Seasonal distribution of Aeromonas hydrophila in river water and isolation from river fish

The seasonal distribution of Aeromonas hydrophila in water and recovery rate from live river fish was investigated. The highest isolation rates of A. hydrophila occurred in water during the late winter followed by a progressive decline in density during the summer and monsoon seasons. The organism was recovered from fish throughout the period from which it was concluded that they form a reservoir which is unrelated to their density in water. The enterotoxigenicity of some environmental strains was tested in suckling mice and rabbit ileal loop.     

Genetic diversity and antimicrobial susceptibility of motile aeromonads isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) farms

A total of 22 motile Aeromonas strains were detected in 48 (18.53%) of 259 fish and 6 (10.71%) of 56 water samples obtained from seven commercial rainbow trout ( Oncorhynchus mykiss , Walbaum) farms in the province of Mersin, Turkey. These strains were identified by conventional microbiological techniques and by using an ID32GN system. Of these isolates 20 (91.3%) were identified as Aeromonas hydrophila and 2 (8.7%) as Aeromonas sobria . While 8 of the A. hydrophila strains were isolated from water samples, 12 isolates were from fish samples. Whereas A. hydrophila strains were found in all farms, A. sobria was detected in only two farms. Genetic diversity by arbitrarily primed polymerase chain reaction (AP-PCR) and antimicrobial sensitivity tests were carried out on eight A. hydrophila isolates obtained from water samples, and isolates from seven A. hydrophila and one A. sobria from fish samples. The AP-PCR band patterns of motile aeromonads demonstrated weak similarity to the A. hydrophila reference strain ATCC 7966. Five A. hydrophila strains in the water samples displayed genetic similarity, but three others were different. Aeromonas hydrophila isolates from fish samples possessed slight similarities, and A. sobria was genetically distant to all A. hydrophila strains. An antimicrobial sensitivity test of 16 isolates revealed that 100% were sensitive to gentamicin, 87.5% to sulphamethoxazole–trimethoprim, 62.5% to enrofloxacin, 43.8% to oxytetracycline, 37.5% to neomycin, 18.75% to streptomycin and 6.25% to erythromycin. All isolates were resistant to novobiocin.     

Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina

The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.     

Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina.

The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.     

Prevalence and distribution of Aeromonas hydrophila in the United States.

The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.     

Prevalence and distribution of Aeromonas hydrophila in the United States.

The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.     

Factors affecting river entry of adult Atlantic salmon in a small river

In this study, effects of stock origin, fish size, water flow and temperature on time of river ascent of adult Atlantic salmon Salmo salar were tested. Brood stocks were collected in eight Norwegian rivers situated between 59 and 69° N. The fish were reared to smolts, individually tagged and released in the River Imsa, south-west Norway (59° N). Adults from all stocks approached the Norwegian coast concurrently, but Atlantic salmon ≥70 cm in natural tip length entered coastal water slightly earlier during summer than smaller fish. Atlantic salmon <70 cm, however, ascended the river significantly earlier and at lower water flow and higher water temperature than larger fish. Although largest in size, the fish from the northern populations (62–69° N) ascended the River Imsa almost 1 month earlier than those from the south (59–60° N). They seemed less restricted by the environmental factors than the fish originating from the more southern rivers. There was no apparent trend among years in time of river ascent. Maximum ascent per day occurred at water discharges between 12·5 and 15 m³ s⁻¹ and at water temperatures between 10 and 12·5° C. There was a significant positive correlation between water flow and river ascent during the first part of the upstream run from July to September with best correlation for September, when multiple regression analysis indicated that water temperature had an additional positive effect. Stock origin, fish size and water discharge were important variables influencing the upstream migration of Atlantic salmon in small rivers.     

Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China?

