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1.
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2.
Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re‐attenuated S. agalactiae (named TFJ‐ery) was developed from a natural low‐virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ‐ery were determined. The results indicated that TFJ‐ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ‐ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ‐ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ‐ery at a dose of 5.0 × 107 CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post‐vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post‐vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ‐ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.  相似文献   

3.
Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real‐time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS‐s/IGS‐a, which targets the 16S‐23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post‐injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post‐injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish.  相似文献   

4.
Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 108 cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 106, 107 or 108 cfu/fish of F. psychrophilum FPG 101, the 108 cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R2 = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log10 gene/copies and >3.0 log10 gene copies/reaction, respectively, following infection with 108 cfu/fish.  相似文献   

5.
This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross‐reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC‐PCR and REP‐PCR) and MALDI‐TOF‐MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI‐TOF‐MS is the most promising method for typing and characterization of V. salmoninarum.  相似文献   

6.
Streptococcus agalactiae is a Gram‐positive facultative intracellular bacterium that leads to severe economic loss of tilapia worldwide. Previous studies demonstrated that CD40 contributes to host protection against intracellular injection. In this study, CD40 was characterized from Nile tilapia (Oreochromis niloticus), named OnCD40. Sequence analysis showed that open reading frame of OnCD40 was 933 bp, containing a single peptide, a transmembrane domain and four cysteine‐rich domains. The qRT‐PCR revealed that OnCD40 was expressed in all examined tissues with the most abundant ones in spleen and thymus. After S. agalactiae stimulation, the expression of OnCD40 was significantly induced in most of the detected organs. Moreover, OnCD40‐overexpressing fish elicited significant protection against subsequent S. agalactiae challenge; approximately 10000‐fold fewer bacteria were detected in spleen of OnCD40‐overexpressing fish in comparison with control fish. Thus, CD40 had protecting function in Nile tilapia against intracellular pathogens.  相似文献   

7.
Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non‐encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild‐type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host–pathogen interactions.  相似文献   

8.
A feeding trial was conducted to investigate the effect of different levels of Bacillus subtilis LT3‐1 in diets on growth, immune parameters, intestinal morphology and disease resistance in genetically improved farmed tilapia, Oreochromis niloticus. Fish (46.91 ± 0.17 g) were fed with a basal diet supplemented with B. subtilis LT3‐1 at 0 (B0), 3.8 × 1010 (B1), 7.6 × 1010 (B2), 1.14 × 1011 (B3) and 1.52 × 1011 (B4) CFU kg?1 for 6 weeks. The results showed that the weight gain of fish in B1 group was significantly enhanced compared to that in B0 group (p < 0.05). The addition of B. subtilis significantly affected serum biochemical indices (total protein, albumin, aspartate aminotransferase, alkaline phosphatase). Besides, the haematocrit, total counts of red and white blood cells, as well as the serum catalase and lysozyme activities, were increased, whereas the serum malondialdehyde, the serum immunoglobulin M and complement three contents were reduced. Parameters for intestinal morphology suggested a healthier intestine for the fish fed B. subtilis‐supplemented diets than fish fed the control diet. The survival rate after Streptococcus agalactiae challenge increased in tilapia fed with B. subtilis. The present study demonstrated B. subtilis can effectively improve growth, immunological status and resistance against S. agalactiae infection in tilapia farming.  相似文献   

9.
Nile tilapia (Oreochromis niloticus) farming is an economic activity that is soaring in the whole world. Septicemia due to Streptococcus agalactiae is the main disease impacting fish farming. The aim of this study was to compare the gut microbiome of healthy animals and animals experimentally infected with S. agalactiae strain 21171A. The microbiome was established with 16S ribosomal DNA next‐generation sequencing (NGS). One hundred Nile tilapias, with an average weight of 35 g, were distributed into two groups. Fifty fish from the challenged group were orally inoculated with 100 μl of a bacterial solution containing 1.98 × 103 CFU/ml of S. agalactiae strain 21171A, while 50 controls were orally inoculated with sterile saline. After the experiment, 24 fish from the challenged group and 27 fish from the control group were analysed. For both groups, bacteria attached to the mucosa (M) and present in faeces (F) were analysed. The mean of the number of taxa identified in the infected group (M + F) (45.87 ± 30.13) was lower than in the control (M + F) (67.70 ± 21.10) (p < .01). Nineteen bacterial taxa were more abundant in faecal samples from the infected group when compared with the control group (p < .01). Thirty‐nine taxa were associated with mucosa samples from the challenged group when compared to the control samples (p < .01). No OTU was associated with healthy samples. The results demonstrate that the infection with S. agalactiae reduces the variability of the gut microbiota. Moreover, some bacteria proliferate during the infection.  相似文献   

10.
The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 101 amplicon copies per assay (equivalent to 2 × 10–9 ng/µl) using bacterial DNA and of 1.44 × 101 gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.  相似文献   

