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1.
Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 102 CFU mL?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 103 CFU g?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.  相似文献   

2.
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   

3.
Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD50 dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.  相似文献   

4.
Vibrio anguillarum, which is part of normal microflora on fish, is the causative agent of vibriosis in aquaculture. It is speculated that V. anguillarum does not affect the host in most situations, but can cause a severe disease once the host is compromised. In the study reported herein, skin‐injured and intestine‐injured zebrafish, Danio rerio, were established as a model to mimic the natural infection caused by V. anguillarum when fish suffered an injury to a mucosal surface. Our results showed the lethal dose to 50% of the population (LD50) of skin‐injured zebrafish was 6.8 × 103 colony‐forming unit (CFU)/mL, which was much lower than intestine‐injured zebrafish (1.9 × 106 CFU/mL) or non‐injured zebrafish (5.5 × 106 CFU/mL). With the quantitative polymerase chain reaction and immunohistochemical analysis, we found that V. anguillarum proliferated rapidly in the skin and muscle after the bacteria entered into the host via the skin injury. The bacteria were subsequently transported to the immune organs and then caused a systemic infection in the fish. However, mortality of skin‐injured zebrafish significantly decreased if the fish were allowed to heal. These results indicate that minimizing injury to the mucosal surfaces of fish, especially the skin, will reduce infections caused by V. anguillarum.  相似文献   

5.
During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non‐O1/non‐O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South‐East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix‐assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization‐associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR+, rtxA+ and hlyAET+ genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.  相似文献   

6.
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   

7.
Growth, biofilm formation, antagonism and residence time in green seawater tanks maintained under fish rearing conditions of Phaeobacter 27‐4 were studied in commercial biofilters made from plastic, sintered glass and ceramic. Phaeobacter reached 108–109 CFU cm?3 and formed rosettes in all materials, but a multilayer biofilm was only observed in the ceramic biofilters. In sterile seawater, plastic and ceramic biofilters reduced Vibrio anguillarum and V. splendidus concentration in one‐two Log after 24–48 h, showing 102–103 CFU mL?1. Sintered glass biofilters only inactivated V. anguillarum. In Marine Broth, sintered glass and ceramic biofilters inhibited V. anguillarum growth in two‐three Log, showing 104–105 CFU mL?1 after 24 h. Plastic biofilters reduced V. anguillarum concentration in one Log after 48 h. V. splendidus growth was only inhibited by sintered glass and ceramic biofilters in one‐two Log, showing 107 CFU mL?1 after 24 h. Phaeobacter also diminished biofilters colonization by the pathogens, both in seawater and in MB. Phaeobacter residence time in green seawater tanks maintained under fish rearing conditions was longer with sintered glass and ceramic biofilters. The latest showed the lowest detachment and, after 11 days, Phaeobacter (106 bacteria·cm?3) covered more than 80% of biofilters total culturable bacteria. DGGE profiles showed that Phaeobacter biofilters stabilizes the green seawater bacterial microbiota.  相似文献   

8.
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9.
Since 2011, high mortality rates and symptoms consistent with vibriosis have been observed in farmed amberjack (Seriola dumerili) in Japan. To identify 41 strains isolated from diseased amberjack, a multilocus sequence analysis using nine concatenated genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) was conducted. Twenty‐seven strains were identified as Vibrio harveyi, suggesting an epidemic of V. harveyi infection in amberjack farms. Other strains were identified as Vibrio anguillarum, Vibrio owensii and Photobacterium damselae subsp. damselae. To develop an efficient diagnostic method for vibriosis in amberjack, a multiplex PCR system was developed to identify V. anguillarum, V. harveyi and P. damselae subsp. damselae. The method successfully discriminated between these three bacterial species, with amplification products of 350 bp for V. anguillarum, 545 bp for V. harveyi and 887 bp for P. damselae subsp. damselae and can be used for diagnosis in aquaculture farms.  相似文献   

