Ping Ouyang

Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish ( Ictalurus Punctatus ) against Streptococcus iniae

Interleukin-8 (IL-8) as an important cytokine involving in inflammatory and immune response, has been studied as effective adjuvants for vaccines in mammals. However, there are fewer reports about the characterization and adjuvant effects of IL-8 in fish. In this study, cloning and sequence analysis of IL-8 coding region of channel catfish (Ictalurus punctatus) were conducted, mature IL-8(rtIL-8) was expressed and evaluated for its adjuvant effects on the immunoprotection of subunit vaccine encoding α-enolase (rENO) of Streptococcus iniae from several aspects in channel catfish. The results showed co-vaccination of rENO with rtIL-8 enhanced immune responses including humoral and cellular immunity, with higher relative percent survival(RPS,71.4%) compared with the moderate RPS of rENO alone(50%) against S. iniae infection at 4 week post vaccination. While rtIL-8 failed to maintain long-lasting immune protection, only with RPS of 26.67% in rENO + rtIL-8-vaccinated fish compared with that of rENO alone(20%) at 8 week, signifying that IL-8 hold promise for use as potential immunopotentiator in vaccines against bacterial infections in fish, whereas it is insufficient to extend the immunoprotection for long time, and further studies are required to understand the mechanisms of IL-8 used as an adjuvant and seek for more effective way to strengthen the adjuvanticity of IL-8.

Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection

qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA.

Adjuvant Immune Enhancement of Subunit Vaccine Encoding pSCPI of Streptococcus iniae in Channel Catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund’s Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1β, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.

The molecular mechanism of G 2 /M cell cycle arrest induced by AFB 1 in the jejunum

Aflatoxin B1 (AFB1) has potent hepatotoxic, carcinogenic, genotoxic, immunotoxic and other adverse effects in human and animals. The aim of this study was to investigate the molecular mechanism of G2/M cell cycle arrest induced by AFB1 in the jejunum of broilers. Broilers, as experimental animals, were fed 0.6 mg/kg AFB1 diet for 3 weeks. Our results showed that AFB1 reduced the jejunal villus height, villus height/crypt ratio and caused G2/M cell cycle arrest. The G2/M cell cycle was accompanied by the increase of ataxia telangiectasia mutated (ATM), p53, Chk2, p21 protein and mRNA expression, and the decrease of Mdm2, cdc25C, cdc2, cyclin B and proliferating cell nuclear antigen protein and mRNA expression. In conclusion, AFB1 blocked G2/M cell cycle by ATM pathway in the jejunum of broilers.

A study on the expression of apoptotic molecules related to death receptor and endoplasmic reticulum pathways in the jejunum of AFB 1 -intoxicated chickens

Aflatoxin B1 (AFB1) is a common contaminant of poultry feeds in tropical and subtropical climates. Early researches have well established the hepatotoxic, carcinogenic, and immunotoxic effects of AFB1 on humans and animals. Recently, it has been shown that AFB1 could cause the up- or down-alteration of mitochondrial pathway molecule expression. However, the information on the expression of death receptor and endoplasmic reticulum molecules in the jejunal apoptosis induced by AFB1 were unavailable. So the present study was conducted to explore the expression of apoptotic molecules related to death receptor and endoplasmic reticulum in the jejunal cells of chickens exposed to AFB1 diet for 3 weeks. Total of 144 one-day-old chickens was randomly divided into two groups, namely control group (containing 0 mg/kg AFB1) and AFB1 group (containing 0.6 mg/kg AFB1). Histopathological observation and microscopic quantitative analysis revealed morphological changes in the jejunum such as the shedding of the mucosal epithelial cells in the apical region of villi along with the decrease of villus height, villus area and villus/crypt ratio in the AFB1 group. Both TUNEL and flow cytometry assays showed that AFB1 intake induced excessive apoptosis of jejunal cells. Quantitative real-time PCR test displayed the general upregulation of death receptors (FAS, FASL, TNF-α and TNF-R1), endoplasmic reticulum signals (GRP78 and GRP94) as well as initiator and executioner caspases (CASPASE-10, CASPASE-8 and CASPASE-3) in the jejunum of AFB1-intoxicated chickens. Its the first study demonstrating that AFB1 induced apoptosis of chickens’ jejunum accompanied by the alteration of death receptor and endoplasmic reticulum molecule expression.

Outbreak of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout in China.

