Spiral conformation of Vibrio cholerae as determined by scanning electron microscopy of elongated cells induced by cephalexin treatment.

The elongated cells of Vibrio spp. induced by cephalexin treatment were examined by scanning electron microscopy. The results showed that Vibrio cholerae has a twisted cell body and a right-handed spiral conformation and that the cell bodies of V. parahaemolyticus and V. alginolyticus are straight rather than curved.     

STUDIES IN THE DYNAMICS OF HISTOGENESIS : II. TENSION OF DIFFERENTIAL GROWTH AS A STIMULUS TO MYOGENESIS IN THE ESOPHAGUS.

Esophageal Development. 1. The region of most active mitosis per mm. of cross-section in the esophagus is the entodermal epithelial tube. The mitotic figures follow a spiral path in the manner of a left-handed helix from the cephalic to the caudal direction. 2. The region of least active growth per mm. of cross-section in the esophagus is the mesenchyme surrounding the epithelial tube. 3. The helicoidal activity of the epithelial tube causes a vortical reaction in the surrounding mesenchyme. The mesenchymal whirlpool represents a reaction to the spirally grooving epithelial tube. 4. In embryos 9.5 to 14 mm. in length the esophageal epithelial tube grows relatively more rapidly in width than in length. During this period the myoblasts which form the inner, close spiral, muscle coat of the esophagus are becoming rapidly differentiated in the outer condensed margin of the mesenchymal maelstrom. 5. The nuclei, first spherical then oval, and finally rod shaped with rounded ends, are drawn out in the direction of the circumference of the mesenchymal rim which is directed tangentially. 6. The cytoplasm is also drawn out in the direction of the mesenchymal rim of the vortex. The elongated rows of isolated granules appear which subsequently, by confluence, form the myofibrillæ. These cytoplasmic derivatives are elongated in the direction of the circumference of the vortex. 7. Between the epithelial tube and the myoblastic rim at the periphery of the mesenchymal whorl is found the embryonic connective tissue. From this direct observation the conclusion is made that an optimum tensional stress stimulus is necessary to elicit the formation of muscular tissue at the circumference of the mesenchymal vortex. Consequently, the formation of a specific derivative from a pluripotent mesenchymal cell is due to the fortuitous circumstance of position. 8. In embryos from 14 to 24 mm. in length, the esophagus grows relatively more rapidly in length than in width. This elongation is due to two factors; first, the descent of the stomach, and, second, the resistance to diametrical growth presented by the inner close spiral musculature. The epithelial tube, still the dominant zone of mitotic activity, pursues the lines of least resistance, and consequently growth in length takes place. This is due to the shifting of the planes of cell division on account of the compression of the inner, close spiral, muscle coat. 9. The undifferentiated mesenchyme peripherad to the inner, close spiral musculature is elongated and the histogenetic changes in muscular formation are gradually taking place between 14 and 24 mm. A very attenuated, outer, elongated, spiral, or longitudinal muscle coat is detected in the esophagus of a 24 mm. pig embryo. 10. The characteristic intestino-colic flexure is a torsional reaction of the mesenchyme. The mesenchymal cells are thrown into a left-handed helicoidal series, corresponding to the activity in the epithelial tube. The right-handed helicoidal reaction of the mesenchyme, therefore, is due to the left-handed helicoidal growth of the epithelial tube. 11. The normal asymmetry of the abdominal viscera as well as the position of the gut is dependent upon the clockwise reaction of the stretched mesenchymal cell. These cells are stretched by the left-handed helicoidal growth of the epithelial tube. One factor producing situs inversus viscerum could be the reversal of the spiral growth of the epithelial tube resulting in a reaction of the mesenchyme in a direction opposite, namely counterclockwise, to that which occurs normally.     

Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences

Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera.     

