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1.
Two extracellular metalloproteases were purified from a culture filtrate derived from Aeromonas salmonicida ssp. salmonicida . One enzyme, leucine aminopeptidase (LAP), which had a molecular mass 37 kDa, hydrolysed aminoterminal l -leucine and l -phenylalanine. The activity was inhibited by 1,10-o-phenanthroline, but not by EDTA. The addition of excess Zn2+ to an o-phenanthroline-inhibited enzyme restored most of its activity. The peptidase was temperature stable, and had an optimum temperature and pH of 60 °C and 8, respectively. The other enzyme, metalloprotease 3 (MP3), which had a molecular mass 20 kDa, was an endoprotease, and hydrolysed azocoll and hide powder-azure, but not gelatine. The MP3 enzyme had an optimum temperature and pH of ≈40 °C and 7.5, respectively, and a cationic isoelectrical point.  相似文献   

2.
Abstract. Extracellular hacmolytic activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the membrane-active enzyme glyccrophospholipidrcholcstcrol acyltransferase (GCAT). About 10% of the total haemolytic activity was due to a high molecular mass complex of LPS and GCAT (mol. mass >1000kDa), containing 35–50% neutral sugars and 1.5–2.0% protein. Some haemolytic activity (30–40% of total), corresponding to 50–70kDa by gel filtration, also contained GCAT-activity and may represent aggregated forms of GCAT. However, about 50% or more of the haemolytie activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the hacmolytic activity of both GCAT and GCAT-LPS. A transposon-produccd serinc protease negative mutant of the same A. salmonicida strain showed reduced haemolytic activity. The mutant produced a 38-kDa GCAT proform of low hacmolytic activity. The proform was processed by autogenous scrinc protease to a highly hacmolytic 26-kDa molecule with pl 6.3, similar to GCAT of the parent strain. The weakly haemolytic GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.  相似文献   

3.
Abstract. A collection of 130 strains of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida isolated from diseased salmonids in Denmark, Norway, North America and Scotland has been characterized with regard to protein patterns, antibiotic resistance and exoprotease activity. Whole cell and outer membrane protein profiling could distinguish three different profiles in A. salmonicida. Eight outer membrane proteins were demonstrated (49, 40, 38, 37, 33, 31, 30 and 29 kDa). One protein profile was deficient in a 38 kDa outer membrane protein and instead contained an outer membrane protein of 37 kDa which was not detectable among the other protein profiles. Strains with the 37 kDa outer membrane protein showed multiple low-level antibiotic resistance towards cephalothin, penicillin, chloramp-henicol, tetracycline and quinolones. In addition, these strains were exoprotease deficient. Strains with the 37 kDa protein were unable to degrade cattle and trout serum proteins and displayed a delayed degradation of casein. Haemolysis on cattle blood agar plates was similarly delayed. In vivo examination of extracellular products from a normal protein profile strain and one with the 37 kDa outer membrane protein demonstrated major differences in pathological effects in rainbow trout. The strain possessing the 37 kDa outer membrane protein produced almost no pathological effects while the normal protein profile strain produced typical furuncles.  相似文献   

4.
Monoclonal antibodies (mabs) against native epitopes of Aeromonas salmonicida strain F216.1/83-secreted proteases were isolated by means of a Protease-Capture-Assay. Ten antibodies reacted with the 70-kDa serine protease as judged from the molecular mass and enzymatic behaviour of the recognized antigen. Eight other mabs bound gelatinolytic antigens which lacked caseinolytic properties and possessed some characteristics of a zinc-dependent metalloprotease. The molecular mass of these mab-defined, immunologically cross-reactive antigens were estimated to be predominantly 108, 90 and 57 kDa. By comparing the antigen recognition and epitope mapping profiles among anti-serine protease- and anti-metalloprotease-mabs, at least six and five different epitope specificities were demonstrated, respectively. Both panels of mabs were shown to recognize the two types of exoproteases in culture filtrates of strain MT004 and of several other typical A. salmonicida strains.  相似文献   

