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The susceptibility to oxolinic acid of 48 stains of Aeromonas salmonicida was established using the disc diffusion and agar dilution protocols of Alderman and Smith (2001) [Alderman, D., Smith, P., 2001. Development of draft protocols of standard reference methods for antimicrobial agent susceptibility testing of bacteria associated with fish disease. Aquaculture 196, 211–243.]. Both methods generated data that had a bimodal distribution. With respect to the agar dilution method an unambiguous breakpoint MIC of 0.25 mg/l allowed all strains to be classified as susceptible or tolerant. The equivalent figure for the disc diffusion data was 30 mm. Spearman's rank correlation analysis demonstrated a high degree of correlation ( r = 0.89) between the zone diameters and the log of the MIC values for members of the strain set. The breakpoints estimated in this work are compared to those estimated by other workers from data generated from different protocols. The issue of the clinical relevance of the breakpoints generated from the distribution of susceptibility data is discussed.

Two protocols designed to detect Aeromonas salmonicida, one based on an enzyme linked immunosorbent assay (ELISA) and one on a polymerase chain reaction (PCR), had previously performed well in extensive laboratory-based validation studies. These proxy methods were used together with direct culture to examine 223 kidneys of wild salmonid fish and 35 sediment samples collected from their habitat. Direct culture failed to detect the organism in any of the kidney samples. ELISA generated positive results from 23.3% of the kidneys and PCR from 20.6% but the degree of agreement between the two proxy methods was not greater than would have been predicted by chance (p>0.05). A total of 60% of the samples generating a positive response in ELISA were negative by PCR and 56% of PCR-positive samples were negative by ELISA. The overall concordance was, therefore, only 27%. With respect to the sediment samples, ELISA generated positive results from 53% and PCR from 33%. Again, the degree of agreement between the two proxy methods was not significantly greater than chance and the concordance was only 24%. The data generated by the two proxy methods investigated in this work was employed in a comparative validation of their application to the detection of A. salmonicida in these sample matrices. The hypothesis that both the PCR and ELISA protocols used in this work can validly be used in this way is clearly inconsistent with the data obtained. This analysis could not, however, discriminate between the hypotheses that neither of the methods possessed validity for these applications or that one of them possesses considerably greater validity than the other. The extensive laboratory-scale validation studies to which the two proxy methods used in this work have been submitted are discussed. It is argued that these laboratory studies provided no grounds for predicting the lack of validity that was detected when these proxy methods were applied to field samples. The results presented in this work provide strong evidence that laboratory-scale studies are not adequate to validate the application of proxy methods to field samples.

The survival of Aeromonas salmonicida was determined in sediment–water containing laboratory microcosms. Survival was estimated by calculating the decimal reduction times (DRT) of colony forming ability (cDRT) on Coomassie brilliant blue tryptone soya agar (TSA) (CBBA). Two sources of sediment and water were used in these experiments. One source, an outflow of a freshwater lake, produced sediments that were acidic and rich in humic acids. The other, a local river, gave sediments that were neutral and low in humic acids. The decimal reduction times in microcosms constructed from the humic acid-rich materials were of the order of 0.3 days, whereas those determined in microcosm, composed of river-derived materials, were approximately 40 days. This 100-fold difference in the decimal reduction times was demonstrated to be a function of the sediment source rather than the source of the water. The estimates of survival of A. salmonicida outside its host represent a critical element in any assessment of the risk of furunculosis transmission. The demonstration here, that such survival is highly matrix dependent, demonstrates that it is unlikely that a single figure could be used as an estimate of such survival in any formal quantitative risk assessment process.

Two laboratory microcosms, constructed of sterilised sediment and water taken from different locations, were seeded with Aeromonas salmonicida. A non-quantitative PCR, a heterogeneous, non-competitive, sandwich ELISA and colony formation were employed to generate survival data. In one microcosm, all three analytical methods generated positive results over the full 279 days of the incubation of the system at 18 °C. In the second microcosm, constructed with humic acid-rich material, colony formation was undetectable after 1 day but the PCR and ELISA methods generated positive results for up to 269 days. Direct injection into fish of material from the two microcosms both seeded with a passaged strain of A. salmonicida was used to determine the disease-producing potential of the bacteria in these systems. A direct correlation was observed between the presence of colony-forming units and the ability to produce furunculosis in test fish. None of the samples that generated positive PCR and ELISA signals but in which colony formation was not detected proved capable of inducing disease in fish. The significance of these data for the application of proxy methods in epizootiological studies is discussed.

