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.     

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.     

Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida,by enzymatic and serological methods

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.     

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.     

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.     

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.     

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.     

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.     

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.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.     

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.     

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.     

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.     

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.     

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.     

Atypical Aeromonas salmonicida infection in the black rockfish, Sebastes schlegeli Hilgendorf, in Korea

Cultured black rockfish, Sebastes schlegeli, suffered mass mortalities during winter 2008 and spring 2009 in Korea, showing clinical signs of ulcer lesions and haemorrhages over their body surface. The aetiological agent was identified as Aeromonas salmonicida (strains RFAS-1, -2 and -3), which is a non-pigmented, slow-growing bacterium. Phenotypes of RFAS strains showed variation, while 16S rRNA, gyrB, rpoD, dnaJ and recA gene sequences of all the strains were affiliated to A. salmonicida. In particular, vapA gene sequences of the strains were most closely related to one of the five subspecies of A. salmonicida subsp. masoucida (=KCCM 40239(T) ). LD(50) values of RFAS-1 for intraperitoneal and intramuscular injection were 1.5 × 10(5.25) and 1.5 × 10(6.4) cfu/rockfish, respectively. However, A. salmonicida strains KCCM 40239(T) and SAS-1, which originate from masou and chum salmon, respectively, were not pathogenic to black rockfish. RFAS strains, possessing A-layer protein on their surface, exhibited β-haemolytic activity against rockfish erythrocytes and capability to survive in rockfish serum, which seem to be associated with virulence.     

Antibiotic resistance of Aeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland

Abstract. Antibiotic sensitivity patterns of 304 isolates of Aeromonas salmonicida from 229 outbreaks of furunculosis among salmon in Scotland between 1988 and 1990 were investigated. Fifty-five per cent were resistant to oxytetracycline and 37% resistant to oxolinic acid. Multiple resistance was common (52%) and 18 out of 19 antibiograms which were found in the first year recurred in the succeeding year. More than a quarter of the outbreaks were associated with two or more A. salmonicida variants distinguishable by their antibiotic sensitivity patterns. The implications of these findings in the control of furunculosis are considered.     

Putative virulence factors of atypical Aeromonas salmonicida isolated from Arctic charr,Salvelinus alpinus (L.), and European grayling,Thymallus thymallus (L.)

Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.     

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.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .