Study on the Quantity Dynamic Changes of Heterobacteria and Vibrios in Larvae Industrialized Culture System

[ Objective ] The dynamic change of heterobacteria and vibrios in larvae industrialized culture system was studied to provide scientific reference for healthy cultivation of shrimp. [ Method ] The heterobacteria, vibrios and pathogenic vibrio parahaemolyticus were monitored in larvae industrialized culture system. [ Result] The heterobacteria, vibrios and pathogenic vibrio parahaemolyticus were the most in fertilized eggs of shrimp but the least in nauplius, then their number would increase with growth. During whole rearing period, both boterobacteria in larvae, vibrios in water would increase by one order of magnitude, while both vibrios in larvae and heterobacteria in water would increase by two orders of magnitude. There were many heterobacteria and vibrios but few vibrio parahaemolyticus in living bait. The correlation coefficients between larvae and heterobacteria and vibrios in water were 0. 704 and 0. 840 in culture system respectively, while the correlation among heterobacteria, vibrios in living bait and larvae, water were weak or negative. [ Conclusion ] There was a dynamic relation between water and larvae in rearing period, and restrictly control of culture condition would restrain the occurrence of disease caused by vibrio parahaemolyticus, besides that bacteria number in bait was not obviously correlated with bacteria nubmer in culture system.

Occurrence and distribution of vibrios in fishes and shellfishes in coastal waters of Hong Kong

The summer occurrence and distribution of vibrios in the fishes and shellfishes in the coasal waters of Hong Kong were investigaed. A total of 69 strains of vibrios were isolated from all samples examined. The strains, along with 10 reference strains were classified with the technique of numerical taxonomy bared on 54 characters and 62 of the 69 strains fell into 5 major phena, identified as V. paraheamolyticus (30 strains), V. alginoloticus (23 strains), V. choloerae (3 strains), V. harveyi (2 strains) and V. fluinalis (4 strains). Among them, V.paraheamolyticus and V. alginolyticus were the predominant species in the fishes, shellfishes and the coastal waters of Hong Kong and comprised 43. 5 % and 33. 3 % of the total Vibrio spp. isolates respectively. Meanwhile, 3 strains of non-Ol V. cholerae were isolated from oyster and it was the first time to record V. cholerae non-Ol in seawater or from shellfishes in Hong Kong. These results highlighted the potential risks of food poisoning associated with raw or undercooked seafood.

Pathogenicity of Vibrios in Fish: an Overview

Bacteria of the genus Vibrio are ubiquitously distributed in the marine environment. Due to the rapid expansion of intensive mariculture and the consequent deterioration of culture conditions, more and more Vibrio spp. have been recognized as pathogenic agents in outbreaks of vibriosis, a serious epizootic disease affecting most wild and farmed fish species worldwide, which has become the most important limiting factor for the development of intensive mariculture industry. Attempts have been made to understand the pathogenicity of vibrios in host fish with the ultimate aim of elucidating the best means for disease control. After an extensive literature survey of the recent advances in the field of fish vibriosis, the pathological changes, virulence factors and associated potential pathogenic mechanisms, transmission routes and related environmental factors involved in outbreak of vibriosis, as well as the controlling strategies are reviewed in the present paper.

Halophilic Vibrios in the waters of Xiamen Harbor and Daya Bay

The distribution featires, spotes composition and seasonal variation of halophilic Vibrios in 7 stations ofXiamen Harbor and 9 stations of the Daya Bay were studied. The counts of Vibrios were analyzed with the most probable number(MPN) technique. The media TCBS was used to isolate Vibrios, and API20E system employed to identifythe Vibrios. The results reveals that the density Of Vibrios in the Daya Bay ranged from 30. 0 x 105 to 2. 4 x 105 celldin-3 and the average density was 6. 61 x 103 cell·din-3, and that in Xiamen Harbor was 2. 3 x 102-2. 4 x 105 cen·din-3 and 7. 8 x 103 cell·din-3 on the average. The number of Vibrios varied seasonally with the water temperature,and was heher in the summer than in the autumn. The Vibrio species in the two bays mainly included Vibrioalginolyticus, V. parahaemolytic-us, V. fluvialis, V. vulnificus, V. mimices and V. metshnikovii and V.alginolyticus was the predominant spotes.