The significance of Aeromonas hydrophila in association with disease outbreaks in aquaculture production in the Zhejiang province of China was investigated. Bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs. PCR and traditional biochemical methods were used for identification of A. hydrophila. Out of 69 motile aeromonads, 35 isolates were identified as A. hydrophila by biochemical tests. However, 6 of those were not identified as A. hydrophila by a species specific PCR method. Serotyping revealed 2 dominant serotypes (O9 and O97) among A. hydrophila isolates. The data presented show that approximately 42% of the motile aeromonads isolated from disease outbreaks among various fish species were A. hydrophila. It is noteworthy that A. hydrophila accounted for more than 50% of the isolated aeromonands isolated from crucian carp Carassius carassius and Wuchang bream Megalobrama amblycephala with haemorrhagic septicaemia. Although this species was the most frequently isolated organism from internal organs of diseased fish and crabs in the present study, other motile Aeromonas spp. were also found. The PCR assay was useful in preventing misidentification of A. hydrophila, which may occur when only phenotypic tests are employed.     

Solubilization of fish proteins using immobilized microbial cells

Cells of Bacillus megaterium, Aeromonas hydrophila, and Pseudomonas marinoglutinosa were immobilized in calcium alginate. The immobilized cells secreted protease when held in fish meat suspension in water. The enzyme synthesis by the entrapped cells was supported by small amounts of soluble nutrients present in the meat. The secreted protease solubilized the fish meat, solubilization being optimum at pH range of 7.5 to 9.5 and at 50 degrees C. Under these conditions immobilized B. megaterium was most efficient giving 30% solubilization of the meat, followed by A. hydrophila (18%), while immobilized P. marinoglutinosa was less effective. The optimum ratio of fish meat to beads was about 4:3 for B. megaterium and A. hydrophila. The beads had a storage life of 30 days at 4 degrees C. The results suggested potential for use of immobilized microbial cells having extracellular protease activity to enhance solubility of waste proteins. A prototype reactor with beads holding assembly was fabricated which could recover the beads from the meat slurry after the treatment.     

Habitat utilisation by fish community in a short-term regulated river reservoir

Short-term streamflow regulation (hydropeaking) affects the ecology of regulated rivers. We examined the longitudinal and temporal changes occurring in fish assemblages in a hydropeaking single river reservoir between two power plants by using electrofishing along the shoreline, hydroacoustics and test fishing in the open water. A longitudinally changing fish community was found among bottom-dwelling fish in the fast-flowing and highly disturbed upstream part of the reservoir progressing to generalists and pelagic fish in the lentic and most stable environment at the downstream end. The fish assemblage showed temporal patterns as fish density increased during night-time darkness and also towards autumn. Our work provides evidence for gradient effects of flow regulation and contributes to awareness of the effects of disturbance (flow/habitat variability) on biological systems.     

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus , was studied using A. hydrophila strain A-3500. After comparison of four different infection methods, subcutaneous injection was selected. Different test temperatures were also tested and higher mortality was observed at 17 and 25°C during a 15-day period. SDS-PAGE analysis of outer membrane proteins prepared from A. hydrophila cultured at 10, 17, 25 and 32°C in formulated salt water showed different protein profiles. For example, a 40-kDa band was found only at 17 and 25°C. Phagocytic rates of A. hydrophila by goldfish macrophages at 10, 17, 25 and 32°C were 20.46 ± 2.07, 16.15 ± 1.39, 15.94 ± 1.85 and 22.22 ± 2.49%, respectively. The results indicated that temperature affects both the cell membrane structure of A. hydrophila and phagocytic activity of goldfish macrophages, resulting in varying fish mortality when infected at different temperatures.     

Environmental stress and bacterial infection in channel catfish, Ictalurus punctatus Rafinesque

Channel catfish, Ictalurus punctatus , were injected intraperitoneally with a sublethal dose of Aerornonas hydrophila and then stressed for 144 h by being maintained either in a dissolved oxygen concentration of 1·5 mg/1, 1·2 mg/1 total ammonia, and/or 6·5 mg/1 free CO₂ with a continuous inflow of water. A significant difference in percentage of mortality was noted between treatments ( P < 0·05). The trunk kidneys of surviving stressed fish had significantly higher total bacterial counts than non-stressed controls. A. hydrophila was isolated from 67% of the stressed fish and 9% of the control fish. Edwardsiella tarda , apparently endemic in the population, was isolated from 43% of the stressed fish and 7% of the control fish. Histopathological lesions were in the gills, liver, spleen, trunk kidney, and head kidney of stressed fish, but not control fish.     

