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.     

Aeromonas salmonicida in Finland: pathological problems associated with atypical and typical strains

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.     

Effects of potassium aluminium sulphate (alum) used in an Aeromonas salmonicida bacterin on Atlantic salmon, Salmo salar L.

Abstract. Potassium aluminium sulphate (alum) was tested for use as an adjuvant in an Aeromonas salmonicida bacterin injected into Atlantic salmon, Salmo salar L. Potential side-effects of visceral adhesions, diminished growth and toxicity, as well as adjuvant effects, were examined. Both alum and the bacterin alone induced inflammation and adhesion formation when injected into the peritoneal cavity. When bacterin and alum were combined, these effects were compounded. Growth was not affected by any of the injected treatments. The acute toxicity of the bacterin was reduced from 33.7 to 0.3% mortality by the addition of alum. Alum appeared to enhance protection against challenge, but there were no statistically significant differences in protection between injecting bacterin alone or injecting bacterin with alum.     

杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种PCR检测方法的建立

杀鲑气单胞菌(Aeromonas salmonicida)是一种重要的鱼类致病菌,可以感染多种海淡水鱼类。杀鲑气单胞菌包括5个亚种,目前常用的生理生化特征和16S rDNA序列分析方法很难实现亚种的快速精确区分。为实现杀鲑气单胞菌亚种的快速鉴定和检测,针对我国常见的杀鲑气单胞菌杀鲑亚种(A. salmonicida subsp. salmonicida)和杀日本鲑亚种(A. salmonicida subsp. masoucida),本研究开发了其特异性的PCR检测方法。根据Gene Bank已公布的杀鲑气单胞菌基因组信息,选择杀鲑亚种phoB基因和杀日本鲑亚种LOC111476736基因作为目标基因,根据其序列设计特异性引物,进一步对PCR反应的退火温度、引物浓度、dNTPs浓度、Mg2+浓度和酶浓度5个方面进行了优化,并测试了该方法的特异性、敏感性和应用效果。结果显示,2对引物分别可以扩增出杀鲑气单胞菌杀鲑亚种522 bp的phoB特异性基因片段和杀日本鲑亚种515 bp的LOC111476736特异性基因片段。杀鲑亚种特异性引物最适退火温度为64 ℃,10 µmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/µL KOD酶的最适添加量分别为1.5、2、1.5和0.5 µL。杀日本鲑亚种特异性引物最适退火温度为64 ℃,10 µmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/µL KOD酶的最适添加量分别为0.75、1、1.5和0.5 µL。以鳗弧菌(Vibrio anguillarum)、美人鱼发光杆菌(Photobacterium damselae)、杀鱼爱德华氏菌(Edwardsiella piscicida)、杀鲑气单胞菌其他亚种等14种其他水产病原菌或常见环境菌为模板进行PCR检测,均无特异性条带。该方法对杀鲑气单胞菌杀鲑亚种的检测灵敏度为12.8 CFU/反应(菌体)或17.6 fg/反应(DNA),对杀鲑气单胞菌杀日本鲑亚种的检测灵敏度为23.8 CFU/反应(菌体)或27.2 fg/反应(DNA)。利用杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种分别对大菱鲆(Scophthalmus maximus)进行人工感染实验,感染后取病鱼组织进行PCR检测,结果显示,本方法可以从感染后的大菱鲆中分别检测到相应病原。综上所述,本研究建立了杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种的特异性PCR检     

Evaluation of an experimental Aeromonas salmonicida epidemic in chinook salmon, Oncorhynchus tshawytscha (Walbaum)

To determine the dynamics of the transmission of Aeromonas salmonicida ssp. salmonicida infection, chinook salmon, Oncorhynchus tshawytscha, were exposed to bacteria by cohabitation. The latent period (time between exposure and infectivity) was determined by exposing a group of chinook salmonid fingerlings to A. salmonicida by bath, then, at daily intervals, by holding five exposed (donor) fish with approximately 50 naive fish for 24 h. The latent period was 3 days post-infection and the time period between the initial exposure to bacteria and the beginning of bacterial shedding was 4.5 days for the same animals. The prevalence and intensity of infection in the donor fish, to which recipient fish were exposed, i.e. the level of exposure, was highly correlated with the development of disease in recipient (susceptible) chinook salmon (r2 = 0.57). An experiment was conducted to determine the daily progress of infection and development of a furunculosis epidemic among recipient fish by cohabiting a single exposed fish with 43 unexposed salmon. At daily intervals, all fish (in seven treatment tanks and one control tank daily) were sacrificed and tested for the presence of A. salmonicida in the kidney (n = 3520). Over 10 days, mean prevalence among recipient fish reached 75% and disease related mortality exceeded 50%. Bacterial concentrations in the water continued to increase over the duration of the experiment in concert with the number of infected animals present in the population.     

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.     

