Search

Experimental work done with the immunization against several fish pathogens is described. The agents studied included Flexibacter columnaris, Aeromonas salmonicida, Vibrio anguillarum and Infectious Hematopoietic Necrosis Virus (IHNV). At the present, it appears that vaccines prepared against IHNV and bacterins against V. anguillarum show potential for use in aquaculture.

Four bacteria commonly associated with diseases of fish (Aeromonas hydrophila complex, Aeromonas salmonicida, Flexibacter columnaris-like organisms and Pseudomonas fluorescens) were evaluated in a serial dilution system for susceptibility to four antibiotics commonly used in the treatment of bacterial fish diseases (chloramphenicol, erythromycin, furpyrinol and oxytetracycline). Findings were expressed as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Furpyrinol was found to have the lowest MIC and MBC against A. hydrophila complex, A. salmonicida and F. columnaris-like organisms. MIC and MBC's for oxytetracycline were complicated by the presenceof R-plasmids, but were less than 4.0 mcg/ml for those without plasmids. Pseudomonas fluorescens was susceptible only to oxytetracycline. MIC for all groups of organisms using chloramphenicol was generally less than 16.0 mcg/ml. Susceptibility to erythromycin by these groups of organisms was greater than 16 mcg/ml.

The study examined pathological changes to the gill structure caused by bacterial gill disease infection, and consequent alterations in cardiac performance. Experimental transmission of the disease was successfully obtained by exposing 40 healthy eels to 5 × 107 cells/ml of cultured Flexibacter columnaris for 2 h. Hyperplasia of the gill epithelium and successive fusion and degeneration of the secondary gill lamellae were observed. Significant changes in heart rate were closely related to the degree of impairment of gill blood circulation. During the first day after exposure, a noticeable bradycardia was registered together with the formation of a hyperplastic gill epithelium. The successive degenerative processes of the secondary gill lamellae (fusion, edema, and blood congestion) resulted in the establishment of a compensatory tachycardia during the second and following days. The extensive literature available on bacterial gill disease points out that the heavy mortality in infected fish is caused by impeded oxygen uptake by the gills. Nevertheless, this may not be the only factor causing death. The results of this investigation suggest that the impaired gill vascular blood circulation also causes severe changes in heart performance. Owing to the close relationship between gill blood circulation and cardiac performance, it may be hypothesized that the interaction of the two phenomena results in the death of fish.

A carp disease, initially diagnosed as carp erythrodermatitis (CE), has occurred enzootically since 1972 at a hatchery near Lelystad, The Netherlands. Gliding bacteria belonging to the Cytophaga family were repeatedly isolated from external lesions in affected carp. These isolates were examined, and compared with reference strains of Flexibacter columnaris, formerly known as Chondrococcus columnaris or Cytophaga columnaris. Comparative tests included cell morphology and motility, growth under various environmental conditions, physiological characteristics, agglutination, determination of DNA base composition and antibiograms. All carp isolates that produced strongly rhizoid colonies on basal agar medium were identified as Flexibacter columnaris. Freshly isolated strains of F. columnaris were pathogenic to carp, producing ulcers surrounded by a red zone, and the bacteria were re-isolated from infected fish. For these reasons the carp disease at Lelystad hatchery is diagnosed as columnaris disease (Davis, 1922); an earlier diagnosis of carp erythrodermatitis (Bootsma, 1973) should be reconsidered. Further investigations, not reported in this paper, have confirmed that carp erythrodermatitis and columnaris disease are separate diseases with different aetiologies.

The survival of Flexibacter columnaris was studied in a variety of waters containing different concentrations of 4 cations. The survival of F. columnaris over a period of 7 days was found to be optimal at the concentrations of 0.03% NaCl, 0.01% KCl, 0.002% CaCl2·2H2O, and 0.004% MgCl2·6H2O, respectively. The best survival was obtained in the formulated water medium containing the combination of the four cations at their respective level of optimum concentration. The survival of F. columnaris in the formulated water was very high and almost similar to the survival in tap water; whereas, distilled water did not support the long-term survival of F. columnaris. The survival of F. columnaris in the media containing single salt at their optimal concentrations were similar to each other but notably lower than the survival in the formulated water and tap water.

Antibody synthesis, in response to vaccination with a 0.1-ml (1.8 × 108 cells/ml) intraperitoneally injected, heat-killed strain of Flexibacter columnaris, was employed to investigate the effect of tritium irradiation (0, 0.04, 0.4, 4.0, 40 rads total dose for 20 days during embryogenesis) on development of the primary immune response in 5-mo rainbow trout, Salmo gairdneri, reared under essentially pathogen-free conditions. Specific agglutinins to F. columnaris, determined 1-wk pre vaccination, and 3, 5, 7, 9, and 11 wk postvaccination increased rapidly in both control and irradiated fish following vaccination. Agglutinin levels in irradiated fish were suppressed to 50% of control levels at 40 rads during the 9th wk, and 50% of control levels at 4.0 rads during the 11th wk. Electrophoretic separation of serum proteins of both control and irradiated–vaccinated fish demonstrated four major protein fractions. Densitometry analyses demonstrated that fraction IV increased significantly in percent of total protein following antigenic stimulation, suggesting that fraction IV represents the specific humoral antibody to F. columnaris. The relative percent of total protein contained in fraction IV was significantly reduced in irradiated–vaccinated fish. Key words: antibody synthesis, agglutination assay, vaccination, Flexibacter columnaris, tritium, Salmo gairdneri, electrophoresis, serum protein, densitometry, primary immune response

