• Volume 36,Issue 3,2012 Table of Contents
    Select All
    Display Type: |
    • >PAPERS
    • Seasonal changes of bacterial community composition in the scallop culture area of Liuqinghe Bay studied by PCR-DGGE

      2012, 36(3):407-413. DOI: 10.3724/SP.J.1231.27767

      Abstract (3108) HTML (0) PDF 1.56 M (2114) Comment (0) Favorites

      Abstract:The seasonal changes of bacterial community composition in the scallop culture area of Liuqinghe Bay in Qingdao were analyzed in this study.Five liters of water samples from the 0.5 m depth were collected each time every month from May 2009 to April 2010,except February,and the one liter water samples were filtered by sterilized micro-filtration membranes to collect the micro-organisms within the size of 0.22 μm to 3.00 μm.Total DNA of the samples were extracted by CTAB method,the bacterial 16S rDNA V3-V5 variable sequence was amplified by PCR,the amplified sequences were separated by DGGE(denaturing gradient gel electrophoresis)techniques,and the DGGE fingerprint was analyzed by software Quantity One.Totally,36 bands with different positions were showed.The most abundant bacterial composition was found in January,and the least was found in March.Based on the the UPGMA cluster analysis,the monthly samples were grouped into 2 seasonal types:March to May,June to January.A total of 20 dominant DGGE bands were successfully cloned and sequenced.Blast results showed that the similarities between these cloned sequences and the sequences in the GenBank were all over 94%.The 20 bands belonged to α-Proteobacteria,β-Proteobacteria,γ-Proteobacteria,Bacteroidetes,Actinobacteria,and Verrucomicrobia,respectively.

    • The relationship of Vibrio parahaemolyticus densities and water quality factor during oyster culture

      2012, 36(3):414-421. DOI: 10.3724/SP.J.1231.27656

      Abstract (2805) HTML (0) PDF 1.22 M (2326) Comment (0) Favorites

      Abstract:Vibrio parahaemolyticus(VP) is a pathogen of bacterial food poisoning associated with shellfish.VP,which is detected both in human and fish,has even higher concentrations in oysters and other filter-feeding shellfish.In order to study the relationship of VP densities versus water quality factor including water salinity,temperature,pH and dissolved oxygen in the course of oyster culture,we detected total VP densities in the oyster and water quality factor at the oyster farm in Yangxi,Guangdong from March to November in 2009.The results showed that the detection ratio of total VP in oysters was 94.38%.The water temperature was correlated positively with total VP in oysters,but the salinity was correlated negatively with total VP in oysters.The dissolved oxygen and pH didn’t show correlation with total VP in oysters.Based on the regression analysis,a quadratic relationship for VP densities versus water temperature and salinity was found.The regression equation of VP densities versus water temperature was:LgVP=0.093T-0.685(R2=0.336);The regression equation of VP densities versus salinity was:LgVP=1.199+0.116S-0.004S2(R2=0.217);The best fit regression equation of VP densities versus water temperature and salinity was LgVP=-1.941+0.116T+0.058S-0.001S2>(R2=0.414).The results of this paper may provide some reference data for the risk analysis of VP during oyster culture.

    • Molecular cloning and expression analysis of the IL-1β gene in Mystus macropterus

      2012, 36(3):321-328. DOI: 10.3724/SP.J.1231

      Abstract (3092) HTML (0) PDF 1.49 M (2022) Comment (0) Favorites

      Abstract:Interleukin-1β(IL-1β)is a key mediator of inflammation and orchestrates a variety of immune responses by initiating gene expression.IL-1β was not identified from nonmammalian species until recently.However,no IL-1β genes have been reported from Siluriformes fishes class in China.In this study,we have cloned and sequenced the IL-1β in Mystus macropterus by RT-PCR and RACE-PCR methods.It contains 1 194 nucleotides,including 3′UTR of 224 nucleotides,5′UTR of 82 nucleotides,and an open reading frame with 888 nucleotides encoding a 296 amino acid peptide with MW of 33.84 ku and PI of 5.00.Compared with eight vertebrates species,IL-1β in M.macropterus shared the highest identity(79.0%)with that in Ictalurus punctatus,and the lowest identity(22.0%)with that in Gallus gallus.Phylogenetic tree analysis based on some IL-1β amino acids indicated that IL-1β amino acids in M.macropterus were clustered closely with Ictalurus punctatus.Expression analysis indicated that the IL-1β gene of M.macropterus was detected in muscle,brain,spleen,head kidney,kidney,stomach,skin,liver and intestine in varying degrees,and increased significantly in head kidney and spleen on 1 day after injection with Aeromonas hydrophila and returned to the normal level 15 d later.The result indicated that M.macropterus IL-1β plays a critical role in the host-pathogen interaction.

