高铁酸钾对几种常见鱼类病原菌的杀菌效果测定

试验测定了高铁酸钾(K2FeO4)溶液对8种常见鱼类病原菌的MIC(最小抑菌浓度,minimal inhibitory concen-tration,MIC)和MBC(最低杀菌浓度,minimal bactericidal concentration,MBC),并研究了不同浓度的K2FeO4溶液对几种病原菌的杀灭效果。结果显示,K2FeO4对温和气单胞菌(Aeromonas sobria)、河弧菌(Vibrio flurialis)、弧菌I组淡水亚组弧菌(Vibriogroup I freshwater

嗜水气单胞菌和河弧菌二联疫苗对鲫的免疫效果

在采用正交试验设计优化了 Aeromonas hydrophila和 Vibrio fluvialis二联全菌疫苗制备工艺的基础上 ,研究了采用该工艺所制备的疫苗对鲫的免疫保护效果。结果表明注射免疫鲫4周后 ,抗 A.hydrophila和 V.fluvialis血清凝集效价分别为 :1∶ 4— 1∶ 3 2和 1∶ 2— 1∶ 1 6 ,平均都为 1∶ 1 0 ,但对攻毒均具有有效的保护作用。浸泡免疫、高渗浸泡免疫或加多糖浸泡免疫在免疫 4周后 ,对 A.hydrophila攻毒均无保护作用 ;然而 ,

鲢鳙鱼流行性传染病的流行病学和细菌病因的初步研究

1990年3～10月,作者对湖北、湖南,河南三省部分地区的28个养殖场进行了鲢鳙鱼流行病学调查和致病菌的初步研究。结果显示:在调查范围内发病率达60%以上;受感染的除鲢、鳙外,还有鲫、鳊(鲂)、鲤、草鱼以及其它野杂鱼、河蚌;分离到138株细菌,经鉴定可归为三类,早期(3～4月)出现的鲁克氏耶尔森菌(Yersinia ruckeri)、中后期(5月下旬以后)出现的产气单胞菌(Aeromonas sp.)及弧菌(Vibrio sp.)。结合水质环境条件分析,致病细菌是继发侵入者,原发病因除水体的理化因子外,不

鳗鲡弧菌病病原菌的分离与鉴定

从患弧菌病的鳗鲡的肝脏分离到编号为E-3-11、E-10-1和E-11-1的三株菌。用此菌人工感染鳗鲡能出现相似的病症,并从肝、肾又重新分离到这种菌,证实为该病的病原菌。这些菌株的特性基本一致,经鉴定为鳗弧菌(Vibrio anguillarum)。

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides

Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

A cyclophilin A inducible expressed in gonad of zhikong scallop Chlamys farreri

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds, interacts with a variety of proteins and therefore regulates their activities. One CypA (designated CfCypA) cDNA was cloned from Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CfCypA consisted of 1,248 nucleotides with a canonical polyadenylation signal sequence AATAAA, a poly (A) tail, and an open reading frame (ORF) of 495 nucleotides encoding a polypeptide of 164 amino acids. The deduced amino acid sequence shared high similarity with CypA from the other species, indicating that CfCypA should be a new member of the CypA family. Quantitative real-time (RT) PCR was employed to assess the mRNA expression of CfCypA in various tissues and its temporal expression in haemocytes and gonad of scallops challenged with Vibrio anguillarum. The mRNA transcripts of CfCypA could be detected in all the examined tissues with highest expression level in gonad. After bacterial challenge, the expression level of CfCypA was almost unchanged in haemocytes, but up-regulated in gonad and increased to the peak (22.59-fold; P < 0.05) at 4 h post-injection, and then dropped to the original level at 8 h post-injection. These results indicated that CfCypA was constitutive expressed in haemocytes, but could be induced in gonad, and perhaps played a critical role in response to the bacterial challenge in gonad.

Identification of a C-type lectin from the bay scallop Argopecten irradians

C-type lectins are Ca2+ dependent carbohydrate-recognition proteins that play crucial roles in the invertebrate innate immunity, such as nonself recognition, activation of proPO system, antibacterial activity, promotion of phagocytosis and nodule formation. In this study, a novel C-type lectin of bay scallops Argopecten irradians (Ai Lec) was identified using expressed sequence tag (EST) and RACE techniques. The Ai Lec cDNA encoded a polypeptide of 171 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 150 amino acids. The deduced amino acid sequence of Ai Lec was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 131 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. The expression of Ai Lec transcript was dominantly detected in the hepatopancreas and slightly detected in the haemocytes of normal scallops. 6 h after Vibrio anguillarum-challenge and 8 h after Micrococcus luteus-challenge, the temporal expression of Ai Lec mRNA in hemocytes was increased by 4.4- and 3.6-folds, respectively. The results suggested that Ai Lec was a constitutive and inducible acute-phase protein and might be involved in immune response to Gram-negative and Gram-positive microbial infection in bay scallop A. irradians.

Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis)

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3' and 5' complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleus tridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

CDNA cloning and mRNA expression of the lipopolysaccharide- and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri

Lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP) play a crucial role in the innate immune response of invertebrates as a pattern recognition protein (PRP). The scallop LGBP gene was obtained from Chlamys farreri challenged by Vibrio anguillarum by randomly sequencing cDNA clones from a whole body cDNA library, and by fully sequencing a clone with homology to known LGBP genes. The scallop LGBP consisted of 1876 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 440 amino acids with the estimated molecular mass of 47.16 kDa and a predicted isoelectric point of 5.095. The deduced amino acid sequence showed a high similarity to that of invertebrate recognition proteins from blue shrimp, black tiger shrimp, mosquito, freshwater crayfish, earthworms, and sea urchins, with conserved features including a potential polysaccharide-binding motif, a glucanase motif, and N-glycosylation sites. The temporal expression of LGBP genes in healthy and V. anguillarum-challenged C farreri scallop, measured by real-time semiquantitative reverse transcription polymerase chain reaction (PCR), showed that expression was up-regulated initially, followed by recovery as the stimulation cleared. Results indicated that scallop LGBP was a constitutive and inducible acute-phase protein that could play a critical role in scallop-pathogen interaction. (C) 2004 Elsevier B.V. All rights reserved.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

MARINE ORGANISMS ATTACHED TO SEAWEED SURFACES IN JIAOZHOU BAY, CHINA

Scanning electron microscopy of the surfaces of the seaweeds Laminaria japonica, haploid Porphyra yezoensis, Ulva pertusa and the diploid conchocelis of P. yezoensis and P. haitanensis revealed Vibrio and Micrococcus to be abundant on the surfaces of U. pertusa and P. yezoensis. Vibrio, Flavobacterium, Pseudomonas, Staphylococcus, Bacillus, Corynebacterium and other genera were isolated from the surfaces of L. japonica.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal. level at 9 h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian. (c) 2007 Elsevier Ltd. All rights reserved.