Bacterial inhibitors affecting proteases produced by Aeromonas salmonicida, and the occurrence of such inhibitors in furunculosis vaccines

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.     

Isolation and characterisation of two extracellular metalloproteases from Aeromonas salmonicida ssp. salmonicida

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.     

Effects of temperature on biochemical reactions and drug resistance of virulent and a virulent Aeromonas salmonicida

Abstract. Incubation temperatures of 11°, 18° and 28° did not substantially affect biochemical reactions of either virulent or avirulent forms of Aeromonas salmonicida subspecies salmonicida. The only change observed, amygdalin fermentation, was positive at 11° and 18° but negative at 28°C. Several isolates utilized sucrose, a characteristic not normally recognized for A. salmonicida subspecies salmonicida. Antimicrobial susceptibility screening indicated resistance to novobiocin increased at the higher incubation temperatures. Standardized drug sensitivity testing procedures and precise zone diameter interpretive standards for bacterial fish pathogens are needed.     

Transmission of protease genes into a protease-deficient mutant of Aeromonas salmonicida in river sediments

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.     

Cloning, characterization, and insertional inactivation of a major extracellular serine protease gene with elastolytic activity from Aeromonas hydrophila

The gene ahpA from Aeromonas hydrophila AG2 encoding an extracellular serine protease, named AhpA, was cloned in pUC18 plasmid. Nucleotide sequence analysis revealed an open reading frame of 1875 bp encoding a 625 amino-acid protein with a molecular weight of 67 567 Da. The gene ahpA was efficiently expressed in Escherichia coli C600 and in the non-proteolytic A. salmonicida masoucida , which was able to overproduce the 64-kDa protease found in the culture supernatant. The N-terminal amino acid sequence of the purified protein revealed a perfect match with the deduced DNA sequence starting at AAT (Asn-25), indicating that AhpA is synthesized as a pre-enzyme with a 24-amino-acid signal peptide and a 601-amino-acid mature extracellular protease. Purified protease had an optimum pH of 7.5 and its activity was strongly inhibited by PMSF, a serine protease inhibitor. The protease hydrolysed casein and elastin. The amino acid sequence of AhpA was highly homologous to A. salmonicida serine protease AspA. Inoculation of A. hydrophila ahpA mutant into trout suggests that the major AhpA secreted protease is not essential for virulence.     

Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida,by enzymatic and serological methods

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.     

Partial purification and characterization of extracellular metalloproteases from Aeromonas salmonicida ssp. salmonicida

Abstract. Aeromonas salmonicida ssp, salmonicida is shown to produce several extracellular proteins having gelatinolytic activity. Among the six isolates tested, two (NCMB 1102 and 84–14–R) produced both high (89–100 kDa) and low molecular (37 kDa) weight gelatinases, while the other four demonstrated only the 89–100 kDa forms. The low molecular form (metalloprotease 1: MP 1, 37 kDa) was isolated by ammonium sulphate precipitation, hydrophobic, ion exchange and size exclusion chromatography. The isolated enzyme was inhibited by the metal-chelating agents o-phenantroline and EDTA, and by excess Zn ions, and thus was defined as a metalloprotease. Its pH-optimum was 7–5, optimal activity was at 40°C and its pI 5.2. Specificity studies demonstrated cleavage of gelatin and azocoll, but not casein.     

Influences of Aeromonas salmonicida lipopolysaccharide, prednisolone and water temperature on plasma protein composition in salmonids

To further characterize the putative role of constitutive and inducible plasma proteins in innate resistance to furunculosis, the present authors compared the alterations in profiles of plasma proteins in resistant and susceptible salmonids, i.e. rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), respectively. Rainbow trout were injected with prednisolone acetate and exposed to higher water temperature (18 °C versus 10 °C), or injected with purified lipopolysaccharide (LPS) from a virulent strain of Aeromonas salmonicida , and plasma components were examined by two-dimensional polyacrylamide electrophoresis . Two days after A. salmonicida LPS exposure, rainbow trout had a four- to five-fold increase in concentrations of plasma proteins composed of p48, p19 and p16 subunits, and a significant decrease in a 100-kDa protein group. Consistent elevation or depletion of proteins corresponding to previously reported rainbow trout A. salmonicida LPS-binding pentraxins and lectins in plasma were not observed. Brook trout exposed to A. salmonicida LPS did not have any consistent plasma protein changes. There were no significant alterations in major plasma proteins following temperature shock and prednisolone acetate administration in rainbow trout plasma. These studies demonstrate that rainbow trout with LPS-induced sterile inflammation have few alterations in major plasma proteins or LPS-binding proteins, and do not exhibit the spectrum of acute phase changes induced by inflammation in mammals.     

