Dominant pathogenic species of mesophilic aeromonads isolated from diseased and healthy fish cultured in Poland

Aeromonas isolates were collected from cultured fish, characterized phenotypically and identified to species using 16S rDNA. The pathogenicity of all isolates was assayed on the basis of haemolytic and proteolytic activity and challenge tests were performed for isolates from healthy fish. A total of 131 Aeromonas isolates were obtained and identified as follows: A. hydrophila (13), A. bestiarum (23), A. salmonicida (motile biogroup) (19), A. caviae (2), A. sobria (18), A. veronii bt. sobria (42), A. jandaei (1), A. encheleia (11) and A. allosaccharophila (2). All isolates of A. hydrophila and A. bestiarum and most isolates of A. salmonicida and A. veronii were classified as pathogenic. Aeromonas hydrophila was isolated only from diseased trout except for one isolate obtained from carp fry. The other potentially pathogenic Aeromonas species were present in diseased as well as healthy fish. The pathogenicity of isolates from healthy fish was correlated with their enzymatic activity and was also tested by challenge experiments. The dominant pathogenic species were A. veronii bt. sobria, A. bestiarum and A. salmonicida in common carp and A. hydrophila in rainbow trout.     

Efficacy of specific antibody against agglutinating Aeromonas salmonicida strains on infectivity and vaccination with inactivated protease

Efficacy of specific antibody on serum resistance and adhesion was investigated using a pathogenic strain of Aeromonas salmonicida A-7301 (which was autoagglutinative, haemagglutinative and protease production positive), a protease-deficient, non-pathogenic mutant NTG-1 induced from A-7301 (autoagglutinative and haemagglutinative), and a non-pathogenic strain GH-7501 (non-agglutinative, non-haemagglutinative and protease positive). A-7301 could grow and produce protease extracellularly in the presence of rainbow trout anti-A-7301 serum, resulting in a considerable reduction of the antibody titre. NTG-1 similarly grew, but the titre scarcely decreased. GH-7501 could not survive in this medium. A-7301 and NTG-1 possessed a high capacity to adhere to the surface of fish monolayer cell cultures, whereas GH-7501 lacked the capacity. The capacity for adhesion was not inhibited by the antibody. Although live NTG-1 cells were ineffective as a live vaccine, sockeye salmon receiving protease fraction (obtained from extracellular products of A-7301 by DEAE-cellulose column chromatography) inactivated with normal serum, suffered only a low mortality when challenged with A-7301. Thus, although the antibody specific to autoagglutinating cells showed no effects on serum resistance and adhesion, which are involved in the infectivity of this pathogen, the possibility of protease as an effective protective antigen was demonstrated.     

Siderophore pattern of fish-pathogenic Vibrio anguiliarum, Aeromonas spp. and Pseudomonas spp. from the German Baltic coast

Abstract A Total of 355 strains of fish-pathogenic bacteria, including Vibrio anguillarum, Aeromonas hydrophila, A. Caviae, Pseudomonas fluorescens and P. putida , were examined for the presence of hydroxamate-or phenolate-type siderophores. These strains were isolated from diseased rainbow trout, Oncorhynchus mykiss (Walbaum), from fish farms in the Baltic Sea off the coast of the former German Democratic Republic. The production of siderophores was demonstrated in bioassays and siderophore-pattern analysed using several indicator strains. All strains tested grew under conditions of iron limitation. The strains of Vibrionacease produced at least a hydorxamate-type siderophore detected by the bioassay with A. flavercens JG-9. Only a few V. anguillarum , but 50% of A. hydrophila produced a phenolate-type siderophore. Seventy percent of V. anguillarum , 33% if A. hydrophila , 40% of A. caviae , 53% of P. fluorescens and 50% of P. putida promoted the growth of the indication strain S. typhimurium SR 1001, which can use iron by 2,3-dihydroxybenxoic acid. The pseudomonads tested produced either hydroxamate- or phenolate-type siderophores, or both.     

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum , A. hydrophila , A. hydrophila subsp. dhakensis , A. salmonicida subsp. salmonicida , A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10–25 bands, of which major bands were seen in the region of 32.5–47.5 and 62–83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83–173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.     