An analysis is presented of some of the data available concerning the epizootics of furunculosis that occurred in commercial fish farms in Norway and Scotland between 1984 and 1995. The primary aim of these analyses was to establish the plausibility of the hypothesis that the introduction of oil-adjuvanted vaccines (OAVs) was the major factor that resulted in the ending of these epizootics. The limitations of the epizootiological data available are recognised and, as a consequence, the analyses are limited to a comparison of the time course of these epizootics and the timing of the introduction of OAVs. With respect to the Scottish data, the decline in the furunculosis epizootic was shown to have preceded the introduction of the OAVs. In Norway, there was a closer temporal coincidence between the introduction of the OAVs and the decline in the epizootic. There were, however, grounds for suspecting that at least some decline in the epizootic in Norway occurred before the introduction of OAVs in that country. It is argued that the assumption that OAVs were the cause of the decline in the furunculosis epizootics is unwarranted. It is also suggested that the general acceptance of the existence of this causal link has inhibited investigation of other factors involved in both the rise and the fall of these epizootics.

On 15 occasions, during and after a therapeutic treatment of fish in an Atlantic salmon farm, oxytetracycline concentrations were determined in samples of blue mussels collected at two sites in the vicinity of the farm. Oxytetracycline was not detected in any samples of mussels collected 20 m from the cage block at a depth of 1 m. Those mussels sampled from immediately under one cage (Cage 65) at the farm (10–11 m depth) contained a concentration of 10.2 μg oxytetracycline g−1 of soft tissue on the last day of treatment. After the end of oxytetracycline administration the concentration detected in these samples declined exponentially (r2 = 0.94) with a half-life of approximately 2 days. At the time of the treatment the farm was stocked with 144 tonnes of fish and 186 kg of oxytetracycline were administered during a ten day treatment. In muscle samples taken from fish (n = 5) in Cage 65, after 8 days of therapy the mean oxytetracycline concentration was 1.3 ± 0.9 μg g−1. Three days after the end of the therapy the top 2 cm of the sediments beneath Cage 65 contained 9.9 ± 2.9 μg g−1 (n = 5). These tissue and sediment concentrations lie within the ranges of values that have previously been reported for such treatments. It is argued that residues present in filter feeding bivalves that occur as a consequence of the therapeutic use of oxytetracycline in marine fish farms are unlikely to present a significant human health hazard.

The concentrations of oxytetracycline and the frequencies of oxytetracycline resistant microorganisms were determined in 11 samples taken from the sediments in the vicinity of a block of fish cages at a marine salmon farm. The cage block contained 10 tonnes of Atlantic salmon smolts and a total of 20 kg of oxytetracycline were administered during the 12 day treatment. Samples cores were collected by divers 5 days after the end of the period of therapy and the top 2 cm of each core was analysed. HPLC analysis was able to quantify the oxytetracycline concentrations in three of the six samples taken directly under the cage block. The mean concentration under the cage was between 0.65 and 1.2 μg g −1 ( n = 6) depending on the values attributed to samples where the concentrations were below the level of quantitation (1.2 μg g −1 ). In the five samples taken from locations not directly under the cage block oxytetracycline was only detected in the sample taken adjacent to, and down current from, the cage block. This sample was collected 10 m to the west of the cage block and contained 4.2 μg g −1 oxytetracycline. These data indicate that oxytetracycline was confined to an area of the sediment which was smaller in extent than the area of the cage block itself. The frequencies of resistance to oxytetracycline in the microflora cultured from the samples were determined by differential plating on 2216V media, containing 25 and 100 μg ml −1 oxytetracycline. Analysis of eighty three samples from sites free of overt human influence demonstrated that the background levels of resistance at these two selection concentrations were 1.3 ± 1.3% and 0.4 ± 0.6%, respectively. Elevated frequencies of resistance were detected in samples from a wider area than the cage block. The median frequency of resistance in the samples ( n = 6) taken from directly under the cage block was 1.4% at 100 μg ml −1 and 5.3% at 25 μg ml −1 . In the samples ( n = 5) taken from outside the cage block the frequencies were 5.3% at 100 μg ml −1 and 8.5% at 25 μg ml −1 . There was no correlation between the concentration of oxytetracycline in a sample and the frequency of resistance that was determined in the culturable microflora in that sample.