Vaccination in three different ways against vibriosis of Seriola dumerili caused by Vibrio hollisae

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili （amberjack） suffering from vibriosis. Healthy S. dumeriIi were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1：40 at 7 d, and reached its peak of 1：320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of 〈1：10, 1：20, 1：160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities （P〈0.05）. Upon challenge with virulent strain, the relative percent survival （RPS） of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.

Interaction of Haematopoietic Tissue Cultures of the Dublin Bay Prawn,Nephrops Norvegicus(L.),with the Causal Agent of Luminous Vibriosis Vibrio Harveyi

Vibrio harveyi cells(dose>103 cells mL-1)and extracellular products(ECP;>25μg mL-1 of total protein concentration)destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure.Cytopathic effects(CPE)started after 4 h of exposure to the bacterial cells,with some granularity in the cytoplasm,mostly in cells in the outer periphe-ry of the explant growth.At the end of the infection,a considerable number of nuclei remained attached to the substrate,apparently unaffected.Following exposure to ECP,initial deterioration was observed at 2 h with the presence of granularity in the cytoplasm of<20%cells,and few cells displayed small vacuoles around the nuclei.Parallel results were obtained using whole animal experiments,with V.harveyi cells being lethal to nephrops within 24 h.

Immune response of shrimp(Penaeus monodon)against Vibrios furnissii pathogen

Objective:To analyse experimental infection and immune system of shrimp(Penaeus monodon)against Vibrios furnissii(V.furnissii).Methods:Experimental animals were collected and acclimatized by maintaining specific temperature,pH and salinity to avoid mortality.Shrimps were experimentally infected with V.furnissii and their immune responses were monitored.After the infection all the shrimps were monitored for any symptoms,death rate in 0,12,24,36,48 h.Then haemolymph were collected and tetrahydrocannabinol,phenol oxidase,nitroblue tetrazolium and lysozyme were monitored in every 12 h at the interval of 48 h.Results:Shrimps infected by live V.furnissii had showed gradual increase in tetrahydrocannabinol,phenol oxidase activity,nitro-blue-tetrazolium and lysozyme activity comparing with the killed and control.Conclusions:The live V.furnissii had showed infection in the shrimp immune system.The live V.furnissii shows infection in experimental shrimps comparing with killed V.furnissii.So the V.furnissii in nature cause the infection in shrimp Penaeus monodon immune system.This report could be applied to control of the infection in shrimp hatchery.

Ecology of antibiotic resistant vibrios in traditional shrimp farming system(bhery)of West Bengal,India

Objective:To study the ecology of antibiotic resistant bacteria with emphasis on sucrose negative vibrios in water and sediments samples of traditional shrimp farming system(bhery)in West Bengal,India.Methods:The vibrios were isolated from traditional shrimp farm samples on thiosulphate citrate bile salt sucrose agar and sucrose negative bacterial strains were used as biomarkers to assess the frequency of antibiotic resistance.Results:The incoming water brought presumptive vibrios ranging from 5.50×10^(1)to 1.00×10^(3)mL in to the bhery,and there appeared to build up vibrios in the culture system with days of culture,as there was about 9 fold increase in vibrios.The levels of vibrios were observed to be moderately higher in outlet water and ranged between 4.15×10^(2)and 4.15x10^(3)mL.The counts of vibrios in pond sediment was found to be 1.00x10^(2)-4.90×10^(3)g;while in inlet(2.00×10^(2)-4.20×10^(4)g)and outlet(3.00×10^(2)-6.85×10^(3)g)their levels were observed to be higher than the pond sediment.Thirteen different Vibrio species were encountered in traditional shrimp culture system and all vibrios were sensitive to chloramphenicol,followed by ciprofloxacin and gatifloxacin(98.24%),gentamicin(95.61%)and other antibiotics.The multiple antibiotic resistance(MAR),i.e.,resistance to at least two antibiotics,was noticed among 43.85%of the sucrose negative vibrios and 41.86%of the sucrose negative non--vibrios.All vibrios harveyi strains exhibited MAR.Although no antibiotic was used in the bhery,the prevalence of MAR in 44%of the sucrose negative vibrios and nonvibrios is a cause of concern.The MAR index was higher in inlet water and sediment samples.The MAR observed in biomarker strains of pond water and sediment(40%)was comparable to those of inlet samples,thus confirming the fact that incoming water was the major source of antibiotic resistant bacteria.Conclusions:It seems that the shrimp culture in bhery does not favour the proliferation and spread of antibiotic resistant bacteria.