    • The pharmacokinetics of ivermectin in Carassius auratus

      2012, 36(3):422-428. DOI: 10.3724/SP.J.1231.2012.27581

      Abstract (3391) HTML (0) PDF 1.18 M (1936) Comment (0) Favorites

      Abstract:Pharmacokinetics of ivermectin in Carassius auratus was studied following a single oral or intramuscular administration at dose of 0.4 mg/kg.After following these two different administration routes,IVM concentration-time cruves in most tissues of C. auratus exhibited multi-peak phenomenon.After intramuscular administration,noncompartmental pharmacokinetic parameters were as follows:Cmax=0.445 mg/L, Tmax=48 h,t1/2z=524.2 h, MRT(0-∞ )=788 h and AUC(0-∞ )=289.2(mg/L)·h.After oral administration,non-compartmental pharmacokinetic parameters were as follows:Cmax=0.264 mg/L, Tmax=8 h, t1/2z=153.9 h,MRT(0-∞ )=269.78 h,and AUC(0-∞ )=83.77(mg/L)·h.Comparing parameters of these two administration routes,we found that the rate of absorption and elimination following the oral administration was relatively higher than that following the intramuscular administration,while the bioavailability of the latter was higher.The AUC0-600 values in different tissues of C.auratus have same sequence:gonad>blood>kidney>hepatopancreas>muscle.IVM shows some kind of accumulative effect in both gonad and kidney,as high IVM concentration and slow elimination rate and long MRT in these two tissues,especially in ovary.When water temperature is 25 ℃,we suggest IVM withdrawal period in C.auratus no less than 25 or 15 days following a single intramuscular or oral administration respectively at dose of 0.4 mg/kg.As drug withdrawal period had significant relationship with both water temperature and administration dose,so drug withdrawal period should be adjusted properly according to the practical aquaculture conditions.

    • Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody

      2012, 36(3):329-335. DOI: 10.3724/SP.J.1231.27810

      Abstract (2784) HTML (0) PDF 4.12 M (1607) Comment (0) Favorites

      Abstract:The high-affinity Na+/glucose contransporter Sglt1 is one of the important members of the sodium:solute symporter family(SSF),belonging to the homologous family 5(SLC5).The Sglt1 plays an important role in accumulating glucoses from intestinal or kidney epithelial cells against an adverse concentration gradient and maintaining the adjustment of metabolism.So far,few studies on Na+/glucose cotransporter have been reported in the freshwater fishes.This research would develop special antibodies of Sglt1,which could supply the foundation for the research of glucose metabolism in Cyprinus carpio intestines on molecular level.To study the molecular mechanism of glucose metabolism in freshwater fishes,the full-length cDNA of sglt1 in C.carpio with an ORF of 1 977 bp was cloned and the antigenicity of Sglt1 was first predicted.92(544-637)amino acids with strong antigenicity and immunogenicity were selected as target section of sglt1 in C.carpio and were cloned into plasmid pET-32a(+)vector which had restriction sites for EcoRⅠ and HindⅢ.The rcombinant plasmid named pET-32a(+)-sglt1-P was transformed into E.coli Rosetta.The Sglt1-P fusion protein of approximately 30 ku was highly expressed in E.coli Rosetta after being induced with IPTG.The purified fusion protein was used as antigen to immunize New Zealand Rabbits with ear margin veins by subcutaneous injection with power.The results of ELISA showed that the titer of the antiserum was about 1:105.The Sglt1-P polyclonal antibody was used to determine the expression of Sglt1 protein in C.carpio intestines through immunohistochemical method.The result revealed that the antibody performed high affinity and specificity and could be applied to study the expression and locating of Sglt1 in the C.carpio.The Sglt1-P polyclonal antibody also could serve as an important research tool to study Sglt1 expression and transshipment activity of C.carpio. Meanwhile,the Sglt1-P polyclonal antibody could also be used to exploratively study the expression location and quantity of the Sglt1 transporters in other fishes.

    • Prokaryotic expression,purification and immune efficacy of VP6 protein of grass carp reovirus

      2012, 36(3):429-435. DOI: 10.3724/SP.J.1231.27803

      Abstract (2535) HTML (0) PDF 1.80 M (2236) Comment (0) Favorites

      Abstract:In order to study the immuno-protective efficacy of GCRV-VP6 subunit vaccine,the recombinant expression vector pET28a(+)-VP6 was constructed by cloning vp6 gene of GCRV into the prokaryotic expression plasmid pET-28a(+).The results of SDS-PAGE and Western-blotting showed that the molecular mass of the recombinant VP6 protein was approximately 43 ku.The recombinant protein reached 20% of the total bacterial proteins,and was mainly in the form of insoluble bodies.The grass carp(60-120 g in body weight and 14-20 cm in body length)was immunized with 500 μg purified VP6 protein by Ni2+ affinity chromatography and antibody titers in the blood of fish were determined by means of indirect agglutination reaction on the 14th,21st,28th,49th and 70th days post-vaccination.The results showed that the specific antibody could be detected on the 14th day,reached peak on the 21st day and could be still detected on the 70th day after immunization.The fish in vaccinated group and control group were challenged with GCRV on 21st days after vaccine delivery and the relative survival rate in the vaccined group was 100%.These results provided important academic foundation for research and development of GCRV genetic vaccine.

