[show abstract][hide abstract] ABSTRACT: Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.
Journal of Fish Diseases 03/2012; 35(3):193-201. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flavobacterium psychrophilum infections cause high mortality among rainbow trout, Oncorhynchus mykiss, fry in Danish fish farms and hatcheries. Hatcheries based entirely on bore-hole water recirculation systems have been suggested as a possibility for eliminating F. psychrophilum or at least keeping the amount of this bacterium low. The occurrence of the bacterium in a bore-hole water recirculation system was compared with a combined bore-hole water and stream water flow-through system in a hatchery where outbreaks of rainbow trout fry syndrome caused by F. psychrophilum often occurred. Broodfish, unfertilized and fertilized eggs, eyed eggs and fry, as well as water samples from the tanks/troughs with broodfish/fry, were examined. Suspect yellow bacterial colonies were either confirmed or rejected as F. psychrophilum by growth characteristics and by PCR. As both virulent and less virulent F. psychrophilum isolates are known, isolates were characterized. The isolates were ribotyped and grouped according to ribotyping patterns. Representatives of the groups were serotyped. Fry isolates were very homogeneous whereas isolates from broodfish were heterogeneous, whether the isolates originated from external surfaces of the fish (mucus from skin and gills, haemorrhages and ulcers) or internal organs. Flavobacterium psychrophilum was isolated from broodfish in both water systems; 56% of investigated broodfish from the borehole/flowthrough system and 36% from the recirculation facility harboured the bacterium. In the recirculation system, the bacterium was isolated from fish (ulcers, milt, liver, abdominal cavity) kept in the system for 11 months. Flavobacterium psychrophilum was found in milt and ovarian fluid as well as on the surface of fertilized eggs, but not inside the eggs. Fry also harboured F. psychrophilum, but in the water recirculation system the bacterium was first isolated from the fry after they had been graded. Flavobacterium psychrophilum was found regularly in other parts of the hatchery (outside the recirculation facility), including at the time of grading, suggesting that the occurrence of F. psychrophilum in the fry recirculation facility was due to contamination from the borehole/flow-through hatchery. It is suggested that the combination of bore-hole water recirculation systems and good management procedures (including egg disinfection) is a possible method for hatcheries to avoid disease outbreaks due to F. psychrophilum.
Journal of Fish Diseases 12/2008; 31(11):799-810. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many pathogens control production of virulence factors by self-produced signals in a process called quorum sensing (QS). We demonstrate that acyl homoserine lactone (AHL) signals, which enable bacteria to express certain phenotypes in relation to cell density, are produced by a wide spectrum of Aeromonas salmonicida strains. All 31 typical strains were AHL producers as were 21 of 26 atypical strains, but on a strain population basis, production of virulence factors such as protease, lipase, A-layer or pigment did not correlate with the production and accumulation of AHLs in the growth medium. Pigment production was only observed in broth under highly aerated conditions. Quorum sensing inhibitors (QSIs) are compounds that specifically block QS systems without affecting bacterial growth and 2 such compounds, sulphur-containing AHL-analogues, reduced production of protease in a typical strain of Aeromonas salmonicida. The most efficient compound N-(heptylsulfanylacetyl)-L-homoserine lactone (HepS-AHL), reduced protease production by a factor of 10. Five extracellular proteases were detected on gelatin-containing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and 3 of these were completely down regulated by HepS-AHL. Hence, QSIs can curb virulence in some strains and could potentially be pursued as bacterial disease control measures in aquaculture.
Diseases of Aquatic Organisms 01/2008; 78(2):105-13. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To profile the quorum-sensing (QS) signals in Yersinia ruckeri and to examine the possible regulatory link between QS signals and a typical QS-regulated virulence phenotype, a protease.
Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) showed that Y. ruckeri produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-L-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-L-homoserine lactone (3-oxo-C9-HSL), were produced. 3-oxo-C8-HSL was detected in organs from fish infected with Y. ruckeri. Protease production was significantly lower at temperatures above 23 degrees C than below although growth was faster at the higher temperatures. Neither addition of sterile filtered high-density Y. ruckeri culture supernatant nor the addition of pure exogenous AHLs induced protease production. Furthermore, three QS inhibitors (QSIs), sulfur-containing AHL analogues, did not inhibit protease production in Y. ruckeri.
Exogenous AHL or sulfur-containing AHL analogues did not influence the protease production indicating that protease production may not be QS regulated in Y. ruckeri.
The array of different AHLs produced indicates that the QS system of Y. ruckeri is complex and could involve several regulatory systems. In this case, neither AHLs nor QSI would be likely to directly affect a QS-regulated phenotype.