一株克氏原螯虾病原——霍乱弧菌的分离鉴定

从尾扇边缘溃烂的克氏原螯虾中分离到一株优势菌LK-18,并对该菌株进行种属鉴定和致病性分析。通过菌落形态观察、16S rRNA测序、血清型分析及基因特性鉴定对细菌的类别进行初步判断,并采用浸泡感染、肌肉注射和腹腔注射等方式进行人工感染实验对细菌的致病性进行评估。经16S rRNA测序分析和4个管家基因(atp A、pyr H、rec A、gyr B)串联分析,该菌株最终被鉴定并命名为VibriocholeraeLK-18。V. choleraeLK-18经PCR鉴定不属于O1和O139血清型,但含hly毒力基因和几丁质分解相关的几丁质酶(chitinase,Chi)基因。人工感染实验虽未能重现烂尾症状,但从克氏原螯虾烂尾组织中分离的V. choleraeLK-18具有致病性,能够引起克氏原螯虾的死亡。研究结果表明霍乱弧菌LK-18是一种致病菌,不仅扩大了非O1/O139群霍乱弧菌的感染宿主范围,同时预警霍乱弧菌有导致克氏原螯虾养殖业暴发疾病的可能,因此要提前做好该疾病的预防工作,从而避免霍乱弧菌感染带来的损失。

Molecular Characterization and Antibacterial Activity of a G-Type Lysozyme in Yellow Drum(Nibea albiflora)

Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.

Transcriptome analysis reveals immune-related genes in tissues of Vibrio anguillarum-infected turbot Scophthalmus maximus

Turbot Scophthalmus maximus is an important mariculture fish species with high economic value.However,the bacterial diseases caused by Vibrio anguillarum infection bring huge economic losses to the turbot aquaculture industry.To understand the immune response of the turbot against V.anguillarum infection and to explore novel immune-related genes,the transcriptome analysis of turbot spleen and gills were conducted after V.anguillarum infection.Differentially expressed genes(DEGs)were identified in spleen and gill of the turbot amounted to 17261 and 16436,respectively.A large number of immunerelated DEGs were enriched in cytokine-cytokine receptor interaction signaling pathway,and the others by the kyoto encyclopedia of genes and genomes(KEGG)enrichment.The gene ontology(GO)classification analysis revealed that V.anguillarum infection had the greatest effect on biological processes and cellular components.Twelve immune-related DEGs were identified in the spleen(cstl.1,egfl6,lamb21,v2rx4,calcr,and gpr78a)and gills(ghra,sh3gl2a,cst12,inhbaa,cxcl8,and il-1b)by heat map.The proteinprotein interaction(PPI)networks were constructed to analyze the immune mechanism.The results demonstrate that the maturation and antigen processing of major histocompatibility complex(MHC)class II molecule,and calcitonin-or adrenomedullin-regulated physiological activity were important events in the immunity of turbot against V.anguillarum infection.In the gills,the protein interactions in TGF-βsignaling pathway,production of inflammatory factors,and endocytosis regulation were most significant.Our research laid a foundation for discovering novel immune-related genes and enriching the knowledge of immune mechanisms of turbot against V.anguillarum infection.

