Transcriptional regulation of extracellular copper zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus and WSSV infection

The cDNA encoding an extracellular copper zinc superoxide dismutase (LvECSOD) was cloned from the hepatopancreas of white shrimp Litopenaeus vannamei. It consisted of 915 bp nucleotides with an open reading frame corresponding to a deduced protein of 178 amino acids. The LvECSOD contains a putative signal peptide of 16 amino acids, two potential N-linked glycosylation sites (N(115)GTA and N(135)ITG) and a copper zinc superoxide dismutase family signature sequence (G(162)NAGaRvACctI(173)). It was found that four copper binding sites, four zinc binding sites and two cysteines involving in the formation of the disulfide bridge were conserved in the protein. LvECSOD shared 33-58% identity to ECSODs from other organisms. Expression analysis revealed that LvECSOD mRNA was widely distributed in all the tissues examined. When the shrimp challenged with Vibrio alginolyticus or white spot syndrome virus (WSSV), expression of LvECSOD mRNA in the hepatopancreas and hemocytes was mediated responsively. Our results suggested that LvECSOD was implicated in the immune response induced by V. alginolyticus and WSSV.     

The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei

White shrimp, Litopenaeus vannamei, which had been injected with chitin at 4, 6 and 8 microg g(-1) or chitosan at 2, 4 and 6 microg g(-1), were challenged with pathogen Vibrio alginolyticus at 2 x 10(6) colony-forming units (cfu) shrimp(-1) and then placed in seawater of 34 per thousand. The survival of shrimp that received chitin or chitosan at either dose was significantly higher than that of control shrimp after 1 day, and at the termination of the experiment (6 days after the challenge). In another experiment, the total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, and phagocytic activity to V. alginolyticus were measured when L. vannamei (10.4 +/- 0.7 g) were injected individually with chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1). L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased significantly its THC and respiratory burst after 2 days. L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) still maintained significantly higher phenoloxidase activity after 6 days. L. vannamei received chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased its phagocytic activity against V. alginolyticus after 1 day, respectively. It is therefore concluded that L. vannamei that received chitin at 6 microg g(-1) or chitosan at 4 microg g(-1) or less increased its immune ability and resistance to V. alginolyticus infection.     

Fucoidan effectively provokes the innate immunity of white shrimp Litopenaeus vannamei and its resistance against experimental Vibrio alginolyticus infection

In this study, we examined the effect of fucoidan on the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus infection. Fucoidan induced degranulation, caused changes in the cell morphology, and increased activation of prophenoloxidase (proPO) and the production of superoxide anions in vitro. Shrimp that received fucoidan via immersion at 100, 200, and 400 mg l^?1 after 3 h showed haemocyte proliferation and a higher mitotic index of haematopoietic tissue. In another experiment, the haemocyte count, phenoloxidase (PO) activity, and respiratory bursts (RBs) were examined after the shrimp had been fed diets containing fucoidan at 0 (control), 0.5, 1.0, and 2.0 g kg^?1 for 7–21 days. Results indicated that these parameters directly increased with time. The immune parameters of shrimp fed the 1.0 g kg^?1 diet were significantly higher than those of shrimp fed the 2.0 g kg^?1 diet after 14 and 21 days. Phagocytic activity and the clearance efficiency against V. alginolyticus were significantly higher in shrimp fed the 1.0 g kg^?1 diet compared to those of shrimp fed the 0, 0.5 and 2.0 g kg^?1 diets. In a separate experiment, shrimp that had been fed diets containing fucoidan for 21 days were challenged with V. alginolyticus at 10⁶ colony-forming units shrimp^?1. Survival rates of shrimp fed the 1.0 and 2.0 g kg^?1 diets were significantly higher than those of shrimp fed the 0 and 0.5 g kg^?1 diets for 96–120 h. We concluded that fucoidan provokes innate immunity of shrimp as evidenced by haemocyte degranulation, proPO activation, and the mitotic index of haematopoietic tissue, and that dietary administration of fucoidan at 1.0 g kg^?1 enhanced the immune response of shrimp and their resistance against V. alginolyticus infection.     

'Bright-red' syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi

Since July 2005, recurrent outbreaks of vibriosis have occurred in shrimp farms in northwestern Mexico. Moribund Litopenaeus vannamei associated with mass mortalities were lethargic and displayed red discoloration spots on their abdomen, and hence were called 'bright-reds' by farmers. Shrimp submitted for diagnosis were examined using wet tissue mounts, bacteriological assays and their respective minimum inhibitory concentration (MIC), and histology. A dominant yellow bacterial colony was isolated in thiosulphate citrate bile salts-sucrose (TCBS) agar and identified by molecular methods as Vibrio harveyi strain CAIM 1792. Pathogenicity of the V. harveyi strain was demonstrated in L. vannamei. The lowest MIC against Vibrio isolates from bright-red shrimp was obtained with enrofloxacine (3.01, SD = 5.96 pg ml(-1)). Histology detected severe necrosis in lymphoid organ tubules, muscle fibers, and connective tissue, as well as melanization and hemocytic nodules associate with microcolonies of Gram-negative bacilli. Bacteria from severely affected shrimp were dispersed from the haemocoel to other tissues causing a systemic vibriosis. The data indicate that V. harveyi strain CAIM 1792 is the cause of bright-red syndrome (BRS) and represents a threat to the Mexican shrimp farming industry.     

Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing

A gram-negative, rod-shaped bacterium identified as Vibrio alginolyticus was isolated from diseased Litopenaeus vannamei (also called Penaeus vannamei) in Taiwanese culture ponds. The diseased shrimp displayed poor growth, anorexia, inactivity, reddish pleural borders of antennae, uropods and telson, opaque and whitish musculature, and mortality. In histological preparations, melanized hemocytic granulomas were observed in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature. Six isolates of Vibrio were collected from diseased shrimp at 3 farms, and these were evaluated for characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics. The results indicated that the isolates belonged to a single species that grew in 1 to 8% NaCl, at 10 to 40 degrees C and on TCBS (thiosulfatecitrate-bile sucrose) agar, and that gave positive catalase, O/F (Oxidation/Fermentation), lysine decarboxylase, gelatinase and cytochrome-oxidase tests. Identification of CH003 (1 of 6 isolates) was confirmed by PCR assay for V. alginolyticus (expected amplicon 1486 bp). The 16S rDNA sequence (GenBank accession number AY373027) gave 99.9% sequence identity to V. alginolyticus (GenBank accession number X74690). The calculated 96 h LD50 dose of the isolated strain was 3.0 x 10(5) colony forming units (CFU) shrimp(-1) (6.6 x 10(4) CFU g(-1)).     

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.     

The immune stimulatory effect of sodium alginate on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4-11.3 g) were injected individually with sodium alginate at 10, 20 or 50 microg g(-1). No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 microg g(-1). However, L. vannamei injected with sodium alginate at 20 microg g(-1)increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 microg g(-1)maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2x10(5)colony-forming units (CFU) shrimp(-1)and then placed in seawater of 34 per thousand. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 microg g(-1)or more and increased its immune ability and resistance from V. alginolyticus infection.     

Apoptosis plays an important role in experimental rabies virus infection.

Cultured rat prostatic adenocarcinoma (AT3) cells infected with the challenge virus standard (CVS) strain of fixed rabies virus showed characteristic morphologic features of apoptosis, evidence of oligonucleosomal DNA fragmentation, and expression of the Bax protein. CVS-infected Bcl-2-transfected AT3 cells did not demonstrate these features. Adult ICR mice inoculated intracerebrally with CVS showed morphologic changes of apoptosis, DNA fragmentation, and increased Bax expression in neurons, with changes most marked in the hippocampus and cerebral cortex. Ultrastructurally, some neurons demonstrated morphologic features more typical of necrosis. These studies provide evidence that apoptosis plays an important role in the pathogenesis of rabies virus infection.     

Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

Haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured in white shrimp Litopenaeus vannamei juveniles (12.3 +/- 1.2 g) which had been fed diets containing sodium alginate at 0.5, 1.0, 2.0 g kg(-1) after five months. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phenoloxidase activity, respiratory burst and SOD activity, but decreased GPX activity significantly. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phagocytic activity and the shrimp fed a diet containing sodium alginate at 0.5, 1.0 or 2.0 g kg(-1) had increased clearance efficiency to V. alginolyticus. In another experiment, L. vannamei, which had been fed control diet, or sodium alginate-containing diets after 5 months, were challenged with V. alginolyticus at 2 x 10(6) colony-forming units (CFU) shrimp(-1) and then placed in seawater of 15 per thousand. The survival of shrimp fed a diet containing 2.0 g kg(-1) after one day, and the survival of shrimp fed diets containing sodium alginate at 0.5 and 1.0 g kg(-1) after 2-4 days increased significantly, as compared to that of shrimp fed control diet. It is therefore concluded that administration of sodium alginate in the diet at 2.0 g kg(-1) or less could enhance the immune ability of L. vannamei and increase its resistance to V. alginolyticus infection.     

The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle

The white shrimp Litopenaeus vannamei (8.0-14.4 g) was examined for haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity, and clearance efficiency to the pathogen Vibrio alginolyticus in relation with moult cycle (postmoult, A, B; intermoult, C; premoult, D(0)/D(1)D(2)/D(3)). Granular cells were the highest at C and D(0)/D(1)stage, and the lowest at A stage. Hyaline cells and THC (total haemocyte count) were higher at C stage, but lower at postmoult stages. Phenoloxidase activity was the highest at C stage, and the lowest at A stage. Respiratory burst was lower at A stage. Phagocytic activity of shrimps against V. alginolyticus decreased significantly at postmoult and premoult stages. Additionally, the clearance efficiency of shrimps to V. alginolyticus was significantly lower for shrimps at A stage than those at C stage. In another experiment, L. vannamei at different moult stages were injected with tryptic soy broth (TSB)-grown V. alginolyticus (1x10(5)cfu shrimp(-1)) and then held in 34% seawater. After 10 h, the mortality of V. alginolyticus-injected shrimps was significantly higher for shrimps at postmoult stage than those at intermoult stage. Over 48-120 h, the mortality of V. alginolyticus-injected shrimps was 50.0%, 33.3% and 40.0% at postmoult, intermoult and premoult stage, respectively. It is concluded that L. vannamei showed a decrease in resistance at A stage through a reduction of its haemocyte count, phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency against V. alginolyticus.     