Identification of cultured Aeromonas salmonicida by an indirect dot blot immunoassay

Abstract. A method is described which improves both the specificity and paracticability of immune identification of Aeromonas salmonicida. The modified assay employs antisera raised against outer membrane proteins (OMP) of A. salmonicida cells and is carried out as a dot blot test on nitrocellulose membranes. Performance of the test with 55 non- A. salmonicida bacterial isolates from fish and water revealed weak cross reactivity in five cases. However, these cross reactive only occur at very high antigen concentrations and can be overcome by adequate dilution.     

Prophylactic effect of β(1,3)-D-glucan (laminaran) against experimental Aeromonas salmonicida and Vibrio salmonicida infections

Immersion and intraperitoneal vaccination are the two most common and successful methods for vaccination of fish (Holm & Júrgensen 1987; Lillehaug 1989). Effective vaccines and the improvement of environmental conditions have dramatically reduced the amount of antibiotics used in aquaculture in Norway from 48.5 t in 1987 to 1.03 t in 1996. Nevertheless, the labour intensive process of vaccination and the often undesirable side-effects of the adjuvants incorporated in injectable vaccines (Anderson 1992; Mulvey, Landolt & Busch 1995; Midtlyng, Reitan, Speilberg 1996), have resulted in a search for alternative approaches to achieve immune stimulation. Hence. there is growing interest in the development of immunomodulators that do not cause or have only minor unwanted effects on the host and preferably can be given as a feed additive. It is widely acknowledged that the beneficial (or in some instances undesirable) effects of immunomodulators such as b-glucans and bacterial products in an infection are mainly a result of their stimulatory effects on macrophages (Johnston 1978; Chung & Secombes 1988; Júrgensen, Lunde & Robertsen 1993).     

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.     

Aeromonas salmonicida: development of resistance to 4-quinolone antimicrobials

Abstract. This paper examines aspects of the resistance of Aeromonas salmonicida isolates to the major fish farming antimicrobials. The antimicrobial resistance of an extensive chronological series of isolates appears to reflect the changing use of different compounds in fish farming since 1969. Repetitive challenge by 50% MIC concentrations of 4-quinolone antimicrobials against selective isolation of viable colonies resulted in a 7·2 × increase in resistance in 12 generations, demonstrating the likely outcome of drug misuse.     

Concentration and detection of Aeromonas salmonicida from hatchery water

Abstract. A method using 1-MDS electropositive filters for the concentration of Aeromonas salmonicida from hatchery water was developed. The procedure consisted of passing hatchery water at ambient pH through the filters followed by the elution of the adsorbed bacteria in a small volume of 3% beef extract solution (pH 10.0). A 300-fold reduction in volume of hatchery water and an average recovery of 35% of the seeded bacteria was achieved.     

Immunogenicity of vaccines from a virulent and an a virulent strain of Aeromonas salmonicida

Abstract. Injected vaccines consisting of formalin-killed cells and extracellular antigens prepared from a virulent and an avirulent strain of Aeromonas salmonicida were tested for their efficacy in protecting juvenile coho salmon. Oncorhychus kisutch (Walbaum). Against experimental furunculosis following active and passive immunization. Sera used in the passive immunization experiments were raised in subadult coho salmon and in rabbits. Results indicated that the avirulent strain was inferior to the virulent strain in its immunogenicity for coho salmon. Thus, even though avirulent cells possessed at least one immunogen–an immunogen that was show in passive immunization experiments to be well recognized by rabbits–the immunogen was only inefficiently protection when vaccinated with avirulent cells. Furher, extracellular antigens of the avirulent strain were not protective for coho salmon even though they elicited the production of anti- A. salmonicida agglutinins. In contrast, the killed cells and extracellular antigens of the virulent strain were both immunogenic in coho salmon. By passively immunizing coho salmon with rabbit sera raised against the virulent and the virulent strain produced at least two immunogens, only one of which was shared in common with the avirulent strain. The extra immunogen possessed by the virulent strain is presumably responsible for its superior immunogenicity in coho salmon; the immunogen had the properties of a protein and is believed to be the A-protein.     

The protective effect of humic‐rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.)

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic‐rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic‐rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite‐derived humic‐rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic‐rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic‐rich water from the recirculating system, the synthetic humic acid or the Leonardite humic‐rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic‐rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.     