    • Effects of heat stress on nonspecific immune parameters and HSP70 gene expression in Taisho Sanke koi(Cyprinus carpio)

      2012, 36(3):336-342. DOI: 10.3724/SP.J.1231

      Abstract (2748) HTML (0) PDF 1.24 M (2024) Comment (0) Favorites

      Abstract:In order to explore the mechanism of koi’s anti-stress,the study was performed to examine the effect of continuous heat stress on non-specific immune parameters and HSP70 gene expression in Taisho Sanke koi under the laboratory conditions.Samples for detections of respiratory burst,complement protein 3(C3),superoxide dismutase(SOD),malondialdehyde(MDA)and relative expression content of heat shock protein 70(HSP70)were taken before stress,2,6,10,14,18,22 and 26 h after stress,respectively.The results were as follows.Respiratory burst activity decreased after stress,and it decreased significantly after stress 2,10,18-26 h(P<0.05).The content of serum C3 was increased slightly 2 h after stress.Compared to before stress,the level of serum C3 decreased 6-26 h after stress,and it decreased significantly 14-22 h after stress(P<0.05).The level of serum SOD had the trend of augment,but not significantly(P>0.05).The concentration of serum MDA increased after stress,and it increased significantly after stress 10-18 h(P<0.05).Compared to before stress,the relative expression content of HSP70 gene significantly increased by 5.93 times 2 h after stress and 2 times 26 h after stress(P<0.05).6-22 h after stress,the relative expression content of HSP70 gene had no significant difference from that before stress(P>0.05).These results indicated that heat stress affected non-specific immune parameters,and lowered non-specific immunity of Taisho Sanke koi.The expression of HSP70 gene was regulated by heat stress.HSP70 induced by heat stress played a role of stress protection to koi.

    • The effect of transgenic rice carrying antimicrobial gene of shrimp on feed spoilage and disease control in GIFT

      2012, 36(3):436-443. DOI: 10.3724/SP.J.1231.27677

      Abstract (2505) HTML (0) PDF 7.47 M (1544) Comment (0) Favorites

      Abstract:Recently,more and more studies focus on the application of antimicrobial peptides to control bacterial diseases.For the first time,we report the using of plant bioreactor to develop functional feed additives in China.During our previous work,the penaeidin 3-2 gene from the shrimp Fenneropenaeus chinensis was transformed into rice Aichi ashahi. Tilapia feed which contained different levels of the transgenic or non-transgenic rice bran were tested for the effect of antimicrobial peptide expressed by rice plant on feed spoilage and disease control.The results of total bacteria and mold count showed that penaeidin 3-2 expressed in rice could effectively restrain growth of microorganisms in feed and bran.Another experiment was conducted to study the effect of different levels of transgenic rice bran(10%,20%,30%,respectively)on survival rate of tilapia infected by Aeromonas hydrophila.The results showed that survival rates of tilapia fed transgenic rice bran were significantly higher than those fed non-transgenic rice bran.In-depth study of the mechanism of enteritis control by transgenic bran was conducted by studying the gut microbial community and intestinal structure.The results showed that transgenic rice bran restrained growth of Escherichia coli, but enhanced lactic acid bacteria proliferation.And the intestinal structure of those fish fed transgenic bran was more intact than the control.So it is indicated that the disease control effect could be in connection with the change of gut microbial community and protective effect of intestinal structure.But it is noteworthy that the excessive addition of transgenic rice bran(30%)could cause epithelial cells degeneration.

    • Construction and evaluation of short hairpin RNA interference vector targeting CP> gene of Epinephelus coioides nervous necrosis virus

      2012, 36(3):343-349. DOI: 10.3724/SP.J.1231

      Abstract (2724) HTML (0) PDF 1.97 M (1969) Comment (0) Favorites

      Abstract:The short hairpin RNA interference sequences targeting CP gene of redspotted grouper nervous necrosis virus were designed by using online designing software of Invitrogen Co.,Ltd.The structure consisted of 19 nt sense strand,-4 nt loop and -19 nt antisense strand.These sequences are synthesized,annealed,and cloned into pENTRTM/U6 to construct the shRNA interference vectors named pshRNA-124,pshRNA-896 and pshRNA-NNV,respectively.Then the expression vector of pEGFP-CP and each of the three shRNA interference vectors are co-transfected into FHM cells by using Lipofectamine 2000.Then the expression and inhibition of EGFP are observed by using fluorescent microscope.The mRNA expression level of CP gene is anaylised by using real time RT-PCR.The result shows that pshRNA124,pshRNA896 and pshRNANNV all suppress the EGFP expression with inhibitory efficiency of 47%,68% and 51%,respectively.The pEGFPCP gene silencing efficiencies of pshRNA-124,pshRNA-896 and pshRNA-NNV at mRNA level are 60%,96% and 55%,respectively,significantly higher than that of control(P<0.05).It demonstrates that all of the three shRNAi vectors have interference effects and can interfere with the expression of EGFP,of which pshRNA-896 is the best one.The result above suggests that shRNAi vectors targeting CP gene of redspotted grouper nervous necrosis virus has been successfully constructed,which will be a basis for further study of the function of CP gene with RNAi technique.