Journal of Applied Microbiology 03/2007; 102(2):363-74. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the study was to characterize 30 unrelated tetracycline resistant Aeromonas spp. from fish farms where none of the tetracycline resistance genes tet(A–E) were detected by multiplex PCR in a previous study. By cloning, sequencing and designing alternative PCR primers the tetracycline resistance gene tet(E) was found in (27/30) 90% of the previously negatively tested Aeromonas spp. The strains originated from farmed fish, water and sediment in Denmark (n=27) and Canada (n=3). The tet(E) gene was in several cases located on large horizontally transferable plasmids (approx. 150 kb) capable of interspecies transfer to Escherichia coli. This is the first report of horizontally transferable tet(E). Additionally, 15 motile Aeromonas strains with previously identified tetracycline resistance genes were tested, and this on one hand verified former findings of tet(E), but also identified more than one tet gene in some strains.The use of multiplex PCR for detection of tet(E) should be done with caution. tet(E) seems to occur frequently in Aeromonas spp. from fish farms and may spread horizontally among bacteria in the aquatic environment.
[show abstract][hide abstract] ABSTRACT: A total of 133 strains of Aeromonas salmonicida ssp. salmonicida, isolated from a wide variety of sources, were characterized by pulsed-field gel electrophoresis patterns. Sixteen profiles were demonstrated, with one profile being predominant in samples from all the countries and species of fish. Our results suggest a clonal distribution of this subspecies, with a predominant clone being responsible for most of the outbreaks worldwide.
[show abstract][hide abstract] ABSTRACT: The plasma concentrations of florfenicol (FF) were measured during a 10-day oral administration of this agent to fish in a cage in a commercial salmon farm. The therapy was initiated as part of a management response to an outbreak of winter ulcer disease associated with a Moritella viscosa infection. Twenty healthy, 20 moribund and 10 dead fish were sampled 24 h after the administration of medicated feed on the eighth day of the therapy. Quantifiable concentrations of FF were detected in all 20 healthy fish with a mean plasma concentration of 3.0 ± 1.8 mg/ml. These data are consistent with previously published data and with parallel laboratory trials performed in this work. The mean weight of these healthy fish was 324 ± 89 g but M. viscosa was isolated from only one of them. With respect to the moribund fish, FF could not be detected in 18 of the 20 fish analysed and the mean weight of these fish was 196 ± 48 g. M. viscosa was isolated from 19 of the 20 moribund fish.The mean MIC value for FF against 14 of the M. viscosa isolated was 0.24 mg/l and it is argued that, in the context of this therapy, these bacteria should probably be classified as sensitive to FF. The analysis of the patterns of mortality before, during and after the period of therapy did not provide any unambiguous evidence that the therapy was, however, effective in reducing death in the cage population.The data generated in this work and in a previous study of winter ulcer disease is used to develop a tentative model of the epizootiology of this disease in post-smolts. In both studies there was a strong correlation between the weights of fish and their health. This suggests that, within a cage population, infection by M. viscosa, ulceration and death are largely confined to a sub-population that has adapted poorly to the environment of the marine cage. Further, as this poorly adapted sub-population is not feeding, it is unlikely that orally administered antimicrobial therapy would represent a cost-effective way of limiting losses.
[show abstract][hide abstract] ABSTRACT: Forty strains of Flavobacterium psychrophilum were tested for the production of siderophores using the universal Chrome Azurol S (CAS) assay. The majority of the strains (85%) were CAS positive (CAS+) and some (15%) were CAS negative (CAS-). The cryptic plasmid pCP1 was carried by all positive strains and was lacking from negative strains. While a weak catechol reaction was detectable in CAS+ culture supernatants, the CAS reaction was, to some extent, heat sensitive, questioning whether the positive reaction was caused only by siderophores. The ability to grow in vitro under iron-restricted conditions did not correlate with the CAS reactivity, as growth of both CAS+ and CAS- strains was similarly impaired under iron restriction induced by 2,2 dipyridyl. Suppressed growth under these conditions was restored by addition of FeCl3, haemoglobin and transferrin for both CAS+ and CAS- strains.