Gill necrosis is a widespread disease of common carp in Europe. Much effort has been concentrated in the investigation of its aetiology during the last 15 years. The results of a three-year research project are presented here and the role of bacteria is demonstrated. This paper shows the development of the disease and the relationship between environmental stress and bacterial invasion. Both healthy and diseased carp populations were monitored for the presence of Flexibacter columnaris . Gill necrosis was reproduced experimentally using Flexibacter columnaris isolates. Based on the bacteriological investigations, artificial infection and field monitoring, three stages of gill necrosis could be distinguished. The first stage is initiated and maintained by environmental stress (probably ammonia, pH, temperature or any toxins). At this stage, Flexibacter columnaris is seldom present on the gills if at all. The gills are pale or dark purple. The second stage starts when Flexibacter columnaris invades the damaged gill (only at water temperatures above 20°C). The infected part of the gill is necrotized and covered with a grey-white coating. The third stage occurs if the fish survives bacterial invasion. Flexibacter columnaris with its grey-white coating disappears and the site of the invasion remains distorted. Different stages of gill necrosis may appear in the same fish population.

Carotenoid pigments were purified from a previously undescribed, red, halophilic spirochete (spirochete RS1), and from Spirochaeta aurantia strain J1. Both spirochetes are facultative anaerobes and produce pigments when growing aerobically. The major pigments of the two spirochetes were identified by means of chromatographic analysis, absorption spectroscopy, hydride reduction, acetylation and silylation experiments, and mass spectrometry. It was concluded that the major pigment from spirochete RS1 was 4-keto-1',2'-dihydro-1'-hydroxytorulene. This conclusion was further supported by infrared spectroscopy and additional analytical data. The evidence showed that the major pigment from S. aurantia was 1',2'-dihydro-1'-hydroxytorulene. Chromatographic and spectrophotometric evidence indicated that this pigment was also present, as a minor carotenoid component, in spirochete RS1. These pigments have been previously detected almost exclusively in gliding bacteria, such as species of Flexibacter, Stigmatella, and Myxococcus. The occurrence of 4-keto-1',2'-dihydro-1'-hydroxytorulene and 1',2'-dihydro-1'-hydroxytorulene in both spirochetes and gliding bacteria may have significance with respect to the evolutionary development of these organisms.

The combination of pasteurization and filtration provided reliable, sterile, natural seawater and estuarine water confirmed by sterility tests. Filtering seawater from the Gulf Stream through 0.1 μm pore membrane filter with 0.45 μm prefilter was not an effective method of sterilization. Flexibacter sp. readily passed through the filters. Pasteurization of the water for 2 h at 70°C before filtration eliminated the Flixibacter contamination. Both manipulations resulted in an increase of dissolved organic carbon, dissolved organic and inorganic nitrogen. Dissolved reactive silicon increased if the pasteurization was accomplished in glass vessels.

The structures assigned to the two Flexibacter pigments flexixanthin (3,1′-dihydroxy-3′,4′-didehydro-1′,2′-dihydro-β,ψ-caroten-4-one) and deoxyflexixanthin (1′-hydroxy-3′,4′-didehydro-1′,2′-dihydro-β,ψ-caroten-4-one) have been confirmed by synthesis of the latter, and of dehydroflexixanthin (3,1′-dihydroxy-2,3,3′,4′-tetradehydro-1′,2′-dihydro-β,ψ-caroten-4-one), the autoxidation product of the former.

Six media commonly used for cultivation of Flexibacter columnaris were divided into two categories based on the presence or absence of certain salts. Growth was limited in Cytophaga, tryptone yeast extract or tryptone yeast infusion medium, none of which contain salts. Growth responses in Chase, Shieh and Liewes media containing salts provided improved cell yields and the Shieh and Liewes preparations were best. Comparisons of generation time were made between cells grown in Shieh and Liewes media. The generation time of F. columnaris, strain 238 grown in the Shieh broth was 147 min compared to 206 min in the Liewes broth. Glucose, pyruvate and citrate were not required for the cultivation of F. columnaris.

The ability of Flexibacter BH3 to adhere to solid surfaces and to overcome the horizontal drag involved in gliding across the surfaces was considered in terms of the Stefan adhesion principle. The extracellular slime produced by Flexibacter BH3 was suitable as a Stefan adhesive because it exhibited viscous properties characteristic of a linear colloid, increasing the adhesiveness of the bacterium but allowing translational motion across the surface. The water-soluble slime was a glycoprotein, containing glucose, fucose, galactose and some uronic acid. Vesicles and tubules on the outer surface of Flexibacter BH3 possessed trilaminar membranes, contained 2-keto-3-deoxyoctonate (KDO), and showed identity with phenol-extracted lipopolysaccharide (LPS) in gel-diffusion tests. Sections of Flexibacter BH3 gliding on a gold film overlaying an agar medium reveraled a highly convuluted cell envelope outer membrane, portions of which closely conformed to the microcontours of the gold surface. Possible mechanisms of gliding are discussed in relation to this close association with solid surface features, to the finding that flexibility and spiral motion are not essential for gliding, and to evidence revealing the extrusion of slime in advance of “pathfinder” bacteria.

Direct fluorescent antibody technique (FAT) was used in the detection of Flexibacter maritimus in experimentally infected black sea bream fry, Acanthopagrus schlegeli, by smear plus immersion. The sensitivity of the technique was compared with the conventional media culture method. Tissue sections of the skin, gill, liver and kidney were stained with fluorescent antibody. By culture method, tissues were separately homogenized, serially diluted and plate counted using Cytophaga agar in 100% seawater. Fluorescent antibody technique detected F. maritimus in all tissues from 15 min until all experimental fish died after 48 h. Whereas by culture method, the bacteria was detected in the skin only from 15 min to 3 h, in the gills from 1 h to 12 h, and in the internal organs from 15 min to 12 h but could not be detected thereafter because of the proliferation of other bacteria. Thus, FAT proved to be a more useful technique than the plate culture method in the diagnosis of induced Flexibacter infection in black sea bream fry.

