Abstract:In order to investigate the epidemic and the antibiotic resistance of Streptococcus agalactiae isolated from tilapia (GIFT Oreochromis niloticus) cultured in Huizhou, Zhaoqing, Zhuhai, Zhanjiang cities of Guangdong province, the relationship between beta-lactamase gene and the resistance to penicillin in S. agalactiae was further analyzed. Isolates were identified by amplified specific gene cfb and 16s rDNA; In this study, k-b method was adopted to determine the antimicrobial sensitivity of isolates; the distribution of beta-lactamase gene in strains was detected by PCR assay; and the relationship between beta-lactamase gene and penicillin drugs was analyzed by statistic 6.0. The results showed that the ranking of positive rate of S. agalactiae carried in tilapia was: Huizhou (46.46%) > Zhanjiang (43.24%) > Zhaoqing (17.30%) > Zhuhai (4.17%). Drug sensitive tests showed that all isolates from the four cities were highly resistant to penicillin (with the resistant rate of 94.29%) and sulfamethazine (with the resistant rate of 86.40%), but were sensitive to enrofloxacin (with the resistant rate of only 3.99%). The diversity of the 9 beta-lactamase genes was highly distributed in the isolates. The relevance between the beta-lactamase genes and antibiotic resistance showed that the SAG0658 was significantly correlated with the resistance to ampicillin, suggesting that the SAG0658 might play an important role in the resistance of S. agalactiae to ampicillin. However, there was no significant correlation between the nine beta-lactamase genes and the resistance to penicillin, indicating that there might be other mechanisms underlying the resistance of S. agalactiae to penicillin.

Abstract:To evaluate the immune effect of an oral vaccine of Edwardsiella ictaluri on channel catfish, E. ictaluri were killed by formalin and sodium alginate was used to prepare the oral microsphere vaccine. The experimental animals were randomly divided into four groups, including E. ictaluri oral microspheres vaccine group, formalin inactivated vaccine group, microspheres without antigen group and control group. After oral immunization, the effect of the vaccine was evaluated by serum non-specific immune parameters, antibody titers and relative percent survival detection. Results showed the serum lysozyme activity, total superoxide disumutase (T-SOD) activity and alternative pathway complement (ACH50) activity significantly increased in vaccinated channel catfish, showing the influence of the relativity gene transcription of the Ictucurus punctatus induced by these oral vaccines respectively by real-time PCR. The results showed that the I. punctatus fed the E. ictaluri oral microsphere vaccine can acquire longer time of nonspecific imumunity. The serum agglutination titers peaked to 1:16 at 5th week. The specific antibodies could still be detected at 7th week. The relative percent survival of channel catfish was 60.7%, far higher than formalin inactivated vaccine group (14.3%) and microspheres without antigen group (10.7%). From the results of real-time PCR, it is found that in the E. ictaluri oral microspheres vaccine group, the expression of immunity genes increased more notably than other groups during the 48 hours after injection, especially in the kidney and spleen. In conclusion, the oral microspheres vaccine of E. ictaluri was able to enhance the non-specific immune function and effective for preventing E. ictaluri disease.

Abstract:This study investigated the pathogenic bacteria of Clarias fuscus with fulminant death in Nanning City, Pubei County and Yulin City of Guangxi, and analysed the effect of the six virulence genes on its pathogenicity. Pathogen was isolated from the heart, liver and some other parts of the diseased fish by using conventional method, then an artificial infection test was measured on the pathogenicity of the isolated strains. Identification of the isolates was confirmed by API 20NE analysis and 16S rRNA gene sequencing, including a PCR assay of detecting six kinds of virulence genes of the isolates. Results showed that five pathogenic strains were isolated from the diseased fish, including three strains of Aeromonas hydrophila and two strains of Aeromonas sobria. The three strains of Aeromonas hydrophila shared the highest similarity (99.8%) with Aeromonas hydrophila standard strain ATCC 7966 (CP000462). Meanwhile the two strains of Aeromonas sobria shared the highest similarity (99.9%) with Aeromonas sobria standard strain NO.106 (AB472903.1). Among the five strains, detection rates of six virulence genes were as follows. Hundred percent of the strains carried gene Act and Aer. Eighty percent carried gene ahal, hly and Alt. Only twenty percent of the strains carried gene ahp. The five Aeromonas strains contained three virulence genotypes in all, including the main genotype Act⁺ahal⁺hly⁺Alt⁺ahp^-Aer⁺ of three strains (accounting for 60% of all the strains), as well as the genotype Act⁺ahal⁺hly⁺Alt⁺ahp⁺Aer⁺ and Act⁺ahal^-hly^-Alt^-ahp^-Aer⁺ of one strain (each accounting for 20% of all the strains). Strain which carried all the six detected virulence genes had the highest pathogenicity, whereas the one carrying only Act and Aer had the minimal pathogenicity. Gene ahp played an important role in pathogenicity of strains, and the pathogenicity of pathogenic bacteria resulted from the synergistic reaction of multiple virulence genes.