Journal of Fish Diseases 08/2005; 28(7):391-8. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the present study was to investigate the production of quorum sensing signals (specifically acylated homoserine lactones, AHLs) among a selection of strains of Gram-negative fish bacterial pathogens. These signals are involved in the regulation of virulence factors in some human and plant-pathogenic bacteria. A total of 59 strains, representing 9 different fish pathogenic species, were tested against 2 AHL monitor bacteria (Agrobacterium tumefaciens NT1 [pZLR4] and Chromobacterium violaceum CV026) in a well diffusion assay and by thin-layer chromatography (TLC). Representative samples were further characterized by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HR-MS). AHLs were produced by all strains of Aeromonas salmonicida, Aeromonas hydrophila, Yersinia ruckeri, Vibrio salmonicida, and Vibrio vulnificus. Some strains of atypical Aeromonas salmonicida and Vibrio splendidus were also positive. Aeromonas species produced N-butanoyl homoserine lactone (BHL) and N-hexanoyl homoserine lactone (HHL) and 1 additional product, whereas N-3-oxo-hexanoyl homoserine lactone (OHHL) and HHL were detected in Vibrio salmonicida. N-3-oxo-octanoyl homoserine lactone (OOHL) and N-3-octanoyl homoserine lactone (OHL) were detected in Y. ruckeri. AHLs were not detected from strains of Photobacterium damselae, Flavobacterium psychrophilum or Moritella viscosa. AHLs were extracted from fish infected with Y. ruckeri but not from fish infected with A. salmonicida. In conclusion, the production of quorum sensing signals, AHLs, is common among the strains that we examined. If the AHL molecules regulate the expression of the virulence phenotype in these bacteria, as shown to occur in some bacterial pathogens, novel disease control measures may be developed by blocking AHL-mediated communication and suppressing virulence.
Diseases of Aquatic Organisms 07/2005; 65(1):43-52. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.
Journal of Fish Diseases 02/2005; 28(1):39-47. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plasma oxolinic acid (OXA) concentrations were measured in fish from a cage of farmed rainbow trout (Oncorhynchus mykiss) 1 day after the termination of medication. The fish were experiencing significant mortalities and following a diagnosis of vibriosis, OXA had been orally administered at 50 mg/kg for 6 days over a 9-day period. Samples from healthy fish (n=20), moribund (n=26) and dead fish (n=10) were analysed by HPLC. There was a dramatic difference in the OXA concentrations between healthy and moribund fish. In the moribund group, none of which showed signs of recent feeding, 85% of the fish had OXA levels below the LOQ (0.005 mg/l). In contrast, 95% of the healthy fish had OXA concentrations >0.015 mg/l and the mean OXA concentration (±standard deviation) was 0.156±0.152 mg/l.The mean OXA concentrations detected in the healthy fish in the farm were similar to those achieved in 30 laboratory held rainbow trout (O. mykiss) following the administration of OXA under similar conditions of salinity, temperature and dosing regimen. In these laboratory held fish, the mean plasma OXA concentration was 0.133±0.068 mg/l. The major difference between the distributions of OXA concentrations in the farm and laboratory populations was in the extent of fish to fish variation observed. In the healthy farmed fish, the percentage coefficient of variation (%CV) was 97% compared to a %CV of 51% in the laboratory held fish.The patterns of the daily mortality in the farmed population were analysed from 20 days before the initiation of therapy to 20 days after its completion but these data failed to provide unambiguous evidence as to the success or other wise of the therapeutic intervention. The moribund fish examined at the end of the therapy showed signs of systemic disease but from the majority, no bacteria were isolated. Strains of Vibrio anguillarum were isolated from some dead and moribund fish and these had a MIC of 0.0625 mg/l. It was, therefore, not possible to use the data generated in this work on the OXA plasma concentrations, the efficacy of the therapy and the MIC values of the infecting bacteria, to investigate the validity of any formula for estimating breakpoint MIC values.
[show abstract][hide abstract] ABSTRACT: Plasma concentrations of oxytetracycline (OTC) were established in two Atlantic salmon (Salmo salar) pre-smolts populations after they had received OTC medicated feed at a rate of 75 mg OTC/kg over 10 days. One population was experiencing an epizootic of furunculosis in a commercial freshwater farm and the other was held in a laboratory. Both populations were maintained at approximately 13 °C. The mean plasma concentration in 26 health farm fish was 0.25±0.06 and the 80th percentile was 0.21 mg/l. The mean concentration for 26 laboratory fish was 0.21±0.06 mg/l with an 80th percentile of 0.15 mg/l.The validity of setting a breakpoint minimum inhibitory concentration (MIC) at a quarter of these plasma concentrations was investigated. The MIC of the Aeromonas salmonicida isolated from the farmed fish (n=7) was 0.5 mg/l and the breakpoints generated by application of the 4:1 ratio were in the range 0.03125–0.0625 mg/l. These breakpoint values would, therefore, predict that the therapy should have had no beneficial effect and that any strain of A. salmonicida with MIC>0.0625 mg/l must be considered as resistant. A consideration of the pattern of the mortalities before and during the period of therapy suggests that the therapy was probably beneficial. Thus, the data obtained in this work suggest that the application of the 4:1 ratio is not a valid method of generating meaningful breakpoint MIC values.Published values for the MIC of OTC against A. salmonicida and the plasma concentrations achieved after oral administration of OTC medicated feed were applied to investigate the validity of the application of the 4:1 ratio. Breakpoints generated by the application of this ratio to these data would suggest that OTC could never have had any value in combating A. salmonicida infections. As this conclusion is contrary to experience, it is argued that examination of the published data reinforces the conclusion that the 4:1 ratio has little value in the oral therapy of fish disease.
