Evaluation of profiles of Aeromonas salmonicida as epidemiological markers of furunculosis infections in fish

Abstract. Strains of Aeromonas salmonicida subsp. salmonicida , the agent of furunculosis disease of salmonid fish, have fairly uniform plasmid patterns. Of 35 strains examined by agarose gel electrophoresis, 28 had a pattern consisting of four small plasmids (4.2, 3.6, 3.5, 3.3 Mda) and a larger plasmid. The larger plasmid was most often 50–56 Mda, but it was larger in some strains. In the remaining seven strains, the same general profile was seen, but one of the small plasmids was missing. An additional plasmid was present in six strains. The pattern seen in 30 strains collected from Ontario fish over an 8-year period did not differ significantly from five reference isolates from other locations. Plasmid profiles of A. salmonicida strains appear too uniform to provide a useful epidemiological tool. The non-pigmented. atypical strains of A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida, A. media , and brown-pigmented strains of A. hydrophila had different plasmid DNA profiles, which were distinct from those of typical isolates of A. salmonicida subsp. salmonicida . Antibiotic susceptibility patterns, determined by the agar dilution method, were uniform for most typical strains. A non-transferable resistance to tetracyclmes was found in two Ontario isolates, but antibiotic resistance was relatively uncommon among the Ontario isolates.     

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.     

Production of the lethal acetylcholinesterase toxin by different Aeromonas hydrophila strains

Abstract. Seven fish pathogenic isolates of Aeromonas hydrophila , one A. sobria and one A. caviae were investigated for production of the fish lethal acetylcholinesterase toxin (AcChE-toxin). Western blotting was used for screening the ECP of these strains with a rabbit antiserum prepared against the purified toxin of strain B₃₂ and all the isolates (except A. sobria ) gave positive results with different patterns of bands. The AcChE-toxin appears to be secreted as a protein of high molecular weight which is stable at −20°C, and in 90% of the strains tested, it appears to be split into lower molecular weight fragments by the action of other components present in the ECP. The smallest, stable and highly active fragment has a MW of 15kDa.     

Amoxycillin resistance in Scottish isolates of Aeromonas salmonicida

Abstract. Eighty isolates of Aeromonas salmonicida , recovered from separate outbreaks of furunculosis in farmed and wild salmon in Scotland during 1988 and 1989, were examined for susceptibility to the β-lactam antibiotic amoxycillin. Susceptibility was determined in terms of minimum inhibitory concentration (MIC). All of the A. salmonicida subsp. salmonicida isolates investigated were susceptible to amoxycillin, with MICs of 0.30–1.50mg1^-1. All of the A. salmonicida subsp. achromogenes isolates tested were resistant to amoxycillin, with MICs in excess of 500mgl^-1. The A. salmonicida subsp. achromogenes produced a β-lactamase enzyme with a pI of approximately 8.0. The enzyme was inducible and its production was unaffected by plasmid curing with ethidium bromide, suggesting that resistance was chromosomal rather than plasmid mediated.     

不同来源嗜水气单胞菌外膜蛋白基因检测及序列分析

采用PCR方法,对分离于发病宽体金线蛭(Whitmania pigra)、中国林蛙(Rana temporaria chensinensis)、牙鲆(Paralichthys olivaceus)、草鱼(Ctenopharyngodon idellua)、青鱼(Mylopharyngodon piceus)、鲤(Cyprinus carpio)的42株致病性嗜水气单胞菌(Aeromonas hydrophila)进行了外膜蛋白(OMP)基因的检测;将代表菌株的外膜蛋白基因通过DNAStar软件与GenBank中报道的4株嗜水气单胞菌(FJ437030.1、AF183931.1、AF276639.2、CP000462.1)和1株温和气单胞菌(A.sobria,FJ437029.1)参考菌株进行了核苷酸同源性比较分析。结果表明,所检不同来源嗜水气单胞菌的OMP基因携带率为100%;所测代表菌株的OMP基因与参考菌株的同源性在86.0%～99.2%之间。所测得的不同来源嗜水气单胞菌OMP基因的同源性98.3%～100%,其中分离于宽体金线蛭的HTQ010505-1与青鱼的HL060811-4两菌株间同源性高达100%。     

Characterisation of Aeromonas strains and species by pulsed field gel electrophoresis and principal components analysis

Aeromonas genomes were investigated by restriction digesting chromosomal DNA with the endonuclease Xba I, separation of restriction fragments by pulsed field gel electrophoresis (PFGE) and principal components analysis (PCA) of resulting separation patterns. A. salmonicida salmonicida were unique amongst the isolates investigated. Separation profiles of these isolates were similar and all characterised by a distinct absence of bands in the 250kb region. Principal components analysis represented these strains as a clearly defined homogeneous group separated by insignificant Euclidian distances. However, A. salmonicida achromogenes isolates in common with those of A. hydrophila and A. sobria were shown by principal components analysis to be more heterogeneous in nature. Fragments from these isolates were more uniform in size distribution but as demonstrated by the Euclidian distances attained through PCA potentially characteristic of each strain. Furthermore passaging of Aeromonas isolates through an appropriate host did not greatly modify fragment separation profiles, indicative of the genomic stability of test aeromonads and the potential of restriction digesting/PFGE/PCA in Aeromonas typing.     

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.     

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.     

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.     