Abstract:The pharmacokinetics and tissue elimination of oxolinic acid (OA) in the Pacific white shrimp, Litopenaeus vannamei, were investigated following oral administration via feed by HPLC method. The minimum inhibitory concentrations (MIC) of OA on the pathogenic Vibrio were detected. The Pharmacokinetics/ pharmacodynamics (PK/PD) parameters were calculated, and dosage regime and withdrawal time were developed. The haemolymph OA concentration vs. time after oral administration via feed was best described by a twocompartment model with first absorption following a single dose of 30 mg/kg. The peak concentration (C_max), peak time (T_max), area under curve (AUC_0-24) and elimination half-life time (t_1/2z) for hemolymph were 14.70 mg/L, 2 h 244.6 mg/L, and 18.56 h, respectively. Peak concentrations(C_max)in muscle, hepatopancreas and gill were 4.11 mg/kg, 17.20 mg/kg and 7.01 mg/kg, respectively; elimination half-life time (t_1/2z) were 10.71 h, 12.31 h and 16.75 h, respectively. The OA MICs of 132 Vibrios isolates varied mainly from 0.15 to 1.25 μg/mL with MIC₅₀ of 0.62 μg/mL and MIC₉₀ of 1.25 μg/mL. The ratios of C_max/MIC₉₀ and AUC_0-24/MIC₉₀, which were two important indices for PK/PD relationship, were 11.76 and 195.7, respectively. The results showed that OA dosage of 30 mg/kg by medicated feed could effectively combat bacterial diseases caused by the Vibrios.

Abstract:The mucosal surfaces of fish, including skin, gill and gastrointestinal tract, contain numerous poorly studied immune substances that act as the first line of defence against invading pathogens.The fish body’s mucosal surfaces are protected from pathogens, physical and chemical attacks by the gel-like extracellular matrix (mucus) which is synthesized, stored and secreted by mucous cells. In order to better understand the vaccine-induced immune responses of mucous cells, histological and histochemical techniques were used to investigate the dynamic changes of the numbers and types of mucous cells in mucosa-associated lymphoid tissues, including skin, gills, foregut, midgut and hindgut, in Paralichthys olivaceus after immersion immunization with inactivated Edwardsiella tarda. The results of hematoxylin and eosin (HE) staining, alcian blue and periodic acid Schiff reaction (AB-PAS), indicated that the type Ⅰ mucous cells containing PAS-positive neutral mucopolysaccharide and type Ⅲ cells containing neutral mucopolysaccharide and a little AB-positive acidic mucopolysaccharide in skin, type Ⅰ, Ⅲ, and Ⅳwhich contained acidic mucopolysaccharide and a little neutral mucopolysaccharide in gill, type Ⅰ in foregut mainly, and type Ⅰ, Ⅲ, Ⅳ and Ⅱ which contained acidic mucopolysaccharide in midgut and hindgut, were observed before immunization. After immunization with inactivated E. tarda, the mucous cell numbers were increased over time and were peaked on day 3 post immunization (p.i.) in skin, gill, foregut and midgut and on day 5 p.i. in hindgut; and then were decreased to the level of control groups; Additionally, the amount of typeⅠmucous cells was reduced while the amount of type Ⅱ, Ⅲ and Ⅳ mucous cells was increased, revealing that the components of mucous cells have been changed from neutral mucopolysaccharide to acidic mucopolysaccharide due to the immunization with E. tarda. The results of AB staining showed that the mucous cells containing acidic mucopolysaccharide were increased after immunization, which were mostly sulfated acid mucopolysaccharide-containing mucous cells. These results will provide valuable information for the researches on the mucosal immunity in fish.

