The in vitro antimicrobial activities of oxolinic acid, flumequine, sarafloxacin, enrofloxacin, and oxytetracycline against strains of bacteria pathogenic to fish (Aerononas salmonicida subsp. salmonicida, atypical A. salmonicida, Vibrio sabnonicida, Vibrio anguillarum, and Yersinia ruckeri) were determined at two different incubation temperatures, 4 and 15°C, by a drug microdilution method. The main objective of the study was to examine the effect of incubation temperature on the in vitro activities of 4-quinolones and oxytetracycline against these bacteria. When tested against A. salmonicida subsp. salmonicida, all of the quinolones examined had MICs two-to threefold higher at 4°C than at 15°C. Similarly, 1.5-to 2-fold higher MICs were recorded for all of the quinolones except sarafloxacin at 4°C than at 15°C when the drugs were tested against V. salmonicida. In contrast to those of the quinolones, the MICs of oxytetracycline were two-to eightfold lower at 4°C than at 15°C against all of the bacterial species tested. Of the antimicrobial agents tested against the bacterial species included in the study, enrofloxacin was the most active and oxytetracycline was the least active. Sarafloxacin was slightly more active than flumequine and oxolinic acid, especially against oxolinic acid-resistant A. salmonicida subsp. salmonicida strains.In recent years, the 4-quinolone antimicrobial compounds oxolinic acid and flumequine, in addition to oxytetracycline, and sulfadiazine-trimethoprim, have been the most frequently used antimicrobial agents in Norwegian aquaculture (18,23).Substantial development of oxolinic acid and tetracycline resistance in bacteria pathogenic to fish (2, 19, 29, 31) has led to a need for new antimicrobial drugs for control of bacterial fish diseases in aquaculture. Aminopenicillins, several sulfonamide-trimethoprim combinations, chloramphenicol analogs such as thiamphenicol and florfenicol, and potent fluoroquinolones have all been proposed and tested as potential new drug candidates (2,3,12,16,21,24,25,30).Several of the new fluoroquinolones generated during the last decade (32) show increased inhibitory (6, 15) and bactericidal (4) activities in vitro against many bacterial pathogens, compared with the old 4-quinolones, such as nalidixic acid and oxolinic acid. Studies have also revealed that the newer compounds show increased potency (2, 25), as well as more effective bactericidal activity (3, 21), against bacteria pathogenic to fish.Along the Norwegian coastline, there are significant seasonal and geographic seawater temperature variations, from just above zero in the winter to well above 18°C in the summer at the same site. The most common bacterial fish diseases in Norway, furunculosis, vibriosis, cold water vibriosis, bacterial kidney disease, yersiniosis, and infections caused by different strains of atypical Aeromonas salmonicida, may all occur at a wide range of temperatures.The general influence of temperature on the pharmacoki-
A total of 264 bacterial strains tentatively or definitely classified as Vibrio anguillarum were examined. The strains were isolated from diseased or healthy Norwegian fish after routine autopsy. With the exception of five isolates from wild saithe (Pollachius virens), the strains originated from nine different species of farmed fish. The bacteria were subjected to morphological, physiological, and biochemical studies, numerical taxonomical analyses, serotyping by slide agglutination and enzyme-linked immunosorbent assay, DNA-plasmid profiling, and in vitro antimicrobial drug susceptibility testing. The results of the microbiological studies were correlated to anamnestic information. The bacterial strains were identified as V. anguillarum serovar 01 (n = 132), serovar 02 (n = 89), serovar 04 (n = 2), serovar 08 (n = 1), and not typeable (n = 1) as well as Vibrio spkndidus biovar I (n = 36) and biovar II (n = 1), Vibrio tubiashii (n = 1), and Vibrio fischerii (n = 1). V. anguillarum serovar 01 or 02 was isolated in 176 out of 179 cases of clinical vibriosis in Atlantic salmon (Salmo salar). V. anguillarum serovar 01 was the only serovar isolated from salmonid fish species other than Atlantic salmon, while V. anguillarum serovar 02 was isolated from all marine fish suffering from vibriosis. A 48-Mda plasmid was isolated from all V. anguillarum serovar 01 isolates examined. Serovar 02 isolates did not harbor any plasmids. Resistance against commonly used antibiotic compounds was not demonstrated among V. anguillarum isolates. Neither V. splendidus biovar I nor other V. anguillarum-related species appeared to be of clinical importance among salmonid fish. However, such bacteria were isolated from diseased turbot (Scopthalmus maximus) and sea bass (Dicentrarchus labrax). The precise role of these bacteria as fish pathogens has to be elucidated. Cell and culture morphology, cell motility, hemolysis patterns, and especially alginate-degrading ability were found to be suitable characteristics for the differentiation of V. splendidus biovar I within the arginine decarboxylase-positive group of Vibrio species. Luminescence could not be demonstrated among V. splendidus isolates. Vibriosis due to Vibrio anguillarum is one of the most important bacterial infections in fish throughout the world (1, 19). The disease has great importance, particularly for marine fish farming (4, 15, 55). Infections have been reported also among farmed bivalve mollusks and crustaceans (11, 12). V. anguillarum and closely related bacterial species are commonly found in estuarine and coastal marine habitats and can readily be isolated from different environmental sources (31-34, 52, 65, 67). These bacteria constitute part of the normal microflora of healthy marine fish (34, 41, 45). On the basis of biochemical and serological differences, two distinct biotypes of V. anguillarum were described (6, 8, 19). Biotype 2 was later differentiated as a new species, Vibrio ordalii (54). A total of 10 different 0 serovars (01 to 010) and additional 0 subgroup...