    • Effects of digestive tract probiotics on immune enzyme activity and anti-WSSV ability of Litopenaeus vannamei

      2012, 36(3):444-450. DOI: 10.3724/SP.J.1231.27592

      Abstract (2775) HTML (0) PDF 1.65 M (1908) Comment (0) Favorites

      Abstract:A 20 days feeding experiment was conducted to study the effects of probiotics on immune enzyme activity(alkaline phosphatase AKP,a cidic phosphatase ACP,superoxide dismutase SOD,catalase CAT and lysozyme UL)and anti-WSSV ability of Litopenaeus vannamei. Diets were supplemented with Photobacterium damsela (live,≥1011 CFU/g or inactivated,1 g/kg)or Bacillus firmus (live,≥1011 CFU/g or inactivated,1 g/kg).Controls were fed non-supplemented diet.In this experiment,feeding the bacteria species(inactivated P.damsela; live Bacillus firmus)significantly increased serum ACP,AKP,SOD and CAT compared with controls(P<0.05),at the same time,their cumulative morality were(49%?15%)and(53%?12%)respectively,which were significantly increased compared with controls(P<0.05).The present results suggested that the diets supplemented with B. firmus (live) or P. damsela (inactivated)could improve immune enzyme activity and anti-WSSV ability of shrimp,and the probiotics with the function of preventing disease could be used as one of effective ways of biological prevention and control to WSSV.

    • Cloning,identification and molecular characteristics analysis of simA gene of a Streptococcus iniae strain DGX07 from channel catfish

      2012, 36(3):350-358. DOI: 10.3724/SP.J.1231

      Abstract (2857) HTML (0) PDF 1.45 M (2067) Comment (0) Favorites

      Abstract:The RAPD analysis of Streptococcus iniae virulent strain DGX07 isolated from channel catfish(Ictalurus punctatus)was conducted by PCR with Primer p14(5′-GATCAAGTCC-3′).The simA gene of DGX07 was amplified by PCR with specific primers and cloned into pMD19-T vector.The positive plasmid was selected and identified by PCR and digestion of double restriction enzyme(BamHⅠ+XhoⅠ).Molecular characterization analysis of simA gene was performed by bioinformatics tools.The results showed that S.iniae simA amino acid sequence was highly conservative and with 100% homology to the simA and simB of S.iniae isolated.The polypeptide contained a signal peptide comprising 41 amino acids and Bap31 and Gram_pos_anchor functional domains.The polypeptide had some important sites related to post-translational modification,including 22 phosphorylation sites and 2 N-glycosylation sites.The hydrophilic regions were larger than hydrophobic regions in polypeptide chain.The S.iniae simA was extramembranous protein with multiple antigen advantaged regions.The analysis of codon bias indicated the codon usage frequency of S.iniae simA different,and the codon bias of S.iniae simA preferred the yeasts.Gene accession number:JF330100.

    • WSSV stress on the immunoe-related enzyme and ultrastructure of hepatopancreas in juvenile Cherax quadricarinatus

      2012, 36(3):451-458. DOI: 10.3724/SP.J.1231.27627

      Abstract (2744) HTML (0) PDF 2.88 M (1944) Comment (0) Favorites

      Abstract:In present study,the enzymatic activities of the peroxidase(POD),lysozyme(LSZ),superoxide dismutase(SOD)and phenoloxidase(PO)have been monitored by use of biochemical techniques.Meanwhile,the transmission electron microscopy(TEM)method has been applied to compare the morphological changes in hepatopancreas of juvenile redclaw crayfish, Cherax quadricarinatus, under two experimental conditions:one manual injection of immune polysaccharide; two White Spot Syndrome Virus(WSSV)stress exposure.Four experimental groups have been set up in this study:control group[with PBS(pH 7.4)injection]; experimental group Ⅰ(with WSSV injection); experimental group Ⅱ(with immune polysaccharides injection); experimental group Ⅲ(with immune polysaccharides injection first and with WSSV injection after 48 hours).The results of biochemical studies showed that:in comparison with the control group,the activities of POD,PO,LSZ in experimental group Ⅰ were decreasing significantly(P<0.01)in 72 hours.In contrast,the activity of SOD in experimental group Ⅰ was increasing first and then decreasing,the enzyme activity was significantly lower(P<0.01)in 72 hours; in the experimental group Ⅱ,the enzymatic activities of POD,PO,LSZ,SOD were all increasing first and then decreasing.Their enzymatic activities were all higher than the control group(P<0.05).In detail,the SOD and LSZ activity reached the threshold after 24 hours immune polysaccharide injection,the POD had the highest activity in 48 hours,the maximum activity of PO reached after 12 hours injection; experimental group Ⅲ,the enzymatic activities of POD,PO,LSZ,SOD were all higher than experimental group Ⅰ(P<0.05)after 72 hours exposure.However,in comparison with the control group,the SOD,POD,LSZ activities were decreasing,with only PO enzymatic activity increasing first and then decreasing,four enzymes,activities were all decreasing significantly(P<0.01)in 72 hours in contrast with the control group.Four enzymatic activities in experimental group Ⅱ is greater than control group and enzyme activity in experimental group Ⅲ is greater than experimental group Ⅰ and this proves a direct evidence that the immune polysaccaride injection can improve the resistance of juvenile prawn to WSSV and be able to actively suppress the infection with WSSV.Moreover,the morphological study in hepatopancreas of C.quadricarinatus showed that:in control group,the hepatopancreas is composed of liver-tubules,the liver-tubules is made up of simple columnar epithelium cells and basement layers,with the simple columnar epithelial cells the neat microvilli on the surface and the liver-tubules epithelial cells structurally-complete.In experimental group Ⅰ,the mitochondria were seriously harmed and had abnormality;the karyotheca disassembly,substance external flow in nucleolus and cell nucleus become torn,at the same time the cell nucleus had the chromosome heterochromatin phenomenon.In addition,more serious of the rough endoplasmic reticulum becoming split liminated structure and ribosome is to reduce in experimental group Ⅰ; In experimental group Ⅱ,the cell nucleus chromatin uniform distribution on the rough endoplasmic reticulum(RER)ribosome showed an increase; In experimental group Ⅲ,RER become tumescence and few split.Meanwhile,the number of vesica increased,and the vacuole were also found increased.Certain portion of mitochondria turned into abnormality,regular ellipse or length structure mitochondria develop into irregular.Our results indicate that WSSV stress can damage the ultrastructure of hepatopancreas in juvenile C.quadricarinatus, and further influence the normal physiological function of C.quadricarinatus.Present study by biochemical techniques and TEM method further shows that the immune polysaccharides can promote the resistance of juvenile prawn to the WSSV infection.