Abstract:In the recent years, the amount of wild-caught Procambarus clarkii has declined rapidly and it was under threat by white spot syndrome virus (WSSV). The absolute quantification of a virus in the host tissue is of great significance to understand the pathogenicity of the virus. However, quantitative analysis of WSSV in the tissues of infected P. clarkii was not reported. In this report, the epidemic of WSSV in red swamp crayfish from five cities in Hubei province was investigated. More than 80% of red swamp crayfish were WSSV positive when the DNA extracted from gills of red swamp crayfish were subjected to PCR assay. In addition, we also investigated the presence of WSSV antigen in the infected red swamp crayfish by Western blot using WSSV-VP28 protein specific serum generated in rabbit. The results showed that a specific band correlated with WSSV-VP28 was present in WSSV PCR-positive samples, but not in WSSV PCR-negative sample. To better quantify the amount of WSSV in the infected tissues, we established an absolute quantitative real-time PCR assay (qRT-PCR) for WSSV using recombinant plasmid pGEX-5x-1-VP28. Briefly, plasmid pGEX-5x-1-VP28 containing WSSV-VP28 gene was purified and quantified. Certain amount of pGEX-5x-1-VP28 was diluted serially and used as templates for qRT-PCR using primers specific for VP28 gene amplification. In doing so, a standard curve for the absolute quantification of WSSV was made. The amounts of WSSV genome that existed in the tissues were further measured by the qRT-PCR using the same above-mentioned primers specific for VP28 gene amplification. The absolute amounts of WSSV genomes in the tissues were obtained by comparison of the values of the qRT-PCR with those in the standard curve. Six tissues of red swamp crayfish, including gill, stomach, intestine, haemolymphocyte, hepatopancreas and heart, were sampled and used for WSSV quantification by the absolute qRT-PCR assay. The results showed that the highest most amounts of WSSV genome (about 10⁸ copies/mg) were detected in the gill, stomach and intestine, followed by haemolymphocyte (about 10⁷ copies/mg), hepatopancreas (about 10⁶ copies/mg). The lowest amount of WSSV was observed in the heart of infected red swamp crayfish, indicating that the replication of WSSV was tissue dependent. The high infection rate of WSSV in the crayfish cultured in the ponds (more than 80% of red swamp crayfish were WSSV positive) indicated that the cultured red swamp crayfish was persistently infected with WSSV.

Abstract:In order to establish a detection method suitable for different strains of OsHV-1,the primers were designed based on the nuclei acid sequence of the conserved DNA polymerase of OsHV-1. A nested PCR detection method (P-nPCR) was established by optimization of the annealing temperatures of the primers and protocols of PCR. Then, both P-nPCR and C-nPCR were employed to test the infection status of the samples collected from different years and hosts. Our results indicated that the detection limits of the P-nPCR detection method was about 100 copies/μL of recombinant plasmid containing OsHV-1 genes. P-nPCR was more specific than C-nPCR in the detection of different variants of OsHV-1, and resulted in a higher prevalence of OsHV-1 for the same samples. In conclusion, a P-nPCR detection method was developed to detect different variants of OsHV-1 infection. The high specificity of P-nPCR to OsHV-1 ensured that different variants of OsHV-1 could be detected as early as possible, which will provide reliable technical support for the detection and epidemiology studies of OsHV-1.

Abstract:A large number of Carassius auratus gibelio and Hypophthalmichthys molitrix young fishes died in the polyculture ponds. In order to ascertain the etiology, tissue injury and provide disease prevention and control measures, macroscopic and microscopic examinations, bacteriological detecting, virological detecting, histopathological observation and drug susceptibility test were operated on the diseased fish. The results showed that there were a few Apiosoma sp. and Trichodina sp. on the body surfaces of diseased fish, which would not cause death and the symptoms of hyperemia and hemorrhage. No other parasitical and fungal pathogens were found. Isolated strains from diseased fish were identified as A. hydrophila by their morphological observation, physiological and biochemical characteristics, and sequence analysis of the 16S rRNA gene. According to the present situations of C. auratus gibelio and H. molitrix viral diseases, one pair of specific primer for PCR amplification of Cyprinid herpesvirus 2(CyHV-2) DNA polymerase gene was used to detect CyHV-2 in diseased fish. CyHV-2 was detected only in diseased C. auratus gibelio. The aseptic supernatants of homogenated tissues from naturally diseased C. auratus gibelio and H. molitrix were respectively used for the artificial infections. Only the former caused healthy C. auratus gibelio to display the clinical sign of hyperemia and to die. It was concluded from the results that A. hydrophila was the main pathogen of diseased C. auratus gibelio and H. molitrix and CyHV-2 was the secondary pathogen of the diseased C. auratus gibelio. There were similar histopathological changes in both of the diseased H. molitrix infected only with A. hydrophila and diseased C. auratus gibelio infected with mixed infections of A. hydrophila and CyHV-2. There were specific histopathological changes in each kind of diseased fish. Granular degeneration of liver and kidney appeared in the mild pathologic lesions and the nuclei of necrotic cells were mainly karyolysis in diseased H. molitrix. Hyaline degeneration of liver appeared in the mild pathologic lesions, the nuclei of necrotic cells were mainly karyopyknosis and karyorrhexis and the hypertrophied cell nuclei with marginated chromatin appeared in the kidney and spleen in diseased C. auratus gibelio. The main organs lost their functions as the result of undergoing the pathological processes from degeneration to necrosis and both kinds of diseased fish died eventually. The results of antimicrobial susceptibility tests suggested that Norfloxacin and Florfenicol could be used to prevent and control A. hydrophila infection. CyHV-2 vaccine and ecological farming methods could be used to prevent and reduce the viral infection and viral development in diseased C. auratus gibelio.