Action of selected antibiotics on four common bacteria associated with diseases of fish

Abstract. Four bacteria commonly associated with diseases of fish ( Aeromonas hydrophila complex, Aeromonas salmonicida, Flexibacter columnaris -like organisms and Pseudomonas fluorescens ) were evaluated in a serial dilution system for susceptibility to four antibiotics commonly used in the treatment of bacterial fish diseases (chloramphenicol, erythromycin, furpyrinol and oxytetracycline). Findings were expressed as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Furpyrinol was found to have the lowest MIC and MBC against A. hydrophila complex, A. salmonicida and F. columnaris-like organisms. MIC and MBC's for oxytetracycline were complicated by the presenceof R-plasmids, but were less than 4.0 mcg/ml for those without plasmids. Pseudomonas fluorescens was susceptible only to oxytetracycline. MIC for all groups of organisms using chloramphenicol was generally less than 16.0 mcg/ml. Susceptibility to erythromycin by these groups of organisms was greater than 16 mcg/ml.     

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.     

Inhibition of epidermal migration in the skin of rainbow trout Salmo gairdneri Richardson in the presence of achromogenic Aeromonas salmonicida

Abstract. Experimental infection of artificially-induced skin ulcers in rainbow trout Salmo gairdneri Richardson with an achromogenic strain of Aeromonas salmonicida induced a syndrome similar to that observed in cyprinid fishes. Sequential histological observations showed that the presence of the bacteria completely inhibited any migration of peripheral epidermal cells and subsequent under-running of the ulcer resulted in its expansion, as peripheral epidermis sloughed.     

Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar , rainbow trout, Oncorhynchus mykiss , and turbot, Scophthalmus maximus , on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida . Four of these cultures, which were identified tentatively as A. hydrophila , Vibrio fluvialis , Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida . There was no indication of serum or mucus antibodies to A. salmonicida , but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the fish.     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

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.     

The bacterial flora of rainbow trout, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs and its relationship to developmental success

Abstract. The bacterial flora of rainbow, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs was studied during incubation. Few bacteria were considered to exist internally, although Pseudomonas species and Aeromonas hydrophila were isolated. During incubation, large numbers of bacteria approaching 500 colony forming units/mm² accumulated around egg surfaces. Both rainbow and brown trout eggs placed in a through-flow system were colonized mainly by Pseudomonas sp. and A. hydrophila , whilst rainbow trout eggs in a separate recycling system were dominated by a Cytophaga species. Glass beads were also incubated (as an inert surface) and found to support significantly lower numbers of bacteria. Egg mortalities were recorded daily and found to differ significantly between both fish species and water conditions. Statistical analysis indicated a potentially significant correlation between hatching success and numbers of surface bacteria.     

Translocation of viable Aeromonas salmonicida across the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum)

The pathogenic bacterium Aeromonas salmonicida is the causative agent of the destructive disease furunculosis in salmonids. Horizontal transmission in salmonids has been suggested to occur via the skin, gills and/or intestine. Previous reports are contradictory regarding the role of the intestine as a route of infection. The present study therefore investigates the possibility of bacterial translocation across intestinal epithelia using Ussing chamber technology, in vitro. Intestinal segments were exposed for 90 min to fluorescein isothiocyanate-labelled pathogenic A. salmonicida. Sampling from the serosal side of the Ussing chambers showed that bacteria were able to translocate across the intestinal epithelium in both the proximal and distal regions. Plating and subsequent colony counting showed that the bacteria were viable after translocation. During the 90 min exposure to A. salmonicida, the intestinal segments maintained high viability as measured by electrical parameters. The distal region responded to bacterial exposure by increasing the electrical resistance, indicating an increased mucus secretion. This study thus demonstrates translocation of live A. salmonicida through the intestinal epithelium of rainbow trout, suggesting that the intestine is a possible route of infection in salmonids.     

Carrier state in furunculosis: secondary infection of trout with different Aeromonas salmonicida strains results in advantage for the primarily harboured one

Abstract. On the basis of previous observations, in vivo experiments were conducted in rainbow trout, Oncorhynchus mykiss (Walbaum), to detect possible competition between strains of Aeromonas salmonicida differing in their pattern of resistance to antimicrobial drugs. Serial infections, or reinfection of naturally-infected trout, were induced by intramuscular injection. Surviving fish were artificially stressed, and all dying fish were examined using selective media to assess the type of latent infection. Secondary infections with virulent strains always resulted in mortality and reisolation of the secondary strain in pure culture. However, when trout having undergone the two successive infections were stressed, the primary strain was always reisolated. Primary strains seem to be able to survive in unknown locations within cells or tissues, regardless of their virulence or growth potency. Although a protective effect conferred by latent infection to secondary challenge could not be clearly proven, such mechanisms resemble what occurs in premunition and could be of epidemiological interest.     

Aeromonas salmonicida subsp. salmonicida: correlation of protein patterns, antibiotic resistance, exoprotease activity, haemolysis and pathological lesions produced in vivo

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.     

