Oxidative Stress Induced By Spring Viraemia Of Carp Virus And Saprolegniasis

In recent years, many studies have shown that a variety of microbial infection can induce oxidative stress, and oxidative stress is one of the pathogenic mechanisms of microbial infection. The relevance between microbial infection and oxidative stress has received more and more attention. While, little was known about the relationship between microbial infection of aquatic animals and oxidative stress. In this study, we used two kinds of common pathogenic microorganisms in aquatic animals, Spring Viraemia of Carp Virus(SVCV) and Saprolegnia to investigate the relationship between microbial infection of aquatic animals and oxidative stress.The major results are summarized as following: 1.Oxidative stress induced by SVCV in Epithelioma Papulosum Cyprini cells.In this study, SVCV was used to infect the Epithelioma Papulosum Cyprini cells at MOI of 0.1 and 1, then several biomarkers of oxidative stress were measured at 0, 3, 6, 12 and 24 h post-infection. Oxidative stress occured after 1m M H2O2 stimulated EPC cells for 24 h was used as the positive control. The biomarkers include oxidative levels: reactive oxygen speices(ROS), protein carbonyl, 8-hydroxy-2-deoxyguanosine(8-OHd G) and malondialdehyde(MDA); and antioxidant levels: total antioxidant capacity(T-AOC), superoxide dismutase(SOD), glutathione(GSH), glutathione peroxidase(Gpx) and catalase(CAT). Meanwhile, real-time PCR was performed to study the transcriptional levels of the glutathione synthetase(GSS) gene, the catalase(CAT) gene, and the superoxide dismutase 1(SOD-1) gene in EPC cells infected by SVCV. Finally EPC cells were were pre-treated with 500μM H2O2 for 2 h and the transcriptional levels of SVCV G gene were analyzed quantificationally after infection with SVCV(0.1MOI). The results demonstrated that: 1. oxidative levels in EPC cells: ROS and protein carbonyl commenced to increase upon SVCV infection and returned to normal levels at 12 h post-infection following with a higher increasment at 24 h post-infection, 8-OHd G increased significantly until 24 h post-infection and MDA showed no significant change; antioxidant levels in EPC cells: T-AOC increased significantly until 24 h post-infection with SVCV, GSH and the transcriptional levels of GSS gene increased significantly until 24 h post-infection(1MOI), CAT and the transcriptional levels of CAT gene commenced to decrease upon SVCV infection and returned to normal levels at 12 h post-infection following by a significant decrease at 24 h post-infection, SOD and the transcriptional levels of SOD-1 gene commenced to increase upon SVCV infection and returned to normal levels quickly at 6h post-infection until 24 h post-infection, Gpx increased significantly until 24 h post-infection with SVCV; oxidative/ antioxidant levels in EPC cells: ROS/T-AOC commenced to decrease upon SVCV infection and reduce to a minimum level at 12 h post-infection following by a higher increase at 24 h post-infection. In total, EPC cells infected with SVCV could produce a series of ROS and caused oxidative damage of DNA and proteins seriously. 2. The levels of ROS in EPC cells pre-treated with 500μM H2O2 for 2 h increased significantly. It suggested that EPC cells were situated in the state of oxidative stress. And the transcriptional levels of SVCV G gene decreased significantly after EPC cells were infected by SVCV at MOI of 0.1.2.2.Oxidative stress induced by Saprolegniasis in Common CarpIn this study, plasma from common carps infected with Saprolegniasis was collected. Several biomarkers of oxidative stress were measured. The markers include the total antioxidant capacity(T-AOC), lipid peroxidation- malondialdehyde(MDA), superoxide dismutase(SOD), glutathione(GSH), glutathione peroxidase(Gpx) and catalase(CAT), was estimated by measuring. Compared with the control group, some biomarkers, including T-AOC, SOD and CAT of the common carps infected with Saprolegniasis, were decreased significantly(p<0.01), and the plasma Gpx was increased significantly(p<0.05). Other biomarkers of plasma MDA and GSH showed no significant change after the infection with Saprolegniasis. Totally, oxidative stress has occurred upon Saprolegniasis infection. The histopathology damage of liver, spleen, and kidney tissues from diseased fish were observed by hematoxylin and eosin(HE) staining. The results suggested that liver cells of the common carps infected with Saprolegniasis was presented with the symptom of acute necrosis, and became vacuolization seriously; spleen and kidney cells was presented with inflammations of different severity, senescence and apoptosis. All the experimental results showed that the antioxidant levels of the common carps infected with Saprolegniasis significantly reduced, which could not effectively remove ROS produced in the carp cells and the oxidative stress was induced. The occurrence of oxidative stress caused oxidative damage of DNA, lipids and proteins, which destroyed the normal physiological function of the carp cells, and resulted in the tissue damage. Meanwhile, oxidative stress induced the inflammation and the occurrence of apoptosis, further aggravated the tissue damage in the common carps.This study provided the theoretical foundation for further research on the pathogenic mechanism of microbial infection in aquatic animals, which can provide new strategies for the prevention and therapeutic of aquatic animal diseases.