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1.
Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface–related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.  相似文献   

2.
The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.  相似文献   

3.
柱状黄杆菌(Flavobacterium columnare)是一种世界范围的水产动物致病菌,是中国重要养殖鱼类草鱼(Ctenopharyngodon idellus)、鳜(Siniperca chuatsi)等烂鳃病的病原。本研究以1972年从患"烂鳃病"草鱼上分离的两株冻干柱状黄杆菌G4和G18菌株为研究对象,并将G4株再次分离纯化得纯化菌株,命名为G4R3。对草鱼鱼苗浸泡攻毒结果显示,G4R3的LD50至少比G18的高3个数量级,因此G4R3为"强毒株",G18为"弱毒株"。利用蛋白质组学方法分析柱状黄杆菌强毒株G4R3和弱毒株G18的胞外蛋白,经过双向电泳并结合图像分析,共发现了34个点是差异表达的蛋白。胶内酶解、肽质量指纹图谱和串联质谱分析后,鉴定出其中的7个蛋白点,代表滑动蛋白K、腺酐甲硫氨酸合成酶和一种可能的膜蛋白等3种蛋白,它们可能是柱状黄杆菌的毒力因子。  相似文献   

4.
Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5–60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.  相似文献   

5.
Channel catfish, Ictalurus punctatus (Rafinesque), goldfish, Carassius auratus (L.), striped bass, Morone saxatilis (Walbaum), and Gulf sturgeon, Acipenser oxyrinchus desotoi Vladykov, were acclimatized to fresh water or salinities of 9.0‰ or less and then exposed to Flavobacterium columnare (formerly known as Flexibacter columnaris ), the bacterial pathogen that causes columnaris disease. None of the fish acclimatized to 3.0 or 9.0‰ salinity died, and all deaths in lower salinities occurred between 1 and 5 days after exposure to F. columnare . Mortality was 97.7% in fresh water and 67.1% in 1.0‰ salinity for channel catfish (model SE, 1.8) and 66.5% in fresh water and 40.8% in 1.0‰ salinity for goldfish (model SE, 1.2); and 96.9% in fresh water and 61.7% in 1.0‰ salinity for striped bass (model SE, 1.8). After exposure to F. columnare , none of the Gulf sturgeon died. Flavobacterium columnare was isolated from the skin and gills of all fish dying during the experiments, but was not isolated from survivors in fresh water and 1.0‰ salinity 21 days after bacterial exposure. In vitro growth of bacteria was significantly higher in 1.0 or 3.0‰ salinity than in control medium (0.3‰ salinity). However, in vitro adhesion of bacteria was reduced with increasing salinity, which could explain the lower mortality of fish at higher salinities.  相似文献   

6.
Specific antibody responses to Flavobacterium columnare (isolate ATCC 23463T) were characterized in plasma and mucus of tilapia following intraperitoneal (i.p.) injection or immersion immunization with formalin-killed sonicated or whole cell preparations. Fish (30 per treatment) received a primary immunization and were booster immunized 4 weeks later. An enzyme-linked immunosorbent assay was developed for detection and quantification of specific anti-F. columnare antibody, and it was found that formalin-killed sonicated cells in Freund's complete adjuvant (FCA) injected i.p. stimulated a significant systemic antibody response within 2 weeks (mean titre 11,200) which increased to 30,600 following secondary immunization. At 10 weeks post-immunization, the mean titre remained significantly elevated above the controls. Antibodies were also observed in cutaneous mucus of fish immunized i.p. with formalin-killed sonicated cells in FCA at 6 and 8 weeks post-immunization (mean titres 67 and 33, respectively). Although some individual fish responded, mean plasma and cutaneous mucus antibody titres were not significantly greater than controls in any of the other treatment groups. The results of this study demonstrate that tilapia can mount a significant humoral response in plasma and cutaneous mucus to F. columnare, but i.p. immunization with FCA is required to elicit this response.  相似文献   

7.
Abstract. Thrombosis of minute vessels is a common feature of acute furunculosis in Atlantic salmon. Crude extracellular products (F.CP) of Aeromans, salmonicida injected into the dorsal aorta of cannulated fish elicited an activation of the coagulations systems of the fish When screened with a number of chromogenic substrates, the ECP protease revealed a factor-X like activity. Thus, it may he hypothesized that the ECP induces thrombosis by entering the fish coagulation systems at factor X. and that thrombus formation is a way of creating a site of infection.  相似文献   