一株牙鲆源哈维氏弧菌的分离鉴定及抗性研究

自秦皇岛昌黎养殖的患病牙鲆(Paralichthys olivaceus)体内分离获得一株革兰氏阴性菌,对其形态特征、生理生化特性及16S rDNA序列进行分析,鉴定其为哈维氏弧菌(Vibrio harveyi);对分离菌株进行了30种抗菌药的药敏试验和11株益生菌的拮抗试验,得出此分离菌株对米诺环素、左氧氟沙星、头孢他啶、氟苯尼考、头孢曲松、诺氟沙星、环丙沙星、强力霉素、复方新诺明等13种药物高度敏感,对克林霉素、青霉素、氨苄西林、林可霉素、苯唑西林等9种药物表现为不同程度的耐药性;地衣芽孢杆菌和枯草芽孢杆菌2种益生菌在一定浓度下对其具有拮抗作用。

Molecular Cloning of clpX Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

According to the clpX gene sequence of Vibrio alginolyticus HY9901,a pair of specific primers were designed,and the full length was cloned by PCR and subjected to bioinformatics analysis.The results showed that the clpX gene was 1281 bp in length and encoded 426 amino acids.Its molecular structure formula was C 3842 H 6405 N 1281 O 1598 S 260,with a theoretical protein molecular weight of approximately 1044473.4 kDa and a theoretical pI value of 5.04.The clpX gene was predominantly situated within the cytoplasm,exhibiting unstable and hydrophilic protein characteristics.It possessed a signal peptide cleavage site,lacked a transmembrane region,and was not associated with any KEGG metabolic pathway.Additionally,it possessed 2 glycine phosphorylation sites,a CAMP-dependent protein kinase phosphorylation site,a C-terminal amidation modification site,6 protein kinase C phosphorylation sites,7 microbody C-terminal target signal sites,and an ATP/GTP site.The clpX phylogenetic tree was constructed using the MEGA 5.0 software via the neighbor-joining method.The results demonstrated that the clpX of V.alginolyticus exhibited up to 100%affinity with the clpX of Vibrio spp.The single subunit 3D structure model of the ClpX protein was obtained using the SWISS-MODEL program.A structural and functional analysis of the protein revealed the presence of three distinct ClpX structural and functional domains.In the prediction of secondary structure,the proportions ofα-helix,random coil,β-sheet and extended strand were 40.38%,37.09%,5.40%and 17.14%,respectively.The analysis of the ClpX protein through the STRING database revealed that the proteins interacting with the ClpX protein were Tig,Atpd,Hflb,Msrb-2,Rpod,Clpp,Clpa,Lon-1,Hfq,and ANP63951.1.A computational analysis of the ClpX protein identified a number of post-translational modification sites,including phosphorylation,acetylation,ubiquitination,glycosylation,methylation,S-palmitoylation,and lactylation.The significance of this study is to analyze the function of the clpX gene and establish a robust foundation for subsequent investigations into the mechanism of the clpX gene in Vibrio alginolyticus.

Gene Cloning and Bioinformatics Analysis of phoR Gene from Vibrio alginolyticus HY9901

PhoR is a histidine kinase in a two-component regulatory system that regulates phosphorus metabolic pathways and undertakes the key mission of information transmission in pathogenic bacteria.The full-length phoR gene was successfully cloned from the Vibrio alginolyticus HY9901 strain.A comprehensive analysis of the cloned gene was conducted using bioinformatics.Sequence analysis revealed that the total length of the phoR gene(GenBank accession No.:KJ958404.1)is 1299 bp,with the coding region containing a total of 432 amino acid residues.The phylogenetic tree of PhoR revealed that it belongs to the same subclade as V.diabolicus.The SMART program was employed for the purpose of functional domain prediction,which revealed that PhoR possesses three major functional domains:PAS(amino acids 98-166),HisKA(amino acids 205-272),and HATPase_c(amino acids 317-429).

