The acetylcholinesterase ichthyotoxin is a common component in the extracellular products of Vibrionaceae strains

In previous work, it was reported that a strain of Aeromonas hydrophila (B₃₂) produces the most potent lethal toxin with neurotoxic activity described so far for fish. In the present study, the presence and distribution of this acetylcholinesterase toxin lethal for fish were determined in extracellular products (ECP) of 42 Vibrionaceae strains using both immunological and colorimetric methods. This neurotoxin was shown to be present in the majority of the ECP from the Aeromonas and Vibrio strains tested and is responsible for the specific acetylcholinesterase activity. Also, although the Western blot and Ouchterlony techniques are valid as qualitative methods for the detection of this toxin, the Western blot procedure was 100-fold more sensitive than the Ouchterlony technique.     

Two immunologically distinct types of protofilaments can be identified in Natrialba magadii flagella

A rabbit antiserum to the 15-kDa acetylcholinesterase toxin neutralised the lethal effect of the 15-kDa toxin of Aeromonas hydrophila when injected into trout. However, immunisation of fish with the 15-kDa toxoid failed to induce an antibody response, and a higher molecular mass form of this toxin was purified from the extracellular products with the aim of inducing an immune response in fish. The optimal conditions for production of extracellular products by A. hydrophila strain B32 were studied to increase the concentration of this protoxin. The extracellular products were fractionated by molecular exclusion chromatography to yield a purified protoxin with an estimated molecular mass of 45 kDa by SDS-PAGE and which gave a positive reaction in Western blotting with the rabbit anti-15-kDa toxin serum. Since the 45-kDa protoxin showed lower specific acetylcholinesterase activity than the active 15-kDa toxin, the behaviour of the active site was studied using specific inhibitors. This 45-kDa protoxin was 13.3-fold less toxic than the 15-kDa toxin and induced antibody production in fish.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

点状气单胞菌引起虹鳟感染症及其病原学机制的初步研究

对山西省朔县省虹鳟试验场1987年8月在亲鱼中爆发的一次流行病进行了病原菌分离、培养、毒力试验和详细的生理生化测定,确定点状气单胞菌为其病原菌。并对该菌胞外产物的致病性进行了研究,通过测定其溶血活性、酪蛋白酶活性、对鱼致死性以及对其引起的组织病理学变化的观察,初步确定了胞外产物与该菌侵袭力的相关性。     

Presence of skin permeability factors in the extracellular products ofYersinia ruckeri

In this study we have demonstrated the presence of permeability factors (PF) in the extracellular products (ECP) of 16 strains of the bacterial fish pathogenYersinia ruckeri. The production and detection of these factors were dependent on the growth media and the ECP extraction procedure. The best results were obtained with a cellophane plate method and use of brain-heart infusion agar (BHIA) medium. The loss of necrotizing activity after heating the samples at 80°C for 15 min indicates the thermolabile nature of the toxin. In addition, this activity was not correlated with the production of hemolysins and cytotoxins by theY. ruckeri strains.     

Pathogenicity of atypical Aeromonas salmonicida in Atlantic salmon compared with protease production

The pathogenicity of extracellular products (ECPs) from 24 atypical Aeromonas salmonicida strains was studied with respect to : lethality in Atlantic salmon, pathogenic effect in muscle, haemolytic activity, cytotoxicity in two fish cell lines and proteolytic activities. Furthermore, the relationship between lethality of ECPs and mortality caused by bacterial challenge was examined. Correlation was demonstrated between the pathogenic properties and proteolytic activities of the ECPs. Cytolytic (GCAT) activity comparable with that of the typical reference strain used (NCMB 1102) was not detected in ECPs of any of the atypical strain tested. An extracellular metallo-caseinase, AsaP1, was linked with lethal toxicity and a strong pathogenic effect. Furuncular-like lesions were produced by ECPs containing AsaP1 activity. One strain produced a lethal toxin which was neither caseinolytic nor with GCAT comparable activity. The examined atypical strains form at least three distinct groups based on different virulence mechanisms and extracellular proteases.     