Abstract:In order to detect the antigenicity of moonlighting protein EF-Tu (Elongation Factor Tu), EF-Tu gene from Streptococcus agalactiae HN0303 isolated from tilapia was cloned. The related properties of EF-Tu protein were predicted and its phylogenetic tree was also constructed. The rEF-Tu protein was obtained by prokaryotic expression systems and purified by Ni-NTA-Sefinose Column. The purified rEF-Tu protein immunized rabbits were used to obtain the polyclonal rabbit anti-rEF-Tu sera for antigenicity detection. The results showed that EF-Tu gene had an ORF with 1197 bases, encoding 398 amino acids with a C₁₉₃₃H₃₀₉₆N₅₃₂O₆₁₅S₁₁ formula, 43.981 ku molecular mass, and a 4.749 theoretical isoelectric point. Futhermore, the deduced amino acids comprised phosphorylation sites, but did not contain the transmembrane domain and signal peptide sequence. Three conserved domains, namely EF-Tu, EF-Tu-II and EF-Tu-III existed in the deduced amino acids via NCBI Conseverd Domains tool. Phylogenetic analysis revealed an exaggerated degree of homology with other S.agalactiae EF-Tu protein. Additionally, high antigen index of the deduced amino acids was predicted using DNAstar-Protean, which means it can form numerous epitopes. rEF-Tu proteins formed into inclusion bodies were found in the pellet and an about 66.4 ku band was observed by SDS-PAGE. Moreover, Western Blot analysis showed that rabbit anti-rEF-Tu sera can combine the tropina with recombinant protein specifically. Adhesion test suggested that rabbit anti-rEF-Tu sera prevented S. agalactiae HN0303 adhering the EPC(Epithelioma papulosum cyprini) with a decrease of 79.99%±2.43%. In this study, our results showed that rEF-Tu possesses nice antigenicity and the rabbit anti-rEF-Tu sera can inhibit the S. agalactiae HN0303 adhesion obviously, which suggested that the EF-Tu protein may become a subunit vaccine candidate against S. agalactiae in tilapia.

Abstract:As a pivotal signaling mediator of Toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. To study its biological function in mandarin fish (Siniperca chuatsi), the expression profiling of the gene and its role in immune responses to the infection of infectious spleen and kidney necrosis virus (ISKNV) and S. chuatsi Rhabdovirus (SCRV) were investigated. Based on the unigene sequences of IRKA4 gene which was obtained from the transcriptomic results of S. chuatsi, specific primers were designed and the complete IRKA4 gene (named ScIRAK) was cloned and sequenced by SMART-RACE. Bioinformatics analysis demonstrated that the CDS of ScIRAK4 gene was 1389bp, encoding a 462 amino acid with an N-terminal death domain (DD) and a central protein kinase domain (PKc). The transcription profiles of IRAK4 in the tissues of S. chuatsi were characterized by fluorescent quantitative RT-PCR. The results showed that the highest mRNA expression was found in the liver (P<0.05), followed by that in the muscle, blood, brain and stomach (P<0.05). The transcriptions of the IRAK4 in the spleen of mandarin fish infected with ISKNV or SCRV were furtherly analyzed. The mRNA of ScIRAK4 was down-regulated in the mandarin fish infected with ISKNV and the lowest transcription was observed at 24 h post infection (P<0.01). By contrast, the mRNA of ScIRAK4 was up-regulated in the mandarin fish infected with SCRV and the higest transcription was observed at 12h post of the infection (P<0.01). These findings suggest that ScIRAK4 plays a crucial and different role in the immune responses of Mandarin fish infected with different viruses.

Abstract:The aim of this paper is to investigate the impact of high temperature stress on serum biochemical parameters and histopathology of Nile tilapia Oreochromis niloticus infected by Streptococcus agalactiae. The average weight of Nile tilapia was (62.0±3.33) g. They were cultured on two temperature levels: 29 ℃ (the optimal water temperature for the growth of tilapia) group and 33 ℃ (high temperature stress) group. All fish were acclimated for one week in the laboratory then artificially infected by S. agalactiae. The cumulative mortality was recorded at different time post infection; Blood and tissue samples were respectively collected at 0, 12, 24, 48, 96 and 120 h after infection. Then they were examined. The result showed that cumulative mortality in 33 ℃ group was significantly higher than that of 29 ℃ group; Glutamic-pyruvic transaminase(ALT) activity of fish in 33 ℃ group was significantly higher than those fish in 29 ℃ group at 96 h after infection, while glutamic-oxaloacetic transamnase(AST) activity of fish in 33 ℃ group was significantly higher than those fish in 29 ℃ group at 48 h after infection; Potassium-ion (K⁺) and sodion (Na⁺) of fish at 120 h after infection were significantly higher and lower respectively than those fish prior to infection in 33 ℃ group, but that were not significantly different compared with those fish prior to infection in 29 ℃ group. Creatinine (CREA) activity of fish in 33 ℃ group was significantly higher than those fish in 29 ℃ group at 12 h after infection. Albumin/globulin(A/G)index of fish in 33 ℃ group was significantly lower than those fish in 29 ℃ group at 120 h after infection. Alkaline phosphatase (AKP) activity of fish was increased with the progress of infection in 29 ℃ group, while it was increased and then decreased in 33 ℃ group, but the peak time in 33 ℃ group was earlier than that in 29 ℃ group. Superoxide dismutase (SOD) activity was increased first and then decreased, finally it was increased once again. Histopathology showed that hepatic cord arranged irregularly, while the spleen and renal glomerular hemorrhaged slightly under high temperature stress; The spleen was severely congested at 12 h after infection and they were remarkably broken and obviously amyloidosis at 24 h after infection in both groups. Renal glomerular atrophy, degeneration and necrosis in renal tubule epithelial cell were obvious at 48 h after infection in both groups. Macrovesicular steatosis and hyaline degeneration in hepatocyte were commonly observed at 96 h after infection in 33 ℃ group, but the same histopathological changes in liver and kidney were only found at 120 h after infection in 29 ℃ group. These results indicated that increased susceptibility of Nile tilapia to S. agalactiae resulted from the immunosuppression caused by high temperature stress. Spleen might be the first target organ specified to this pathogen. The injury of liver was accelerated by high temperature stress and liver damage was more serious in the late infection.

