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1.
In spring of 2011, an epidemic outbreak of torafugu with high mortality occurred in an aquafarm with marine industrial recirculation aquaculture system (MIRAS) in Yantai, Shandong Province, China. The diseased fish showed anorexia, haemorrhaging and festering fin and skin and swelling internal organs. Forty‐five dominant bacterial isolates were obtained from the diseased fish, and were found to belong to 12 species according to 16S rRNA gene sequences. One strain from each species was selected to test the pathogenicity, and five strains were showed to be virulent to zebrafish. Whereas Enterovibrio nigricans Fr42 was highly virulent with the LD50 of 7.8 × 104 CFU g?1, Photobacterium swingsii Fr23, Vibrio owensii Fr40, V. harveyi Fr51 and V. rotiferianus Fr71 were moderately virulent with the LD50 of 1.7 × 106 to 8.4 × 106 CFU g?1. Both the bacteria and their extracellular products of the five strains were found to show phospholipase, caseinase, gelatinase, amylase and/or lipase activities. The production of N‐acyl homoserine lactones (AHLs) of the five strains was detected by three different AHLs biosensors, and three of them were found to produce AHLs by at least one kind of biosensor. This is the first study describing various opportunistic bacterial pathogens of fish cultured in MIRAS in China.  相似文献   

2.
Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.  相似文献   

3.
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4.
A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 104 ± 1 × 104 CFU g?1 (without enrichment) and 40 ± 10 CFU g?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.  相似文献   

5.
The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 106, 107 and 108 CFU g?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 107 CFU g?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.  相似文献   

6.
Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.  相似文献   

7.
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8.
A marine antagonistic bacterium, JG1, was isolated from rearing water of healthy turbot (Scophthalmus maximus) in Qingdao, China. Strain JG1 was Gram‐negative, straight rod and motile by polar flagella. The colony, when cultured for 24 h under room temperature, produced a yellow pigment. On the basis of morphological, physiological and biochemical characteristics, along with 16S rDNA sequence analysis, JG1 was identified as Pseudoalteromonas flavipulchra. JG1 had good inhibitory effects on several bacterial pathogens of aquaculture in the genus Vibrio (V. anguillarum, V. alginolyticus, V. campbellii, V. harveyi, V. mimicus, V. parahaemolyticus and V. tubiashii) and Aeromonas (A. hydrophila and A. salmonicida). No mortality occurred 14 days after zebra fish and 7 days after mantis shrimp were intraperitoneally injected with JG1 at 106 CFU per animal, and 7 days after scallop and clam were immersed in JG1 at 107 CFU mL?1. A good antagonistic effect on several bacterial pathogens and nontoxicity to the above‐mentioned animals make JG1 a potential probiotic in aquaculture. In addition, a fast detection technique based on polymerase chain reaction amplification of the gyrB gene was established to allow us to determine the fate of JG1 when it is applied in aquaculture in the future.  相似文献   

9.
Enteric septicaemia of catfish (ESC) caused by Edwardsiella ictaluri is becoming an increasing problem in aquaculture and has been reported worldwide in a variety of fish species. This study reports ESC in hybrid catfish, Clarias macrocephalus (Günther) × Clarias gariepinus (Burchell), cultured in southern Thailand. The bacteria were identified as E. ictaluri by conventional and rapid identification systems, as well as by genetic and phylogenetic characterization. Analysis of 16S rRNA indicated 100% homology to the 16S rRNA sequence of several E. ictaluri strains in GenBank. Plasmid profiles demonstrated 4.0‐ and 5.6‐kb plasmids, compared with the 4.8‐ and 5.6‐kb plasmids in the US isolates, and representative genes of three of the four known pathogenicity islands of US isolates were present. Serologically, lipopolysaccharide (LPS) purified from the Thai isolates was not recognized by a monoclonal antibody against the LPS of US isolates. Fish experimentally infected with E. ictaluri showed 23–100% mortality within 14 days with a 168‐h LD50 of 6.92 × 107 CFU mL?1 by immersion and a 96‐h LD50 of 1.58 × 106 CFU fish?1 by intraperitoneal injection. Examination of tissue sections obtained from both naturally and experimentally infected fish indicated that infection of hybrid catfish with E. ictaluri produced lesions in several organs including liver, kidney, spleen, heart and brain. Histopathology findings included cellular necrosis, focal haemorrhage, infiltration of lymphocytes and multifocal granulomatous inflammation in the infected organs.  相似文献   

