Clinical, microbiological and epidemiological findings in recent outbreaks of goldfish ulcer disease due to atypical Aeromonas salmonicida in south-eastern Australia

Abstract. The spread of goldfish ulcer disease (GUD) from Victoria to New South Wales, Australia, and the first isolation of Aeromonas salmonicida from wild goldfish are reported. Cultural, biochemical and protein SDS-PAGE characteristics of these recent isolates are compared with those of existing Australian isolates, with strains recovered from goldfish in Italy and the USA (atypical strains) and with strain ATCC 14174 (typical strain). The Australian isolates were identical and closely resembled the exotic atypical strains. Although there were several biochemical differences between the atypical isolates and the typical ATCC 14174 strain, the results of SDS-PAGE confirmed that these strains were closely related. The homology of the Australian and overseas strains recovered from goldfish supports the common view that A. salmonicida was introduced first into Australia with diseased goldfish in 1974. The three widely separated outbreaks of GUD reported here confirm that an atypical strain of A. salmonicida is now endemic in Australia.     

Non-pigment-producing isolates of Aeromonas salmonicida subspecies salmonicida: isolation, identification, transmission and pathogenicity in Atlantic salmon, Salmo salar L.

Four non-pigment-producing isolates and two pigment-producing isolates of Aeromonas salmonicida sp. salmonicida were isolated from the head-kidney of diseased farmed Atlantic salmon, Salmo salar L. The cultural, morphological and biochemical features of the isolates were compared with those of reference strains. Injection and cohabitation experiments were performed. The only difference between the non-pigment-producing isolates and the pigment producing reference strains of A. salmonicida ssp. salmonicida was the inability of the former to produce pigment. In the injection experiments, the investigated non-pigment-producing isolate produced a significantly higher mortality compared with the mortality caused by the reference strain, whereas no difference in mortality was detected in the cohabitation experiments.     

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.     

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.     

Aetiology of an ulcerative disease in goldfish, Carassius auratus (L.): characterization of the causative agent

Abstract. A bacterium resembling Aeromonas salmonicida and determined to be the aetiologic agent of a cutaneous ulcerative disease in goldfish Carassius auratus (L.) was further characterized in this study.
Forty-five isolates of the bacterium (43 from the United States and one each position (moles % guanine plus cytosine) and DNA homology. The bacteriological atypical A. salmonicida previously described. Several important biochemical characteristics distinguished the goldfish isolates from typical A. salmanicida , but the DNA binding experiments indicated a high degree of relatedness between the goldfish isolates and typical A. salmonicida strains.     

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.     

vapA (A‐layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes

Sequence variation in a region of the virulence array protein gene (vapA; A‐layer) was assessed in 333 (‘typical’ and ‘atypical’) isolates of the fish pathogenic bacterium Aeromonas salmonicida. Resulting similarity dendrograms revealed extensive heterogeneity, with nearly all isolates belonging to either of 14 distinct clusters or A‐layer types. All acknowledged A. salmonicida subspecies (except ssp. pectinolytica, from which no vapA sequence could be obtained) were clearly separated, and notably, all isolates phenotypically identified as ssp. salmonicida formed a distinct and exclusive A‐layer type. Additionally, an array of un‐subspeciated atypical strains formed several equally prominent clusters, demonstrating that the concept of typical/atypical A. salmonicida is inappropriate for describing the high degree of diversity evidently occurring outside ssp. salmonicida. Most representatives assessed in this study were clinical isolates of spatiotemporally diverse origins, and were derived from a variety of hosts. We observed that from several fish species or families, isolates predominantly belonged to certain A‐layer types, possibly indicating a need for host‐/A‐layer type‐specific A. salmonicida vaccines. All in all, A‐layer typing shows promise as an inexpensive and rapid means of unambiguously distinguishing clinically relevant A. salmonicida subspecies, as well as presently un‐subspeciated atypical strains.     

Evaluation of cross protection by vaccines against atypical and typical furunculosis in Atlantic salmon, Salmo salar L.