10.
This study was performed to evaluate the effect of dietary chitosan on haematology, innate immunity and protection against Vibrio anguillarum in Asian seabass, Lates calcarifer. A basal diet supplemented with 0, 5, 10 and 20 g chitosan kg?1 diet was fed to the four different groups for 60 days. The haematological (total erythrocyte count, total leucocyte count, total serum protein, albumin, globulin and albumin‐globulin ratio) and innate immune parameters (phagocytic ratio, respiratory burst, serum lysozyme and serum bactericidal activities) were monitored at fortnight interval to assess the effect of chitosan feeding in Asian seabass. All the studied haematological and innate immune parameters were increased significantly (P ≤ 0.05) in chitosan‐fed groups in comparison with control. However, the group fed diet containing 10 g chitosan Kg?1 feed showed highest haematological and innate immune parameters on 45th day in comparison with other groups. Moreover, the fish fed the diet containing 10 g chitosan Kg?1 feed had significantly higher post‐challenge survival (75.56 ± 4.44%) on the 30th day following V. anguillarum challenge. Therefore, this study suggests that chitosan at 10 g kg?1 diet could be used as prophylactic in Asian seabass culture to enhance the protection against any possible infection by V. anguillarum.  相似文献   

11.
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   

12.
Swollen abdomen disease (SAD) seriously threatens the aquaculture of turbots and flounders. Two dominant bacterial strains, FS1 and FS2, were isolated from the livers and kidneys of fish with diagnosed SAD. Applications of biochemical analyses, sequence analyses of 16S ribosomal RNA gene and heat shock protein 60 gene revealed two distinct pathogenic bacterial species, Edwardsiella tarda and Vibrio alginolyticus. These two hypothesized SAD‐causing pathogens were validated by challenge trials on flounder, Paralichthys olivaceus. Challenges with E. tarda and V. alginolyticus demonstrated lethal dose 50 (LD50) values at 1.51 × 105 colony‐forming units (CFU) and 1.05 × 105 CFU, respectively. To develop a rapid SAD diagnosis method in flounders and turbots, a multiplex polymerase chain reaction (PCR) assay method was developed to simultaneously detect E. tarda and V. alginolyticus. Our multiplex PCR assay successfully detected as low as 105 CFU/mL E. tarda and V. alginolyticus in flounders and turbots. No other common fish pathogens were detected with the multiplex PCR, suggesting a high specificity of this assay. The multiplex PCR assay developed in this study showed a great reliability in detecting SAD‐causing bacterial pathogens. Further optimization of this assay may contribute to the development of a novel SAD diagnosis tool in aquaculture.  相似文献   

13.
The aim of this study was to screen Bacillus strains from the guts of Scophthalmus maximus, Paralichthys olivacues, Epinephelus coioides and Clupanodon punctatus, for use as probiotics in aquaculture. Eight Bacillus strains were screened, and strain M001 was selected for probiotic study based on its antagonistic activity against multiple aquatic bacterial pathogens including Vibrio anguillarum, V. campbellii, V. vulnificus, V. parahamolyticus, Streptococcus sp. and Edwardsiella tarda. M001 was identified as B. amyloliquefaciens based on the biochemical tests and 16S rRNA gene analysis. In vitro experiments revealed that M001 was able to grow at a wider range of temperature, pH and salinity and was capable to use turbot mucus as nutrient for growth. Additionally, M001 isolate greatly inhibited the growth of V. anguillarum by producing antibacterial substances and was acid tolerance, non‐antibiotic resistance and non‐harmful. Thereafter, the potential probiotic effect of M001 was tested in turbot by dietary administration of M001 at a dose of 108 CFU g?1 diet for 42 days. No significant differences of weight gain, specific growth rate and feed ratio were found in the M001‐diet group of fish compared with control fish, but which increased, respectively, by 5.5%, 4.7% and 7.0% after 42 days of feeding. Several digestive enzyme activities were found to increase significantly in the M001‐diet group, including protease and amylase activities in hepatopancreas, protease activity in intestine and lipase activity in stomach (P < 0.05). Sera superoxide dismutase activity and total protein content (P < 0.05) were also increased significantly (P < 0.05) in the M001‐diet group. The challenge experiment showed that the M001‐diet group of fish showed a relative per cent of survival of 62.7% against V. anguillarim infection. The Bacillus M001 identified from this study has good potential to provide vibriosis control as probiotic feed additive for turbot aquaculture.  相似文献   

