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Inhibition of vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish 总被引:17,自引:0,他引:17
Gram L Melchiorsen J Spanggaard B Huber I Nielsen TF 《Applied and environmental microbiology》1999,65(3):969-973
To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strain Pseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect of P. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth of V. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not. P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1, 000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykiss Walbaum) to P. fluorescens AH2 at a density of 10(5) CFU/ml for 5 days before a challenge with V. anguillarum at 10(4) to 10(5) CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 10(7) CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12 degrees C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont. 相似文献
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《Cell differentiation and development : the official journal of the International Society of Developmental Biologists》1990,29(2):123-128
A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 μs, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the F1 offspring assayed. In F2 offspring obtained by mating transgenic F1 fish, 88% were transgenic. 相似文献
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Effect of Cecropin B and a Synthetic Analogue on Propagation of Fish Viruses In Vitro 总被引:2,自引:0,他引:2
Abstract Cecropins and other natural antimicrobial peptides are widely distributed in animals from insects to mammals. These
proteins have been shown to be major constituents of the innate immune systems of animals for nonspecific defense of the host
against various bacteria and parasites. Therefore, exploitation of this natural innate defense system may lead to the development
of effective methods for protecting fish from invasion by microbial pathogens. Recently, we have demonstrated that the introduction
of cecropin transgenes into Japanese medaka (Oryzias latipes) conferred resistance to infection by fish bacterial pathogens.
Aside from a few reports documenting the antiviral effect of antimicrobial peptides including cecropins against mammalian
viruses, there is no evidence for the effect of these peptides against fish viruses. In this article we present results of
in vitro characterization of native cecropin B and a synthetic analogue, CF17, against several important fish viral pathogens—namely,
infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), snakehead rhabdovirus (SHRV), and
infectious pancreatic necrosis virus (IPNV). Upon coincubation of these peptides and viruses, the viral titers yielded in
fish cells were reduced from several fold to 104-fold. Direct disruption of the viral envelope and disintegration of the viral
capsids may be involved in the inhibition of viral replication by the peptides. Results of our studies demonstrate the potential
of manipulating antimicrobial peptide genes by transgenesis to combat viral infection in fish. 相似文献
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The alternative and classical complement pathways of channel catfish (Ictalurus punctatus), most importantly the classical complement pathway which requires antibody, demonstrated bactericidal activity against two Gram-negative bacterial fish pathogens, Flexibacter columnaris and Vibrio anguillarum. This shows the importance of antibody-mediated bactericidal immunity in fish by the classical complement pathway in providing protection following immunization or natural infections. 相似文献
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噬菌蛭弧菌对鱼类常见致病菌裂解作用的研究 总被引:12,自引:0,他引:12
调查了北京地区25份水样,其中24份检出噬菌蛭弧菌。本次试验选用4株鱼类主要致病菌为宿主菌,检出的蛭弧菌对上述4种细菌的裂解范围有所不同。其中嗜水气单胞菌可被全部检出的蛭弧菌裂解(24/24),其他3株菌仅部分被裂解,依次为肠型点状气单胞菌(17/24),荧光假单胞菌(9/24),鳗弧菌(7/24)。本次试验直接从水样中检出6株对4种宿主菌均有裂解作用的蛭弧菌,为进一步利用蛭弧菌防治鱼类常见细菌性疾病提供了可用资料。 相似文献
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The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis 总被引:1,自引:0,他引:1
Mingqiang Qiao Tiina Immonen Olli Koponen Per E.J. Saris 《FEMS microbiology letters》1995,131(1):75-80
Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cmt-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 103 transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 102 transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens. 相似文献
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J K Lu T T Chen C L Chrisman O M Andrisani J E Dixon 《Molecular Marine Biology and Biotechnology》1992,1(4-5):366-375
