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1.
Lactic acid bacteria (LAB) were isolated from adult, wild‐caught and farmed seabass (Lates calcarifer) intestines for evaluation as possible probiotics using the well agar diffusion method. Five LAB isolates (designated as LAB‐1–5) were found to inhibit Aeromonas hydrophila, a known seabass pathogen. Median lethal concentrations (LC50) of A. hydrophila on juvenile seabass were measured in aquaria. Median lethal concentration values of 7.76, 7.47 and 7.26 log10 CFU mL?1 for 72, 96 and 120 h, respectively, were found. Juvenile seabass (0.6±0.2 g) were cultured in aquaria and fed individual LAB‐1–5 fortified feeds with 7 log10 CFU g?1 LAB. Seabass fed LAB‐4 fortified feed had significantly greater growth (P<0.05) than fish fed other feeds. Seabass fed LAB‐4 also had greater survival, but this was non‐significant (P<0.05). Challenge tests of LAB‐4 fed seabass with A. hydrophila at ～7 log10CFU mL?1 yielded significantly greater survival compared with control seabass (P<0.05). Aeromonas hydrophila infections in seabass were confirmed by observing disease manifestation and by immunohistochemistry techniques. LAB‐4 was preliminarily identified using lactic acid analysis, biochemical and physical characteristics. It was further identified using 16S rDNA sequencing. LAB‐4 was identified as Weissella confusa (identity of 99%). GenBank accession number for the 16S rDNA sequence for LAB‐4 was AB023241.  相似文献   

2.
Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 107 CFU fish?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.  相似文献   

3.
Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues—eye, oesophagus, gills and skin—as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.  相似文献   

4.
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5.
Abstract Stable isotope analysis of fishes is often performed using muscle or organ tissues that require sacrificing animals. Non‐lethal sampling provides an alternative for evaluating isotopic composition for species of concern or individuals of exceptional value. Stable isotope values of white muscle (lethal) were compared with those from fins and scales (non‐lethal) in walleye, Sander vitreus (Mitchill), from multiple systems, size classes and across a range of isotopic values. Isotopic variability was also compared among populations to determine the potential of non‐lethal tissues for diet‐variability analyses. Muscle‐derived isotope values were enriched compared with fins and depleted relative to scales. A split‐sample validation technique and linear regression found that isotopic composition of walleye fins and scales was significantly related to that in muscle tissue for both δ13C and δ15N (r2 = 0.79–0.93). However, isotopic variability was significantly different between tissue types in two of six populations for δ15N and three of six populations for δ13C. Although species and population specific, these findings indicate that isotopic measures obtained from non‐lethal tissues are indicative of those obtained from muscle.  相似文献   

6.
An inactivated betanodavirus, red‐spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 °C water temperature by a single intraperitoneal injection of formalin‐inactivated RGNNV. Fish immunized at vaccine doses of 108.5, 108.0, 107.5, 107.0 and 106.5 TCID50 per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post‐immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre <1:80). When fish were challenged with RGNNV (105.0 and 104.0 TCID50/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 108.5, 108.0, 107.5 and 107.0 TCID50 per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 107.5 TCID50 per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 106.5 TCID50 per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 107.0 TCID50 per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.  相似文献   

7.
Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.  相似文献   

8.
Infectious haematopoietic necrosis (IHN) and viral haemorrhagic septicaemia (VHS) are OIE‐listed and notifiable viral fish diseases which are controlled by eradication and surveillance programmes globally. The present study provides improved RT‐qPCR procedures based on recently described OIE protocols. Improvements comprise the design of a new TaqMan® probe, replacing a TaqMan® MGB probe that turned out to show impaired binding. Reason for this is SNPs detected in the nucleoprotein N gene sequences of IHNV strains targeted by the RT‐qPCR. Furthermore, the IHNV and VHSV RT‐qPCR assays were realized as one‐step and one‐run procedures supplemented by an endogenous control system. The IHNV and VHSV RT‐qPCR assays are characterized by a technical sensitivity of 19 and 190 gene equivalents (cRNA) and an analytical sensitivity of 2–7 and 13 TCID50/ml, respectively. For verification purposes, 105 IHNV and 165 VHSV isolates and several non‐targeted viral and bacterial pathogens were included and returned adequate results. However, in field samples divergent results left 14 samples of 154 undetected for IHNV and one sample of 127 for VHSV using cell culture. The study shows that RT‐qPCR assays ensure facilitated and reliable testing on IHNV and VHSV in eradication and surveillance programmes.  相似文献   