Pathological activities of Yersinia ruckeri, the Enteric Redmouth (ERM) bacterium

Abstract The adherence and invasive capacities as well as the pathobiological activities exhibited by Yersinia ruckeri were examined. Although adhesive ability was dependent on the cell-line employed, all the strains showed moderate adhesion and invasiveness in the salmon cell-line CHSE-214. With regard to the extracellular products (ECP) all of them were strongly toxic for fish with LD₅₀ ranging from 2 to 9.12 μg protein per g fish. In addition, all the ECP samples showed caseinase, gelatinase, amylase, lipase and phospholipase activities, hydrolysed esculin and displayed hemolytic activities for trout, salmon, sheep and human erythrocytes. Heat treatment (100°C for 10 min) caused the loss of all these biological activities except the hydrolysis of gelatin. On the other hand, SDS-PAGE analysis of the LPS and protein components of the ECP revealed variations among strains depending on the serotype. The lack of lethal effects of the LPS present in the ECP was also demonstrated.     

The quantitative relationship of lethality between extracellular protease and extracellular haemolysin of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.)

An additive relationship of lethality between purified protease and haemolysin of the extracellular products (ECP) of Aeromonas salmonicida was demonstrated by i.p. injection in Atlantic salmon (Salmo salar L.). The lethal toxicity of the combinations of protease and haemolysin follow a linear regression line y = -54.54x + 2400. The LD50 of protease and haemolysin when injected separately was 2400 ng/g fish and 44 ng protein/g fish, respectively.     

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme.

An extracellular lethal toxin produced by Aeromonas salmonicida was purified by fast-protein liquid ion-exchange chromatography. The toxin is composed of glycerophospholipid:cholesterol acyltransferase (GCAT) (molecular mass, 25 kilodaltons) aggregated with lipopolysaccharide (LPS), the GCAT/LPS complex having a molecular mass of about 2,000 kilodaltons, estimated by gel filtration chromatography. The toxin is lethal for Atlantic salmon (Salmo salar L.) at a concentration of 0.045 micrograms of protein per g of body weight. The toxin is a hemolysin (T-lysin, active on fish erythrocytes), leukocytolysin, and cytotoxin. Antiserum to the purified toxin neutralized the lethal toxicity of the crude extracellular toxins, indicating this toxin to be the major lethal factor produced by A. salmonicida. In the crude extracellular products, small amounts of free GCAT were also present. This has been purified, and its activities and properties have been compared with those of the GCAT/LPS complex. The presence of LPS did not influence the GCAT activity of the enzyme with egg yolk or phosphatidylcholine (lecithin) as a substrate, but the specific hemolytic activity and lethal toxicity was about eightfold higher in the complexed form. Furthermore, the free GCAT was more susceptible to proteolytic and heat inactivation than was the GCAT/LPS complex. Recombination of LPS (phenol extracted from extracellular products of A. salmonicida) with free GCAT enhanced the hemolytic activity, lethal toxicity, and heat stability of the latter but did not influence its lecithinase activity. In native polyacrylamide gel electrophoresis, the GCAT/LPS complex and the recombined GCAT-LPS both showed a high-molecular-mass band which did not enter the gel, while the free GCAT produced a single band with low molecular mass. In isoelectric focusing gels, the GCAT/LPS and recombined GCAT-LPS produced a nonfocusing smear with pIs from pI 5.0 to 5.8, while the free GCAT produced a single band with pI 4.3. These data show that free GCAT can combine with LPS to produce a high-molecular-mass complex with enhanced toxicity and heat stability compared with those of free GCAT, similar to the preexisting GCAT/LPS complex, and indicate that the LPS moiety of the toxin plays an active role in toxicity.     