The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were examined in the white shrimp Litopenaeus vannamei (10.3+/-1.5 g) injected individually with hot-water extract of Gracilaria tenuistipitata at 4 or 6 microg g-1. L. vannamei receiving hot-water extract of G. tenuistipitata at either dose increased significantly its THC, phenoloxidase activity, and respiratory burst after 2 days. L. vannamei received hot-water extract of G. tenuistipitata at 6 microg g-1 increased its phagocytic activity and clearance efficiency to V. alginolyticus after 1 day. In another experiment, L. vannamei which had been injected with hot-water extract of G. tenuistipitata were challenged with V. alginolyticus at 2x10(6) colony-forming units (cfu) shrimp-1 and then placed in seawater of 34 per thousand. The survival of shrimp that received hot-water extract of G. tenuistipitata at 6 microg g-1 was significantly higher than that of shrimp that received saline and the control shrimp after 3 days, and at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei receiving the hot-water extract of G. tenuistipitata at 6 microg g-1 or less increased its immune ability and resistance to V. alginolyticus infection.     

Effect of saponin immersion on enhancement of the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The haemocyte count, phenoloxidase (PO) activity, specific alpha(2)-macroglobulin (alpha2-M) activity, respiratory burst, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, phagocytic activity, and clearance efficiency against Vibrio alginolyticus were examined when the white shrimp Litopenaeus vannamei (10.42+/-2.0g) were immersed in seawater (34 per thousand) containing different concentrations of saponin (0, 0.5, 1 and 2mgL(-1)) for 24, 48 and 72h. Hyaline cells (HC), the total haemocyte count (THC), specific alpha2-M activity, respiratory burst, SOD activity, and GPx activity directly increased with the saponin concentration, whereas PO activity was inversely related to the saponin concentration. White shrimp L. vannamei that were immersed in saponin at 1 and 2mgL(-1) showed increased phagocytic activity and clearance efficiency to V. alginolyticus over 48-72h. In another experiment, shrimp immersed in seawater containing different concentrations of saponin after 72h were challenged with V. alginolyticus at 3.2x10(5) colony-forming units (cfu)shrimp(-1), and then placed in seawater. The survival rate of shrimp immersed in seawater containing saponin at either dose was significantly higher than that of control shrimp after 12h, as well as at the termination of the experiment (5days after the challenge). It was therefore concluded that L. vannamei immersed in water containing saponin at 2mgL(-1) or less exhibited an immunomodulatory effect, as well as protection against V. alginolyticus infection.     

A peroxide-induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis

In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA-binding protein MrgA. We have identified the P-type metal-transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon. Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region. Physiological studies implicate ZosA in Zn(II) uptake. ZosA functions together with two Zur-regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis. ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur-regulated transporters. A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation. ZosA is important for resistance to both H2O2 and the thiol-oxidizing agent diamide. This suggests that increased intracellular Zn(II) may protect thiols from oxidation. In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance. Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA. Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.     

Autophagy plays an important role in protecting Pacific oysters from OsHV-1 and Vibrio aestuarianus infections

Recent mass mortality outbreaks around the world in Pacific oysters, Crassostrea gigas, have seriously affected the aquaculture economy. Although the causes for these mortality outbreaks appear complex, infectious agents are involved. Two pathogens are associated with mass mortality outbreaks, the virus ostreid herpesvirus 1 (OsHV-1) and the bacterium Vibrio aestuarianus. Here we describe the interactions between these 2 pathogens and autophagy, a conserved intracellular pathway playing a key role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters.     

Human low-density lipoprotein receptor plays an important role in hepatitis B virus infection

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV vaccine is effective to prevent new HBV infection but does not offer therapeutic benefit to hepatitis B patients. Neither are current antiviral drugs curative of chronic hepatitis B. A more thorough understanding of HBV infection and replication holds a great promise for identification of novel antiviral drugs and design of optimal strategies towards the ultimate elimination of chronic hepatitis B. Recently, we have developed a robust HBV cell culture system and discovered that human apolipoprotein E (apoE) is enriched on the HBV envelope and promotes HBV infection and production. In the present study, we have determined the role of the low-density lipoprotein receptor (LDLR) in HBV infection. A LDLR-blocking monoclonal antibody potently inhibited HBV infection in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) as well as in primary human hepatocytes. More importantly, small interfering RNAs (siRNAs)-mediated knockdown of LDLR expression and the CRISPR/Cas9-induced knockout of the LDLR gene markedly reduced HBV infection. A recombinant LDLR protein could block heparin-mediated apoE pulldown, suggesting that LDLR may act as an HBV cell attachment receptor via binding to the HBV-associated apoE. Collectively, these findings demonstrate that LDLR plays an important role in HBV infection probably by serving as a virus attachment receptor.