Infectious pancreatic necrosis virus (IPNV) is a member of the Aquabirnavirus genus, which caused mass mortality (nearly 100%) in farmed rainbow trout (Oncorhynchus mykiss) in aquaculture farms in 2016, China. Major clinical signs included decreased appetite, mucous-like stools, and darkened pigmentation. Pathological changes in moribund fish were observed, such as marked vacuolar degeneration of the pancreatic cells with pyknotic nucleus and decreased zymogen granules, severe hemorrhage in the liver, and tumidness of respiratory epithelium in gills. In addition, the tissue fluid of diseased fish could produce a cytopathic effect (CPE) in RTG-2 cells. The presence of specific 206bp fragments by the reverse transcription-polymerase chain reaction (RT-PCR) using tissue homogenate of diseased fish and supernatant of infected cells revealed that IPNV could be confirmed. The pathogenicity test of cell culture supernatant detected cumulative mortality of 80%, and the clinical symptoms observed in the moribund and dead fish were similar to the naturally infected fish. Furthermore, the sequence analysis of VP2 gene showed that the isolated virus strain belonged to genogroup 1, and 97% homology with the Mexican IPNV isolate was found. To our knowledge, this is the first report on IPNV natural infection in the southwest of China.

Interleukin-8 holds promise to serve as a molecular adjuvant in DNA vaccination model against Streptococcus iniae infection in fish

DNA vaccines had been widely used in animal models against various viral infections, while it was not so convincing for many infectious diseases especially bacterial disease in aquaculture. Interleukin-8(IL-8) as one of the CXC chemokines, its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, recombination plasmid pcDNA3.1/IL-8(pcIL-8) was conducted and the capacity of IL-8 as molecular adjuvant was explored from several aspects by co-injecting with a DNA vaccine encoding a-enolase(pcENO) against Streptococcus iniae infection in channel catfish. The results suggested that co-injection of pcIL-8 with DNA vaccine increased the innate immunity and specific antibody levels, as well as increased the immune-related genes involving in pro-inflammatory response, humoral and cellular immunity. Moreover, pcIL-8 enhanced the immunoprotection of pcENO with the relative percent survival(RPS) of 60% to 80% against S.iniae infection at 4 week post vaccination(p.v.), with the significantly higher RPS of 73.33% in pcENO+pcIL-8 group compared with that of pcENO alone(53.33%) at challenge test of 8 weeks p.v. Taken together, these results indicate pcIL-8 as a molecular adjuvant co-injected with DNA vaccine not only improves the immunoprotection but also maintains long period of immunity for channel catfish against S.iniae infection. Our study signifies that IL-8 holds promise to serve as a potential adjuvant in DNA vaccines against bacterial infections for long time.

Genome Sequence of the Fish Pathogen Yersinia ruckeri SC09 Provides Insights into Niche Adaptation and Pathogenic Mechanism

Yersinia ruckeri is the etiologic agent of enteric red mouth disease (ERM), a severe fish disease prevailing in worldwide aquaculture industries. Here we report for the first time the complete genome of Y. ruckeri (Yersinia ruckeri) SC09, a highly virulent strain isolated from Ictalurus punctatus with severe septicemia. SC09 possesses a single chromosome of 3,923,491 base pairs, which contains 3651 predicted protein coding sequences (CDS), 19 rRNA genes, and 79 tRNA genes. Among the CDS, we have identified a Ysa locus containing genes encoding all the components of a type III secretion system (T3SS). Comparative analysis suggest that SC09-Ysa share extensive similarity in sequence, gene content, and gene arrangement with Salmonella enterica pathogenicity island 1 (SPI1) and chromosome-encoded T3SS from Yersinia enterocolitica biotype 1B. Furthermore, phylogenetic analysis shown that SC09-Ysa and SPI1-T3SS belong on the same branch of the phylogenetic tree. These results suggest that SC09-Ysa and SPI1-T3SS appear to mediate biological function to adapt to specific hosts with a similar niche, and both of them are likely to facilitate the development of an intracellular niche. In addition, our analysis also indicated that a substantial part of the SC09 genome might contribute to adaption in the intestinal microenvironment, including a number of proteins associated with aerobic or anaerobic respiration, signal transduction, and various stress reactions. Genomic analysis of the bacterium offered insights into the pathogenic mechanism associated with intracellular infection and intestinal survivability, which constitutes an important first step in understanding the pathogenesis of Y. ruckeri.