斑点叉尾■溃烂症的病原鉴定和致病特性

为探明斑点叉尾[鱼回](Ictalunes punctatus)溃烂症的病因,从4尾患鱼肝脾中分离纯化出4株优势菌株,并进行病原鉴定、毒力基因检测、动物回归感染和药敏试验。4株优势菌经鉴定并命名为杀鲑气单胞菌无色亚种(Aeromonas salmonicida subsp achromogenes)X-G1,杀鲑气单胞菌杀鲑亚种(A.s subsp salmonicida)X-P2、X-P3和嗜水气单胞菌(A.hydrophila)X-P4。15℃时,杀鲑气单胞菌X-G1、X-P2和X-P3的世代时间(约14 min)均小于嗜水气单胞菌X-P4(约20 min);25℃时,杀鲑气单胞菌X-G1、X-P2和X-P3株的世代时间(约20 min)均大于嗜水气单胞菌X-P4株(约16 min)。X-G1株可检到弹性蛋白酶、溶血素和甘油磷脂胆固醇酰基转移酶等3种毒力基因;X-P2株仅可检到弹性蛋白酶1种毒力基因;X-P3株可检测到弹性蛋白酶、溶血素、细胞毒性肠毒素、丝氨酸蛋白酶、酯酶、气溶素和甘油磷脂胆固醇酰基转移酶等7种毒力基因;X-P4株可检测到鞭毛、弹性蛋白酶、气溶素、细胞毒性肠毒素、热不稳定性肠毒素、丝氨酸蛋白酶和溶血素等7种毒力基因。分离株X-G1、X-P2、X-P3和X-P4在15~17℃水温下腹腔注射攻毒的半数致死浓度(LD 50)依次为0.49×10^4、0.78×10^4、0.53×10^4、3.84×10^4 CFU/g;而在23~26℃水温下测得的LD 50依次为1.48×10^4、1.80×10^4、0.82×10^4、0.68×10^4 CFU/g。分离株混合感染比单一株感染均表现出更强的致死能力。分离菌株对多西环素、恩诺沙星、氟苯尼考均敏感,但因患病鱼不能摄食药饵而导致治疗失败。     

褐牙鲆腹水症病原菌的分离鉴定及其灭活疫苗的研制

从患腹水病褐牙鲆体内分离致病菌,通过生理生化和分子生物学方法进行鉴定;该菌的16S rDNA序列与标准株同源性达99.8%,证明该菌为嗜水气单胞菌.制备灭活疫苗后分别注射小鼠和牙鲆,测定其免疫效果.试验结果表明,用所制备的灭活疫苗免疫小鼠,其保护率达到90%,免疫牙鲆的保护率达到80%;采用渗透压差法浸泡免疫牙鲆在攻毒后发病率仅为6%,攻毒后保护率为85%;通过间接ELISA检测表明受免动物体内产生了较高水平的抗体;剖检及病理切片结果表明免疫过的动物内脏健康无异样,而未免动物肝脏充血,肠腔有积水.说明该疫苗安全有效.     

Plasma ladderlectin concentration following sterile inflammation and Aeromonas salmonicida subsp. salmonicida infection

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.     

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.     

日本鳗鲡败血腹水病病原研究

2000年3月中旬，自福建仙游某日本鳗鲡（Anguilla japonica)养殖场患败血腹水症的发病日本鳗鲡的肝脏及腹水处分离到四株细菌，经鉴定其中一株为迟钝爱德华氏菌（dwardsiella tarda)，一株温和气单胞菌（Aeromomas sobria)和两株嗜水气单胞菌（Aeromonas hydrophila)溶血试验，小鼠攻毒试验显示，上述四株细菌都有较强制 毒力，综合分析，上述菌株可能是引发这次日本幼鳗败血腹水病之病原菌。     

PCR扩增特异性16SrDNA和溶血素基因检测致病性嗜水气单胞菌

根据已发表的气单胞菌16S rDNA基因序列及气单胞菌气溶素(aerolysin)基因序列，设计了2对引物，建立了检测致病性嗜水气单胞菌的PCR方法。通过对12株气单胞菌的检测，发现16S rDNA引物具有高度的特异性，仅对嗜水气单胞菌扩增阳性。而Aero基因引物检测结果与采用生物学方法(鲜血平板法)检测的结果符合率为97．2％，且具有高度的敏感性，可检测最低1fg的模板。将16S rDNA与Aero基因结合PCR方法检测致病性嗜水气单胞菌与用致病性嗜水气单胞菌检测试剂盒的符合率为94．4％。该方法的建立为致病性嗜水气单胞菌的检测提供了一种简便、快速的途径，是一种比较实用的致病性嗜水气单胞菌的检测方法。