In Iceland, farmed salmonids are vaccinated against A. salmonicida ssp. achromogenes (Asa), which causes atypical furunculosis and is endemic in local waters . Classical furunculosis, caused by A. salmonicida ssp. salmonicida (Ass), was not diagnosed in this country until June 1995. In the present study, protection in experimental challenges against atypical and classical furunculosis in Atlantic salmon vaccinated with an autogenous Asa bacterin (Iceland Biojec.OO, IBOO), a commercial furunculosis vaccine (Biojec.1500), or a mixture of both vaccines was compared. The results showed that both vaccines gave protection against an injection challenge with Asa. However, better protection was obtained with the IBOO (homologous) vaccine. Infection of Asa by cohabitation could not be established in fresh water. Fish vaccinated with Biojec.1500 or with both vaccines simultaneously were equally well protected against Ass in a cohabitation challenge. On the other hand, no protection against classical furunculosis was achieved in fish vaccinated by IBOO alone.     

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.     

Protection against atypical furunculosis in Atlantic halibut,Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.     

First description of atypical furunculosis in freshwater farmed Atlantic salmon,Salmo salar L., in Chile

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that the atypical isolates belong to two genetic groups that were associated with the geographical origin.     

Effect of three dietary oils on disease susceptibility in Arctic charr (Salvelinus alpinus L.) during cohabitant challenge with Aeromonas salmonicida ssp. salmonicida

Arctic charr ( Salvelinus alpinus L.) were fed diets based on a commercial recipe supplemented with either linseed, soybean or marine oil prior to cohabitant challenge with Aeromonas salmonicida ssp. s almonicida . Mortality varied significantly between the three dietary groups. Highest mortality (48%) was observed in fish fed the marine oil and the lowest mortality (20%) was in the group fed soybean oil. Transmission electron microscopy (TEM) examination of the digestive tract of uninfected fish demonstrated substantial numbers of bacterial cells between microvilli. However, only a few bacteria were recovered that were associated with the microvilli of infected fish. Immunocytochemical staining/labelling investigations using TEM and an immunogold method were performed on mid-gut segments of fish fed the marine oil diet and showed augmentation of goblet cells and the presence of A. salmonicida ssp. salmonicida in the gastrointestinal tract of diseased fish after challenge with the pathogen. It is suggested that the gastrointestinal tract could be an infection route of A. salmonicida ssp. salmonicida. The greater prevalence of goblet cells supports the suggestion that sloughing off mucus is a protective response against bacterial infections. These results make an important contribution to our understanding of how nutrition can affect the disease resistance of fish.     

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.     

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。     

Characterization of Edwardsiella tarda strains isolated from turbot, Psetta maxima (L.)

The biochemical, serological and molecular characteristics of a group of 21 Edwardsiella tarda strains isolated from turbot, Psetta maxima, in two different areas of Europe were analysed and compared with a total of 13 strains of this bacterial species with different geographical and host origins. All the turbot isolates were biochemically identical to the E. tarda strains included as reference. The use of different techniques including microagglutination, dot blot and Western blot of lipopolysaccharides allowed us to determine that all the turbot isolates constitute an homogeneous and distinctive serological group. Genetic analysis by randomly amplified polymorphic DNA (RAPD) analysis demonstrated that although the E. tarda strains from turbot were compiled in a unique group using the primers P3 and P6, two clonal lineages could be detected when oligonucleotides P4 and P5 were employed.     

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.     

Differences in resistance of carp, Cyprinus carpio L., to atypical Aeromonas salmonicida

Abstract. The degree of resistance to an atypical strain of Aeromonas salmonicida , the causative bacterium of carp erythrodermatitis, was examined in two strains of carp, Cyprinus carpio L.: a Polish line. R3, sixth generation of conventional inbreeding (full-sib matings); and a Hungarian line. R8, fifth generation of conventional inbreeding. Comparisons were made between and within the two strains. Results showed a significant difference ( P < 0·001) in the degree of resistance, with the Hungarian carp showing greater resistance than the Polish carp. Differences within each strain were also observed indicating a maternal influence on resistance. Two transferrin genotypes in three genetic combinations were identified (DD, DG, GG) but were not found to influence resistance.     

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.     

发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。