Characteristics of Z-DNA helices formed by imperfect (purine-pyrimidine) sequences in plasmids

The capacities of three synthetic sequences to adopt left-handed helices were evaluated in recombinant plasmids. The sequences consisted of very short runs of (CG)n (n = 2-4) interspersed with runs of alternating A.T base pairs and/or with regions of non-alternating base pairs. The plasmids were studied by two-dimensional gel electrophoresis to determine the natures of the conformational transitions and their free energies of formation. These results coupled with analyses with chemical (diethyl pyrocarbonate, osmium tetroxide, and bromoacetaldehyde) and enzymatic (S1 nuclease, T7 gene 3 product, and MHhaI) probes indicated that the entire sequence was adopting a left-handed helix in each case. In one of these sequences, Z-DNA formation necessitated the retention of the anti conformation of one of the guanines in a region of non-alternation. In a sequence which contains out-of-phase regions of alternation, our results indicate the formation of a separate left-handed helix in the central (CG)2 region, thus forming two Z-Z junctions. In summary, we conclude that only very short regions of alternating CG are necessary to effect the B to Z transition and that this conformational change can be transmitted through non-alternating regions. A set of empirical rules governing the characteristics of the B to Z transition and the types of left-handed helices in supercoiled plasmids was derived from studies on a systematic series of 17 plasmids.     

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.     

Rapid direct determination using combined separation by prepared immunomagnetic and flow cytometry of Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD), was originally isolated from coho salmon Oncorhychus kisutch in the USA. Bacterial cold-water disease has since been spreading throughout Japan and has caused serious damage to populations of ayu Plecoglossus altivel in many farms and rivers. The rapid method of detecting for F. psuchrophilum is requested, however, traditional methods are laborious because of complicated assay procedures. In this study, a rapid method of detecting F. psychrophilum was developed using a modified method of flow cytometry (FCM) analysis and immunomagnetic separation (IMS). Magnetic iron, in small particles, was prepared by the reaction of a mixture of ferric and ferrous ions under alkaline conditions. The particles were coated with antiserum against F. psychrophilum by dextran. Polyclonal antibodies (anti-F. psychrophilum) conjugated with fluorescein isothiocyanate (FITC) were reacted with F. psychrophilum, and then prepared immunomagnetic were applied using IMS, followed by FCM determination. A good correlation was observed between the cell numbers determined by the FCM method and the traditional method in the range of 10(2)-10(8) cells ml(-1). The FCM analysis could count cells within 1min, and the total analysis time, including sample preparation, was less than 2 h.     

Energetic and structural inter-relationship between DNA supercoiling and the right- to left-handed Z helix transitions in recombinant plasmids

We have evaluated the B to Z conformational transitions in supercoiled recombinant plasmids containing different lengths of (dC-dG) described in the preceding paper. The sodium chloride-induced right- to left-handed transition in a small segment of the plasmids caused a relaxation of (-) supercoils which was monitored by electrophoretic mobility changes of individual topoisomers on agarose gels containing NaCl at concentrations up to 5.0 M. The number of supercoils relaxed was proportional to the length of the (dC-dG) segment in the plasmid in good agreement with theoretical values. A short B/Z junction region (less than 5 base pairs) was inferred. The stability of the Z conformation in (dC-dG) segments of the plasmids had a strong length dependency; shorter lengths were less stable. Ten base pairs of (dC-dG) was insufficient to allow a Z conformation under the conditions studied. Supercoiling imparts a substantial favorable free energy to the Z conformation, reducing the NaCl concentration necessary to cause the transition. The relationship of supercoiling with the NaCl concentration necessary to cause a B leads to Z transition suggests that supercoiling alone is sufficient to stabilize the Z conformation at physiological salt concentrations. These results support the notion that left-handed DNA has an important biological role.     

Mode of cell growth of Malassezia (Pityrosporum) as revealed by using plasma membrane configurations as natural markers

The mode of cell growth of Malassezia was studied by freeze-fracture using the plasma membrane configurations of this organism as natural markers. The plasma membrane of the mature cell bodies of M. pachydermatis had a ring swelling, and on each side of the ring, one set of straight and spiral grooves and circumvallate bulgings. The cell always divided at the ring swelling (M. pachydermatis) or depression (M. furfur), soon followed by budding there. A new set of similar configurations formed on the bud. In all the 12 strains of Malassezia studied, the spiral grooves in the mother and bud parts were both left-handed but opposite in the direction of elongation. By comparing distances between the spiral grooves in short and long buds and in mothers, the bud tip was suggested as the major, and adjacent regions as the minor, sites of wall growth. Some characterizations of the plasma membrane invaginations, especially in relation to the mode of cell growth, were also described.     