5.
Abstract. Outer membranes and lipopolysaccharides of the marine fish pathogens Vibrio salmonicida and Vibrio anguillarum were isolated. SDS-PAGE profiles of purified LPS preparations from V. salmonicida revealed a broad low molecular weight band, whereas V. anguillarum LPS profiles demonstrated both a low-molecular band and several weaker high-molecular weight bands. Hydrolysis of V, salmonicida and V. anguillarum LPS separating the polysaccharide chain from the lipid A part and subsequent gel-chromatography suggests a polysaccharide molecular weight of ca. 1000 ('rough type' LPS) and ca. 6000 ('smooth type' LPS), respectively. Western blot of V. salmonicida outer membrane preparations and purified lipopolysaccharides and subsequent immunostaining with mouse monoclonal antibodies was performed. Eleven out of 15 monoclonal antibodies made against V. salmonicida cells reacted with one broad antigen-band in the low molecular weight region of both outer membrane and LPS profiles, corresponding to the LPS region. The previously reported outer surface antigen, VS-P1 from V. salmonicida , was observed to carry LPS epitopes as revealed by binding of monoclonal antibodies to VS-P1 as well as purified LPS preparations. These results strongly suggest that the VS-P1 antigen is a complex of both protein and LPS molecules.  相似文献   

6.
Abstract. Atlantic salmon, Salmo salar L., responded to intraperitoneal injection of formalin killed Vibrio salmonicida or live infectious pancreatic necrosis virus ( ipnv ) by producing specific antibodies. The antibody titre varied significantly within the group tested. Western blot analysis demonstrated that high-titre antisera recognized two major bacterial antigens with molecular weights of 12–15 kD and 22–27 kD. In addition, a few narrow bands with higher molecular weights were observed. An antiserum raised against IPNV recognised two major antigens corresponding to the structural proteins of the virus. E lisa and Western blot analysis showed that the immune serum raised against Vibrio salmonicida reacted slightly with Vibrio anguillarum , whereas no reaction to Yersinia ruckeri or Aeromonas salmonicida was detected. Indirect elisa and an elisa competition assay revealed that the immune serum raised against the N1 serotype was specific for this serotype of ipnv . The results demonstrate that Atlantic salmon has a humoral immune system capable of producing antibodies which discriminate between related bacterial antigens and between different serotypes of a virus.  相似文献   

7.
Abstract. Specificities of polyclonal salmon antisera made against the fish pathogens Vibrio salmonicida and Vibrio anguillarum were studied. Using ELISA and Western blot techniques, antisera made against V. salmonicida or V. anguillarum serovar 1 demonstrated high responses against the homologous bacterium or its isolated LPS. In contrast, antisera obtained after immunization with V. anguillarum serovar 2 displayed low antibody titres against homologous antigens. Elcctrophoretic transfer of SDS-PAGE separated V. salmonicida LPS antigen to nitrocellulose strips and subsequent immunostaining with salmon antisera revealed a strong reaction exclusively in the low molecular weight region (<14kD). On the other hand, immunoblots of V. anguillarum LPS preparations using salmon immunesera raised against this species showed a heterogenous staining pattern ranging from high to medium LPS-size. In addition, most of the salmon antisera made against V. anguillarum serovar 2 also reacted with a low molecular weight LPS antigen band.  相似文献   

8.
Abstract. Eighty isolates of Aeromonas salmonicida , recovered from separate outbreaks of furunculosis in farmed and wild salmon in Scotland during 1988 and 1989, were examined for susceptibility to the β-lactam antibiotic amoxycillin. Susceptibility was determined in terms of minimum inhibitory concentration (MIC). All of the A. salmonicida subsp. salmonicida isolates investigated were susceptible to amoxycillin, with MICs of 0.30–1.50mg1-1. All of the A. salmonicida subsp. achromogenes isolates tested were resistant to amoxycillin, with MICs in excess of 500mgl-1. The A. salmonicida subsp. achromogenes produced a β-lactamase enzyme with a pI of approximately 8.0. The enzyme was inducible and its production was unaffected by plasmid curing with ethidium bromide, suggesting that resistance was chromosomal rather than plasmid mediated.  相似文献   