8.
Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare.  相似文献   

9.
A 10-week feeding trial was conducted to evaluate the growth and physiological condition of F1 amago salmon Oncorhynchus masou ishikawae juveniles obtained from broodstock that had been selected for growth on a low fish-meal (LFM) diet. F1 juveniles from broodstock showing superior or inferior growth on a LFM diet (LFM-S, LFM-I), and those from broodstock showing intermediate growth on a fish meal-based (FM) diet (FM-M), were fed a LFM diet and a FM diet. In fish fed the LFM diet, growth of LFM-S was significantly better than FM-M at 3 weeks; however, this superiority in growth disappeared at the end of the 10-week trial. Growth of LFM-I was significantly lower than LFM-S throughout the feeding trial. Growth of fish fed the FM diet was not significantly different among groups, and higher than fish fed the LFM diet. Improvements in the blood hemoglobin concentration and morphological conditions of the distal intestine and liver were observed in LFM-S fed the LFM diet. Although the effect of selective breeding was limited in F1 amago salmon, the present results suggest that continuous selection for growth with a LFM diet has potential as a method to adapt to the limited supply of fish meal.  相似文献   

10.
Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.  相似文献   

11.
Juvenile chum salmon Oncorhynchus keta originating from rivers along the Pacific coast of Japan migrate northwards to the Sea of Okhotsk, typically passing off Konbumori, near the easternmost part of Hokkaido Island. We used daily-increment analysis of otoliths to back-calculate the growth rates [mean daily growth rate in fork length (FL)] of 369 juveniles (56–146 mm FL) originating from various rivers southwest of Konbumori, and sampled at Konbumori between 2005 and 2014. We examined differences in growth rate in terms of differences in distance from the source of juveniles, their river or region of origin, to Konbumori, and FL at time of collection. The results show that juvenile chum salmon originating from distant sources tended to grow faster than those from more proximal sources, likely contributing to larger FLs in the former. Growth rates of larger fish (≥ 90 mm FL) differed little (medians: 0.64–0.68 mm/day) among regions of origin, whereas those of smaller fish tended to be low among fish originating from more proximal regions (20–126 km from Konbumori). These results suggest that fish migrating from more distant rivers were better able to survive and to reach Konbumori by achieving or exceeding a certain growth rate.  相似文献   

12.
Nonspecific responses of Japanese eels to environmental stress were monitored by assaying various lytic activities in eel epidermal extract. In fish maintained at 10 and 30 °C for up to 10 days, epidermal proteolytic activities due to serine protease and aminopeptidase and hemolytic activity varied within a 2-fold value range. Other proteolytic activities, due to cathepsins B and L, in the fish at 30 °C increased for up to 8 days and were 3.4 and 2.9-fold over those in fish maintained at 10 °C, respectively. This was accompanied by a 3.0-fold increase in bacteriolytic activity. Other forms of stress were exerted on the fishes by immersing them in a suspension of Flavobacterium columnare or giving them intraperitoneal injections of Edwardsiella tarda over 72 h. Although serine protease and aminopeptidase activities and hemolytic activity in the fishes exposed to F. columnare changed marginally, and were similar to those in the control fish, cathepsins B and L activities in the infected fishes increased more than 1.5-fold over their initial values over a 48 h period, along with a 4.5-fold increase in bacteriolytic activity. No marked change was detected in any of the lytic activities of the fishes exposed to E. tarda. These findings indicate that epidermal cathepsins B and L probably participate in bacteriolysis associated with Japanese eel skin and that their activities are elicited by environmental stimuli and may be an important nonspecific response of eels. Abbreviations: Cbz – carbobenzoxy; MCA – 4-methylcoumaryl-7-amide.  相似文献   

13.
A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L−1 of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L−1 significantly ( P  < 0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro , the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 μg mL−1 (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection.  相似文献   

14.
The Norwegian aquaculture of Atlantic salmon (Salmo salar L.) is hampered by ulcerative disorders associated with bacterial infections. Chronic ulceration may provide microenvironments that disturb the normal microbial biodiversity of external surfaces. Studying the composition of microbial communities in skin ulcers will enhance our understanding of ulcer aetiology. To achieve this, we tested marine farmed Atlantic salmon and sampled the base and edge of ulcers at the end of winter (April) and end of summer (September), in addition to skin mucus of healthy individuals. In order to assess microbiota associated with the host and obtain insight into the environmental ecology, we also sampled sea water, the sediment layer underneath the farm facility and the distal intestine of Atlantic salmon. The skin microbiota of Atlantic salmon was different from that of the surrounding water. Residential Tenacibaculum and Arcobacter species persistently dominated the cutaneous skin and ulcer mucus surfaces of Atlantic salmon during both winter and summer periods. The intestinal microbiota was dominated by Mycoplasma with an increase in Aliivibrio and Alcaligenes abundance in the intestine of fish with ulcerative disorder at the end of winter. These findings suggest the presence of resilient microbes in the mucus surfaces of Atlantic salmon.  相似文献   

15.
Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 106 CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.  相似文献   