    • Differential expression of immune-related genes from skin of zebrafish induced by the infection of Citrobacter freundii

      2012, 36(3):359-366. DOI: 10.3724/SP.J.1231

      Abstract (2883) HTML (0) PDF 2.34 M (1850) Comment (0) Favorites

      Abstract:With water pollution aggravated and intensive aquaculture spread,the occurrence of opportunistic infections has become a significant health problem in farmed fish.Citrobacter freundii is an enterobacterium commonly isolated from diseased and healthy animals,including human,mammals and fish,which is considered to be an opportunistic pathogen.Skin is an essential protective barrier for fish and functions as a first line of defense against invading pathogens,but its molecular mechanism remains unclear.The objective of this study is to screen the differential gene expression in response to pathogen infection in skin of fish,and provide a basis for understanding the fish skin immune mechanism to the pathogen at the molecular level.Here,a strain of bacteria was isolated from intestine in Ctenopharyngodon idellus,which was identified by using the morphological observation,physiological and biochemical characteristics,and 16S rDNA sequence analysis.The results showed that the isolated strain was identified species levels as Citrobacter freundii,and animal tests showed that the strain was lethal to zebrafish.Immune response in skin of fish analyzed by using Citrobacter freundii induced zebrafish as a model,which applied the Affymetrix zebrafish cDNA microarray hybridized to the skin tissues.Total RNAs were isolated from the skin tissues of the zebrafish and labeled with biotin,and hybridized to zebrafish cDNA gene chips.The expression profiles from the hybridization to 15617 genes in zebrafish cDNA array were analyzed by the GeneChip Operating Software(GCOS 1.4).Out of 15617 genes in zebrafish cDNA chips,total of 88 immunerelated genes were identified to be significantly expressed in the skin tissues,of which 74 genes are upregulated and 14 are downregulated in the zebrafish skin tissues.Furthermore,the differential expression genes could be categorized into eight functional groups by using the Gene Ontology(GO)method,including complement activation,acute phase response,defense immune response,platelet activation,coagulation factors,response to stress and stimulus,apoptosis,antigen processing and presentation,cell adhesion and migration,etc.In addition,the analysis of the pathways showed that the MAPK(hsp70,daxx,nfkbiab),JAK/STAT(ifn1),and TGF-β(thbs1) signaling pathways may be involved in the immune responses of skin in fish.The Affymetrix zebrafish cDNA microarray is useful for identifying the functional genes involved in the skin immune defense of fish,and future functional analysis of these genes may contribute to understanding the mechanisms of skin immune responses and offer a scientific reference for the Citrobacter-zebrafish model of bacterial pathogenesis.

    • Effect of feeding modes of Ala-Gln on growth and resistance to crowding stress of Cyprinus carpio var. jian