9.
Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post‐smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 102 to 10?4 50% end‐point tissue culture infectious dose (TCID50) mL?1 sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post‐smolts was <10?1 TCID50 mL?1 by bath immersion (4 h at 10 °C). The peak shedding rate for IPNV following intraperitoneal challenge using post‐smolts was estimated to be 6.8 × 103 TCID50 h?1 kg?1 and occurred 11 days post‐challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.  相似文献   

10.
A novel permanently growing brain cell line from koi (Cyprinus carpio L.) (KB cell line) was established, and its suitability for detection of koi herpesvirus (KHV) was demonstrated in this study. The KB cell line was optimally maintained at 27°C in Leibovitz's L‐15 medium supplemented with 10% foetal bovine serum (FBS). It was subcultured more than 100 times, and chromosome analysis revealed that 51.54% of KB cells at passage 80 maintained the abnormal diploid chromosome number 2n = 96 while the modal chromosome number was 2n = 100. The cell line was cryopreserved in liquid nitrogen at ?196°C and was recovered from storage after 1 year with good cell viability and vitality. The results of virus isolation demonstrated that KB cells were susceptible to KHV, which was shown by the presence of an obvious cytopathic effect and abundant virus particles. The viral titres of KHV in KB reached 105.73TCID50/0.1 ml within 7 days. Immunofluorescence and Western blot assays confirmed that KB replicated KHV. The newly established KB cell line will serve as a useful tool to elucidate KHV disease (KHVD) pathogenesis.  相似文献   

11.
A total of 210 European seabass fry with an average body weight of 0.51 ± 0.03 g were divided into seven experimental treatments (three replicates per treatment). The seven experimental diets (45.64% CP) were formulated to contain synthetic astaxanthin (SA; 0.1 g kg?1), marigold flower meal (MFM; 0.2 g kg?1) or crab waste meal (CM; 10 g kg?1), with or without sodium taurocholate (ST; 0.4 g kg?1); the control diet contained no supplementation. The experiment was conducted for 8 weeks. The results show that seabass fry fed the diets containing different sources of carotenoids, especially with ST, demonstrated higher growth efficiency, survival (%), feed efficiency, protein and ether extract contents of fish body. The best results were found with DietMFM+ST. Whole‐body carotenoid levels increased significantly with all supplementation treatments; the highest level was observed with SA, followed by fish fed DietMFM and DietCM, especially with ST co‐supplementation. Moreover, thiobarbituric acid‐reactive substances were significantly reduced in fish fed the diets containing different sources of carotenoids compared to the control diet. The total antioxidant status significantly increased in fish fed DietSA and DietCM alone or with ST compared to the control group. In conclusion, these results reveal that DietMFM+ST is the most suitable treatment for improving growth, feed efficiency, and the protein and ether extract contents of seabass fry compared to other sources of carotenoids. The best oxidant/antioxidant balance and carotenoid content were achieved using SA, CM and MFM with ST supplementation.  相似文献   

12.
In this study, the susceptibility of turbot juveniles to two betanodavirus strains was assessed, a RGNNV/SJNNV reassortant (Ss160.03) and a SJNNV strain. The reassortant isolate exhibits a slightly modified SJNNV CP, with two amino acid substitutions in the C‐terminal domain (positions 247 and 270). To analyse the role of these residues as virulence and host determinants in turbot, three recombinant strains (rSs160.03247, rSs160.03270, rSs160.03247+270) harbouring site‐specific mutations in the CP sequence were also tested in experimental trials. Moderate mortalities (up to 50%) were recorded at 18 °C in the fish challenged with the Ss160.03 strain, whereas low mortalities (17%) were observed in the group challenged with the SJNNV strain. A slight decrease (around 10%) was observed in the mortalities caused by the mutants rSs160.03247 and rSs160.03270, whilst the mutation of both positions reduced mortality by more than half of that observed in fish challenged with the wild strain. These results are confirmed by the replication in brain tissues, because whereas the wild strain was detected from 5 to 30 dpi and reached the highest viral load, the recombinant virus harbouring both mutations was not detected in the brain until 20 dpi and with a moderate viral load.  相似文献   