16.
Cutaneous mucus is the first physical and chemical barrier of fish. This slime layer is secreted by mucous cells located in the epidermis and is mainly composed of glycoproteins that have their origin in the diet. Therefore, food deprivation can potentially change the abundance and glucidic nature of skin mucous cells, thus changing the mucus properties. To test this hypothesis, we conducted an experiment with Atlantic salmon, Salmo salar L. Changes in the number and glucidic nature of epidermal mucus cells were analysed using standard techniques. The outcome of this study shows that food deprivation caused a rapid decrease in the density of epidermal mucous cells in Atlantic salmon. Lectin histochemistry revealed a change in the presence and stainability of some sugar residues in the mucous cells of unfed fish compared with fed fish. Given that the primary reason for mucus secretion in fish is for protection against infections, we speculate that the changes in the mucus properties caused by nutritional stress may affect their disease resistance. This fact is particularly important for fish that spend a period of time deprived of food, either as a part of their natural life cycle, or as part of farming practices.  相似文献   

17.
为了解苏丹鱼MR(Lh MR)的结构特点及其在抗感染免疫反应中的作用,本研究克隆并分析了LhMR基因,采用实时荧光定量PCR(qRT-PCR)技术检测了LhMR在正常及柱状黄杆菌感染苏丹鱼组织中的表达。结果显示,LhMR开放阅读框4296 bp,编码1431个氨基酸(aa)。LhMR的氨基酸序列和分子结构与其他物种高度相似,胞外1个富含蓖麻类β型三叶草的结构域(RICIN)、1个纤连蛋白Ⅱ型结构域(FNⅡ)、8个串连的C型凝集素样结构域(CLECTs)、1个跨膜区和1个胞内区。通过qRT-PCR检测显示,LhMR在脾脏、体肾、心脏、脑、皮肤、肌肉、鳃、肝脏、后肠、前肠和中肠11种组织中均有表达,其中脾脏中表达量最高;经柱状黄杆菌感染苏丹鱼后,脾脏、肠、体肾和鳃中LhMR基因的相对表达量显著升高。通过免疫组织化学(IHC)检测发现,在脾脏、肾脏和鳃中的LhMR蛋白表达水平提高。通过苏木精-伊红染色病理组织切片表明,在体肾、肠、肝脏和鳃中均有不同程度的病变。体肾的肾小管有大量的血细胞浸润,上皮细胞发生坏死;肠壁变薄,组织大量弥散坏死;肝脏组织中有多个空泡;鳃小片肿胀、混乱、坏死和脱落。研究表明,LhMR在苏丹鱼感染柱状黄杆菌免疫反应中发挥重要作用,为苏丹鱼柱形病的防控提供新的参考。  相似文献   

18.
Skin secretions of fishes constitute a rich source of proteins with a broad spectrum of antimicrobial properties. We report here the characterization of proteases from skin mucus of tambacu, an economically important Neotropical hybrid fish. The effects of pH on the proteolytic activities of the mucus acting on various substracts – hemoglobin, casein, bovine serum albumin (BSA) and ovalbumin (OVA) – were tested. Optimal pH values for protease activity on hemoglobin were 4.5 and 8.5, on casein, 8.5, on BSA, 5.0 and 7.5, and on ovalbumin, 4.5 and 6.5. The proteolytic activity was inhibited on all of these substrates in the presence of specific inhibitors: caseinolytic activity was inhibited by inhibitors of serine and metalloproteases; hemoglobinolytic activity was inhibited by serine, aspartic and metalloproteases inhibitors; albuminolytic activity on BSA was inhibited by serine and aspartic proteases inhibitors, and on ovalbumin, by cysteine and aspartic proteases inhibitors. Gelatin zymography revealed that the skin mucus of tambacu consisted primarily of serine and metalloproteases. Hemoglobin zymography showed one proteolytic band inhibited by EDTA, whereas casein zymography showed two proteases inhibited by serine proteases inhibitors. We were able to identify all classes of proteases in the mucus from the skin of tambacu. These, and these results suggest that the proteolytic activities of the skin mucus of fish may play an important role in the defense against microorganisms and ectoparasites.  相似文献   

19.
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20.
Variability in pathogenicity of Flavobacterium columnare makes disease treatment difficult because there is currently no way to easily recognize those strains that warrant aggressive treatments. In order to identify suitable virulence markers, 17 isolates of F. columnare were cultured from six different fish species. The DNA from all isolates was analysed using random amplified polymorphic DNA (RAPD). Bootstrap analysis of the RAPD data produced a tree with three major groups supported by bootstrap scores of 80-100%. Virulence of the isolates was determined by bath exposure of channel catfish, Ictaluruspunctatus (Rafinesque), and golden shiners, Notemigonus crysoleucas (Mitchill), to broth cultures of F. columnare. In channel catfish, 13 of 17 isolates produced 100% mortality within 48 h post-exposure. All isolates of cyprinid fish origin clustered in a single RAPD group. At least two of the four isolates that were not virulent in channel catfish were of cyprinid fish origin. There was a wide variation in cell morphology between isolates with lengths of cells or cell chains ranging from 3 to 11 microm, even under identical culture conditions. Most of the shorter or single cell isolates fell into a single RAPD group. No clear association was identified between virulence and any other characteristic, including RAPD group.  相似文献   

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