Toxicity and immunogenicity of Aeromonas salmonicida extracellular products to salmonids

Abstract. Aeromonas salmonicida extracellular products (ECP) were fractionated by gel filtration chromatography. Three protein fractions were found. The first and second fractions showed low and high haemolytic activity, respectively, whereas the third fraction showed protcolytic activity. By intramuscular injection into juvenile rainbow trout, the median lethal doses of the first, second and third fractions were calculated at >669, 152 and <1514μg/g body weight, respectively. Cytopathic effects of the fractionated ECP to coho salmon lymphocytes were observed in vitro. The first and second fractions caused cell deformation, nuclear granulation and cytoplasmic streaming after 3h. No cytologic effects with the third fraction were observed. A mixture of the first and third fractions, a mixture of the second and third fractions, and non-fractionated ECP caused nuclear granulation and cytoplasmic streaming after 3Qmin. Using immunodiffusion analysis, the first and second fractions formed a single precipitating line each against white-spotted char anti-ECP sera, but the third fraction did not formed a precipitating line against the antisera.     

养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2 和Mg2 可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。     

Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge

Three groups of Atlantic salmon, Salmo salar L., were exposed to live, colony-forming, radiolabelled Aeromonas salmonicida bacteria in a bath challenge: (1) fish with artificial wounds; (2) fish with a reduced epidermal mucus layer caused by removal of the mucus layer on two occasions by a swabbing procedure; and (3) a control group of untreated fish. Fish were killed 2, 6 and 24 h after challenge, and radioactivity (cpm g^–1) was measured in the blood, mucus, skin, wound area, gills, anterior kidney, posterior kidney, spleen, midgut and hindgut. The highest levels of radioactivity were measured in the wound areas and in the gills. There was a significant positive correlation between the levels of radioactivity in the gills and blood, and between the mucus and skin at 2 h post-challenge. Two hours after the bath challenge, live A. salmonicida bacteria were found in the blood of fish in the 'swabbed' and 'artificial wound' groups, and not in the control group. Twenty-four hours after the bath challenge, the kidney of fish from all groups contained viable bacteria, whereas the blood was negative.     

患病细鳞鱼杀鲑气单胞菌的分离与鉴定

从患病细鳞鱼(Brachymystax lenok)的病变组织处分离到1株致病菌,经过分离培养,生化鉴定,16 S rRNA序列分析和人工感染实验确定该病原菌为杀鲑气单胞菌无色亚种(Aeromonas salmonicida subsp.achromogenes)。采用20种药物进行药敏分析,结果显示:分离菌株对阿米卡星、哌拉西林、恩诺沙星等9种抗生素敏感;对环丙沙星、诺氟沙星等4种抗生素中度敏感;对卡那霉素、青霉素、氨苄西林等7种抗生素耐药。     

The A-layer protein of Aeromonas salmonicida: further characterization and a new isolation procedure

Abstract. The predominant cell surface protein (A-protein) of Aeromonas salmonicida has been purified by a method utilizing a glycine/hydrochloridc extraction from whole cells and HPLC/ion exchanger (DEAE) columns. This procedure yielded two LPS-frec molecules (a 40- and a 50-kDa form) both shown to contain A-protein determinants. The former appears to be a digest product of the latter, as a serine protease produced by A. salmonicida was shown to process the 50-kDa form into a 40-kDa molecule in vitro. The A-layer protein was shown to contain one isoform, although multiple isoelectric forms appeared as preparative artifacts, probably due to deamidation. The A-layer protein and LPS arc the most significant surface antigens recognized by the Atlantic salmon B-lymphocytes or antibodies. Immunological studies of LPS-free and LPS-containing A-protein preparations were undertaken to test whether the two components behave like antigenie competitors or whether the LPS moiety could adjuvant the antibody response against the A-protein. The latter was shown to be the case.     

绿鳍马面鲀与许氏平鲉杀鲑气单胞菌病原的分离和鉴定

2018年和2019年,山东省烟台市蓬莱市一养殖场工厂化养殖的绿鳍马面鲀(Thamnaconus septentrionalis)和许氏平鲉(Sebastes schlegeli)发病死亡,主要症状为嘴部溃疡、红肿和出血。从发病鱼内脏中均可分离到大量形态一致的优势菌,分别命名为2018TS-1和2019SS-1,分离菌株经16S rRNA测序、生理生化鉴定和vapA基因分析确定为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida subsp. masoucida)。人工感染结果显示,2018TS-1和2019SS-1分别能引起绿鳍马面鲀和许氏平鲉的死亡,被感染鱼呈嘴部红肿症状,与自然发病症状一致,其半数致死量分别为1.78×105和0.89×105 CFU/尾。本研究首次报道了国内工厂化养殖绿鳍马面鲀和许氏平鲉感染杀鲑气单胞菌的病例,是目前人工养殖绿鳍马面鲀的首个疾病报道,也是继大西洋鲑(Salmo salar)、大菱鲆(Scophthalmus maximus)和裸盖鱼(Anoplopoma fimbria)等品种后,在山东省海水养殖鱼类中再次发现杀鲑气单胞菌杀日本鲑亚种的感染。本研究结果丰富了杀鲑气单胞菌杀日本鲑亚种的感染宿主范围,也为绿鳍马面鲀和许氏平鲉养殖的病害防控提供依据。