9.
Abstract. Cultures of Aeromonas salmonicida , subspecies salmonicida and achromogenes , produced considerable quantities of inhibitors that affected extracellular bacterial proteases (endopeplidases), as well as some animal trypsins (from pig, salmon and trout) included in this study. Electrophoretic separation of these inhibitors revealed a complex of three to four single factors which were similar for the two subspecies. One of the factors only inhibited protease from the homologus subspecies, while another only affected enzyme from the other subspecies (cross-wise inhibition). Both subspecies produced a factor which inhibited animal trypsins only. Subspecies salmonicida also possessed a factor which inhibited most of the examined proteases unspecifically. In young cultures (2–3 days at 15°C), the inhibitors were demonstrable both in the disintegrated cell material and in cell-free culture filtrate. The activity of the factor which only inhibited the protease from subspecies salmonicida could be increased considerably by moderate heating of the material. The effect of inhibitors produced by other relevant Gram-negative bacteria on the proteases of the A. salmonicida subspecies included was more limited. Considerable quantities of inhibitors against proteases of the subspecies salmonicida and achromogenes were demonstrated in cell-free filtrates of two commercially available vaccines against furunculosis in fish.  相似文献   

10.
Resistance of Aeromonas salmonicida to amoxicillin   总被引：3，自引：0，他引：3  
Abstract. Isolates of Aeromonas salmonicida were obtained from farmed Atlantic salmon, salmo salar L., with amoxicillin minimum inhibitory concentrations (MICs) of 2·5–10 μg ml-1 Several antibiotic sensitivity patterns were found among the isolates including resistance to the four UK-licensed antibacterial drugs. Combination of clavulanic acid with amoxicillin was not effective, but all isolates were sensitive to florfenicol and to a cephalosporin, the latter having very low MICs.  相似文献   

11.
Abstract. The transformation of Aeromonas salmonicida with DNA fragments from bacterial cell-free sonicates was investigated with intraspecific, interspecific band intergeneric fish pathogenic bacteria including Aeromonas salmonicida, Aeromonas hydrophila, Pseiidomonas fluorescens and Vibrio anguillarum strains as donor bacteria. A phenotypic marker for transformation was extracellular protease production since a protease-deficient mutant NTG-1 induced from pathogenic A. salmonicida strain A-7301 by mutagenesis was used as a recipient. This mutant was non-pathogenic to rainbow trout. The mutant was incubated with each sonicate at 20°C for 20 days with a nutrient-poor medium containing a trace (5 μg/ml each) of both humic acid and tryptone in the presence of clean river sand (100 g/100 ml medium) corresponding with an environment of rivers. During the incubation, the survival of mutant NTG-1 cells was observed and protease positive NTG-1 cells were isolated from each culture. The protease production of the isolates was due to the transmission of protease genes of the donor strains. The activity of proteases produced by the transformants extra-cellularly was determined. These transformants induced with the sonicates of the parent strain, intraspecific strain and with the sonicates of the interspecific A. hydrophila strain were pathogenic to rainbow trout, whereas the transformants derived with the sonicates of the intergeneric strains P. fluorescens and V. anguiUarum showed non-pathogenicity, although all the donor strains, with the exception of the P. fluorescens strain, were pathogenic. These findings are interesting since they demonstrate that trausformation in A. salmonicida occurs with considerable ease even intergenencally and interspecifically, as well as intraspecifically in river environments, and that there is a large difference in the lethal toxicity of extracellular protease produced by these bacteria.  相似文献   