Hepatoprotective potential of isoquercitrin against type 2 diabetes-induced hepatic injury in rats

Non-alcoholic fatty liver disease is a main complication of type 2 diabetes. Isoquercitrin are employed for antidiabetic therapies, but the effects on liver function and the hepatocytes are unclear. The aim of this study was to investigate the effects of isoquercitrin on the T2DM-induced hepatic injury in rats. Isoquercitrin (10 mg/kg/d, 30 mg/kg/d), sitagliptin phosphate (10 mg/kg/d) was given orally for 21 days. The administration of isoquercitrin at 10 mg/kg/d and 30 mg/kg/d showed a dose dependent. Compare to the negative control (treated with saline), rats medicated with isoquercitrin (30 mg/kg/d) and sitagliptin phosphate (10 mg/kg/d) improved the clinical symptoms, FBG and glucose tolerance, reduced serum ALT, AST and IR, but increased TP, Alb, SOD, GSH, MDA, HDL-C, INS and GLP-1. On histology, Rats of these to groups presented nearly normal liver tissue and Langerhans, degeneration, necrosis and apoptosis were markedly reduced. Instead, hepatocytes showed regenerate. These two groups also showed significant increase in mRNA expression of PKA, AKT, PKCa, InsR and PI3K, and a decrease in DPP-IV mRNA level. These results indicated that treatment with isoquercitrin protects against hepatic injury by T2DM.

Sodium selenite prevents suppression of mucosal humoral response by AFB 1 in broiler’s cecal tonsil

Aflatoxin B1 (AFB1), the most common mycotoxin in human food and animal feed, produces hepatotoxic, genotoxic and immunosuppressive effects in multiple species. Selenium (Se) has emerged as an important element in the dietary prevention of various toxic agents. The present study was designed to scrutinize the protective effects of sodium selenite on the histological lesions and suppression of mucosal humoral response in the cecal tonsil generated by AFB1. A total of 156 one-day-old broilers were divided into four groups and fed on basal diet (control group), 0.6 mg/kg AFB1 (AFB1 group), 0.4 mg/kg Se supplement (+Se group), and 0.6 mg/kg AFB1 + 0.4 mg/kg Se supplement (AFB1+Se group) respectively for 21 days. Our results showed that 0.4 mg/kg Se supplement in broilers diets could improve the AFB1-induced histological lesions in the cecal tonsils including the depletion of lymphocytes in the lymphatic nodules as well as the shedding of microvilli in the absorptive cells. Moreover, Se could restore the decreased number of IgA+ cells and expression levels of pIgR, IgA, IgG, and IgM mRNA induced by AFB1 to be close to those in the control group. These results demonstrated that 0.4 mg/kg supplemented dietary Se in the form of sodium selenite could protect the cecal tonsils from the histological lesions and suppression of the mucosal humoral response provoked by 0.6 mg/kg AFB1. Our study may provide new experimental evidences for better understanding of AFB1-induced damage of mucosal immunity and protective effect of Se against this toxin.Aflatoxin B1 (AFB1), the most common mycotoxin in human food and animal feed, produces hepatotoxic, genotoxic and immunosuppressive effects in multiple species. Selenium (Se) has emerged as an important element in the dietary prevention of various toxic agents. The present study was designed to scrutinize the protective effects of sodium selenite on the histological lesions and suppression of mucosal humoral response in the cecal tonsil generated by AFB1. A total of 156 one-day-old broilers were divided into four groups and fed on basal diet (control group), 0.6 mg/kg AFB1 (AFB1 group), 0.4 mg/kg Se supplement (+Se group), and 0.6 mg/kg AFB1 + 0.4 mg/kg Se supplement (AFB1+Se group) respectively for 21 days. Our results showed that 0.4 mg/kg Se supplement in broilers diets could improve the AFB1-induced histological lesions in the cecal tonsils including the depletion of lymphocytes in the lymphatic nodules as well as the shedding of microvilli in the absorptive cells. Moreover, Se could restore the decreased number of IgA+ cells and expression levels of pIgR, IgA, IgG, and IgM mRNA induced by AFB1 to be close to those in the control group. These results demonstrated that 0.4 mg/kg supplemented dietary Se in the form of sodium selenite could protect the cecal tonsils from the histological lesions and suppression of the mucosal humoral response provoked by 0.6 mg/kg AFB1. Our study may provide new experimental evidences for better understanding of AFB1-induced damage of mucosal immunity and protective effect of Se against this toxin.