Channel catfish, Ictalurus punctatus, continuously exposed to 6 mg/L (ppm) nitrite and intraperitoneally injected with Aeromonas hydrophila had lower bacterial median lethal doses (LD50) than control fish. The clearance rate of injected A. hydrophila was reduced in fish chronically exposed to 5 mg/L (ppm) nitrite. Flexibacter columnaris infections occurred spontaneously in channel catfish exposed to 5 mg/L (ppm) nitrite for 7 days. None of the control fish became infected with F. columnaris.

Twenty-six isolates of gliding bacteria associated with columnaris disease were collected from different species of fish from a wide geographic range. Although there were three phenotypic variations in colony morphology among the strains observed, the growth and biochemical characteristics, as well as, GC content revealed no differences among these isolates. However, when compared by DNA hybridization, there appeared to be three distinct groups based on the DNA homology. Eighteen of the twenty rhizoid strains represented the first group. They were 81-98% homologous with Pacific Northwest strain DD3 of Flexibacter columnaris and included isolates from Canada, Chile, Japan, Korea, ROC and the USA (Atlantic and Pacific Coasts). The mucoid strain K4m was 95% homologous with strain DD3. Taiwanese strains 4G and 5F belonged to the second group and could be classified into new species of gliding bacteria as a result of low DNA homology (

Survival and infectivity of Flexibacter columnaris was found to decline in the presence of other species of bacteria, such as Aeromonas hydrophila (a fish pathogen) or Citrobacter freundii (nonpathogenic to fish). In the in vitro experiment, among the selected combination ratios of the initial density of F. columnaris to A. hydrophila or C. freundii (1 : 1, 10 : 1, and 1 : 10), the survival of F. columnaris was markedly reduced at 1 : 10. In the in vivo experiment, F. columnaris failed to invade loach when the initial density of A. hydrophila or C.freundii was approximately 100 times as much as F. columnaris. In the presence of the competitive bacteria, the density of F. columnaris found in the mucus of fish's body surface did not change during the period of experiment. On the other hand, in the absence of competitive bacterium, the density of F. columnaris in the mucus increased gradually, reached a peak after 24-48 hours and showed visible signs of infection. In the waters sampled from four different aquaculture facilities F. columnaris did not invade loach when the density of F. columnaris added was 1/100 of the total bacteria found in the test water.

A recurring disease known as ‘black patch necrosis’ (BPN) has been the cause of severe mortalities in 0-group and 1-group Dover sole during the period 1974-1978 at the Sea Fish Industry Authority's Marine Cultivation Unit at Hunter-ston, Ayrshire, Scotland. Samples of diseased and healthy Dover sole tissue were examined microbiologically and a long thin filamentous Gram-negative organism, strongly resembling Flexibacter columnar's, was repeatedly isolated from diseased tissue but was not found in healthy tissue. This organism was pathogenic to Dover sole, producing 100% mortality in 96 h at 17.5°C (±2°C) and the organism was reisolated from all the infected fish. The pathogenic isolate and reisolate were examined and compared with reference strain NCMB 1038 of Flexibacter columnarts, formerly known as Chondrococcus columnaris or Cytophaga columnaris. The isolate, reisolate and type culture were identical with respect to cell morphology, oxidase test, catalase production, production of H2S, casein hydrolysis, gelatin liquefaction, hydrolysis of Tween 20, degradation of tyrosine and starch hydrolysis. All bacterial types showed similar patterns of sensitivity to a range of antibiotics. It is proposed that a Flexibacter columnari's-like organism is associated with the condition in farmed Dover sole known as ‘black patch necrosis’.

Phenetic data on almost 600 aerobic, heterotrophic bacteria from a marine fishrearing unit were collected and analysed using numerical taxonomic techniques. Reference strains, representing 42 taxa were included in the analyses. At similarity levels of 85% or above, with analyses prepared from the simple matching coefficient (SSM), 81% of the isolates were recovered in eight major and 43 minor phena. Five of the major phena were equated with Acinetobacter calcoaceticus, Photobacterium phosphoreum and Vibrio spp. (three groups); the three unidentified phena contained Gram negative rods with polar flagella which were considered to be intermediate between Cytophaga/Flexibacter and Flavobacterium (two phena), and Gram variable rods. The surface of healthy turbot (Scophthalmus maximus L.) was populated by a diverse array of bacteria, including Alcaligenes faecalis, Bacillus firmus, Photobacterium angustum,‘Photobacterium logei’ and Pseudomonas fluorescens. Taxa, isolated as pure culture growth from within the lesions of moribund animals, included Alteromonas haloplanktis and unidentified Gram negative, budding bacteria. Vibrio anguillarum was not recovered from any turbot suspected of suffering from ‘vibriosis’.

Capnocytophaga spp. contain a group of unusual sulfonolipids, called capnoids (W. Godchaux III and E. R. Leadbetter, J. Bacteriol. 144:592-602, 1980). One of these lipids, capnine, is 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid; the others are, apparently, N-acylated versions of capnine. The lipids were found, in amounts ranging from 2.5 to 16 mumol of capnoid sulfur per g of cells (wet weight), in two Cytophaga spp. and also in several closely related organisms: several Capnocytophaga spp., Sporocytophaga myxococcoides, two Flexibacter spp., and two Flavobacterium spp. With the exception of the flavobacteria, all of these bacteria have been shown to exhibit gliding motility. The two Flavobacterium spp. belong to a subset of that genus that shares many other characteristics with the cytophagas. Only the Capnocytophaga spp. contained large quantities of capnine as such; in all of the others, most (and possibly all) of the capnoids were present as N-acylcapnines. Capnoid-negative bacteria included some gliding organisms that may not be closely related to the cytophagas: two fruiting myxobacters, a gliding cyanobacterium (Plectonema sp.), Beggiatoa alba, Vitreoscilla stercoraria, Herpetosiphon aurantiacus, and Lysobacter enzymogenes. Nongliding bacteria representing nine genera were also tested, and all of these fell into the capnoid-negative group.