12.
Abstract. Aeromonas salmonicida was studied at fish farms producing salmonid smolts in northern and central Finland from 1982 onwards. Aeromonas salmonicida subsp. salmonicida has been causing epizootics in salmon, Salmo salar L., and trout, S. trutta L., at two coastal farms in northern Finland sinee 1986, involving 1–29% mortality in the fish-rearing units affceted. The disease causes more serious losses of sea trout yearlings and brood fish than of salmon. The achromogenic atypical A. salmonicida proved to be the most common bacterial disease in brown and sea trout at one farm in northern Finland throughout the period, causing constant heavy losses, mainly of fingerlings, especially in 1982–1986. It was found only occasionally in central Finland. Pigment-producing atypical A- salmonicida caused mortality among brown trout at one farm in northern Finland and one in central Finland.  相似文献   

13.
Abstract. The infectivity of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida to Atlantic salmon, Salmo salar L., in sea water was investigated and found to be similar to that reported for fresh water. The minimal infective dose in short duration bath exposures (1–3 days) was 104 colony-forming units (cfu) per ml, while prolonged exposure for three weeks, but not for 1 week, produced infection with 102 cfu/ml. Intragastric intubation of A. salmonicida established infection with doses of >105 cfu. Release of bacteria from dead or morbid infected fish was monitored and found to be in the order of 105–108 cfu/fish/h. These results emphasize the importance of removing dead fish from farm sites.  相似文献   

14.
Proteases secreted by strains of Aeromonas salmonicida   总被引：2，自引：0，他引：2  
Abstract. The proteases secreted by four strains (MT004, 1102, 480 and 480P-) of Aeromonas salmonicida grown in liquid culture have been studied. Strains MT004, 1102 and 480 all show a similar pattern with two types of proteases produced; one of molecular weight 70 000 which is active against casein and gelatin and one (or more) of lower molecular weight (about 20 000) which is (are) active against gelatin but not casein. Strain 480P- produces only the latter type of protease(s). The protease of molecular weight 70 000 is classified as a serine-type protease, but further characterization of the features of its active site has not yet proved possible. The results are discussed in terms of the previously published but often contradictory data on the proteases of A. salmonicida.  相似文献   

15.
Plasma samples obtained from rainbow trout either experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine (Lipogen) were analysed by enzyme immunoassay to evaluate changes in rainbow trout ladderlectin (RTLL) concentrations during the acute phase response (APR). Plasma RTLL concentrations in fish injected with A. salmonicida LPS, vaccine or live A. salmonicida varied over a 10 day period, but did not significantly increase. In contrast, fish experimentally infected with A. salmonicida exhibited a modest, but statistically significant ( P  <   0.05), decrease in RTLL concentration. These studies demonstrate that RTLL is not detectably induced during the trout APR to sterile inflammation or A. salmonicida infection, but plasma concentration of this protein may be reduced during bacterial infection.  相似文献   

16.
Abstract. Strains of Aeromonas salmonicida subsp. salmonicida , the agent of furunculosis disease of salmonid fish, have fairly uniform plasmid patterns. Of 35 strains examined by agarose gel electrophoresis, 28 had a pattern consisting of four small plasmids (4.2, 3.6, 3.5, 3.3 Mda) and a larger plasmid. The larger plasmid was most often 50–56 Mda, but it was larger in some strains. In the remaining seven strains, the same general profile was seen, but one of the small plasmids was missing. An additional plasmid was present in six strains. The pattern seen in 30 strains collected from Ontario fish over an 8-year period did not differ significantly from five reference isolates from other locations. Plasmid profiles of A. salmonicida strains appear too uniform to provide a useful epidemiological tool. The non-pigmented. atypical strains of A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida, A. media , and brown-pigmented strains of A. hydrophila had different plasmid DNA profiles, which were distinct from those of typical isolates of A. salmonicida subsp. salmonicida . Antibiotic susceptibility patterns, determined by the agar dilution method, were uniform for most typical strains. A non-transferable resistance to tetracyclmes was found in two Ontario isolates, but antibiotic resistance was relatively uncommon among the Ontario isolates.  相似文献   

