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.     

Genomic and phenotypic characterization of an atypical Aeromonas salmonicida strain isolated from a lumpfish and producing unusual granular structures

Aeromonas salmonicida strains are roughly classified into two categories, typical and atypical strains. The latter mainly regroup isolates that present unusual phenotypes or hosts, comparatively to the typical strains that belong to the salmonicida subspecies. This study focuses on an uncharacterized atypical strain, M18076‐11, isolated from lumpfish (Cyclopterus lumpus) and not part of the four recognized Aeromonas salmonicida subspecies. This isolate presents an unreported phenotype in the A. salmonicida species: the formation of large granular aggregates. Granules are formed of a heterogeneous mix of live and dead cells, with live cells composing the majority of the population. Even if no mechanism was determined to cause cellular aggregation, small globular structures at the cell surface were observed, which might affect granular formation. Pan‐genome phylogenetic analysis indicated that this strain groups alongside the masoucida subspecies. However, phenotypic tests showed that these strains have diverging phenotypes, suggesting that M18076‐11 might belong to a new subspecies. Also, a pAsal1‐like plasmid, which was only reported in strains of the subspecies salmonicida, was discovered in M18076‐11. This study sheds light on unsuspected diversity in A. salmonicida subspecies and stresses the need of thorough identification when a new strain is encountered, as unique traits might be discovered.     

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     

Use of a genetically attenuated strain of Aeromonas salmonicida to vaccinate salmonid fish

A genetically attenuated strain of Aeromonas salmonicida has been developed that has a complete deletion of the aroA gene (Brivax II), making it suitable for development as a commercial vaccine. Brivax II was effectively cleared from Atlantic salmon, Salmo salar, a species highly susceptible to furunculosis, confirming that it is attenuated. Clearance rate was dependent on the vaccine dose administered, being longer with higher doses. Immunological studies using Brivax II injected in rainbow trout, Oncorhynchus mykiss, confirmed that live vaccines stimulate a greater response, in terms of generating leucocytes able to proliferate to a subsequent encounter with antigen, relative to killed vaccines. Development of strains of Brivax II as carriers of heterologous antigens was also investigated. Escherichia coli -galactosidase was chosen as the model antigen, and three strains containing plasmids with the LacZ gene were constructed (Brivax 12, Brivax 61 and Brivax 107). All three strains were shown to express -galactosidase in vivo in rainbow trout and to be cleared effectively. Interestingly, Brivax 107 was cleared faster than the other two Lac⁺ strains and had the highest level of -galactosidase activity. The two strains expressing lower levels of activity also behaved differently in vivo, in that Brivax 12 accumulated derivatives expressing lower levels of -galactosidase activity, suggesting that mutants are being selected in vivo. The potential advantages of live vaccines over killed vaccines are discussed.     

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.     

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.     

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis)

In non‐salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold‐water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.     

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.     

The development of live vaccines for furunculosis lacking the A-layer and O-antigen of Aeromonas salmonicida

Abstract. Mutants of Aeromonas salmonicida strains lacking either the A-protein, O-antigen or both of these major surface antigens were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), for their suitability as live vaccines (LV). All of these mutants were shown to be attenuated, as fish receiving ∼5 × 10⁷ of the respective strains showed no clinical signs of furunculosis. Immersion vaccination of fish in 5 × 10⁷ cfu ml^-1 of these strains with an identical immersion dose 14 days later resulted in significant protection by all strains from challenge with a heterologous virulent strain of A. salmonicida 5 weeks later. The levels of protection conferred were all greater than or equal to that provided by an injected bacterin using the same vaccination schedule. With one exception, all LV strains that still possessed a functional O-antigen provided protective indices (PI) four- to seven-fold greater than the PI for the fish injected with bacterin. When antibody responses of vaccinated fish were compared, it was found that only vaccination by bacterin gave rise to a measurable agglutinating litre. Western immunoblots using the immune fish sera failed to reveal any major differences in antigen recognition in fish that received any of the vaccines tested. These data suggest that the immune response generated by the use of live vaccine strains is different from that generated by a bacterin, and that these useful mutations may be incorporated into existing furunculosis LVs for further attenuation.     

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 nucleotide sequence of the gene encoding GCAT from Aeromonas salmonicida ssp. salmonicida

The nucleotide sequence of the gene encoding GCAT (glycerophospholipid: cholesterol acetyltransferase) in Aeromonas salmonicida ssp. salmonicida has been determined. The sequence revealed an open reading frame of 1005 bp encoding 335 amino acids, an 18-amino-acid signal peptide and a 317-amino-acid mature polypeptide with a deduced molecular mass of 35 380 Da and an estimated pI of 6.25. Within the encoding region, the homology with the corresponding gene fromAeromonas hydrophila was 92.9% for the nucleotide sequence and 93.7% for the deduced amino acid sequence.     

Direct analysis of starved Aeromonas salmonicida

Survival of Aeromonas salmonicida was monitored during prolonged incubation in either distilled water or lake water. Culturability was determined from colony forming units enumerated on tryptone soy agar, whilst flow cytometry was used for direct analysis of viable cells after staining with fluorescent dyes which differentially stained bacteria in relation to defined cellular properties. Over time, populations of culturable cells steadily declined and were not detected after 10 days incubation in distilled water or 33 days in lake water. Flow cytometric analysis revealed that cellular properties related to viability were lost shortly after culturable cells became undetectable in distilled water. In contrast, those incubated in lake water showed little change in these properties over a 57-day experimental period. The implications of these differences are discussed, and it is concluded that A. salmonicida is capable of remaining intact and active upon prolonged incubation in lake water, although this does not conclusively prove viability.     

Resistance of Aeromonas salmonicida to amoxicillin

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.     

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.     

Serological comparison of selected isolates of Aeromonas salmonicida ssp. salmonicida

Abstract. Eight isolates of Acronionus salmonicida ssp. salmonicida were collected during furunculosis epizootics in North American Pacific coast states and provinces. Both virulent and avirulent forms of each isolate, confirmed by challenge and electron microscopy, were examined. Serological comparisons by cross-absorption agglutination tests revealed no serological differences between isolates. Using the double diffusion precipitin test, a single band was observed when antigen from a sonicated virulent strain was reacted with antiserum against a sonicated, virulent strain absorbed with homologous, avirulent strain. The presence of the single band was eliminated by excess sonication.     

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。     

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.     

Utilization of haem compounds by Aeromonas salmonicida

Abstract. The ability of A-layer-positi ve (A⁺) and A-layer-negative (A⁻) strains of Aeromonas salmonicida to utilize haem sources of iron under conditions of iron-restriction was evaluated. In a plate bioassay, only A⁺ strains of A. salmonicida were able to utilize haem from a variety of sources including haem, haemin, myoglobin, haemoglobin, haemoglobin- haptoglobin and haem-albumin complexes. Trypsin-digestion of whole cells abolished haem- binding, indicating that binding was cell-surface associated, involving a protein binding site or receptor. Competitive binding studies indicated that all haem compounds were bound by a common receptor, which was not iron-regulated and was associated with the presence of the 49-kDa A-layer protein. The ability of both typical A⁺ (siderophore-positive) and atypical A⁺ (siderophore-negative) strains to utilize haem indicated that the mechanism of haem utilization was not siderophore-mediated and that A. salmonicida possesses both siderophore-dependent and siderophore-independent mechanisms to overcome iron-restricted conditions encountered in vivo.