Eight unidentified fish pathogens and 10 strains received as “Flexibacter columnaris,” “Cytophaga psychrophila,” and Flexibacter maritimus were compared with the type strains of all previously described species in the genera Cytophaga and Flexibacter and with seven Flavobacterium species by determining levels of deoxyribonucleic acid (DNA) relatedness (S1 nuclease method) and by performing phenotypic tests. The name Flexibacter columnaris sp. nov. is revived for a DNA relatedness group comprising eight strains that are 75 to 100% related to strain TG 39/87 and 0 to 8% related to all of the other species studied. These strains produce flat rhizoid colonies which adhere to agar, show strong gliding movement, absorb Congo red, reduce nitrate to nitrite, and produce H2S. The guanine-plus-cytosine content of the DNA is 32 mol%. The type strain is strain NCMB 2248 (= ATCC 23463). The name Flexibacter psychrophilus sp. nov. is revived for a DNA relatedness group comprising seven strains that are 90 to 100% related to strain NCMB 1947T (T = type strain) and 0 to 5% related to all of the other species studied. These strains produce circular, convex colonies that have regular or spreading margins and do not adhere to agar, show very slow gliding movement, do not absorb Congo red, do not reduce nitrate, and do not produce H2S. The guanine-plus-cytosine content of the DNA is 33 mol%. The type strain is strain NCMB 1947. Both F. columnaris and F. psychrophilus produce the flexirubin type of pigments, are strongly proteolytic, and do not hydrolyze (or produce acid from) any carbohydrate. Flexibacter maritimus (three strains) was shown to constitute a DNA relatedness group that is 0 to 8% related to all of the other species tested. Furthermore, this organism can be differentiated by phenotypic tests. The presence of F. maritimus in Europe is shown for the first time.

Bisher wurden in den „Gleitenden Bakterien” vom Typ Flexibacter/Cytophaga nur Carotinoide aufgefunden. Chromatographische Trennung des Farbstoffkomplexes aus Flexibacter elegans fuhrte nun zur Isolierung des Hauptpigmentes Flexirubin, fur das Struktur 7 abgeleitet wird. Flexirubin ist der erste Vertreter einer neuartigen Gruppe von Bakterienpigmenten, die sich strukturell durch einen Polyencarbonsaure-Chromophor in Esterbindung mit einem Phenol, das eine lange Alkyl-Seitenkette tragt, auszeichnet. Investigations on Metabolites of Microorganisms, XI: Flexirubin, a Novel Pigment from Flexibacter elegans Up to now only carotinoid pigments have been found in ‘gliding bacteria’ of the Flexibacter/Cytophaga-type. The main pigment of Flexibacter elegans was now isolated by chromatographic separation and named flexirubin. Its structure was deduced to be 7. Flexirubin is the first member of a new class of bacterial pigments. In respect to the chemical structure this class is characterized by a polyene-carboxylic acid chromophore, which is esterified with a phenol carrying a long aliphatic side chain.

Submicroscopic goblet-shaped particles (goblets) were released from the cell envelope of the marine gliding bacterium Flexibacter polymorphus when treated with the nonionic detergent Triton X-100 followed by sonication. The goblets were purified by cesium chloride density gradient centrifugation and exhibited an equilibrium buoyant density of 1.30 g/mL at 23 °C. They were composed of protein and a small amount of carbohydrate (approximately 3.4% by weight). Aqueous suspensions exhibited an absorption maximum in the ultraviolet at a wavelength of 276 nm and a smaller shoulder at 281 nm. Phospholipids were not detected in purified preparations of goblets, though they are known to be prominent constituents of the intact membranes of this microbe. Polyacrylamide gel electrophoresis of goblets solubilized in sodium dodecyl sulfate and 2-mercaptoethanol indicated four major polypeptide components ranging in molecular weight from 13 000 to 80 000. This number of different protein subunits corroborates earlier ultrastructural observations indicating a multisubunit composition.

Channel catfish, Ictalurus punctatus, exhibited passive cutaneous anaphylaxis (PCA) when sensitized with Flexibacter columnaris antiserum produced in channel catfish (the first record of PCA in any fish), but not when sensitized with homologous antiserum against channel catfish virus. Channel catfish did not give delayed skin hypersensitivity responses after immunization with either channel catfish virus or F. columnaris.

Gliding bacteria inflicted fish diseases were prevailing in November and December, but not in May and June. Annual survey of the gliding bacteria revealed that Flexibacter columnaris was common in September and October while other Flexibacter spp. in March and April.The virulence of these bacteria to eel and tilapia were examined. Methods and conditions for their infection were also studied. In running water, F. columnaris was more virulent to eel than to tilapia, while Flexibacter spp. were more virulent to tilapia than to eel. Artificial infection of gliding bacteria was more effective by contact method than by injection method. Mortality rate of fishes infected with gliding bacteria was high when they were kept in standing water rather than running water post-infection.Supplement of ferric ion to the artificially infected fishes shortened their survival time. This was more pronounced in fishes infected by injection. However, human transferrin afforded protection to fish against infection with these bacteria.