Gene constructs consisting of human growth hormone (hGH) gene driven by promoter/regulatory sequence of mouse metallothionein (mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK), or chicken beta-actin (cBA) gene were injected into the cytoplasm of fertilized medaka eggs via the micropyle. More than 49% of the injected embryos survived at hatching. Up to 26% of the survivors showed integration of the introduced gene construct, as determined by polymerase chain reaction analysis and subsequent confirmation by Southern blot hybridization of the genomic DNA. A significant fraction of F1 progeny, derived from crosses between transgenic founders and the nontransgenic individuals, inherited the transgene. Expression of hGH gene was also observed in some of the P1 founders and F1 transgenic progeny carrying mMT-hCG or cBA-hGH gene. Furthermore, the growth performance of the P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1 transgenic progeny was significantly greater than their full sibling, nontransgenic individuals. In addition to the microinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryos of medaka by electroporation using an exponential decay electroporator. Approximately 70% of the electroporated embryos survived at hatching, and 20% of the survived individuals integrated RSVLTR-rtGH2 cDNA into their genomes. These two techniques will greatly enhance the ability to study regulation of gene expression in transgenic animals during differentiation and development. 相似文献
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Jia X Patrzykat A Devlin RH Ackerman PA Iwama GK Hancock RE 《Applied and environmental microbiology》2000,66(5):1928-1932
Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems. 相似文献
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I-SceI meganuclease mediates highly efficient transgenesis in fish 总被引:15,自引:0,他引:15
Thermes V Grabher C Ristoratore F Bourrat F Choulika A Wittbrodt J Joly JS 《Mechanisms of development》2002,118(1-2):91-98
The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection. 相似文献
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Kinoshita M Yamauchi M Sasanuma M Ishikawa Y Osada T Inoue K Wakamatsu Y Ozato K 《Zoological science》2003,20(7):869-875
A particle gun is used in a potential method for introducing foreign genes into fish. In this paper, we report on the stable transmission of a transgene and its expression profile of the F4 generation in the transgenic medaka (Oryzias latipes). We established four transgenic strains, which contained a green fluorescent protein (GFP) gene controlled by a medaka beta-actin promoter, using a particle gun. One more transgenic strain was also generated by microinjection for comparison. In all five strains, the founder was discovered to be mosaic for the transgene. However, from the F1 to F4 generations, transgenes and their expression profiles were stably inherited in the Mendelian manner. The expression profile was common among the five strains regardless of the method for gene transfer: GFP fluorescence became detectable at an early neurula stage. In this stage, the fluorescence was observed ubiquitously in most tissues. As somite developed, GFP fluorescence became intense only in the skeletal muscle and lens but it decreased in other tissues. In adult fish, an intense fluorescence was restricted in the skeletal muscle and lens, while a considerably weak fluorescence was observed in the brain, gill, heart, kidney, spleen, and ovary. From these results, it was concluded that the transgene and its expression profile were stably transmitted to offspring, and thus the particle gun is an effective method for transgenesis in spite of its easiness. 相似文献
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Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater. 总被引:2,自引:1,他引:1 下载免费PDF全文
H Sugita T Asai K Hayashi T Mitsuya K Amanuma C Maruyama Y Deguchi 《Applied microbiology》1992,58(12):4072-4075
Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently. 相似文献
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H Sugita T Asai K Hayashi T Mitsuya K Amanuma C Maruyama Y Deguchi 《Applied and environmental microbiology》1992,58(12):4072-4075
Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently. 相似文献
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Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct 总被引:1,自引:0,他引:1
Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish. 相似文献
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C. M. A. Caipang † N. Hynes † M. F. Brinchmann † K. Korsnes † V. Kiron † 《Journal of fish biology》2008,73(1):115-122