Abstract:To develop serological methods for detecting type Ⅱ grass carp reovirus (GCRV), the genes encoding outer capsid proteins VP4 and VP35 were cloned into the prokaryotic expression vector PGEX-4T-3 respectively. Polyclonal antibodies were generated by immunization of mices with the purified recombinant VP4 and VP35 proteins respectively and titered by ELISA.The specificity of antibodies was determined by Western blot. SDSPAGE results showed that the size of the recombinant VP4 and VP35 proteins was about 98ku and 61ku, respectively. The recombinant proteins mainly existed in the form of inclusion bodies. The titer of antibody against VP4 was up to 1:4×10⁵, the titer of antibody against VP35 was up to 1:10⁶. The results of Western blot showed that antibodies were able to identify both recombinant proteins and corresponding capsid proteins in virus particles. Furthermore, serum antibodies of grass carp infected by JX02 were able to recognize VP4 and VP35 proteins specifically. In this paper, the polyclonal antibodies against VP4 and VP35 were successfully prepared, which laid a foundation for developing a serological diagnostic method for major epidemic strains of GCRV and functional studies of these two viral capsid proteins.

Abstract:Aeromonas veronii is an acute epidemic pathogen of channel catfish (Ictalurus punctatus), causing obvious symptoms to hosts, such as distension of abdomen, enteritis and hemorrhage in multiple organs. The goal of this research was to reveal the pathogenicity of A. veronii extracellular products (ECP), and lay a foundation for vaccine and treatment. The pathogenicity of the extracellular products extracted from a high A. veronii isolated from I. punctatus was investigated in vitro and in vivo. In vitro, A. veronii extracellular products were used to detect the biologic activities and the hemolytic spectrum to a variety of erythrocyte. Then, an in vivo research on challenging the channel catfish with ECP was applied to confirm the pathogenicity. Biologic activities showed that the ECP possess lipase, protease, lecithase, DNAzyme, hemolysin; and the hemolytic effect of ECP showed a high hemolytic activity to fish erythrocyte, while no hemolytic activity in chicken and duck erythrocyte. The in vivo research on channel catfish showed that the ECP from the high A. veronii strain had an obvious pathogenicity to channel catfish, with a death rate of 100%. Diseased fish showed increasing mucus and fade spots on the skin, with eye congestion, red anus, liver hemorrhage, oedema of gill, rough surface of the spleen and intestinal expansion with thinning intestinal wall. Histopathological examination showed that edema, vacuolar degeneration, even necrosis in liver; reticuloendotheliosis spleen with few lymphocytes; and hemorrhage and necrosis of the lamella epithelia. The results of in vitro and in vivo showed that ECP of the A. veronii has a variety of extracellular enzymes, which may contribute to pathogenicity of the channel catfish.

Abstract:Infectious spleen and kidney necrosis virus (ISKNV) causes a disease with high mortality, resulting in significant economic loss to Siniperca chuatsi culture industry in China. To establish ELISA assay to determine virus antigen content of ISKNV vaccine and investigate the pathogenic mechanism of ISKNV major capsid protein (MCP), the monoclonal antibody (MAb) against recombinant MCP protein was developed and the properties were identified. The purified recombinant MCP was injected into BALB/c mice through subcutaneous route for three times. Then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice. Three hybridoma cell lines against ISKNV MCP were screened using indirect ELISA and were identified to be IgG1 subtype, designated as 5F1, 3D9 and 5B4, respectively. The three McAbs had no reaction with SCRV and STIV except ISKNV by the indirect ELISA. The hybridoma cell line 5F1 was selected for ascites preparation. When the recombinant MCP and ISKNV supernatant was used as detective antigen respectively, the titer of ascites was 1:51 200 and 1:400 respectively. Indirect immunofluorescence and Western-blot analysis showed that the McAb 5F1 could recognize authentic MCP protein of ISKNV and working concentration of McAb 5F1 was confirmed. From above, the MAb against MCP of ISKNV was successfully prepared, which laid a foundation for further study.