17.
Abstract. The specificity of the major protease secreted by Aeromonas salmonicida has been explored using a number of proteins and p-nitroanilides as substrates. The 70kDa protease was found to hydrolyse two p-nitroanilides which have been reported to be specific substrates for thrombin. Kinetic parameters (kcat, and Km) were compared for the 70kDa protease and for thrombin as were the effects of a number of inhibitors. The 70kDa protease is able to degrade proteins which have a relatively open structure, for example, caseins or denatured bovine scrum albumin, to small fragments mostly of Mr<2500. However, proteins with a more compact structure are more resistant to the protease. It was concluded that the 70kDa protease shows some of the specificity features of thrombin, although it is less discriminating in its choice of both low and high Mr substrates than thrombin. In preliminary experiments, the 70 kDa protease was found, like thrombin, to decrease the clotting time of rainbow trout blood. The possible physiological significance of these results is discussed.  相似文献   

18.
Abstract. The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sea water under a variety of conditions. Survival in different types of microcosms (glass or dialysis bags); of bacteria grown under both in vivo and in vitro (broth culture) conditions; and in sterile and non-sterile sea water were compared. In all cases, survival was found to be of short duration (<10 days) and did not conflict with the previously stated obligate nature of the pathogen. The spread of furunculosis may depend less on its ability to survive in the environment than on its rate of shedding from infected fish and prevailing hydrographic conditions. Survival was extended and growth occurred in sterile sea water to which nutrients (tryptone soya broth) had been added. However, sea water obtained from beneath a commercial salmon cage, which would have been expected to be nutrient rich, did not prolong the survival of the pathogen. In vivo infectivity studies provided no evidence for the existence of unculturable but infective forms of A. salmonicida subsp. salmonicida which, therefore, validates the use of colony-forming units as a measure of survival.  相似文献   

19.
Abstract The possible mechanism of inactivation of the toxicity of Aeromonas salmonicida extracellular products (ECP) by normal rainbow trout serum was investigated using juvenile rainbow trout. ECP was prepared from culture supernatant by an acetone precipitation method. The ECP was incubated with normal rainbow trout serum at 20°C for 2 h, and the interrelationship between ECP proteolytic activity and immune complex-initiating, haemolytic complement activity (CH50) of normal serum against antibody-sensitized goldfish red blood cells was evaluated. When normal serum was incubated with increasing concentrations of ECP, the CH50 activity of serum decreased. The CH50 activity was completely abolished in serum treated with undiluted ECP. ECP treated with serum was administered to trout intraperitoneally to determine mortality. All the fish receiving untreated ECP (0.05 ml = 0.5 mg protein) alone died within 24 h. When ECP was treated with serum at 1:1 to 4:1 (serum: ECP) in volume a similar high mortality was produced. These inocula possessed high protease activity and no or low CH50 activity. However, mortality decreased and finally no mortality was recorded as ECP was treated with large volumes of serum (9:1 to 19:1). These inocula had lower protease activity and considerably higher CH50 activity. Fish receiving ECP treated with heat-inactivated serum at 19:1 showed 100% mortality. A serum: ECP inoculum derived from fish which had been administered lipopolysaccharide from Salmonella enteritidis and which possessed a low CH50 activity also gave a high mortality when used at 19:1. These results suggest that rainbow trout complement is implicated in the inactivation of toxicity of A. salmonicida ECP.  相似文献   

20.
Four non-pigment-producing isolates and two pigment-producing isolates of Aeromonas salmonicida sp. salmonicida were isolated from the head-kidney of diseased farmed Atlantic salmon, Salmo salar L. The cultural, morphological and biochemical features of the isolates were compared with those of reference strains. Injection and cohabitation experiments were performed. The only difference between the non-pigment-producing isolates and the pigment producing reference strains of A. salmonicida ssp. salmonicida was the inability of the former to produce pigment. In the injection experiments, the investigated non-pigment-producing isolate produced a significantly higher mortality compared with the mortality caused by the reference strain, whereas no difference in mortality was detected in the cohabitation experiments.  相似文献   

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