The present investigation determined the antibacterial activities in the sera of both cultured and wild Atlantic cod Gadus morhua . Serum samples from both groups of fish were quantified for total protein, and their effects against fish pathogens Listonella anguillarum , Aeromonas salmonicida and Yersinia ruckeri were determined using co-incubation assay. Total serum protein concentrations were not significantly different between farmed and wild Atlantic cod. Sera of cultured Atlantic cod significantly decreased growth of L. anguillarum by at least two-log10 reductions in bacterial count, while those of the wild Atlantic cod were able to inhibit the growth of all three fish bacterial pathogens. The present study showed that sera from wild fish possess broader antibacterial activities than cultured Atlantic cod and that these could provide an explanation for the differences regarding their immunity to bacterial infections as well as their health status. 相似文献
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Enhanced Bacterial Disease Resistance of Transgenic Channel Catfish Ictalurus punctatus Possessing Cecropin Genes 总被引:3,自引:0,他引:3
Dunham RA Warr GW Nichols A Duncan PL Argue B Middleton D Kucuktas H 《Marine biotechnology (New York, N.Y.)》2002,4(3):338-344
The cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus promoter, was transferred to the channel
catfish Ictalurus punctatus. Transgenic individuals (P1) were mated to produce individuals (F1) that exhibited enhanced disease
resistance and survival when challenged with pathogenic bacteria. During the epizootic of Flavobacterium columnare in an earthen
pond, the percentage of transgenic individuals containing preprocecropin B construct that survived (100%) was significantly
greater (P <0.005) THAN THAT OF NONTRANSGENIC CONTROLS (27.3%). ALSO, WHEN CHALLENGED IN TANKS WITH EDWARDSIELLA ICTALURI,
THE CAUSATIVE AGENT OF ENTERIC SEPTICEMIA OF CATFISH, THE PERCENTAGE OF TRANSGENIC INDIVIDUALS CONTAINING CATFISH IG LEADER
CECROPIN B CONSTRUCT THAT SURVIVED (40.7%) WAS SIGNIFICANTLY GREATER (P <0.01) THAN THAT OF NONTRANSGENIC CONTROLS (14.8%).
THERE WERE NO PLEIOTROPIC EFFECTS OF THE TRANSGENES, AND GROWTH RATES OF THE TRANSGENIC AND NONTRANSGENIC SIBLINGS WERE NOT
DIFFERENT (P > 0.05). Inheritance of the transgene by the F1 generation, 20.2% to 30.7% was typical of that in studies with
transgenic channel catfish. 相似文献
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Although there have been several studies showing the production of transgenic fish through electroporation techniques, success rates have been low and few studies show germ-line integration and expression. When electroporation has been successful, the device used is no longer commercially available. The goal of this experiment was to find an alternative efficient method of generating transgenic Japanese medaka (Oryzias latipes) using a commercially available electroporation device. The Gene Pulser II and RF module (Bio-Rad Laboratories, USA), along with two reporter gene constructs, were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology, the Gene Pulser II incorporates a direct current (DC)-shifted radio frequency (RF) signal. With this technique, over 1000 embryos can be electroporated in less than 30 min. The plasmid pCMV-SPORT--gal (Invitrogen, USA) was used in the supercoiled form to optimize parameters for gene transfer into single-celled embryos, and resulted in up to 100% somatic gene transfer. Similar conditions were used to generate fish transgenic for both the pCMV-EGFP plasmid (Clontech, USA) and a cytomegalovirus (CMV) driven phytase-EGFP construct. The conditions used were a voltage of 25 V, a percent modulation of 100%, a radio frequency of 35 kHz, a burst duration of 10 ms, 3 bursts, and a burst interval of 1.0 s. Seventy percent of the embryos electroporated with the pCMV-EGFP construct survived to sexual maturity, and of those, 85% were capable of passing the transgene on to their offspring. Transgenic second generation back-crossed (BC2) fry were subjected to Southern blot analysis, which confirmed germ-line integration, and observation for green fluorescence protein, which confirmed protein expression. DC-shifted RF pulses are effective and efficient in the production of transgenic medaka, and germ-line integration and expression can be achieved without linearization of the transgene vector. 相似文献
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Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected. 相似文献
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