Toxicity of the extracellular products ofVibrio damsela isolated from diseased fish

In this work we analyzed the pathogenic in vivo and in vitro activities for both fish and mammals of extracellular products (ECP) of several isolates ofVibrio damsela implicated in disease problems in marine culture. The ECP from all the strains were strongly lethal for fish (LD₅₀ ranging from 0.06 to 3.7 g protein/g fish) and mice (LD₅₀ ranging from 0.02 to 0.43 g protein/g mouse), causing death between 4 and 72 h after inoculation. These ECP samples possessed low proteolytic activity without production of caseinase, gelatinase, or elastase. However, most of them showed remarkable phospholipase and hemolytic activity for sheep, human, and turbot red blood cells. In addition, all the ECP samples were cytotoxic for fish and homoiothermic cell lines. The levels of enzymic and cytotoxic activities were clearly associated with the degree of virulence for fish. Moreover, the enzymic patterns of both live cells and ECP evaluated with the API-ZYM system were very similar among the strains, indicative that most of the activities are associated with exoenzymes.The in vivo and in vitro biological activities were considerably reduced after heat treatment (100°C for 10 min), but not totally lost in the highly virulent strains. Although we have demonstrated that the toxicity of the ECP is not directly associated with their lipopolysaccharides (LPS) content, these compounds could confer some heat-stabilizing effect to the toxic fractions.     

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.     

Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity.

In this work, the enzymatic activities of selected strains of biotypes 1 and 2 of Vabrio vulnificus were analyzed by using conventional methods and the API ZYM system. The toxic activities of extracellular products (ECPs) were further evaluated by in vitro and in vivo experiments. The ECPs of both biotypes (i) showed high-level hydrolytic activities, (ii) displayed cytotoxicity for fish cell lines, and (iii) were lethal for eels. Exotoxins seem to be proteinaceous since heat treatment of ECP samples destroyed their toxicity. Only biotype 2 strains were virulent for cels, suggesting that host specificity must be related to differences in cell surface properties. Infectivity trials with other fish species also revealed that only biotype 2 strains were virulent.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Purification and partial characterization of a cytotonic enterotoxin produced by Aeromonas hydrophila

This report describes the purification and partial characterization of a cytotonic enterotoxin produced by a human diarrheal isolate (SSU) of Aeromonas hydrophila. The extracellular enterotoxin was purified by (NH4)2SO4 precipitation, hydrophobic column chromatography, and chromatofocusing. The highly purified enterotoxin exhibited a molecular mass of 44 kDa and an isoelectric point in the range of 4.3 - 5.5 as determined by chromatofocusing. Western blot analysis using Aeromonas anti-enterotoxin revealed a single band at 44 kDa; however, cholera antitoxin failed to detect the enterotoxin antigen. This non-cholera toxin cross-reactive (non-CTC) enterotoxin was biologically active in vivo as determined by rabbit ligated ileal loop and rabbit skin vascular permeability assays. Biological activity also was in vitro by this toxin as measured by the elongation of Chinese hamster ovary (CHO) cells. The enterotoxic activity associated with this molecule was neutralized completely by homologous antibodies but not by cholera antitoxin. The purified toxin preparation was free of hemolytic and cytotoxic activities as determined by its inability to lyse rabbit red blood cells or damage CHO cells, respectively. Furthermore, this toxin induced the elevation of cAMP in CHO cells suggesting thereby that the mechanism of action of Aeromonas non-CTC enterotoxin may be similar to heat-labile enterotoxins of Escherichia coli and Vibrio cholerae.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.     

Toxicity of Photobacterium damselae subsp. damselae strains isolated from new cultured marine fish

The in vivo and in vitro toxicity of bacterial cells and their extracellular products (ECPs) from 16 strains of Photobacterium damselae subsp. damselae isolated from 7 epizootic outbreaks were evaluated. On the basis of their 50% lethal dose (LD50) values (about 1 x 10(50 CFU), these strains may be considered as moderately virulent. However, their ECPs were strongly lethal for redbanded seabream Pagrus auriga causing fish death within 2 h post-inoculation (protein concentration ranged between 2.1 and 6.41 microg g(-1) fish). The bacterial ECPs tested exhibited several enzymatic activities, such as amylase, lipase, phospholipase, alkaline phosphatase, esterase-lipase, acid phosphatase, and beta-glucosaminidase. These ECPs displayed a strong cytotoxic effect on 4 fish and 2 mammalian cell lines, although this activity disappeared when ECPs were heated at 100 degrees C. The virulence of the strains tested could not be related to the hemolytic activity or to the production of the toxin damselysin. Therefore, another unknown type of toxin could play an important role in the virulence mechanisms of this bacterial pathogen.