13.
Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T‐antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell‐infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK‐1 and EO‐2 cells at temperatures 25–30 °C, and the progeny virus yields were 107.0, 107.4 and 107.7 TCID50 mL?1, respectively. The purified virions were icosahedral particles, 70–80 nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2 month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.  相似文献   

14.
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15.

Sulfated polysaccharides (SP) from seaweeds exhibit numerous biological activities and are less toxic than conventional antiviral drugs. However, little is known about their mode of action. This study aims at extracting the polysaccharides from brown seaweed Sargassum ilicifolium and then evaluating its antiviral activity against fish Betanodavirus. The extracted SP was characterized through FT-IR, 1H NMR, and thermal gravimetric analysis (TGA). The antiviral activity of SP was tested on SGA cell line by syncytia reduction and the polysaccharide represented a prominent antiviral activity to Betanodavirus by reducing the cytopathic effect (CPE) formation in vitro. The study revealed that SP effectively blocked viral attachment and entry. Moreover, during the in vivo challenge experiment in Gambusia affinis, SP treatment showed a significant decrease in the viral titers. Hence, these results indicate that the polysaccharides obtained from S. ilicifolium exhibited the high antiviral potential to Betanodavirus infection in fishes both in vitro and in vivo.

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16.
Three different concentrations (107, 105 and 103 TCID50 ml-1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 107 TCID50 ml-1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 107 and 105 TCID50 ml-1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.  相似文献   

17.
Three different concentrations (107, 105 and 103 TCID50 ml?1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 107 TCID50 ml?1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 107 and 105 TCID50 ml?1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/ brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.  相似文献   

18.
Largemouth bass virus (LMBV) is a recently discovered iridovirus that causes a fatal disease of largemouth bass, Micropterus salmoides (Lacepède). Fish can become infected by waterborne LMBV, but oral transmission of this virus has not been demonstrated previously. Largemouth bass were gavaged with guppies, Poecilia reticulata Peters, which had been injected with LMBV, and then sampled periodically during a 7‐week observation period. The dose of LMBV averaged 105.6 tissue culture infectious doses – 50% cytopathic endpoint (TCID50) per largemouth bass. Five of 24 largemouth bass exposed to LMBV became infected with the virus, but none of the fish had clinical signs typical of LMBV disease. Virus titres in largemouth bass were highest in swim bladder (105.5–9.5 TCID50 g?1) and were 105.2 TCID50 g?1 or lower in cutaneous mucus, head kidney, trunk kidney, spleen, gonad and intestine. These results indicate that LMBV can be transmitted orally to largemouth bass, but further study is needed to determine the factors affecting pathogenicity of the virus.  相似文献   

19.
Cyprinid herpesvirus 3 (CyHV‐3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV‐3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF‐1 and CCB) infected with CyHV‐3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1–4 h post‐infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post‐infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5–7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non‐enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non‐infectious particles, particularly in KF‐1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV‐3 propagation.  相似文献   

20.
Asian seabass (Lates calcarifer) has been designated as a candidate species for open sea cage culture in India. A case of vibriosis in Asian seabass reared in open sea floating cages is reported. Haemorrhage and ulcer were observed grossly in the diseased fish. The pathogen, identified as Vibrio alginolyticus based on biochemical and molecular characterization, was isolated from liver, gill, kidney, brain and blood. Histological examination of the diseased fish showed congestion, haemorrhage and necrosis in vital organs. The LD50 studies showed that the organism was virulent to Asian seabass, and the LD50 value was 103.2 CFU g?1 fish.  相似文献   

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