Kaiyu Wang

First Report of a Ranavirus Associated with Morbidity and Mortality in Farmed Chinese Giant Salamanders (Andrias davidianus)

From February to May 2010, an outbreak of disease occurred amongst farmed Chinese giant salamanders (Andrias davidianus) in Hanzhong County, Shanxi Province, China. Clinical signs included anorexia, lethargy, ecchymoses and swollen areas on the head and limbs, and skin ulceration. The aim of this study was to determine the cause of this disease. Necropsy examination revealed subcutaneous and intramuscular oedema, swollen and pale livers with multifocal haemorrhage, swollen kidneys with multifocal haemorrhage and distended fluid-filled intestines with areas of haemorrhage. Light microscopy revealed intracytoplasmic inclusions suggestive of a viral infection in a variety of organs, as well as degeneration and necrosis of these organs. Electron microscopy of ultrathin sections of the same tissues revealed iridovirus-like particles within the inclusions. Of the six specimens tested, all were positive for ranavirus major capsid protein (MCP) gene. Sequence alignments of the ranavirus MCP gene from these specimens showed 95-98% similarity with published ranavirus data. The virus, provisionally designated as Chinese giant salamander virus (CGSV), was isolated from tissue homogenates of diseased salamanders following inoculation of epithelioma papilloma cyprini cells. Sequence analysis of the MCP genes showed that the isolated virus was a ranavirus with marked sequence identity to other members of the genus Ranavirus. Kochs postulates were fulfilled by infecting healthy Chinese giant salamanders with the CGSV. These salamanders all died within 6-8 days. This is the first report of ranavirus infection associated with mass mortality in Chinese giant salamanders.

Streptococcus agalactiae, an Emerging Pathogen for Cultured Ya‐Fish, Schizothorax prenanti, in China

Streptococcus agalactiae (Group B streptococcus) has emerged as an important pathogen that affects humans and animals, including aquatic species. S. agalactiae infections are becoming an increasing problem in aquaculture and have been reported worldwide in a variety of fish species, especially those living in warm water. Recently, a very serious infectious disease of unknown aetiology broke out in ya-fish (Schizothorax prenanti) farms in Sichuan Province. A Gram-positive, chain-forming coccus was isolated from moribund cultured ya-fish. The goals of this study were to identify the bacterial strains isolated from diseased fish between 2009 and 2011 in Sichuan Province, China, to evaluate the pathogenicity of the pathogen in ya-fish, crucian carp (Carassius carassius) and the Nile tilapia (Oreochromis niloticus); and to determine the susceptibility of the pathogen strains to many currently available anti-microbial agents. The virulence tests were conducted by intraperitoneal injection of bacterial suspensions. In this study, four strains of a Gram-positive, chain-forming coccus were isolated from moribund cultured ya-fish (S. prenanti). The coccoid microorganism was identified as S. agalactiae using a commercial streptococcal grouping kit and 16S rDNA sequencing analysis. Susceptibility of the isolates to 22 antibiotics was tested using the disc diffusion method. All isolates showed a similar antibiotic susceptibility, which were sensitive to amoxicillin, ciprofloxacin, lomefloxacin, chloramphenicol, rifampin, vancomycin, azithromycin, florfenicol, cefalexin, cefradine and deoxycycline and resistant to gentamicin, sinomin (SMZ/TMP), penicillin, tenemycin, fradiomycin and streptomycin. Furthermore, the virulence tests were conducted by intraperitoneal injection of the isolated strain GY101 in ya-fish, crucian carp and the Nile tilapia. This coccus was lethal to ya-fish, Nile tilapia and crucian carp. The mortality rates of infected ya-fish were 100%, 100%, 60% and 20% at doses of 1.0 × 10⁷, 1.0 × 10⁶, 1.0 × 10⁵ and 1.0 × 10⁴ CFU/fish, respectively. The values were 100%, 80%, 60% and 30% for Nile tilapia and 100%, 60%, 10% and 0% for crucian carp. To our knowledge, this is the first report of S. agalactiae as a pathogen of ya-fish.