Abstract:To better understand the pathogenesis of the Ostreid Herpesvirus-1 (OsHV-1) to blood clam, experimental infection was conducted by injecting filtered tissue homogenate into the gastropod of each calm. Experimental clams were randomly allocated into 3 groups: blank control group, negative contral group, OsHV-1 infected group. After the experiment, the temporal distribution patterns of OsHV-1 infection loads in six different tissues were assayed with quantitative PCR (qPCR). No virus was detected in clams injected with sea water and the negative control tissue homogenate. While virus DNA were detected in animals injected with tissue homogenate prepared from naturally infected clams. The virus DNA loads in tissues fluctuated first, and then reached about 106 copies viral DNA per ng of total DNA. Pathological changes including lysed connective tissue, dilation of the digestive tubules, eosinophilic inclusion bodies, nuclear chromatin margination and pyknosis were found in affected animals. Transmission electron microscopy (TEM) revealed herpes-like viral particles within the connective tissue of the mantle. These viral particles were about 90-110 nm in diameter with an electron-dense nucleoid packaged in capsid, which resembled those found in naturally infected clams. Overall, this work demonstrated the pathogenesis of OsHV-1 to blood clams and closely associated with the mass mortality of them. Additionally, our work also indicated the stress reaction after injecting OsHV-1 might play a role in the suppression of OsHV-1 replication, while the mechanisms need to be further studied.

Abstract:Among the three genotypes of grass carp reovirus (GCRV), type II GCRV accounts for the major epidemic of grass carp hemorrhagic disease at present in China. Bioinformatic analysis predicted that VP56 of type II GCRV might function as the fiber-like protein of reoviruses. This study aimed to screen potential host proteins that interact with VP56, which might present clues on the biological role of VP56. VP56 gene was amplified by RT-PCR and was used to generate the bait plasmid, pGBKT7-VP56. Subsequently, cDNA library plasmid of Ctenopharyngodon idella kidney cells was screened to test potential interaction partners in yeast AH109 transformed with pGBKT7-VP56. The positive clones were analyzed by plasmid sequencing and nucleotide sequence blasting. To test whether JAM-A served as a potential interaction partner for reoviral fiber protein in grass carp like in mammalian cells, pGADT7-GcJAM-A vector was constructed for the sake of further validation of its interaction with VP56 protein. Results in this study showed that the bait plasmid pGBKT7-VP56 has no selfactivation in yeast, 9 positive clones were obtained, and 7 cellular proteins and an extracellular matrix protein were suggested to bind VP56, and no interaction between VP56 protein and GcJAM-A protein was detected in yeast cell. These results may pave the way for further functional analysis of VP56 protein.

Abstract:The aim of the present study was to investigate the physical-chemical and biological characteristics of Cyprinid herpesvirus 2 (CyHV-2) and its ultrastructural morphogenesis in vitro. By using the newly established cell line derived from gibel carp brain, designated GiCB, which was highly permissive to the infection of CyHV-2, the physical-chemical and biological characteristics, and growth curve of CyHV-2 were investigated; meanwhile, the susceptibility of available fish cell lines to CyHV-2 infection was compared and the ultrastructural morphogenesis was conducted in this study. The results indicated that the CyHV-2 is sensitive to the treatment of heat, acid (pH 3.0), alkali (pH10.0), chloroform, ether and frozen-thawing. CyHV-2 could not propagate in fish cell lines EPC, RTG-2, Koi-Fin, CIK, CCK and PF-Fin, etc., which were verified by CPE observation and CyHV-2 DNA Nest-PCR assay; According to the growth curve of CyHV-2 in GiCB cells, a log phase increase in virus production was followed by an eclipse period of about 12 h. Virus production peaked around 96 h (10^7.52±0.26 TCID₅₀/mL). The CyHV-2 ultrastructural morphogenesis in GiCB cells presented in three main stages, including virus adsorption and entry, replication and assembly, maturation and budding. This study provided useful information for the further studies on CyHV-2 infection mechanisms, pathogenicity and the control methods of the disease caused by the virus.