The effect of water temperature upon mortality from experimental infection by Flexibacter columnaris and on mean time to death was investigated in juvenile steelhead trout (Salmo gairdneri), coho salmon (Oncorhynchus kisutch), and spring chinook salmon (Oncorhynchus tshawytscha). Eight temperatures increasing from 3.9 to 23.3 C (39 to 74 F) by increments of 2.8 C (5 F) were studied. Fish were infected by the direct contact method whereby a suspension of the pathogen was added to the water. It was found that at temperatures of 9.4 C (49 F) and below, no deaths due to the experimental infection with F. columnaris occurred. At 12.2 C (54 F) mortality varied from 4 to 20% among the three species, and increased progressively with increasing temperature to 100% in steelhead trout and coho salmon at 20.5 C (69 F), and 70% in chinook salmon at that temperature. With all three salmonid species, an inverse linear relationship was found between water temperature and the log10 of the mean number of days from exposure to death. This means that as the temperature increased above 12.2 C (54 F), the disease process was progressively accelerated, resulting in a minimum time to death at 20.5 or 23.3 C (69 or 74 F) and a maximum at 12.2 C (54 F). The results indicate that under the conditions of these experiments, columnaris disease in salmonids was completely suppressed by water temperatures of 9.4 C (49 F) or below.

Autumn—shed leaves of white oak (Quercus alba) and pignut hickory (Carya glabra) were fastened together and incubated in a third order hardwater stream in south—western Michigan. At biweekly intervals, leaves were removed and the microbial populations assessed. Direct counts of bacteria increased logarithmically with time, but viable counts were generally lower. Flexibacter, Achromobacter, Flavobacteria, Pseudomonas, and Cytophaga were isolated from leaves. No apparent bacterial succession was detected. The mycoflora was assessed in five ways, including direct examination and incubations ranging from environment simulating to particle platings on nutrient medium. Although this last technique suggested that common soil and litter fungi dominated, direct examination and environment—simulating incubations showed that aquatic hyphomycetes, particularly Alatospora acuminata, Flagellospora curvula, and Tetracladium marchalianum were the dominant members of the mycoflora. The successional pattern of these fungi on both leaf species is discussed. It is concluded that the aquatic hyphomycetes are actively growing and completing their life cycle in this environment and that the terrestrial fungi are normally present mainly in a dormant state, growing and sporulating only in greatly enriched conditions. The highest ATP levels observed corresponded to the periods of highest fungal frequencies .

Use of a dialysis microchamber has allowed continuous observations on the same set of gliding bacteria during changes in the composition of the perfused medium. This procedure has revealed the presence of an adaptive, cyanide-insensitive metabolic pathway, which allows cyanide-treated Flexibacter BH3 to begin gliding again at a reduced rate when glucose is the substrate. In addition, it has revealed that individual flexibacter cells can maintain their gliding motility for up to 20 h in the absence of exogenous substrate. Gliding in Flexibacter BH3 was prevented by those inhibitors blocking the electron transport process. Inhibitors of glucose metabolism did not prevent motility, since the flexibacters obviously metabolize endogenous substrate under such circumstances. Proton ionophores, which induce membrane depolarization, rapidly inhibited gliding in Flexibacter BH3. This inhibition was irreversible in the case of gramicidin S. Gliding was not inhibited by cytochalasin B or antiactin antibody. High concentrations of Ca2+ were particularly inhibitory to the gliding process. The significance of these results is discussed in relation to a possible mechanism of gliding involving the generation of rhythmical contractions in the outer cell membrane of Flexibacter BH3.

Summary: One hundred and fifty pink-pigmented facultatively methylotrophic bacteria (PPFMs) of the ‘Pseudomonas extorquens’ type, 28 other facultative methylotrophs and 16 non-methylo-trophic marker strains of the genera Pseudomonas, Alcaligenes, Mycoplana and Microcyclus were compared in a numerical phenetic study using 140 unit characters. Data were analysed using the simple matching (SSM) and Jaccard (SJ) coefficients and single, complete and average linkage algorithms. Cluster composition was largely the same with each similarity coefficient and linkage method. Four major and seven minor clusters containing 187 of the 194 strains were defined above the 80% similarity level. The non-pink methylotrophs were recovered in two major, four minor and two single-membered clusters. One of the major clusters could be equated with Microcyclus aquaticus but strains in the other were not identified. Strains in two of the minor clusters were identified as Pseudomonas species but members of the other two minor and one of the single-strain clusters were not positively identified. They had pseudomonad properties but were not closely associated with any marker strains. The other single-membered methylotroph cluster was thought to be a cytophaga or flexibacter. The remaining minor and single-membered clusters contained only marker strains. All the PPFMs were recovered in the two remaining major clusters which were closely related to each other but not to the rest of the organisms studied. The generic assignment of the PPFMs is discussed and the suggestion is made that the genus Methylobacterium may be the most appropriate place for them, despite their apparent inability to utilize methane.

Flexibacter FS-1 filaments are unable to glide on agarose gels or on charge-neutralized glass unless those substrata are supplemented with the charged, cold water-soluble fraction of agar, or a variety of polyanionic polysaccharides derived from plants, yeasts and bacteria. Two graft polymers of xanthan gum also promote gliding motility under these conditions, as do an extracellular product of the bacteria themselves. A number of other polymers and small molecular species are ineffective as supplements. These results are considered in the context of the soil habitat of this Flexibacter. Among a variety of other gliding bacteria tested, several strains of the order Cytophagales also were unable to glide on agarose.