[show abstract][hide abstract] ABSTRACT: Concentrations of oxolinic acid (OXA) were measured in the plasma, muscle, liver, and kidney of 48 Atlantic salmons (Salmo salar) 1 day after the end of an oral administration. OXA was administered over a period of 13 days to control an outbreak of winter ulcer disease in a commercial marine farm. On the basis of their behaviour, the fish were classified as healthy (n=18), moribund (n=20), or dead (n=10). There was a dramatic difference in the OXA concentrations in the healthy fish and those classified as moribund or dead. There was no evidence of bacterial infection in the 18 healthy fish, all of which were shown to be feeding. In these fish, the mean concentrations of OXA (±standard deviation) in the plasma, muscle, liver, and kidney were 0.40±0.36 mg/l, 1.0±0.71 mg/kg, 0.93±0.67 mg/kg, and 1.13±0.87 mg/kg, respectively. Within the healthy group, there were considerable individual fish-to-fish variations in OXA concentrations and the mean coefficient of variation (CV) for the concentrations in the four tissues was 77%. In contrast, all 20 moribund fish showed external lesions and 19 showed signs of systemic infection. Only 2 showed signs of feeding, and the concentrations of OXA were below the limit of quantitation (LOQ) in 68%, 85%, 70%, and 80% of the plasma, muscle, liver, and kidney, respectively. These data suggest that the major function of the therapy was to assist healthy fish to resist de novo infection and that moribund fish had gained little or no benefit from the oral administration of OXA.A numerical description of the concentration of the antimicrobial agent achieved in therapy is necessary to determine the resistance or sensitivity of the bacteria involved in the infection. The degree of fish-to-fish variation in the concentrations of OXA, both within the healthy fish and between healthy and moribund fish, presents difficulties in generating a clinically meaningful description relevant to the whole population. This issue is discussed, and it is suggested that for this application, the minimum concentration achieved by at least 80% of the treated population might represent a useful parameter for describing the concentrations of agents achieved during therapy.The plasma data from this investigation were used to estimate clinically relevant breakpoint minimum inhibitory concentration (MIC) values. The validity of these breakpoint values was discussed with reference to the outcome of the therapy and the susceptibility of the bacteria isolated from infected fish.
[show abstract][hide abstract] ABSTRACT: The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum
Fish & Shellfish Immunology 12/2003; 15(5):387-95. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.
Applied and Environmental Microbiology 10/2003; 69(9):5275-80. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: The medication effect of oxytetracycline on groups of rainbow trout fry experimentally infected with three strains of Flavobacterium psychrophilum was investigated. The infection model was based on intraperitoneal injection of the pathogen and treatment was done using medicated feed resulting in 100 mg oxytetracycline/kg fish for 10 days. The three F. psychrophilum strains had different antimicrobial susceptibilities and successful treatment was only obtained in the trial using a strain with a MICOTC of 0.25 μg/ml. No effect of treatment was seen in the group infected with a strain having MICOTC of 8.0 μg/ml and only little effect was seen when the strain MICOTC was 4.0 μg/ml. This shows that it is valid to predict the treatment efficiency of OTC from in vitro data facing an outbreak of rainbow trout fry syndrome. The importance of doing susceptibility testing is emphasized, and as shown the selection of media for antimicrobial susceptibility testing of F. psychrophilum is important.
[show abstract][hide abstract] ABSTRACT: Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.
Diseases of Aquatic Organisms 12/2002; 52(2):109-18. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.
Fish & Shellfish Immunology 03/2002; 12(2):141-53. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908-4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had "empty" integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetA positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids and tetA among the OTC-resistant aeromonads, tetE and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.
Applied and Environmental Microbiology 01/2002; 67(12):5675-82. · 3.68 Impact Factor