      2012, 36(3):459-465. DOI: 10.3724/SP.J.1231.27569

      Abstract (2740) HTML (0) PDF 1.01 M (2126) Comment (0) Favorites

      Abstract:The trial was carried out on healthy Cyprinus carpio var. jian with the initial weight(33.52?0.17) g in a single recirculating system for 8 weeks.Diet was administered at concentrations 0.0%(control diet)and 0.5%(trial diet)Ala-Gln of dietary dry matter,which were fed to the C. carpio var. jian in five different feeding modes.continuously fed by control diet during eight weeks(Ⅰ,control group),trail diet at an interval of two weeks(Ⅱ),trail diet before four weeks at an interval of four weeks(Ⅲ),trail diet after four weeks at an interval of four weeks(Ⅳ),and continuously fed by trail diet during eight weeks(Ⅴ).Acute crowding stress was implemented at the last of experiment.The trail was conducted to investigate the effects of feeding modes of Ala-Gln on growth and the resisence to acute crowding stress for C. carpio var. jian.Results indicated that growth of different feeding modes of Ala-Gln was significantly higher than that of control group(P<0.05).Specific growth rate of Ⅱ was higher than that of Ⅲ,Ⅳ,Ⅴ groups(P<0.05)and that of Ⅲ was higher than that of Ⅴ group(P<0.05).After acute crowding stress,Serum cortisol reached peak at 0 h and glucose at 1h,serum HSP70 remained high level before 1-12 h then declined,at last,restored the normal level until 48 h.Serum cortisol and glucose were lower in every feeding mode than in control group(P<0.05).The smallest increase of serum glucose was Ⅱ group,The first recovery occurred in Ⅱ and Ⅲ group after stress.Serum HSP70 in every feeding mode was significantly higher than that in the control group(P<0.05),and returned to normal state in 48 h and 72 h.Serum HSP70 in Ⅳ and Ⅴ groups was still significantly higher than the control(P<0.05).In the current experiment,it concluded that adding Ala-Gln could improve the growth and resistance to crowding stress of C. carpio var. jian.There were significant differences of every feeding mode.Given economic efficiency and practicability,interval feeding modes will be suggested in the practical culture of C. carpio var. jian.

    • Replacement of fish meal by corn gluten meal in diets of Scophthatmus maximus

      2012, 36(3):466-472. DOI: 10.3724/SP.J.1231.27590

      Abstract (2895) HTML (0) PDF 1.00 M (2238) Comment (0) Favorites

      Abstract:The corn gluten meal(CGM)was evaluated as a replacement of fish meal(FM)in a practical diet formulated to contain 50% protein and 12.5% lipid.FM was replaced by 0%,12%,25%,38%,and 50.5% of CGM(C0,C12,C25,C38 and C50.5)respectively.And another diet(C50.5CAA)was supplemental with a 1.80% essential amino acid(CAA)mixture(L-lysine:1.2%,L-arg:0.6%)based on the diet C50.5.Juvenile Scophthatmus maximus (12.51?0.02 g)were reared in an indoor,semi-closed recirculating system.Each diet was randomly fed to triplicate groups of 18 fish per tank(300 L)arranged in a completely randomized design.The fish were hand-fed to satiation twice daily for 49 days.At the end of the feeding trial,feed intake(FI),specific growth rate(SGR),feed efficiency ratio(FER)and protein efficiency ratio(PER)were significantly affected by the inclusion of CGM,with decreasing values as inclusion levels of CGM rose.There were no significant differences(P>0.05)in growth performance between fish fed diet C0 and C12.However,fish fed diet containing above 25% CGM had significantly lower(P<0.05)growth than those fed diet C0 and/or C12.Supplementation with CAA significantly improved FER of fish(P<0.05),and FI,SGR and PER of fish also showed a tendency to be improved by supplement with CAA.Body composition,serum triglycerides and serum urea of the fish were not significantly(P>0.05)affected by dietary treatments.Total cholesterol concentration in serum of fish was significantly reduced with increasing dietary CGM level(P<0.05).

    • cDNA cloning and expression analysis of cytMnSOD and mtMnSOD from Macrobrachium nipponense

      2012, 36(3):367-374. DOI: 10.3724/SP.J.1231

      Abstract (2649) HTML (0) PDF 1.76 M (1977) Comment (0) Favorites

      Abstract:The cytMnSOD and mtMnSOD genes were cloned from hepatopancreas of Macrobrachium nipponense using the rapid amplification of cDNA ends(RACE)method for the first time.The full-length cDNA of cytMnSOD was 1 233 bp with a 858 bp of open reading frame(ORF)encoding a 286 amino acids(aa)protein with a 60 aa leader sequence; whereas the full-length cDNA of mtMnSOD was 1 113 bp with a 654 bp of ORF encoding a 218 aa protein with a 21 aa signal peptide sequence in the N-terminus.The calculated molecular masses and estimated pI of translated cytMnSOD and mtMnSOD proteins were 31.33 ku,24.05 ku and 5.62,7.12,respectively.The deduced amino acid sequence of cytMnSOD had 40% of similarity to that of mtMnSOD. Both cytMnSOD and mtMnSOD contained a manganese superoxide dismutase domain(DVWEHAYY),four conserved amino acids responsible for binding manganese(H110,H158,D243,H247 and H48,H96,D180,H184),and two N-glycosylation sites(NHT,NMS and NHT,NLS).The constructed phylogenetic tree indicated that arthropod MnSODs could be classified into two distinct branches,cytMnSODs,and mtMnSODs.The MnSODs from M.nipponense were more closely related to Macrobrachium rosenbergii MnSODs than that from other crustaceans.Real-time RT-PCR analysis showed that both cytMnSOD and mtMnSOD from M.nipponense were expressed in hepatopancreas,muscle,haemocytes,ovarian,mandibular organ and gill,but relatively higher in the hepatopancreas.The levels of both cytMnSOD and mtMnSOD transcripts in hepatopancreas were the highest in stage C among the molt cycle.Injection of pathogenic bacteria Aeromonas hydrophila resulted in the significant increase of cytMnSOD and mtMnSOD expressions 3 h after treatment,indicating that MnSOD may act as an important molecule involved in immune defense against A.hydrophila.