Abstract:In order to investigate the whole-cell proteomics and two-dimensional (2-D) gel maps of Aeromonas hydrophila isolated from the fish in this study, the proteomics analysis of A. hydrophila strain Zf-1 was performed using two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization timeof- flight mass spectrometry (MALDI-TOF/TOF-MS) techniques. A. hydrophila strain was prepared by shaking culture at 28 ℃ for 12 h, and the proteins of whole cell were extracted by using trichloroacetic acid (TCA)/acetone precipitation method. The results showed that 146 protein spots were observed in 2-DE gels performed with 18 cm pH3-10 IPG strips, and it shared matching profiles of about 81% protein expression spots while the 60 μg loading dose, and 7 cm pH3-10 IPG strips were used for isoelectric focusing (IEF) to 20 000 Vh. A total of 23 protein spots were further identified by the peptide mass fingerprinting (PMF) analysis, including Membrane lipoprotein, Molecular chaperones, 30S ribosomal protein S1, Elongation factor, Cytochrome C, etc; Furthermore, 11 enzymes of metabolism related proteins were found to be ATP F0F1 synthase subunit alpha, Dihydrolipoamide dehydrogenase, Enolase, Arginine deiminase, DNA-directed RNA polymerase subunit alpha, Glyceraldehyde-3- phosphate dehydrogenase, Carbamate kinase, Adenylate kinase, Uridine phosphorylase, Nitroreductase, and Peptidylprolyl isomerase. Analysis of protein functional classification with Gene Ontology (GO) of A. hydrophila based on molecular function showed that binding (17.1%), catalytic activity (14.3%), and transferase activity (11.4%) were responsible for the main biological functions. In conclusion, the proteomics analysis and 2-D map were constructed for A. hydrophila strain Zf-1 from the fish in this study, and the results will help to understanding the pathogenic mechanism and treatment of diseases caused by A. hydrophila from fish.

Abstract:Aeromonas schubertii had caused snakehead disease with white spots in internal organs, which resulted in economic losses in snakehead industry. In this study, A. schubertii strain WL-4 which was isolated from the snakehead (Channa maculate ♀×C. argus ♂) was analyzed in relation with its physiology and biochemistry characteristics, its growth characteristics and virulence factors, and its susceptibility was tested against 20 antibiotics. Its physiology and biochemistry characteristics were tested by using bacterial biochemical identification instrument and its growth situation was analyzed at different temperatures, salinities and pH values. In comparison with the type strain, A. schubertii ATCC43700, A. schubertii WL-4 strain was almost same as the type strain in major physiological and biological characteristics. Moreover, 16S rRNA gene of A. schubertii WL-4 strain is 99. 9% similar to that in A. schubertii ATCC43700. The results of growth characteristics showed that the WL-4 strain's optimum growth temperature was 30 ℃, optimal salinity was 5 and optimum pH was 7. In addition, the WL-4 strain exhibited virulence to snakeheads (25±5)g in experimental challenges with LC₅₀ being 1.2×10⁶ CFU/mL. The WL-4 strain was positive for hemolytic activity, and elastase and lipase activity by phenotypic determination. Susceptibility tests, which detected the minimum inhibition concentration (MIC) of 20 antibacterial agents by trace double dilution method, showed that the strain WL-4 was resistant to sulfonamides and amide alcohols, and susceptible to enrofloxacin, norfloxacin, doxycycline, neomycin, ampicillin and so on. In conclusion, biological characteristics of the WL-4 strain were consistent with the occurrence of the disease with white spots in snakeheads' internal organs which were caused by A. schubertii. In addition, the results of antibacterial susceptibility tests may have an applied value in the control of A. schubertii infection agents in aquaculture.

Abstract:To avoid the biosecurity risk of live virus used as a reference material, the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV) was expressed in Pichia expression system to prepare reference protein,then it can be used as reference material in immunological assay. The IHNV-uk glycoprotein gene (IHNVG) was amplified by RT-PCR with the specific primers designed according to the GenBank IHNV gene sequence,then the IHNV-G was cloned to pPICZαA and transformed into GS115 to make gene fusion system of the target gene and yeast. The fusion gene was induced by 1% methanol to get soluble glycoprotein, the molecular weight of protein is than 70ku in SDS-PAGE analysis.and reactinogenicity of expressed protein was determined with goat polyclonal antibodies against IHNV by Western Blot analysis. The reactinogenicity of 0.1531 mg expressed protein was comparable to 0.3125TCID₅₀ IHNV in ELISA analysis, and the expressed protein can exist stably for two months at -20 ℃. So it was proved that the expressed protein can replace IHNV as reference material. The study provides a foundation for the development of virus ELISA test kit.

Abstract:MARCH5, as an E3 ubiquitin ligase, is one of the important immune regulatory factors in mammals. To investigate the immune function of MARCH5A in large yellow croaker, Larimichthys crocea, the full-length cDNA of MARCH5A was identified in this study (named LcMARCH5A). The full-length of MARCH5A cDNA is 1045 bp, containing an ORF of 867 bp which encoded a 288 aa protein with one RINGv domain and four trans-membrane domains. The analysis of phylogenic tree showed that there are 11 MARCH family proteins, same as those in mammals. However, there are many isoforms of MARCH proteins in fishes, and MARCH5A is fish-specific gene copy, and there are some differences in physical and chemical parameters and spatial structure between LcMARCH5A and LcMARCH5B. Tissues distribution showed that MARCH5A transcripts were broadly distributed in all detected tissues, with high expression in blood and heart, moderate in gill and brain, low in kidney and skin as indicated by qRCR analysis. After Cryptocaryon irritans challenge, the mRNA of MARCH5A was significantly up-regulated early in the skin and reached a peak with 9.65-fold to the control at day 2, then down-regulated thereafter. In the gill, spleen and head-kidney, the expression also increased early, and later decreased. These results showed that MARCH5A plays an important role in fish defense against C. irritans infection.