Aetiology of an ulcerative disease in goldfish Carassius auratus (L.): microbiological examination of diseased fish from seven locations

Abstract. A comparative diagnostic study was conducted on goldfish, Carassius auratus (L.), with a cutaneous ulcerative disease from five locations in the United States and one each in England and Japan. Fish were examined for parasites, viruses and bacteria. Fish from all locations examine d were infested by ectoparasites; no single parasite species was common to all locations. No virus-associated cytopathology was observed in fathead minnow (FHM) or adult goldfish (CAR) monolayer cell cultures inoculated with homogenates of cutaneous lesions, kidneys and livers from diseased fish. The only bacterium cultured from fish from all locations was an atypical, often late-pigmenting strain of Aeromonas salmonicida . This organism was isolated from 64 of 83 (77%) of the total lesions cultured and was most prevalent in early lesions. A second commonly isolated organism was Aeromonas hydrophila , which was cultured from fish at four of the seven locations and from 28 (34%) of the total lesions cultured. A. hydrophila was most prevalent in terminal lesions. From these studies it was concluded that A. salmonicida was the probable cause of ulcers noted In the cases examined an d that A. hydrophila was a secondary invader.     

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。     

Cellular components of probiotics control Yersinia ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.     

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.     

嗜水气单胞菌与温和气单胞菌的LAMP检测方法的建立

利用环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）分别建立嗜水气单胞菌（Aeromonas hydrophila,AH）与温和气单胞菌（A.sobria,AS）的快速检测方法。针对嗜水气单胞菌pilin基因、温和气单胞菌zipA基因设计特异性LAMP引物。在恒温条件下利用实时浊度仪对2组引物进行特异性和灵敏度试验,并以琼脂糖凝胶电泳和核酸染料颜色变化对扩增结果进行判定。结果显示,LAMP实时浊度法能够特异地检测嗜水气单胞菌和温和气单胞菌,最低检出限分别为46 fg·mL^-1和320 fg·mL^-1,是普通PCR方法的104倍和102倍;并能应用于已知临床样品检测。该研究建立的嗜水气单胞菌与温和气单胞菌LAMP快速检测方法具有高效、特异、灵敏的特点。     

Extracellular glycerophospholipid:cholesterol acyltransferase from Aeromonas salmonicida: activation by serine protease

Abstract. Extracellular hacmolytic activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the membrane-active enzyme glyccrophospholipidrcholcstcrol acyltransferase (GCAT). About 10% of the total haemolytic activity was due to a high molecular mass complex of LPS and GCAT (mol. mass >1000kDa), containing 35–50% neutral sugars and 1.5–2.0% protein. Some haemolytic activity (30–40% of total), corresponding to 50–70kDa by gel filtration, also contained GCAT-activity and may represent aggregated forms of GCAT. However, about 50% or more of the haemolytie activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the hacmolytic activity of both GCAT and GCAT-LPS. A transposon-produccd serinc protease negative mutant of the same A. salmonicida strain showed reduced haemolytic activity. The mutant produced a 38-kDa GCAT proform of low hacmolytic activity. The proform was processed by autogenous scrinc protease to a highly hacmolytic 26-kDa molecule with pl 6.3, similar to GCAT of the parent strain. The weakly haemolytic GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.     

Cloning and characterization of the haemolysin determinants from Aeromonas hydrophila

Abstract. Two haemolysin genes (AHH4 and AHH-2) of Aeromonas hydrophila ATCC7966 were cloned into a plasmid vector in Escherichia coli K-12. An open reading frame (ORF) of the AHH-1 haemolysin gene was 1734 base pairs (bp). and corresponded to a protein of 577 amino acid residues. Analysis of the deduced amino sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The sequence also included the -10 region and the -35 region of a promoter, and a ribosome- binding site upstream from the ORF. The termination site was located downstream from the ORF. The haemolysin was a thermolabile protein with the predicted molecular mass of 60 kDa. The AHH-1 gene is distributed in various A. hydrophila and A. salmonicida strains. The nucleotide sequence of a 981 bp ORF of the AHH-2 gene was encoded with the predicted molecular mass of 377 kDa polypeptides. The homology of the nucleotide sequence was very low between the AHH-1 and AHH-2 genes, and also with the aerolysin gene cloned by Howard & Buckley (19S6). No leader peptide was found in the N-terminal region of the ORF of the AHH2 gene. The AHH-2 gene was detected in the original strain ATCC7966, but was not detected in other tested strains of A. hydrophila and A. salmonicida.     

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.     

Protection against carp erythrodermatitis following bath or subcutaneous exposure to sublethal numbers of virulent Aeromonas salmonicida subsp. nova

Abstract. A bath challenge system was used to infect carp. Cyprinus carpio L., with Aeromonas salmonicida subsp. nova , the causative agent of carp cruthrodermatitis. Bath-challenged fish became infected with the bacterium exihibitinng typical signs of the disease, Carp that were sublethally bath exposed became infected and exhibited some skin lesions, but after one week, these quickly healed and the animals fully recovered from the infection, Naive fish that had not been previously exposed to the bacterium had mortalities of 100% when infected by the subcutaneous route and 40–60% by the bath route of infection. Carp that received sublethal infections were able to withstand subsequent lethal infection and recover regardless of the route of infection. Sublethally bath-exposed carp were protected from subsequently lethal challenges of A. salmonicida subsp. nova for at least 5 months.     

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.     

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.