Molecular Cloning and Bioinformatics Analysis of sucC Gene of Vibrio alginolyticus Strain HY9901

[Objectives]To clone the sucC gene of Vibrio alginolyticus strain HY9901 and conduct the bioinformatics analysis.[Methods]Based on the sucC gene of V.alginolyticus strain HY9901,specific primers were designed to amplify the full length sequence by PCR and make further analysis.[Results]The theoretical molecular weight of SucC protein was about 41528.45 Da,and the full length was 1167 bp,encoding 388 amino acids.It has no signal peptide and transmembrane region,and has a variety of functional sites.It is predicted that it is mainly located in the cytoplasm,and the ubiquitin and lactate modification sites overlap,and it has high gene homology with Vibrio parahaemolyticus.Theα-helix,random coil and extended strand are the main secondary structures.The similarity between the constructed three-level structure model and the template is high.[Conclusions]This study reveals the structural characteristics and functional potential of SucC protein,and provides a theoretical basis for the study of drug resistance mechanism and prevention strategies.

Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

Molecular Cloning and Bioinformatics Analysis of cyaA Gene of Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to explore the biological functions of cyaA gene of Vibrio alginolyticus.[Methods]With DNA of V.alginolyticus HY 9901 as a template,primers were designed according to the sequence of cyaA gene,and the cyaA gene was amplified by PCR.Bioinformatics analysis was performed.[Results]The cyaA gene of V.alginolyticus HY9901 was 2529 bp in size,and encoded 842 amino acids.The molecular structure of CyaA protein was C_(4358)H_(6745)N_(1171)O_(1286)S_(35).Its theoretical molecular weight was 97.24167 kDa and the theoretical pI value was 5.56.It had no signal peptide and transmembrane domain.CyaA protein had three N-terminal glycosylation sites,one cAMP and cGMP-dependent protein kinase phosphorylation site,nine protein kinase C phosphorylation sites,nine casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,seven N-terminal myristoylation sites,one pentenyl binding site and ten microbody C-terminal localization signal sites.Subcellular localization prediction showed that CyaA protein was mainly located in the nucleus and cytoplasm.Through multi-sequence alignment and phylogenetic tree construction,it was concluded that V.alginolyticus had high CyaA homology with other Vibrio species.cyaA of V.alginolyticus was clustered with Vibrio fluminensis and Vibrio marinisedimini,and they were closely related.The secondary structure of CyaA protein consisted ofα-helixes(43.11%),random coils(38.00%)and extended strands(14.49%).In protein network interaction,it was found that the proteins adjacent to CyaA protein were Crp-2,CpdA,Crr,PtsG-2,ANP67209.1,Crp-1,PykF,Pyk,RelA and Ndk.[Conclusions]This study provides a new idea for formulating strategies for the prevention and control of vibriosis.

创伤弧菌对水产养殖的影响及致病机制

随着水产养殖向集约化和工厂化养殖发展,水产养殖动物病害频繁发生,在诸多病原菌中,创伤弧菌(Vibrio vulnificus)是水产养殖动物的主要病原体之一,给水产养殖业造成巨大损失。文章简要介绍了创伤弧菌对水产养殖动物的影响及致病机理,同时对创伤弧菌疾病预防和治疗提出更环保的治疗方式,以期加强对创伤弧菌的致病性和危害性的重视与研究,有助于合理地进行疾病诊断和采取防控措施,同时为进一步深入研究创伤弧菌的致病机理奠定重要基础。

宁德市南美白对虾副溶血弧菌的分离与药敏试验

为给宁德市南美白对虾(Penaeus vannmei)养殖病害提供精准用药依据,从7个海水养殖场南美白对虾体内分离病原菌,通过形态学观察、生理生化和16s rRNA鉴定,确定其分类地位,并进行药物敏感试验。结果表明,分离的9株病原菌在TCBS琼脂培养基中菌落均呈蓝-绿色,通过革兰氏染色镜检可见为革兰氏阴性菌,呈棒状、弧状、卵圆状,无芽孢。在氧化酶反应、葡萄糖、赖氨酸、鸟氨酸、甘露醇、6%盐胨水、8%盐胨水、动力等8项试验中,结果呈阳性,确定为副溶血弧菌。9株副溶血弧菌对青霉素、新霉素、四环素等3种药物耐受比例较高,对恩诺沙星、呋喃唑酮、万古霉素等6种药物均高度敏感。