11.
Effects of Bacillus cereus BC‐01, Lactobacillus acidophilus LAG01, Clostridium butyricum CBG01 and their combinations as supplementation on the growth performance, digestive enzyme activities and serum non‐specific immunity of hybrid grouper (Epinephelus lanceolatus ♂×E. fuscoguttatus ♀) were assessed. Seven different diets, that is one control diet (basal feed without any probiotics, CT) and six treatment diets containing single B. cereus (Bs), L. acidophilus (Ls) and C. butyricum (Cs) at 1.0 × 109 cfu/kg feed, and also their combinations in equal proportion at 1.0, 2.0 and 3.0 × 109 cfu/kg feed (BLC1, BLC2 and BLC3) were prepared respectively. After 60‐day feeding trial, the final weight, specific growth rate,food consumption, food conversion efficiency and apparent digestibility coefficient of fish in Ls and BLC3 were significantly higher compared with the control (p < .05). The activities of pepsin and trypsin in the intestine of fish for Ls and BLC3 were significantly higher relative to the control (p < .05). Relative to controls, significantly enhanced amylase and lipase activities in proximal intestine except for Cs and BLC1 and lipase activities in distal intestine except for Cs were observed (p < .05). Meanwhile, activities of superoxide dismutase in the serum of fish for all treatments, lysozyme and catalase in Ls and BLC3, and glutathione peroxidase except for Cs were significantly enhanced (p < .05). Based on the above, dietary supplementation of single L. acidophilus at 1.0 × 109 cfu/kg or combination of three strains at 3.0 × 109 cfu/kg for hybrid grouper is recommended.  相似文献   

12.
Streptococcus agalactiae causes a severe systemic disease in fish, and the routes of entry are still ill‐defined. To address this issue, two groups of 33 red tilapia Oreochromis spp. each of 10 g were orally infected with S. agalactiae (n = 30), and by immersion (n = 30), six individuals were control‐uninfected fish. Three tilapias were killed at each time point from 30 min to 96 h post‐inoculation (pi); controls were killed at 96 h. Samples from most tissues were examined by haematoxylin–eosin (H&E), indirect immunoperoxidase (IPI) and periodic acid‐Schiff; only intestine from fish infected by gavage was evaluated by transmission electron microscopy. The results of both experiments suggest that the main entry site of S. agalactiae in tilapia is the gastrointestinal epithelium; mucus seems to play an important defensive role, and environmental conditions may be an important predisposing factor for the infection.  相似文献   

13.
Attempts were made to identify the association between water quality parameters and the presence of Streptococcus agalactiae in cage cultured red hybrid tilapia, Oreochromis niloticus × O. mossambicus. Fish from commercial floating net cage‐culture systems in a river and lake were randomly sampled over a 24‐month period. Swabs from the brains, eyes and kidneys were streaked directly onto blood agar to isolate S. agalactiae. Water temperature, dissolved oxygen, pH, clarity, ammonia, nitrite, sulfide, rate of water flow and depth of water at sampling sites were measured at the same time of fish sampling. The prevalence of fish that were cultured positive to S. agalactiae was significantly higher in lake compared with river. The length and weight of the infected fish were between 9 and 33 cm, and between 20 and 760 g respectively. There was a significant and positive strong correlation between the presence of S. agalactiae and fish mortalities in lake. All water quality parameters showed significant differences between river and lake. However, only water temperature, clarity and pH of lake and the ammonia, temperature and dissolved oxygen in river showed significant correlation with the presence of S. agalactiae in the cultured fish. It was concluded that several unfavourable water quality in the fish farm influencing the presence of S. agalactiae in cultured red hybrid tilapia.  相似文献   

14.
Probiotic potential of the autochthonous bacteria in catla, Catla catla has been evaluated through determination of antagonistic activity (in vitro) of the cellular components of gut bacteria against seven fish pathogens. Altogether 208 strains were isolated, inhibitory activity of the isolates was evaluated through cross‐streaking and 16 primarily selected antagonistic strains were confirmed using the double‐layer method. Four bacteria that showed antagonism against ≥4 pathogens were selected as putative probiotics. The intracellular, extracellular, whole‐cell and heat‐killed cell components exhibited bactericidal activity against the pathogens. In addition, the selected strains were capable of producing different extracellular enzymes, competent to grow in intestinal mucus and could tolerate diluted bile juice. Analysis of 16SrRNA partial gene sequence revealed that both the strains CC1FG2 and CC1FG4 were Bacillus methylotrophicus (KF559344 and KF559345), while the isolates CC1HG5 and CC2HG7 were Bacillus subtilis subsp. spizizenii (KF559346) and Enterobacter hormaechei (KF559347) respectively. Bio‐safety evaluation through intra‐peritoneal injection of the isolates did not induce any pathological signs or mortalities in C. catla. The study confirmed probiotic properties of autochthonous gut bacteria in C. catla and demonstrated potential for using them as bio‐control agents. However, in vivo studies are essential to explore their efficacy in the commercial aquaculture.  相似文献   

15.
In non‐salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold‐water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.  相似文献   