This paper reports the development of protocols for the bath administration of flumequine to Atlantic salmon (Salmo salar L.) smolts during their transfer from a fresh water hatchery to a marine farm in buckets suspended under a helicopter. The fish were treated at a density of 365 kg m−3 in water of pH 6.3 and hardness 13.2 mg 1−1 CaCO3. Serum flumequine concentrations showed a linear increase with respect to time of the bath and were dose dependent. The addition of benzocaine to the baths had little effect on uptake during the first 20 min of the bath. 720 000 smolts with covert stress inducible furunculosis were transported to 20 sea cages during 180 helicopter trips in buckets containing 100 μg ml−1 flumequine and 5.7 μg ml−1 benzocaine. Furunculosis was detected in fish in all sea cages shortly after transfer and the treatment was considered to have failed to control the activation of the covert infections. The pharmacokinetics of flumequine were determined in fish that were introduced to sea water at the termination of a bath treatment in fresh water. This data was compared to that obtained from fish that remained in fresh water following a similar treatment. The introduction of the fish into sea water resulted in a very rapid excretion of flumequine via the intestine. It is argued that this rapid elimination of the antimicrobial agent may have been an important factor in the failure of the bath treatment under commercial conditions.

Tanks (39 cm × 30 cm × 27 cm) containing marine sediment overlaid with various amounts of sterilised commercial fish food pellets and seawater were used to study the emergence of microorganisms capable of forming colonies on TSCA media in both the presence and absence of 25 μg/ml oxytetracycline. All experimental systems were free of oxytetracycline. Initial levels of culturable organisms in the sediments used in these experiments were 7.1 · 10 4 cfu/g of which 0.2% were capable of growth on the oxytetracycline-containing agar. In tanks containing no feed, the number of resistant cfu/g increased slightly, from 1.8 · 10 4 to 5.1 · 10 4 cfu/g, over 70 days incubation at 8.5–12 °C. In the tank containing low levels (1–2 cm depth) of feed, the increase in resistant organisms was from 4.9 · 10 3 to 2.3 · 10 4 cfu/g. There was no significant increase in the relative size of the resistant sub-population in either tank. In the tank containing high levels (16–17 cm depth) of fish feed, the levels of oxytetracycline-resistant cfu' s/g rose from below the limit of detection ( 1 ) to 1.9 · 10 8 and their relative abundance rose to 34% by the end of the experiment ( t = 70 days). The resistant flora isolated from the tanks with high levels of feed were characteristically slowgrowing and resistant to high levels (> 512 μg/ml) of oxytetracycline. Over 50% of these strains were insensitive to oxolinic acid, cotrimoxazole and furazolidone, but the frequency of sensitivity to ampicillin and chloramphenicol was high. In contrast, the flora isolated from the sediment used in all tanks exhibited more rapid growth and lower levels of resistance to oxytetracycline. These strains were more frequently sensitive to the other antimicrobial agents.

Concentrations of oxytetracycline and the frequency of oxytetracycline resistance in the environmental microflora were monitored following the therapeutic use of this agent at a marine fish farm. 529 kg of oxytetracycline were administered over a 24 day period at an average of 1.4 kg per cage per day. Three days after the end of the therapy 4.6 ± 3.7 μg/g oxytetracycline were detected in the sediments and the frequency of resistance in the sediment microflora was 9.0 ± 5.3%. A rise in the frequency of resistance in this flora to 26 ± 8.7% occurred 24 days after the therapy. This rise was not associated with any increase in the concentrations of oxytetracycline in the sediment. At this time the frequency of resistance in the flora isolated from mussels suspended above the sediments (36 ± 8.5%) was significantly ( P = 0.005) higher than that present in the sediment flora. The feed used on the farm 24 days after the end of therapy was shown to contain 4.6 × 10 4 oxytetracycline-resistant cfu/g. The distribution of phenotypic groups in the oxytetracycline-resistant flora in this feed and in the sediments during the peak in resistance were compared with those from other marine environments. These data demonstrated that resistant flora in feed can, under certain circumstances, significantly contribute to the resistant flora detected in sediments under fish cages.