An appraisal of the extracellular toxins of Aeromonas salmonicida ssp. salmonicida

Abstract. Aeromonas salmonicida produces many extracellular enzymes, some of which are known to play an important role in pathogenesis and virulence, while the role of others is presently speculative. The latter group includes amylase, aryl-sulphatase, glucosidases, esterases and lysophospholipase. There are two enzymes which are known to be of prime importance in pathogenesis: a 70-kDa protease (caseinase) and a 25-kDa phospholipase (glycerophospholipid: cholesterol acyltransferase, GCAT). The protease causes extensive tissue liquefaction, activates the blood clotting system and is lethal for fish at 2·4 μg/g fish. It is inhibited by α2-macroglobulin but resistant to all the other serum protease inhibitors. Its role in vivo appears to be as a broad spectrum protease providing amino acids for in vivo growth. The GCAT is mainly present in a high molecular weight complex with LPS. The complex is extremely haemolytic for fish (but not mammalian) erythrocytes. It is the most lethal component of the exotoxins (lethal dose 45 ng/g fish). The complex with LPS confers enhanced toxicity to the GCAT and stability to heat and proteolytic degradation. In vitro , this toxin also has high leucocytolytic and cytolytic (RTG-2) activity. On injection into fish, it causes very little histopathology other than a marked degranulation of eosinophilic granular cells (EGCs) in the gills. Its precise mode of pathogenesis is uncertain and appears complex. The protease and the GCAT/LPS have an additive relationship in respect to lethal doses and mixtures of the two produce extensive liquefactive and haemorrhagic lesions typical of furuncles. The possible relationship of the GCAT/LPS to other less well characterized factors (cytotoxin, leucocytolysin, haemolysin, salmolysin) is discussed.     

Vaccine-induced protective immunity against Aeromonas salmonicida tested in experimental carp erythrodermatitis

Abstract The development and applicability of a dose-controlled experimental infection with atypical Aeromonas salmonicida in carp is described. The proliferation and clinical manifestations of experimentally induced carp erythrodermatitis mimicked a natural infection. An in-vivo assay was used to evaluate the lethal properties of cell-free culture supernatants and a simple serum-free growth medium was devised for maintaining the virulence of the challenge strain. Depending on the inoculation dose, a sublethal (chronic) to a lethal (acute) infection could be induced, and a dose-response relation was observed between A. salmonicida inoculum size and carp mortality. The dose-controlled experimental infection was used as a challenge test for laboratory evaluation of the efficacy of potential vaccine candidates. The vaccine candidates tested, a cell envelope preparation, purified lipopolysaccharide and purified A-layer (ACE) protein showed no protection or only a feeble one at the best, while formalinized whole cells showed a consistent but only moderate protection. In contrast, when concentrated, detoxified culture supernatant was used, the carp were protected against a subsequent lethal challenge. These observations indicate that immunity against A. salmonicida extracellular products is of prime importance.     

Development and characterization of monoclonal antibodies specific for two different exoproteases of Aeromonas salmonicida

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.     

The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products

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.     

大黄鱼源气单胞菌的分离鉴定及致腐表型

为鉴别大黄鱼腐败菌气单胞菌的致腐能力,采用生理生化和分子方法(16S r RNA)鉴定冷藏大黄鱼货架期终点不产H2S的菌株。分析菌株在体外培养条件下的生长、胞外酶活性、三甲胺(TMA)和生物胺形成的腐败表型,将菌株接种在灭菌鱼汁中测定其致腐能力。结果显示,分离株AE03和AE04在0~37°C及0~60的盐度下生长良好,在4和25°C条件下AE03菌株的生长速率显著高于AE04菌株。2株细菌不产H2S,能还原氧化三甲胺(TMAO),分解尿素和液化明胶,氧化酶、赖氨酸、鸟氨酸脱羧酶、精氨酸水解酶等都呈阳性,能利用葡萄糖。结合16S r RNA鉴定发现分离株AE03为杀鲑气单胞菌,AE04为软体动物气单胞菌。在体外培养中,2种气单胞菌均能产生蛋白酶、形成TMA和生物被膜,其中AE03菌株生成各指标的能力高于AE04菌株,同时AE03菌株还具有脂酶活性,产生生物胺,其中尸胺含量较高。气单胞菌AE03在灭菌鱼汁中产生的挥发性盐基氮(TVB-N)显著高于AE04。通过紫色杆菌CV026和根癌农杆菌A136 2种报告菌检测发现,2株气单胞菌是高丝氨酸内酯(AHLs)产生菌,其中AE03菌株产生的AHLs含量显著高于AE04菌株。研究表明,杀鲑气单胞菌AE03为冷藏大黄鱼的强致腐菌,且能分泌高含量的AHLs,研究为从群体感应角度阐明气单胞菌的致腐机理奠定了良好的基础。     

Glycogen, the preferred energy source for extracellular protein formation and growth of Aeromonas salmonicida

Abstract. A study was made of the effect of supplementing a rich 3% (w/v) tryptone soya broth (TSB) medium and a poorer 1.7% (w/v) tryptone-based medium with glucose, maltose and glycogen, as carbon sources, on growth and exoprotein formation by Aeromonas salmonicida. In TSB, glucose inhibited growth and repressed exoprotein formation whilst maltose and glycogen had little effect, up to 20h, when compared with an unsupplemented control. By contrast, in the poorer medium, over a 24-h incubation period, growth was stimulated three-fold by glycogen, and whilst exoprotein formation was low in comparison with that observed in TSB, the greatest production was observed in the presence of glycogen. Extracellular α-amylase was measured in the tryptone medium in the presence of the three carbon sources and the highest level, produced in the presence of glycogen, was 1.6 times that with added maltose whilst none was detectable with glucose present. This pattern was repeated in the case of the maltose-inducible porin, LamB, of the outer membrane.     