Sediment cores were set up to study microbial colonisation and interactions on marine sand grains under enrichment conditions. Cores were enriched with photosynthetic media in the light and dark (PL, PD) and heterotrophic media in the light and dark (HL, HD), and were incubated for 25 days. Sediment chlorophylls were then measured by acetone extraction, viable heterotrophic bacteria by plate counts, and numbers of cells mm−2 sand grain surface by s.e.m. Chlorophyll a occurred in all sediments but was highest in the PL sediment. Bacteriochlorophyll a was only observed in the HL sediment. Heterotrophic viable counts were high in the HL and HD sediments. Dense growth of diatoms and blue-green algae, and a marine fungal Thraustochytrid sp. occurred on PL grains. The blue-green alga Schizothrix was often associated with the diatom Amphora on PL grains. Many different bacteria grew on HL and HD grains and some unusual colony and cell morphologies were recorded (Caulobacter, Flexibacter, polymer strands). Characteristic flakey material sometimes occurred in hollows on grains. The results are discussed in relation to microbial communities in low energy sedimentary environments.

The effect of growth rate, growth phase, pH, and temperature on the permanent adhesion of a glidingFlexibacter sp. and three nongliding bacteria,Pseudomonas fluorescens, Enterobacter cloacae, andChromobacterium sp., to polystyrene substrata was investigated. The permanent adhesion of the flexibacter appeared to be related to growth, as levels of adhesion increased with increased growth rate in continuous culture and declined rapidly with death phase in batch culture. With the three nongliding bacteria, there was no relationship between growth rate and levels of permanent adhesion. The permanent adhesion of the nongliding bacteria was maximum between pH 5.5 and pH 7 and between 20 and 30°C, whereas the adhesion of the flexibacter progressively decreased with increasing temperature and pH. The effect of different nutrient conditions on the gliding motility of the flexibacter across agar was also investigated. Gliding motility was inhibited by increased nutrient concentration and was affected by carbon source. Inhibition appeared to be related to the accumulation of a viscous exopolymer. It is proposed that the differences in the permanent adhesion of the gliding and nongliding bacteria may be related to their adaptation to different ecological niches.

Extensive erosion of external ligament surfaces was associated with the presence of dense and homogeneous populations of very long rod-shaped bacteria within the lesions. The distinctive morphological characteristics of these bacteria suggested their probable affinity with one of the marine genera, Cytophaga, Flexibacter or Flavobacterium. The prevalences of bacterial lesions in both hinge ligament elements and in mantle tissues were estimated for a high-mortality population of juvenile oysters from a commercial hatchery. The ligament resilia of 93% of the oysters were estimated to contain erosive lesions, while only 20% of the ligament tensilia contained lesions. Bacterial infections in mantle tissues were estimated to occur in 47% of the oyster population. Separate morphometric studies using histological material were conducted to test hypotheses concerning both the apparent association between bacterial lesions in ligaments and mantle tissues, and the mechanistic basis of the apparent association. The probability of observing a bacterial lesion in mantle tissues was significantly greater (P

Exposure of young striped bass Morone saxatilis (weight, 8.5–34 g) to a mixture of arsenic, cadmium, copper, lead, and selenium at 4 and 10 times the average environmental concentrations of 1–3 μg/L protected the fish from experimental infection with Flexibacter columnaris, the causal organism of columnaris disease. In four trials, all striped bass died within 7 d after a 2-min exposure to 5 × 106 F. columnaris cells in untreated water. In contrast, no fish died after a single day's exposure to the metal mixture followed by infection with F. columnaris and a second exposure to the metals for seven more days. When striped bass were exposed 5 d to individual metals, copper protected against infection and cadmium offered marginal protection but was slightly toxic after 2 d exposure. Arsenic increased susceptibility to infection, and lead and selenium were without an apparent effect.

SUMMARY: Cytophaga johnsonae and C. aquatilis (ATCC 29551), examined by frozen-surface replica and freeze-etch techniques, displayed undulated and particulated surfaces. They produced copious amounts of extracellular slime when cultured. Cytophaga aquatilis contained large, complex mesosomes in freeze-etched cells whereas C. johnsonae contained none. In non-cryoprotected freeze-etch preparations, C. aquatilis contained two major fracture planes whereas the C. johnsonae cell envelope fractured in three distinct planes. Linear fibres, such as the peripheral fibrils previously described in Flexibacter columnaris, were not observed in the outer membrane of freeze-etched C. aquatilis. Cytophaga johnsonae contained periplasmic linear fibrils; however, these appeared to be associated with the outer surface of the cytoplasmic membrane or an intermediate layer and not with peripheral fibrils.

The flexibacters are a form of gliding bacteria which are often found on the surfaces of solid bodies in fresh and salt water. An individual organism lacks motility in the bulk aqueous phase but glides over a solid surface with its rod-like body aligned with and nearly touching the surface. It has been suggested that this gliding motion in Flexibacter strain BH3 may be caused by waves moving down the outer surface of the rod-shaped cell [2]. This paper is concerned with the fluid mechanical aspects of this form of propulsion.Formulae for the velocity of the organism and for the power dissipation are obtained by using a lubrication theory analysis in the small gap between the bacterium and the wall. It is found that for any progressive waveform there is an optimum distance from the wall at which the flexibacter may maximize its speed for a given power output. Assuming that the flexibacter sits at this optimum distance and taking the waveform to be sinusoidal we calculate the power required for the flexibacter to move at the maximum observed speed. It is found that this power requirement represents only a small fraction of the power available to the cell.

A yellow-pigmented, gram-negative, gliding bacterium isolated from an industrial water spray air humidification system was implicated as a causative agent in several occurrences of lung disease with hypersensitivity pneumonitis-like symptoms. The bacterium, designated WF-164, lacked microcysts or fruiting bodies and had a DNA base composition of 34.8 mol% of guanine plus cytosine. Gliding, flexing, nonflagellated cells measuring 0.3 by 3.5 to 8.9 micron were observed by using light and electron microscopy. Tests to determine utilization of selected carbohydrates revealed an amylolitic, chitinoclastic, noncellulytic bacterium. A number of additional biochemical and physiological tests were performed. DNA homology studies detected a 77.8% similarity to Cytophaga aquatilis (ATCC 29551). Comparisons of cellular fatty acid and carbohydrate contents of isolate WF-164 with a Flexibacter sp., several Cytophaga spp., and Flavobacterium reference strains revealed similar patterns to that of C. aquatilis. On the basis of these characteristics, isolate WF-164 was identified as a new Cytophaga sp.