the background level of resistance to oxytetracycline in sediments free of anthrogogenic influences was determined on 2216 V agar with 25 μg·g −1 oxytetracycline. The mean frequency of resistance in 153 samples taken in Galway Bay was 1.2 ±1.8%. The impact of oxytetracycline therapy on the frequency of resistance in the sediments under a marine fish farm was investigated on two occasions. In the first investigation, oxytetracycline was detected at a concentration of 9.9 ± 2.9 μg·g −1 in the sediments under a cage that received 865 g oxytetracycline per day for 10 days, but no significant rise in resistance frequency was detected. In the second investigation, oxytetracycline was detected at a concentration of 10.9 ± 6.5 μg·g −1 in the sediments under a cage block that received 175 kg oxytetracycline over 12 days. The frequency of resistance reached 16.0 ± 8.9% after the treatment. The frequency declined at an exponential rate ( r 2 =0.89) with a half-life of 26 days. At 73 days after the end of therapy the frequency, in under-cage samples, was not significantly higher than the background level. At the end of the therapy elevated frequencies of resistance were detected up to 75 m from the edge of the cage block and in samples where the levels of oxytetracycline were below the limit of detection (1.2 μg·g −1 ). Thirty-three days after the end of the therapy the frequency of resistance in all samples not directly under the cages was not significantly higher than in samples taken from sediments free of anthropogenic influence.

The concentration of oxytetracycline in the sediment under two adjacent cage blocks in a marine salmon farm was determined following the therapeutic use of the drug. The sediment cores were grey, indicating some build-up of organic material. Infaunal polychaetes were present as were mobile fauna including crabs, starfish and flat fish. There were no significant accumulations of undigested feed pellets under the cages. At one block where 186 kg oxytetracycline was used over 10 days the oxytetracycline concentrations were determined under a single cage that received 8.65 kg oxytetracycline during the treatment. Peak concentrations in the top 2 cm of the sediment were 9.9±2.9 μg·g −1 . This declined at an exponential rate ( r 2 =0.99) with a half-life of 16 days. At the second block the oxytetracycline concentration was measured with a sampling programme designed to determine the horizontal and vertical distribution of oxytetracycline under the whole cage block during and after a treatment where 175 kg of oxytetracycline were used over 12 days. Peak concentrations, in the top 2 cm of the sediments under the cage block, were 10.9±6.5 μg·g −1 and this declined at an exponential rate ( r 2 =0.99) with a half-life of 13 days. Nineteen days after the end of the therapy oxytetracycline was detected at depths of up to 8 cm in the sediment, but the concentration of the antibacterial agent had decreased at all levels in the sediment 14 days later. At the end of the treatment oxytetracycline was detected in an area of the sediment less than twice the area of the cages themselves. Data on current flow and sedimentation rate were used to generate a predictive model of the area of sediment that would be subject to the deposition of both pelleted fish feed and fish faeces. Oxytetracycline was confined to the area of sediment predicted to be subject to feed deposition that was directly under and slightly to the west of the cage block. Oxytetracycline was not detected in the area predicted to be subject to faecal deposition only.

Standard bacteriology and enzyme-linked imrnunosorbent assay (ELISA) were employed to detect Aeromonas salmonicida in samples of hdney, intestine and mucus from pre-smolt Atlantic salmon Salmo salar with stress-inducible furunculosis infections. No A. salrnonicida was isolated by bacteriological culture of samples from 30 unstressed salmon. In the same samples. ELISA detected antigen in 15 intestinal samples, but none in any of the kidney or mucus samples. The frequency of stress-inducible infections in a paired group of Atlantic salmon was 7/29 when the kidneys of the stressed fish were examined bacteriologically and 13/29 when ELISA results from kidney, intestinal content and mucus were combined. If ELISA results from kidneys alone were considered the frequency of detection was 8/20. In 2 separate experiments brown trout enrichment was used to detect A. salmonicida in pre-smolt Atlantic salmon with stress-inducible infections and compared to the standard stress test. The pathogen was detected in the combined mucus, fins and gills of 7/20 and 14/20 fish and in the blood of 3/20 and 2/20 fish respectively. The frequency of stress-inducible infections in paired groups of fish from the same populations was 10/20 and 16/20 respectively. These data suggest that the intestine may be the primary location of A. salmonjcida in salmon with stress-inducible infections and that colonisation of the mucus, fins and gills may also occur. The implications of these findings for the design of methods to eliminate such infections are discussed.