The in vitro susceptibility of Aeromonas salmonicida and other fish-pathogenic bacteria to 29 antimicrobial agents

Abstract. The minimum inhibitory concentrations (MICs) of 29 antimicrobial agents were determined for some representative Aeromonas salmonidda, Yersinia ruckeri and Vibrio isolates. Eleven of them, including three widely used in clinical practice, were further tested against 28–36 recent field isolates of A. salmonidda. The fluoroquinolones were inhibitory at very low concentrations with 50% of A. salmonidda isolates having a MIC of enro-floxacin ≤0·02μg ml-1. The distribution of MICs to the fluoroquinolones and to oxolinic acid were similar indicating possible cross resistance. Amoxycillin, florfenicol, minocycline, rifampicin and the potentiated sulphonamides had a single peak MIC distribution with values of ≤1·25μg ml-1. Minimum inhibitory concentrations of the fluoroquinolones were four or more times greater at 10°C compared with 22°C; this was not so with amoxycillin, florfenicol or the sulphachlorpyridazine-trimethoprim (S-TP) combination. A. salmonicida was rapidly killed at 22°C by the fluoroquinolones and S-TP whereas oxolinic acid and amoxycillin killed within 24h. Florfenicol was bacteriostatic. The therapeutic potential of some of these antibiotics is considered.     

Aetiology of an ulcerative disease in goldfish, Carassius auratus (L.): experimental induction of the disease

Abstract. Infection experiments were conducted to determine the primary aetiology of an ulcerative disease in goldish, Carassius auratus (L.). Goldfish were exposed to atypical Aeromonas salmonicida and A. hydrophila , previousl y isolated from cutaneous ulcers, and to 04 5 μm filtrates of cutaneous ulcers and kidneys from diseased fish. Fish were exposed to each preparation by intraperitoneal, intramuscular or subcutaneous injection and by a method in which a small patch of scales was removed from each side of the fish and the inoculums applied. Most of the fish injected with A. salmonicida died without developing coetaneous ulcers; however, ulcers were induced in five of the ten fish exposed by the scale removal technique. Exposure to ultra filtrates or A. hydrophila did not result in consistent ulcer formation o r death. Additional experiments showed that a 30-min exposure of goldfish, without prior treatment, to water containing 3 × 10⁵ colony forming units (cfu/ml) of A. salnumicida was sufficient to produce cutaneous lesions in nine often fish exposed. Multiple lesions were produced in most fish and A. salmonicida was consistently recovered. Fish exposed by similar waterborne challenge s to 6·2 × 10⁶ cfu/ml of A. hydrophila or to 7·2 × 10⁶ cfu/ml of another lesion isolate identified as a member of the A. hydrophila complex produced no lesions, eve n when scales were removed. The studies demonstrate that atypical A. salmonicida can initiate cutaneous lesion s characteristic of ulcerative disease, while A. hydrophila and an A. hydrophila complex organism cannot.     

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita , and the brain of catla, Catla catla , respectively. The cell lines were maintained in Leibovitz's L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 °C with an optimum of 28 °C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 °C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp . were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.     

A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Abstract. A natural bath challenge method has been developed for furunculosis using Atlantic salmon, Salmo salar L., and rainbow trout, Salmo gairdneri Richardson. Fish were placed in an enclosed, continuously circulating tank system, supplemented with additional oxygen, the temperature raised gradually (overnight) to 15–16°C, a low dose of Aeromonas salmonicida (strain 184/186) introduced into the tank and the challenge maintained for 14 days. The challenge strain was characterized with respect to possible virulence factors and possessed an A-layer, ability to auto-agglutinate, haemagglutinate, adhere to Atlantic salmon cells and resist destruction by serum. No caseinase activity was detected. LD₅₀ for salmon using this method was 1.8×10³ cells per millilitre while trout had an LD₅₀ of 9.5×10⁴ cells per millilitre. Onset of the disease appeared to depend on fish size with larger trout (50 g) taking up to 10 days to show signs of the disease while mortalities in smaller trout (8.5g) commenced on day 1 post-challenge.     

Infectious load of Aeromonas salmonicida subsp. salmonicida during the initial phase of a cohabitant infection experiment with Atlantic salmon, Salmo salar L.

Abstract The abundances of Aeromonas salmonicida subsp. salmonicida in the water and in the surface microlayer was studied during the initial phase of a cohabitant infection experiment with Atlantic salmon, Salmo salar L., smolt. Aeromonas salmonicida was detected in the water samples only until the intraperitoneally infected smolt were dead and had been removed. In the lipid rich surface microlayer, A. salmonicida was detected in high concentrations from the day of the first fish mortality and throughout the rest of the experiment. The significance of the high cell surface hydrophobicity is discussed as a possible reason for enrichment of A. salmonicida at the air-water interface.     

The survival of Aeromonas salmonicida subsp. salmonicida in sea water

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.