    • Dietary non-protein energy sources:growth,physiology and biochemisty by the juvenile Australian redclaw crayfish,Cherax quadricarinatus(Von Martens)

      2012, 36(3):473-480. DOI: 10.3724/SP.J.1231.27726

      Abstract (2618) HTML (0) PDF 1.11 M (1970) Comment (0) Favorites

      Abstract:A growth experiment was conducted to determine the optimal carbohydratetolipid(CHO∶L)ratio for juvenile Australian redclaw crayfish[initial weight(1.72?0.01) g(mean?SD)]cultured in indoor recirculating aquaculture system.Five isonitrogenous[(31.130?0.004) g/kg]and around isoenergetic[(17.490?0.396) MJ/kg]diets with dietary CHO∶L ratios are 10.75∶1,4.81∶1,2.66∶1,1.52∶1 and 0.87∶1,were fed twice a day to apparent satiation.Over 8-week growth trial,the best final weight,weight gain,specific growth rate and feed conversion ratio value(P<0.05)were observed in redclaw crayfish fed diets with a CHO∶L ratio of 2.66∶1.Redclaw crayfish fed either the lowest(0.87∶1)or highest(10.75∶1)CHO∶L ratio tended to produce lower growth and poorer feed conversion ratio.Dietary lipid levels was 40-145 g/kg,the lipase and alkaline phosphatase activities were stimulated by elevated levels of dietary lipid.However,the activities of hexokinase and pyruvate kinase showed opposite trend.The pepsin and intestine amylase activities were significantly(P<0.01)affected by CHO∶L ratios.The higher activity of pepsin were observed when the CHO∶L were 2.66∶1 and 1.52∶1(P<0.05).The intestine amylase activity was significantly increased(P<0.01)as dietary CHO levels(156.3-360.4 g/kg)increased.Based on a secondorder polynomial regression analysis of WG against dietary carbohydrate and lipid levels,growth maximum was observed when the dietary carbohydrate and lipid levels were 268.82 g/kg and 122.20 g/kg,corresponding to a CHO∶L ratio of 2.20∶1.These results indicated that the utilization of dietary lipid and carbohydrate was moderate in redclaw crayfish and the preferential utilization of carbohydrate was higher than that of lipid.

    • Distribution of lymphocystis disease virus receptor27.8 ku in flounder(Paralichthys olivaceus)

      2012, 36(3):375-382. DOI: 10.3724/SP.J.1231

      Abstract (2732) HTML (0) PDF 4.43 M (1709) Comment (0) Favorites

      Abstract:Lymphocystis disease virus(LCDV)is the causative agent of lymphocystis disease,which has infected more than 140 species of marine and freshwater fish belonging to 42 families worldwide and caused great economic losses.Determination of the receptor molecules on the surface of susceptible cells will contribute to the understanding of viral infection,pathogenesis and tissue tropism in hosts,which will also benefit the development of anti-viral therapeutic agents.In this paper,monoclonal antibodies(Mabs)2G11 and 3D9 against 27.8 ku receptor protein of LCDV were applied to detect LCDV receptor in the peripheral blood,leukocyte,gill,stomach,intestine,skin,liver,head-kidney,body kidney,spleen,gonad,brain and heart tissues in flounder(Paralichthys olivaceus)by indirect immunofluorescence assay and immunohistochemical experiment.Results showed that receptor-positive signals were found in cell membranes of leukocytes,gill,gastric mucosal cells,intestinal epidermal cells,epidermis,liver,connective tissue cells in the spleen surface and tubular epithelium behind the headkidney,which indicated that LCDV 27.8 ku receptor protein existed in these tissues.No receptor-positive signals were observed in the body kidney,gonad,brain,heart tissues and red blood cells ofP.olivaceus.We deduced that LCDV entered the fish’s body through binding to the receptor molecules in gill,epidermis and gastrointestinal epithelium,or entered the peripheral blood and infected the leucocyte and then transmitted to other tissues(liver,spleen,head-kidney,etc.)through blood circulation.

    • Molecular and VITEK identification of Vibrio alginolyticus-related isolates

      2012, 36(3):383-390. DOI: 10.3724/SP.J.1231

      Abstract (3180) HTML (0) PDF 1.31 M (2183) Comment (0) Favorites

      Abstract:Vibrio alginolyticus,a member of the core group of the genus Vibrio,also called V.harveyi group,shares high phenotypic and genotypic resemblance with the other species of this group.It is very difficult to identify strains of V.alginolyticus from others.The housekeeping genes and various automated systems based on biochemical characteristics of bacteria provide more alternatives for identification.In this study,we compared the resolution of 16S rRNA gene,toxR gene,pyrH gene and VITEK 2 COMPACT GN card on V.alginolyticus-related isolates.There are multiple copies of 16S rRNA gene in the genome of Vibrio and the intragenomic heterogeneity among different copies might be higher than intergenomic heterogeneity in some strains.These facts make the 16S rRNA gene not suitable for the discrimination of V.harveyi group species.Both of single-copy genes toxR and pyrH show a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity.The toxR gene similarity analyses of eighteen isolates and five reference strains can clearly group them into four species.The phylogenetic tree based on toxR gene shows that strains of individual species clustered together independently,and there are two different clusters within V.alginolyticus,indicating that they have different evolving paths.The similarity and phylogenetic analyses based on pyrH gene come to the same result of toxR gene.The discriminatory power of pyrH gene is slightly lower than that of toxR gene.VITEK 2 COMPACT GN card presents one misidentification among 9 isolates of V.alginolyticus and its species database does not include V.campbellii,V.rotiferianus and V.natriegens.We suggested that toxR gene could be the first choice for fast identification of V.alginolyticus isolates,and pyrH gene may be used for verification.