16.
Adequate enrichment of live prey like Artemia, naturally deficient of essential highly unsaturated fatty acids (HUFA), such as docosahexaenoic acid (22:6n‐3, DHA), is critical for the rapidly developing tissues, survival, normal development and production of good‐quality fingerlings. The aim of the study was to evaluate the effects of a pulse (10–30 dah) of Shewanella putrefaciens Pdp11 (2.5*107 cfu/ml) using Artemia metanauplii as live vector, on its proper lipid profiles and resultant Solea senegalensis body composition and performance. Probiotic administration significantly increased total lipids and specifically n‐3 HUFA levels in Pdp11‐enriched Artemia. The live prey lipid modulation was also reflected in the total lipid contents and fatty acid profiles of Pdp11 sole specimens, which achieved a higher growth performance. A fatty acid multivariate principal component analysis confirmed a neat separation of two groups corresponding to Control and probiotic fish for each age sampled (23, 56, 87 and 119 dah). In addition, a further SIMPER analysis highlighted that the Pdp11 Artemia effect on sole lipid profile was different for each fatty acid and was gradually diluted with age. Results suggest an ability of Pdp11 strain to produce n‐3 HUFA as an effective tool for fish marine larviculture optimization.  相似文献   

17.
The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD50 of 1.24 × 105 and 1.88 × 107 cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.  相似文献   

18.
A high bioflocculant‐producing bacterial strain BX‐13 isolated from an intensive fish pond by multiple subcultures and measured through primary screening and rescreening using kaolin. The strain BX‐13 was identified to belong to the same branch as Bacillus subtilis by morphological, physiological and biochemical, 16S rDNA gene sequencing, Blast homology search and multiple sequence alignments. The bioflocculation potential of BX‐13 and Rhodococcus erythropolis was compared, using water from the intensive pond where BX‐13 was isolated. The results showed the number of colonies in the BX‐13 treatment was higher than that of the R. erythropolis treatment from day 10 to the end of the experiment (p < 0.05). The numbers of both BX‐13 and R. erythropolis in the water body peaked on day 15, with 2.23 × 107 cfu/L and 9.90 × 106 cfu/L respectively. The bioflocculation volume (BFV) in water increased and then stabilized in both BX‐13 and R. erythropolis treatments. The BFV in BX‐13 peaked on day 25 (52.13 ml/L) and R. erythropolis peaked on day 30 (22.63 ml/L). At the end of the experiment, the BFV of the BX‐13 treatment was higher than the R. erythropolis treatment (p < 0.05). In conclusion, B. subtilis BX‐13 has a very strong flocculation effect, and merits further research into its application for water quality regulation in aquaculture systems.  相似文献   

19.
To understand the nutrient utilization efficiency and distribution in indoor fish culture system under treatment of probiotics, this study examined water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with four strains of lactic acid bacteria (LAB) isolated from fishery products. The results showed that the concentration of ammonia nitrogen (NH?‐N) and nitrite nitrogen (NO?‐N) in LAB treatments were all significantly lower (p < 0.05) than in the control group. The nitrate nitrogen (NO?‐N) in LAB treatments showed no significant differences (p > 0.05) compared with control group. The fish feed and water exchange accounted for 79.07% and 17.02% of total N input respectively. For N output, the drainage and residual diets in LAB treatment accounted for 24.50%–25.80% and 1.33%–1.60% respectively, and they were significantly lower than in the control group (27.60% and 2.20% respectively). Fish growth and lost N in LAB treatments accounted for 27.10%–30.50% and 11.00%–18.50% respectively, and they were both significantly higher (p < 0.05) than in the control group (22.30% and 5.30% respectively). The results indicated that the indigenous LAB strains were capable of improving fish growth, and reducing NH?‐N and NO?‐N level (at concentration of 105 cfu/ml) by directly adding in S. maximus culture water. Moreover, specific strains of LAB may increase nitrogen loss by promoting denitrification process in culture system.  相似文献   

20.
Foodborne disease outbreaks caused by raw olive flounders (Paralichthys olivaceus) parasitized with Kudoa septempunctata have been reported in Japan. Origins of olive flounders consumed in Japan vary, being either domestic or imported, and aquaculture‐raised or natural. Although it is unknown whether different sources are associated with different outcomes, it is desirable to identify whether this is the case by determining whether unique K. septempunctata strains occur and if so, whether some are associated with foodborne illness. We here developed an intraspecific genotyping method, using the sequence variation of mitochondrial genes. We collected olive flounder samples from foodborne disease outbreaks, domestic fish farms or quarantine offices and investigated whether K. septempunctata genotype is associated with pathogenicity or geographic origin. The 104 samples were classified into three genotypes, ST1, ST2 and ST3. Frequency of symptomatic cases differed by genotypes, but the association was not statistically significant. Whereas K. septempunctata detected from aquaculture‐raised and natural fish from Japan were either ST1 or ST2, those from fish inspected at quarantine from Korea to Japan were ST3. Our method can be applied to phylogeographic analysis of K. septempunctata and contribute to containing the foodborne disease. The genotype database is hosted in the PubMLST website ( http://pubmlst.org/kseptempunctata/ ).  相似文献   

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