14.
Seven marine cyanobacteria were isolated from two regions of the Gulf of Thailand and evaluated by the agar diffusion method for antibacterial activity. Inhibitory compound was purified from the crude methanol extract and its structure was elucidated based on extensive spectroscopic analysis, including IR, 1D and 2D NMR spectra as well as mass spectrometry. A novel antimicrobial compound produced by the marine cyanobacterium Leptolyngbya sp. LT19 was identified to be a 2‐hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate which has so far never been reported in microorganisms. Biological assays revealed that this novel compound exhibited antibacterial activities against the Gram‐negative, persistent shrimp pathogens, Vibrio harveyi and V. parahaemolyticus with minimal inhibitory concentration of 250–1000 and 350–1000 μg mL?1 respectively.  相似文献   

15.
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16.
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   

17.
With the development of gene technology, expressing heterologous antigens in attenuated bacteria has become an important strategy to design multivalent vaccines. In our previous work, an attenuated Vibrio anguillarum named MVAV6203 was developed and proven to be an efficient live vaccine candidate. In this research, we aimed to express protective antigen glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) of Edwardsiella tarda in attenuated Vibrio anguillarum to establish a multivalent V. anguillarum vector vaccine. Several strategies were compared between low‐ vs. high‐copy plasmid‐mediated antigen expression, in vivo‐inducible vs. constitutive antigen expression and intracellular vs. surface‐displaying antigen expression. Zebrafish, Danio rerio (Hamilton), was applied as the fish model to evaluate the immune protection of the V. anguillarum vector vaccine candidates. Our results demonstrated that V. anguillarum MVAV6203 (pUTatLNG40), which harbours a low‐copy plasmid‐loaded antigen surface display system under the control of a constitutive promoter, presented the best protective efficacy against the infection of Vibrio anguillarum (relative per cent survival, RPS = 85%) and Edwardsiella tarda (RPS = 70%).  相似文献   

18.
Vibrio anguillarum, also known as Listonella anguillarum, is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide. Because of its high morbidity and mortality rates, substantial research has been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease‐prevention strategies. This review summarizes the current state of knowledge pertaining to V. anguillarum, focusing on pathogenesis, known virulence factors, diagnosis, prevention and treatment.  相似文献   

19.
The aro genes in bacteria encode enzymes needed for the biosynthesis of aromatic amino acids, and mutant bacteria that are defective in the enzymes can replicate only a limited number in vertebrates owing to the lack or scarceness of chorismate, through which the mutant bacteria of the aro genes become attenuated. In the present study, the 5‐enolpyruvylshikimate‐3‐phosphate synthase (aroA) gene‐knockout Vibrio anguillarum (ΔaroA V. anguillarum) were generated by the allelic exchange method, and its vaccine potential was evaluated in the olive flounder, Paralichthys olivaceus, by comparing the protective efficacy of a formalin‐inactivated V. anguillarum. The LD50 (50% lethal dose) value of ΔaroA V. anguillarum was 1000 times higher than that of wild‐type V. anguillarum in olive flounder fingerlings, and the growth of ΔaroA V. anguillarum was significantly suppressed by coincubation with nonimmune olive flounder serum compared with that of wild‐type V. anguillarum. The survival rates and serum agglutination titers of fish immunized with ΔaroA V. anguillarum were significantly higher than those of fish immunized with the same amount of formalin‐inactivated V. anguillarum, suggesting that although the inactivated V. anguillarum vaccine can provide a high protection in olive flounder, the protective efficacy can be enhanced by immunization with an auxotrophic mutant ΔaroA V. anguillarum.  相似文献   

20.
To investigate the effect of non‐ionic ammonia (NH3‐N) stress (0.1 and 0.5 mg L?1) on the immunity of Litopenaeus vannamei cultured in long‐term freshwater, the total haemocyte count (THC), the activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase dismutase (SOD) and the content of malondialdehyde (MDA) were determined and further compared with those of seawater shrimps. The results showed that NH3‐N stress significantly reduced THC and the activity of PO and SOD (P < 0.05). Under 0.1 mg L?1 NH3‐N stress, NOS activity increased first and then decreased significantly, while it dropped dramatically under 0.5 mg L?1 NH3‐N stress (P < 0.05). During NH3‐N stress, MDA content increased continuously, and the MDA content in hepatopancreas of freshwater shrimps was higher than that of seawater shrimps. It was concluded that NH3‐N stress significantly influenced the non‐specific immunity and could also upset the balance of antioxidant system of L. vannamei in both freshwater and seawater shrimps. Compared with in seawater, the shrimps in freshwater were more vulnerable to NH3‐N stress because of higher lipid peroxidation and lower immunity.  相似文献   

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