    • Detection of cfa gene and isolation and identification of the pathogenCitrobacter freundii isolated from Portunus trituberculatus

      2012, 36(3):391-398. DOI: 10.3724/SP.J.1231

      Abstract (2796) HTML (0) PDF 1.20 M (2242) Comment (0) Favorites

      Abstract:Portunus trituberculatus,mainly distributed in East Chins Sea and Yellow Sea,is a large marine economic crabs with rich nutritional and medicinal properties.In recent years, P.trituberculatus aquaculture has developed rapidly along the Jiangsu coastal waters,while some epidemic diseases broke out which was attributed to many factors,for example,pathogenic microorganism.In the late autumn of 2009,the cultured P.trituberculatus suffered from serious disease in one farm of Ganyu County in Jiangsu Province.The main symptom displayed as drumble,not feeding,the tissue oedema of liver,gill and muscle;the liver and muscle were rotten in some crabs.Virtually pure dense cultures were obtained from liver,muscle,and lymph fluid of diseased P.trituberculatus,and strong pathogenicity of the isolate strain(JG0911201)to the P.trituberculatus.The phenotypic characteristics of isolate strain were examined,including morphological characteristics,physiological,and biochemical characteristics,the 16S rRNA and gyrB genes were amplified and compared with those sequences deposited in databases.Phylogenetic analysis of 16S rRNA and gyrB genes indicated high homogeneity between the isolate strain and Citrobacter freundii from GenBank database.The 16S rRNA gene fragment length is 1 451 bp(GenBank accession number HQ170626)and gyrB gene fragment length is 1 186 bp(GenBank accession HQ170627)The results showed that the identified isolate strain belonged to C.freundii.Colonization factor antigen was a pathogenic factor of enterobacteriaceae which could produce enterotoxin that was capable of causing death.And the cfa gene(100 bp)could be amplified by PCR.It was concluded that the isolated strain C.freundii carried cfa and provided an effective method for detecting the C.freundii.C.freundii was a conditional pathogen that is gramnegative,bacillus,aerobic or amphimicrobian.And many diseases were attributed to the C.freundii for man,reptiles,amphibians.In recent years,a lot of aquatic animals such as Cherax quadricianalus,Ophicephalus argus,Oreochromis niloticus,Eriocheir sinesis etc.,were also infected by the C.freundii.In order to choose the appropriate antibiotic the drug sensitive was tested,and testing results by paper extension methods for drug sensitive showed that among 48 antibiotics,ciprofloxacin,fleroxacin,ceftriaxone,enoxacin,enrofloxacin,floxacin and levofloxacin had the most inhibitive effects on the strain.The paper reported for the first time that C.freundii could lead to the mass mortality of P.trituberculatus in Jiangsu coastal waters and a detailed study of the pathogen was made,which would be helpful in the disease control and health management in P.trituberculatus cultivation.

    • Molecular epidemiology of Streptococcus agalactiae isolated from tilapia in Southern China

      2012, 36(3):399-406. DOI: 10.3724/SP.J.1231.27743

      Abstract (2879) HTML (0) PDF 1.58 M (2742) Comment (0) Favorites

      Abstract:Streptococcus agalactiae strains were isolated and collected from moribund tilapia in Guangdong,Hainan,and Fujian Provinces,and these isolates were identified by biochemical and molecular biological characterization.In the current study,we compared the phylogenetic relationships among S. agalactiae strains.Furthermore,we compared antibiotic sensitivity,molecular classification and molecular serotype of these strains which were isolated from different years and different geographical regions.Antibiotic sensitivity assays showed that among 20 antibiotics tested,there are 10 antibiotics that had no differences,and the other 10 items showed similar sensitivity.Five most diverse loci(Variable number of tandem repeats,VNTR)were used for the construction of MLVA to evaluate the diversity of these strains.Molecular classification assays showed that all S.agalactiae strains isolated from fish were of the same molecular classification,but the S.agalactiae strain(C918)isolated from dairy cattle was different.Molecular serotype of these isolates using a multiplex PCR reaction,and the results showed that the molecular serotype of S.aglactiae was Ⅰa.However,surface protein genes assays showed that the surface protein of S.agalactiae strains is alpha-C protein except the S.agalactiae(C918)strain.Conclusion:these S. agalactiae isolates isolated from fish are of the same classification by molecular classification assays.Therefore,these results provide a theoretical basis for control of the tilapia diseases and for vaccine development of S.agalactiae in tilapia.

Current Issue


Volume , No.

Table of Contents

Archive

Volume

Issue

Most Read

Most Cited

Most Downloaded