The present authors report the first description of a bacterial disease, Varracalbmi, recorded sporadically among seawater‐reared Atlantic salmon in northern Norway between 1989 and early 1992. The causative organism, a Gram‐negative, non‐motile, capsule‐forming, oxidase‐positive and facultatively anaerobic rod, has not previously been described. The mean water temperatures during the outbreaks varied from 2.5 to 8 °C. The disease was characterized by haemorrhagic and necrotizing pyogranulomatous inflammation which targeted the eye and resulted in panophthalmitis. Accompanying lesions included deep dermal ulcerations, necrotizing and haemorrhagic pyogranulomatous lesions in the visceral organs, and necrosis of the pseudobranch. The proposed name of the disease, Varracalbmi, is the Lappish name for ‘bloody eye’.
An in vitro susceptibility assay of sarafloxacin (A-56620), a new 4-quinolone, was performed against five important bacterial species that are pathogenic to fish. A collection of 44 clinical isolates and five corresponding type strains were included in the study. The objectives were to determine the minimal inhibitory concentrations (MICs) of sarafloxacin by a drug microdilution method and to compare the MIC values at two different temperatures, 4 and 15°C Sarafloxacin was active against all species tested and showed the following mean MIC values at 15 and 4°C, respectively, against the bacterial pathogens investigated: Aeromonas salmonicida subspecies salmonicida, 0.029 and 0.045 Mg/mL; atypical A. salmonicida. 0.053 and 0.041 Mg/mL; Vibrio anguillarum, 0.085 and 0.054 Mg/mL; V. salmonicida, 0.125 and 0.123 Mg/rnL; and Yersinia ruckeri, 0.023 and 0.031 ng/mL. The MICs ranged from 0.0025 /ig/mL (or less) for two strains of A. salmonicida salmonicida to 0.32 Mg/mL for one strain of atypical A. salmonicida and one strain of V. anguillarum. A decrease in antimicrobial activity was observed as the incubation temperature was lowered from 15 to 4°C; however, no significant statistical difference between the measured values was demonstrated.
An unusual form of bacterial gill disease (BGD) was identified which affected five species of cultured salmonids from Canada (i.e. rainbow trout, chinook salmon and Atlantic salmon), Norway (i.e. brown trout) and Chile (i.e. coho salmon). All outbreaks occurred at low water temperatures (< 10 °C) and with clinical presentations distinct from classical BGD, which is caused by Flavobacterium branchiophilum. In contrast to classical BGD, fish did not show marked respiratory distress with flaring of the opercula, the animals did not orientate at the surface of the water column near inflow water or at the margins of the tanks, and the feed response of the fish was varied. While mortality was increased, it was not precipitous as in classical BGD. Eight outbreaks were examined in greater detail using histopathology, scanning electron microscopy, bacteriology and immunohistochemistry. Large numbers of small bacterial rods were seen adhering to the lamellar epithelium of affected gills from all outbreaks. Histologically, the lamellar epithelium appeared swollen, often with evidence of single cell degeneration and exfoliation. In more severe instances, the formation of lamellar synechiae was seen, usually associated with sequestration of bacteria between fused lamellae. By contrast with typical BGD, overt epithelial hyperplasia, lamellar fusion and filamental clubbing were not common sequelae to infection; instead, the end result was shortened and somewhat stubby lamellae covered with swollen epithelial cells. The predominant bacterium recovered from affected gills was a small, Gram‐negative, motile, fluorescent pigment‐producing rod that shared phenotypic characteristics with Pseudomonas fluorescens. Polyclonal antisera prepared against three representative isolates indicated a weak antigenic similarity among them. Immunohistochemistry corroborated this finding, in that the antisera reacted strongly with gill sections containing the homologous bacteria, but not against morphologically similar bacteria in heterologous sections. A Gram‐negative, yellow pigmented bacterium (YPB), identified as Flavobacterium psychrophilum, was also recovered, but only from the gills in the Ontario outbreaks. Antiserum prepared against this YPB indicated an antigenic similarity among isolates recovered from the Ontario outbreaks, but immunohistochemistry failed to recognize antigenically related bacteria on the gills of fish from the other outbreaks. Based on the unusual clinical presentation and the histopathological appearance of the gills, in conjunction with the absence of filamentous bacteria associated with and recovered from affected gills, the present authors have called this condition ‘atypical bacterial gill disease’ or ABGD.
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