Safety and immunogenicity of an oral DNA vaccine encoding Sip of Streptococcus agalactiae from Nile tilapia Oreochromis niloticus delivered by live attenuated Salmonella typhimurium

Attenuated Salmonella typhimurium SL7207 was used as a carrier for a reconstructed DNA vaccine against Streptococcus agalactiae. A 1.02 kb DNA fragment, encoding for a portion of the surface immunogenic protein (Sip) of S. agalactiae was inserted into pVAX1. The recombinant plasmid pVAX1-sip was transfected in EPC cells to detect the transient expression by an indirect immunofluorescence assay, together with Western blot analysis. The pVAX1-sip was transformed by electroporation into SL7207. The stability of pVAX1-sip into Salmonella was over 90% after 50 generations with antibiotic selection in vitro while remained stable over 80% during 35 generations under antibiotic-free conditions. The LD50 of SL/pVAX1-sip was 1.7 × 10(11) CFU/fish by intragastric administration which indicated a quite low virulence. Tilapias were inoculated orally at 10(8) CFU/fish, the recombinant bacteria were found present in intestinal tract, spleens and livers and eventually eliminated from the tissues 4 weeks after immunization. Fish immunized at 10(7), 10(8) and 10(9) CFU/fish with different immunization times caused various levels of serum antibody and an effective protection against lethal challenge with the wild-type strain S. agalactiae. Integration studies showed that the pVAX1-sip did not integrate with tilapia chromosomes. The DNA vaccine SL/pVAX1-sip was proved to be safe and effective in protecting tilapias against S. agalactiae infection.

A recombinant truncated surface immunogenic protein (tSip) plus adjuvant FIA confers active protection against Group B streptococcus infection in tilapia.

PURPOSE Tilapia is an important agricultural fish that has been plagued by Group B streptococcus (GBS) infections in recent years, some of them severe. It is well-known that surface immunogenicity protein (Sip) is an effective vaccine against GBS. EXPERIMENTAL DESIGN Since Sip was not expressed in either E. coli BL21 or E. coli Rosetta, we removed the N-terminal signal peptide and LysM of the virus to produce purified truncated Sip (tSip(1)), which multiplied easily in an E. coli host. The antibodys ability to recognize and combine with GBS was determined by Western-blot and specific staining in vitro. The relative percentage of survival (RPS), antibody titers, bacterial recovery, and pathologic morphology were monitored in vivo to evaluate the immune effects. Freunds incomplete adjuvant (FIA) plus tSip and aluminum hydroxide gel (AH) plus tSip were also evaluated. RESULTS It revealed that tSip mixed with FIA was an effective vaccine against GBS in tilapia, while AH is toxic to tilapia.

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.

Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae, an Effective Protective Antigen in Mice

Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.

OmpN, outer membrane proteins of Edwardsiella ictaluri are potential vaccine candidates for channel catfish (Ictalurus punctatus).

Outer membrane proteins (OMPs) are a class of proteins that reside in the outer membrane of Gram-negative bacteria. OMPs act as epitopes and are potential vaccine candidates. Outer membrane protein N (OmpN) is a component of the outer membrane of Edwardsiella ictaluri (E. ictaluri). In a previous study, the OmpN1-, OmpN2-, OmpN3-encoding genes of E. ictaluri were cloned, and here they were expressed in Escherichia coli. Western blotting showed that these three proteins had molecular weights of ∼60kDa. Channel catfish were immunized with recombinant OmpNs (rOmpNs) and then challenged with E. ictaluri. The results showed that rOmpN1, rOmpN2, and rOmpN3, as well as a mixture of all three proteins (in a ratio of 1:1:1) generated moderate immune protection (relative percentage of survival=62.5, 62.5, 67.5, and 75%, respectively). In an agglutination antibody titer assay, fish antisera showed an antibody titer of 1:128. Furthermore, each of the proteins stimulated high levels of lysozyme activity. In addition, a real-time polymerase chain reaction analysis revealed significant up-regulation of immune-related genes encoding major histocompatibility complex class I (MHC I), MHC II, CD4L, tumor necrosis factor-α, and interferon-γ after 24 and 48h of challenge, compared with the levels stimulated by phosphate-buffered saline. Taken together, we conclude that rOmpNs may elicit immune responses and generate protection against E. ictaluri in channel catfish. Thus, rOmpNs could be promising vaccine candidates against E. ictaluri.

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.

Pathological changes in cultured channel catfish Ictalurus punctatus spontaneously infected with Streptococcus iniae.

The pathological changes present in channel catfish Ictalurus punctatus spontaneously infected by Streptococcus iniae are described. The most consistent gross findings were marked petechial hemorrhages of the skin and congestion of internal organs, particularly the liver, spleen and kidneys. Other features included color fading at the edge of fin rays, enteritis and ascites. Histological examination showed oedema, degeneration and necrotic changes in many organs. Further, hepatitis, splenitis, interstitial nephritis, and meningitis with numerous monocyte and neutrocyte infiltrates were evident. Intact S. iniae cells were seen in macrophages. Apparently, spontaneous S. iniae infection caused acute septicaemia in channel catfish. This is the first histopathological report on channel catfish naturally infected with S. iniae.