of mequindox (MEQ) are associated with oxidative stress and pathological toxicity in the kidney. In this study, we demonstrated long term effects of MEQ on intra- or extra-adrenal renin-angiotensin-aldosterone system (RAAS) in vivo. RAAS plays a major role in aldosterone secretion. High doses of MEQ in the diet for 180 days in male rats led to inhibition of intra- and extra-adrenal RAAS, concident with down-regulation of Na(+)/K(+)-ATPase (NAKA) and mineralocorticoid receptor (MR), the downstream of aldosterone action. Significant changes of malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) in kidney were also observed in the high doses (110, 275mg/kg) groups. The mRNA levels of most subunits of NADPH oxidase were significantly upregulated at low doses (25-110mg/kg) but the upregulation was diminished at higher doses in both kidney and adrenal gland, indicating a complicated and contradictory effect of MEQ on NADPH. These results highlight the complex interactions of drug metabolism, RAAS, NADPH oxidase and oxidative stress in response to MEQ-induced tissue toxicity and aldosterone secretion.

Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats.

Mequindox (MEQ) is a synthetic quinoxaline 1,4-dioxides (QdNOs) derivative which can effectively improve growth and feed efficiency in animals. This study was to investigate the dose-dependent long-term toxicity in the adrenal of male rats exposed to 180 days of MEQ feed. Our data demonstrated that high doses of MEQ in the diet for 180 days led to adrenal damage and steroid hormone decrease, combined with sodium decrease and potassium increase in rat plasma. Significant changes of GSH and SOD in plasma were observed in the high doses (110, 275 mg/kg) groups. At the same doses, MEQ treatment down-regulated the mRNA levels of CYP11A1, CYP11B1 and CYP11B2 which located in mitochondria, but up-regulated mRNA levels of CYP21 and 3beta-HSD which located in endoplasmic reticulum. In conclusion, we reported the dose-dependent long-term toxicity of MEQ on adrenal gland in male rats, which raise awareness of its toxic effects to animals and consumers, and its mechanism may involve in oxidative stress and steroid hormone biosynthesis pathway.