10.
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11.
Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non‐A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport‐induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 106 CFU fish?1) and A. veronii (8.9 × 106 CFU fish?1) killed 100% of experimental fish within 24 h, while a 10‐fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100‐fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.  相似文献   

12.
This study aimed to develop a synbiotic combination with probiotic, Lactobacillus plantarum 7–40 and one of three prebiotics, fructooligosaccharide (FOS), galactooligosaccharide (GOS) and mannan oligosaccharide (MOS). The best in vitro growth was observed when probiotic was cultured in the medium containing either FOS or GOS as the sole of carbon source. The analysis of enzyme activity revealed that GOS induced the highest activities of protease and β‐galactosidase of probiotic. Based on the findings, probiotic + GOS were selected as synbiotic to evaluate if it could promote the growth of white shrimp, Litopenaeus vannamei. For this, four diets, including a basal diet with no GOS or probiotic (control), 0.4% GOS (PRE), 108 CFU probiotic kg?1 (PRO) and 0.4% GOS in combination with 108 CFU probiotic kg?1 (SYN), were fed to shrimp for 60 days, and then the growth performance, intestinal microbiota (including total Vibrio counts, VBCs; and lactic acid bacteria, LAB) and digestive enzyme (including protease, leu‐aminopeptidase and β‐galactosidase) were evaluated. The weight gain (WG) of shrimp fed the PRO did not significantly differ from those of control (p > .05). Shrimp fed the SYN had significantly higher WG compared with the other treatments (p < .05). In addition, the SYN‐fed shrimp had significantly higher LAB and protease, leu‐aminopeptidase and β‐galactosidase activity (p < .05). The lowest presumptive Vibrio count (VBC) was also observed in intestines of SYN‐fed shrimp. Therefore, we suggested that Lac. plantarum 7–40+ GOS can be used as a synergistic synbiotic for shrimp culture.  相似文献   

13.
Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L?1 for A. hydrophila and 50 mg L?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L?1 for A. hydrophila, 100 mg L?1 for V. harveyi, and 125 mg L?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L?1 for A. hydrophila and 200 mg L?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.  相似文献   

14.
Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca2+) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca2+ ion.  相似文献   

15.
Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.  相似文献   

16.
This study was conducted to evaluate the prevalence, potential pathogenicity and antimicrobial resistance of Vibrio isolates from 65 soil/water/fish samples collected from inland saline aquaculture areas. Depending on the sample type, presumptive Vibrio counts ranged from 2.50 to 6.16 log10 CFU/ml (or/g). Among the 119 confirmed Vibrio isolates, Vibrio cholerae was found to most dominant (91.6%) and it was detected in all the samples from inland saline areas. Seven other Vibrio spp. including Vibrio parahaemolyticus and Vibrio vulnificus were also detected. Except one O139 serotype, rest of the V. cholerae isolates were found belonging to non‐O1/non‐O139 serogroups. None of the V. cholerae isolate was found positive for ctx gene. Antimicrobial susceptibility testing against 7 commonly used antibiotics revealed highest resistance (50.4%) against ampicillin. Very high intermediate resistance (87.4%) was also observed against erythromycin. Contrary to previous studies, high susceptibility (>70%) to chloramphenicol, nalidixic acid, tetracycline and trimethoprim was observed in Vibrio isolates obtained in present study. Almost 20% of Vibrio isolates were resistant to two or more antibiotic classes with multiple antibiotic resistance (MAR) index value of ≥0.28. Presence of V. cholerae isolates with very high MAR index value of 0.85 also suggested that these multidrug‐resistant environment isolates could serve as reservoir of antibiotic‐resistant genes in aquatic systems. The presence of multiple drug resistance vibrios in emerging inland saline aquaculture systems emphasizes the need for their routine monitoring for developing the risk assessment and mitigation strategies.  相似文献   