Behavior of an Aeromonas hydrophila aroA live vaccine in water microcosms

Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16 degrees C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.     

Migration of anadromous brown trout Salmo trutta in a Norwegian river

1. Upstream and downstream migrating anadromous brown trout Salmo trutta were monitored daily in fish traps in the River Imsa in south-western Norway for 24 years, from 1976 to 1999. One-third of the fish descended to sea during spring (February–June) and two-thirds during autumn (September–January).
2. In spring, high water temperature appeared to influence the downstream descent. Large brown trout (> 30 cm, chiefly two or more sea sojourns) descended earlier and appeared less dependent on high water temperature than smaller and younger fish. The spring water flow was generally low and of little importance for the descent.
3. In autumn, the daily number of descending brown trout correlated positively with flow and negatively with water temperature.
4. Brown trout ascended from the sea between April and December, but more than 70% ascended between August and October. The number of ascending trout increased significantly with both decreasing temperature and flow during the autumn. This response to flow appeared to be the result of the autumn discharge which is generally high and most fish ascended at an intermediate flow of 7.5–10 m³ s⁻¹ (which is low for the season).
5. In a river like the Imsa with low spring and high autumn flows, water temperature appears to be the main environmental factor influencing the timing and rate of spring descent, while both water temperature and flow seemed to influence the timing and rate of the autumn descent and ascent. These relationships make sea trout migrations susceptible to variation in climate and human impacts of the flow regime in rivers.     

翘嘴鳜病原嗜水气单胞菌分子特征及LAMP检测方法的建立

嗜水气单胞菌(Aeromonas hydrophila)是一种危害鳜鱼养殖生产的重要病原细菌, 为进一步明确该病原菌的分子特征及建立快速检测技术, 实验对引起翘嘴鳜(Siniperca chuatsi)暴发性死亡的病原嗜水气单胞菌进行了致病性、菌株毒力特征研究, 同时以嗜水气单胞菌气溶素基因aerA为分子靶标设计引物, 利用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了病原嗜水气单胞菌的快速检测方法。结果表明, 本次引起翘嘴鳜暴发性死亡的病原嗜水气单胞菌半致死浓度为1.6×106 CFU/mL, 携带aerA等14种毒力基因, 此14种毒力基因可用于其致病性分析及分子检测。以气溶素基因aerA设计引物进行的环介导恒温扩增, 结果显示可扩增出阶梯状条带, 加入SYBR Green I染色后呈现绿色的阳性反应, 而对照组均未出现任何扩增条带且反应体系呈现橙色, 表明LAMP检测方法对于嗜水气单胞菌检测具有很好的特异性; 灵敏度检测的最低检测限为4.6×101 CFU/mL; 10种经人工感染的淡水养殖鱼虾组织匀浆增菌液, 提取DNA后进行LAMP方法检测, 结果均可获得阳性扩增结果, 而对照未染菌组呈阴性, 表明该方法具有较好的应用性, 可应用于嗜水气单胞菌引起的水生动物疾病的检测。     

Incidence of haemolysin-positive and drug-resistant Aeromonas hydrophila in freshly caught finfish and prawn collected from major commercial fishes of coastal South India

The incidence of Aeromonas hydrophila in freshly caught finfish and prawns from four major commercial fish landing sites of coastal South India was studied for a period of one year. Among 514 analysed samples of seafood (410 finfish and 104 prawn), 37% of them (37.3% of finfish and 35.6% of prawn) were contaminated with A. hydrophila. A total of 255 strains of A. hydrophila were isolated. Of the total isolates, about 78.4% of them were producers of haemolysin. All strains were resistant to bacitracin and all were sensitive to chloramphenicol. The results indicate that the strains originated from high-risk sources. The presence of A. hydrophila is an indication of marine contamination. The increasing presence of haemolysin-producing multiple drug-resistant A. hydrophila in fish and prawn may become a potential human health hazard.     