Abstract:To investigate the relationship between sensitivity varieties,mutations,efflux pump activities and crossresistance of selected strains after they were selected. The strains of Aeromonas hydrophila(Ah) from cultured fish sensitive to fluoroquinolone were selected in vitro stepwise exposure to increasing concentration of Norfloxacin (NF) and Enrofloxacin (EN) on solid medium, aiming to gain the fluoroquinolone-resistant isolates. Then genes of gryA and parC from selected strains were amplified and sequenced. The minimal inhibitory concentrations (MICs) of selected and non-selected antimicrobials as well as the MICs after efflux pump inhibition 1-Methyl-2- pyrrolidinone (NMP) was added to each strain were determined by broth microdilution method. Results shows the MICs of EN and NF increased 409.6-and 4096-fold after selection, respectively, while the MICs of non-selected fluoroquinolone and other antimicrobials also changed greatly. Mutations happened in the quinolone resistancedetermining region (QRDR) encoded by gyrA and parC genes after being selected: Ser83→Ile mutations of GyrA and Ser87→Ile/Arg mutations of ParC were identified. After NMP was added, MICs of all selected strains have declined in different degrees. Cross-resistance of selected strains is closely related to the strain, among the selected strains No. 3 and 8 have no cross-resistance to16 kinds of non-selected antibiotics, and the strains selected by EN and NF have nearly no cross-resistance to aminoglycosides, rifamycins and aminoglycosides(except GM), rifamycins, separately. All selected strains have great cross-resistance to tetracyclines and chloramphenicols. The resistant mechanisms of Ah to fluoroquinolone might include the target gene mutations and an active efflux system, and we should also pay more attention to the cross-resisance when choosing antimicrobials.

Abstract:In order to screen and obtain probiotic bacteria for effective protection against Aeromonas hydrophilia NJ-1 infection in aquatic animals, we compared the expression levels of NFκB, TNF-α and IL-10 in ZF-4 cell model co-cultured with five different potential probiotic lactic acid bacteria (LAB) by quantitative PCR. The apoptosis of ZF-4 cells induced by A. hydrophilia NJ-1 infection was also determined in LAB-treated and control groups. Next, Bacillus coagulans 09.712 with best immunomodulative effect in vitro was supplemented into feed and fed to zebrafish for 28 days. The expression levels of cytokines (TNF-α, IL-1β and IL-10) in gut tissue were monitored and the cumulative mortality was recorded for 15 days after challenge. Our results showed that among these five probiotic LAB, treatment with B. coagulans 09.712 could significantly increase the expression levels of NFκB, TNF-α and IL-10 in ZF-4 cells. The apoptosis rate was 23.36%, much lower than other groups. In addition, administration of B. coagulans 09.712 significantly enhanced the levels of IL-1β, TNF-α and IL-10 in gut on the first day post challenge. The expression of cytokines remarkably decreased during the periods after challenge. At day 3-post challenge, the level of IL-1β in B. coagulans 09.712-treated group was lower than that of control, while the level of IL-10 was higher in bacteria treatment. The cumulative mortality of zebrafish fed with bacteria was significantly lower than that of control. Taken together, B. coagulans 09.712 has an effective immunomodulatory effect in vitro and in vivo against infection. Our work may provide new insight into immunomodulative and immunoprotective function of probiotic LAB in zebrafish.

Abstract:To evaluate the effect of quorum sensing (QS) of Aeromonas hydrophila NJ-1 on modulating pathogenic bacterial virulent factor, NJ-1 was co-cultured with Bacillus sp. R1, a AHLs degrading strain. The results showed that under monoculture conditions, the AHL amount of NJ-1 varied with the same trend as the bacteria growth curve. The maximum activity of both AHLs and virulence appeared at 27-30 h, and then decreased with the decline phase. The expression of QseB started at 27 h, and peaked at 48 h, while the expression of LuxS kept at a lower level. Under coculture conditions, AHLs bioactivity was detected only in trace level. Accordingly, the expression of LuxR was not upregulated, and the expression of virulence factors was also distinctly decreased compared to the monoculture. LuxS were upregulated at 36 h, while the expression pattern of QseB was similar to the monoculutre. In conclusion, Bacillus sp. R1 could reduce the virulence of A. hydrophila NJ-1 by the regulatition of the QS of Lux I/R through degrading AHLs activity.