17.
The present feeding trial examined the effect of synbiotic use of Bacillus mycoides and organic selenium (OS) as Sel‐Plex on marron immunity, growth and survival. The marron were cultured in recirculated tanks and fed test diets consisting of a basal diet; basal diet supplemented with B. mycoides (108 CFU g?1 of feed); basal diet supplemented with OS (Sel‐Plex) (0.2 g kg ? 1 of feed) and basal diet supplemented with synbiotic (B. mycoides at 108 CFU g?1 and OS 0.2 g kg ? 1 feed) diet, in triplicate. The effect of the prebiotic OS (Sel‐Plex) on the growth rate of B. mycoides was also studied in vitro. The results suggested that synbiotic use of B. mycoides and OS significantly improved some immune parameters of marron, particularly the glutathione peroxidase, and to some extent total haemocyte counts. However, the synbiotic feed did not synergistically improve marron growth, in fact the use of B. mycoides‐supplemented diet alone demonstrated significantly higher growth in marron compared with the growth of marron fed on other test diets. Supplementation of the basal diet with host origin B. mycoides significantly increased the intestinal bacterial population (3.399 ± 825 CFU g ? 1 of gut) in marron compared with other diets. Organic selenium as Sel‐Plex in Trypticase Soya Broth also confirmed that OS did not increase the amount of growth of B. mycoides and resulted in a lower intestinal bacterial population in the synbiotic diet‐fed marron. In conclusion, synbiotic of OS and B. mycoides may improve a particular immune parameters of marron and to a lesser extent their growth.  相似文献   

18.
The aim of this study was to examine the effects of the immunostimulant combination (IC) containing β‐glucan, A3α‐peptidoglycan, vitamin C and vitamin E on the growth performance, non‐specific immunity and protection against Vibrio harveyi infection in cobia (Rachycentron canadum). Fish were fed diets containing six graded levels of IC (0, 1, 2, 3, 4 and 5 g kg?1 diet) for 8 weeks. The results showed that the survival rate ranged from 81.1 to 84.4% with no significant difference among all the groups (P > 0.05) after the feeding experiment. Dietary IC significantly increased the specific growth rate (SGR), serum lysozyme, alternative complement pathway (ACH50) activity, phagocytosis percentage (PP) and respiratory burst activity of head kidney macrophages of cobia. Moreover, feeding of supplemented diets containing 3.0 g kg?1 IC resulted in significantly lower mortality against the pathogens, V. harveyi compared with the control group. To elevate the growth and immune resistance ability of cobia, the optimal dose of dietary IC administration, determined by second‐order polynomial regression analysis was 3.43 and 2.71 g kg?1 diet, respectively, on the basis of the SGR and mortality after challenge with V. harveyi.  相似文献   

19.
Three in vivo experiments were conducted to measure the effectiveness of commercial Bacillus probiotics on survival and development of Litopenaeus vannamei larvae from nauplii 3–4 (N3–4) to zoea 3 (Z3) stages. Experiment I: Nine commercial Bacillus probiotics were individually added to larvae twice at N6 and Z1 at levels of 2, 20, and 100 mg L?1. Only six of nine products at 20 mg L?1 exhibited higher or significantly higher larval survival (P < 0.05) than the control. Experiment II: Two superior products from the first experiment were administered to larvae with six dose frequencies at 20 mg L?1. For both products, three doses, once at each of N3–4, N6 and Z1 stages, yielded the best larval survival and development rates (P < 0.05), and these were confirmed by enhanced activities of tryptase and amylase. Experiment III: The isolates of these two products, identified as Bacillus subtilis and Bacillus licheniformis, were delivered to larvae singly at concentrations of 1.0 × 108 and 1.0 × 109 CFU L?1, or at the same concentrations by mixing the two equally. At 1.0 × 109 CFU L?1, B. subtilis exerted more beneficial effects on larvae than B. licheniformis or the mixture. Therefore, the optimal dosage and dose frequency of commercial Bacillus products should be evaluated prior to large‐scale application in shrimp hatcheries to avoid futility or even adverse effects, as spore counts and non‐bacterial ingredients are key factors influencing the efficacy of Bacillus probiotics.  相似文献   

20.
Vibrio harveyi, a luminescent Gram‐negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non‐luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence‐related phenotypic traits of the wild‐type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non‐luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non‐luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.  相似文献   

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