SUMMARY: A filamentous, gliding microbe isolated from a marine source is described as a new species of Flexibacter (Cytophagales: Flexibacteraceae). The filaments are typically multicellular. At the end of each cell there is normally a refractile granule of lipid material (not elemental sulphur, as would be the case in a Beggiatoa species). Under certain culture conditions, unicellular elements, helically coiled filaments or, occasionally, branched filaments may be produced. The filaments, which are not photosynthetic, are peach-coloured when grown in light: the pigment is a carotenoid spectroscopically resembling saproxanthin. The guanine + cytosine proportional content in the DNA is 29%. The organism grows in media prepared with sea water or an equivalent saline solution. It can utilize glucose as sole carbon source; glutamate or Tryptone can serve as a suitable source of both C and N. Cobalamin is the only organic micronutrient essential for growth. The generation (doubling) time is 6 h at 22°C.

Summary: The physico-chemical basis for the effects of nutrient conditions on the attachment of four freshwater isolates, Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp. and Flexibacter sp., to hydrophobic (PD) and relatively hydrophilic (TCD) polystyrene surfaces was investigated. Different nutrient conditions and growth rates resulted in changes in the physico-chemistry of the bacterial surfaces, measured either by liquid contact angles on lawns of cells or by hydrophobic and electrostatic interaction chromatography of cells, and in different levels of attachment to the substrata. The phenotypic changes in cell surfaces and the levels of attachment were different for each species. Levels of bacterial adhesion differed for the two substrata, indicating different adhesion interactions with PD and TCD surfaces. Treatment of attached cells with chloramphenicol did not cause detachment of any of the bacteria from PD or TCD, whereas periodate and protease treatment removed some attached cells, the degree of detachment depending on the species. The presence of complex organic molecules, in the liquid phase and conditioning the solid surface, influenced the extent of bacterial attachment, the effect depending on the substratum, organic concentration and bacterial species. The results suggest that changes in nutrient conditions in natural aquatic habitats will affect the attachment of individual bacterial species differently, thus influencing the population structure of developing biofilms.

Heavy mortalities of underyearling Atlantic salmon parr have occurred at the Mactaquac Fish Culture Station in New Brunswick as the result of ‘saddleback’ disease. The organism causing this lesion was isolated and identified as Flexibacter columnaris. This bacterium was transmitted to other salmon parr, where it produced the typical saddle lesion from which it was re-isolated. The condition was more readily transmitted at 20°C than at 15°C. Transmission required entrance to the dermis through a break in the epidermal surface and was unsuccessful in intact fish. The morphology of the saddleback isolate and its histopathological effect on the fish tissues is described. F. columnaris appeared to be restricted to the dermis and underlying muscle of the saddle area when histological techniques were used, although it was also isolated from the kidneys of some affected fish. The gills, kidney and other organs of the fish did not seem to be affected by the presence of F. columnaris.

The therapeutic efficacy of potassium permanganate (KMnO4) was evaluated on fathead minnows (Pimephales promelas) infected with Flexibacter columnaris in organically enriched pond water and in organically depleted tap water. Potassium permanganate was bactericidal in tap water but only temporarily reduced F. columnaris in pond water. Efficacy of KMnO4 in pond water was enhanced by satisfying the demand of organic matter for KMnO4. The most effective therapeutic concentration of KMnO4 for fathead minnows infected with F. columnaris was 4 mg/liter plus the KMnO4 organic demand.

Earlier (Godchaux, W., and Leadbetter, E. R. (1980) J. Bacteriol. 144, 592-602; (1983) J. Bacteriol. 153, 1238-1246) we demonstrated that an unusual class of sulfonolipids are major components of the cell envelope of gliding bacteria of the genus Cytophaga and of closely related genera. One of these lipids, to which we have assigned the trivial name capnine, was purified and was shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid (which might also be named as 1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Though capnine accumulates as such in the cells of some Capnocytophaga spp., most organisms of the Cytophaga-like genera contain, instead, sulfonolipids that are less polar than capnine. These less polar lipids have been purified from a Capnocytophaga sp., a marine Cytophaga sp., Cytophaga johnsonae, and a Flexibacter sp. Acid methanolysis of the lipids yielded both aminosulfonates and a collection of fatty acid methyl esters. The infrared absorption spectra of the lipids indicated that the fatty acids were in amide (and not ester) linkage to the aminosulfonates. In every instance, analysis by mass spectrometry and other methods revealed that most, if not all, of the aminosulfonates obtained by methanolysis were structurally identical to capnine (though small amounts of variants of that compound may be present in some cases). The less polar sulfonolipids are, therefore, predominantly N-fatty acyl capnines, 1-deoxy-1-sulfonic acid analogs of ceramides. The fatty acid methyl esters obtained from the lipids were heterogeneous, but in all cases were rich in hydroxylated fatty acyl groups, which constituted 66 to 95% of the total.