Acoustic assessment of the fish spatio‐temporal distribution during the initial filling of the Three Gorges Reservoir,Yangtze River (China), from 2006 to 2010

The aim of this study was to quantify the fish fauna in the Three Gorges Dam (TGD) and assess the tendency for change as well as for any spatial and temporal distribution. Two types of echo‐sounder equipment (HTI Model 241 and Simrad EY60) were used to detect the fish density and fish spatio‐temporal distribution. Eight surveys were conducted between 2006 and 2010, with the results showing that fish abundance in the Three Gorges Reservoir (TGR) increased yearly from 15.5 to 123 ind./1000 m³, a change apparently correlated with the corresponding increase in the reservoir water level. Horizontally, fish density increased from the lower reaches to the upper reaches. Fish preferred inhabiting the area around the Zhongxian section during winter and the Chongqing section in spring. Vertically, fish density was highest in the lower layer, followed by the middle layer. The difference in fish density in different water layers observed in spring was not as evident as in the winter. Fish target strength throughout the eight investigations showed a skewed distribution with respect to fish size. The overall increasing tendency of fish density and the spatio‐temporal distribution of fish species were closely related to the water level of the TGR during the impounding period. Other influencing factors included water flow rate, water depth, reservoir shape, habitat type, and protection policy.     

Cloning, characterisation and expression of Aeromonas hydrophila major adhesin

Aeromonas hydrophila, an important pathogen in fish, is believed to cause diseases by adhesive and enterotoxic mechanisms. The adhesion is a prerequisite for successful invasion. In this study, the gene of a 43 kDa major adhesin (designated as AHA1) was cloned and expressed. Nucleotide sequence analysis of AHA1 revealed an open reading frame encoding a polypeptide of 373 amino acids with a 20-amino-acid putative signal peptide (molecular weight 40,737 Da). The amino acid sequences of Aha1p showed a very high homology with the other two outer membrane proteins of A. hydrophila. Using the T-5 expression system, this major adhesin Aha1p was expressed in Escherichia coli. The purified recombinant adhesin could competitively inhibit A. hydrophila from invading fish epithelial cells in vitro. Western-blot analysis showed that this major adhesin is a very conserved antigen among various strains of Aeromonas. When used to immunise blue gourami, the recombinant adhesin could confer significant protection to fish against experimental A. hydrophila challenge.     

Electrophoretic and immunochemical analyses of the lipopolysaccharides from various strains of Aeromonas hydrophila.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the lipopolysaccharides isolated from strains of Aeromonas hydrophila which exhibit virulence for fish and which autoaggregate during growth in static broth culture. The lipopolysaccharides contained O-polysaccharide chains of homogeneous chain length. Two of the strains produced a surface protein array, and immunofluorescence and phage-binding studies revealed that a number of these O-polysaccharide chains of homogeneous length traversed the protein array and were exposed on the cell surface. Immunochemical analyses by immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence, and immunoprecipitation with both polyclonal and monoclonal antibodies revealed the presence of three epitopes on the polysaccharide moiety of this homogenous-chain-length lipopolysaccharide morphotype. One epitope was species serogroup specific and reactive by immunoblotting. This epitope was not present on the heterogeneous-chain-length O polysaccharides of nonautoaggregating strains of A. hydrophila examined. The second epitope was conformation dependent and cross-reactive with an epitope on the homogenous-chain-length O polysaccharides of Aeromonas salmonicida lipopolysaccharide. The third epitope was recognized by a monoclonal antibody and appeared to involve that region of the A. hydrophila and A. salmonicida lipopolysaccharide molecules which contained the O-polysaccharide-core oligosaccharide glycosidic linkage.