DNA hybridization experiments showed that there was a high degree of homology amongVitreoscilla strains but not with DNA fromFilibacter limicola. Flexibacter spp were much more heterogeneous indicating a low genetic similarity. These results were also reflected in the membrane fatty acids of the bacteria. TheVitreoscilla strains were very similar with the 16:1ω7c fatty acid being dominant. The membrane fatty acids ofF. limicola were dominated by a15:0 and a17:0 components which provided additional support for its relatedness to the genusBacillus. There was much greater diversity in the fatty acid patterns of theFlexibacter spp.F. aurantiacus, F. ruber andF. elegans shared the common dominant fatty acids 16:1ω7c with theVitreoscilla strains, but this was replaced by the 16:1ω6c acid inF. flexilis. F. ruber was distinguished by the absence of branched odd-chain monounsaturated fatty acids andF. elegans by the dominance of the β-OH i15:0 acid. Precise determination of fatty acid double bond positions and geometry are essential for correct interpretation of increasingly complex ecological and taxonomic data sets.

1. Flexibacter elegans Fx e1 contains 3 pigments; their quantitative proportion is 2.5 : 2.5 : 95%. The absolute pigment content is 0.6–0.8 μg per mg dry cells. 2. Contrary to statements in the literature, these pigments are not carotenoids. This can be deduced from their electronic spectra which lack fine structure and are pH-dependent; from their resistance to photooxidation; from their failure to incorporate 14C-mevalonate; and from their mass spectra which do not show the M-92 and M-106 fragments typical for carotenoids. 3. The main pigment is characterized in detail; it has been named flexirubin. 4. The new pigments are widely distributed among members of the Cytophaga-Flexibacter group and seem to be valuable chemosystematic markers. 5. Specific pigment content of the bacteria does neither change along the growth curve, nor under illumination. Low pH or high phosphate concentrations depress specific pigment content. 6. The taxonomical position of experimental strain Fx e1 is discussed: The organism shows a cyclic change in cell shape. In young cultures there are long and very agile thread cells which fragment later in culture development, until finally only very short rods remain. A redefinition of the genus Flexibacter based on this shape change is proposed.

Flexibacter FS-1, a gram-negative gliding bacterium was grown in liquid culture as long (over 100-μm) filaments. The filaments possessed a triple-track wall which resembled that found in other gram-negative bacteria. Although phase-contrast microscopy indicated that the long filaments were nonseptate, electron microscopy revealed three or four septa along the length of each filament. The septa contained lysozyme-sensitive, electron-opaque material, presumed to be peptidoglycan, sandwiched between cell membranes. The outer triple track wall was not part of the septum. Mesosomes were seen in various areas of the cell and frequently were observed attached to septa in different stages of completion. Studies of the organism in slide culture revealed that individual filaments grew in an exponential fashion and divided in the middle despite the long length and multiseptate condition. When the temperature of a liquid culture growing exponentially with a generation time of 90 minutes was shifted from 30 to 35 C, the filaments fragmented into three or four shorter cells within 120 min. The short cells continued to grow exponentially at 35 C at approximately the same rate as at 30 C. When the culture was shifted back to 30 C, the cells immediately stopped dividing and began to elongate. After a period of 2 to 3 hr, cell division resumed. It is suggested that the shift-up in temperature induced the completion of the cross wall (centripetal growth of the triple-track wall) and cell separation at the sites of previously formed septa, whereas the shift-down in temperature caused a transient inhibition of cross-wall formation but not of growth. Fragmentation was inhibited by sodium azide but took place despite the inhibition of protein synthesis by chloramphenicol or the inhibition of deoxyribonucleic acid synthesis by mitomycin C.

SUMMARY: About 90 strains of gliding microbes (flexibacteria) have been considered. Data obtained by Lewin & Lounsbery (1969) and Mandel & Lewin (1969) have been used by Fager (1969) in a computer programme designed to indicate affinities and thereby possible relationships. Largely on the basis of Fager's analysis, a classification of these organisms is here proposed. Including a few species described elsewhere, 27 species and varieties are distinguished, most of them apparently new. A simplified diagnostic key is presented for their identification. They are assigned to six genera, three (Saprospira, Flexithrix and Herpetosiphon) are recognized primarily on morphological grounds; three others (Cytophaga, Flexibacter and Microscilla), here somewhat re-defined, are based largely on other convenient characteristics.

Co-existent bacteria isolated from the stock culture of Chattonella marina on peptone-yeast extract (Z-CII) agar plates were predominantly classified into four colony types. Among them, tiny pale pinkish colonies were composed mainly of a filamentous bacterium, which was identified as Flexibacter sp. on the basis of bacteriological characterization. The isolates of Flexibacter sp. formed very tiny circular colonies on Z-CII agar, but they produced large thin rhizoidal colonies on yeast extract-enrichment solution (YES) agar. The agar media containing various kinds of peptones, sugars, organic acids or nucleic acid bases yielded very slow growth of bacterial colonies as compared with those on YES agar. The isolates were shown to have an essential vitamin B_ requirement for growth. Maximum growth yield of the isolates was observed at 26℃ and they could not grow above 40℃.

The name Flexibacter maritimus is proposed for a group of 15 bacterial strains isolated from diseased red sea bream (Pagrus major), black sea bream (Acanthopagrus schlegeli), and rock bream (Oplegnathus fasciatus). These bacteria grew in cytophaga medium prepared with seawater but failed to grow in cytophaga medium supplemented with NaCl. The isolates were gram-negative, flexible rods which exhibited gliding motility on wet surfaces. They did not utilize agar, cellulose, or chitin. The mean guanine-plus-cytosine content of the deoxyribonucleic acids of eight selected strains was 31.8 ± 0.4 mol%. Strain R2 (= NCMB 2514) is designated the type strain of the new species.

Short Flexibacter FS-1 cells, generated during logarithmic growth in glucose-salts medium or by filament fragmentation during the transition from log to stationary phase in rich medium, are unable to glide. Motility returns when cells elongate. This strain also dissociates stable, short, nongliding variants.