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.     

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.     

Role of divalent cations on DNA polymorphism under low ionic strength conditions.

We have examined the conformational properties of poly(dG-m5dC) under a variety of low salt conditions and sample preparations. Extensive dialysis against 0.5 mM Na-cacodylate resulted in a left-handed polynucleotide conformation as determined by circular dichroism, in agreement with recently reported results. Similarly, extensive dialysis against Tris-EGTA also led to a left-handed conformation. Dilution of these samples led to a transition to the right-handed conformation. More stringent treatments such as dialysis followed by passage over an ion exchange column also resulted in a right-handed conformation. When these various solutions were examined using atomic absorption spectroscopy, significant levels of Mg+2 were observed (greater than or equal to 190 per 1000 nucleotides) in all samples showing a left-handed form, while much lower levels (less than or equal to 45 per 1000 nucleotides) were found in the low salt samples displaying a right-handed conformation. Addition of MgCl2 to samples in which divalent cations had been almost completely removed led to the reformation of the left-handed form. These results indicate that the left-handed form seen under certain low salt conditions is due to the presence of Mg+2 ions that remain bound to the polynucleotide, even in the presence of EDTA.     

Conformational stability of alternating d (CG) oligomers in high salt solution.

The conformation of d (CG)n oligomers with n = 2,3 has been studied in aqueous solution in the presence of high salt concentration. A minimum n value of three is necessary to obtain a left-handed Z-helix. When d (CG)3 is flanked by three non Z-helicogenic alternating AT sequences the left-handed helix is unstable and a B-type conformation is obtained also at high salt concentration.     

Determination of the DNA conformation of the simian virus 40 (SV40) enhancer in SV40 minichromosomes

The simian virus 40 (SV40) enhancer contains three 8-bp purine-pyrimidine alternating sequences which are known to adopt the left-handed Z-DNA conformation in vitro. In this paper, we have undertaken the determination of the DNA conformation adopted by these Z-motifs in the SV40 minichromosome. We have analyzed the presence of Z-DNA through the change in linkage which should accompany formation of this left-handed conformation. Our results indicate that, regardless of the precise moment of the viral lytic cycle at which minichromosomes are harvested and the condition of the transfected DNA, either relaxed or negatively supercoiled, none of the three Z motifs of the SV40 enhancer exist to a significant extent as Z-DNA in SV40 minichromosomes. The SV40 enhancer adopts predominantly a right-handed B-DNA conformation in vivo.     

Electrophoretic and Western blot analyses of the lipopolysaccharide and glycocalyx of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS) and it has emerged as one of the most significant bacterial pathogens in salmonid aquaculture worldwide. Previous studies have suggested that the O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) of F. psychrophilum is highly immunogenic and may be involved in eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss Walbaum). In the present study, SDS-PAGE and Western blotting techniques were used to analyse the carbohydrate antigens of F. psychrophilum. Our analysis identified two distinct carbohydrate-banding patterns. One banding pattern corresponds with LPS, and we hypothesise that the other carbohydrate-banding pattern is that of the loosely associated glycocalyx of F. psychrophilum. Electron microscopy of F. psychrophilum cells immunogold labelled with a monoclonal antibody specific for this banding pattern supports this hypothesis as the outermost layer of the bacterium was heavily labelled. This is a significant finding because the immunogenic antigens that have been referred to as the O-PS of LPS, and implicated as potential vaccine candidate antigens, appear to be components of the glycocalyx of F. psychrophilum. This research suggests that the glycocalyx of F. psychrophilum may be an important antigen to consider for the development of a vaccine to control CWD and RTFS.     

Short sequences of non-proline residues can adopt the polyproline II helical conformation

The left-handed polyproline II (P(II)) helix is a structure that has been given a great deal of attention lately because of its role in a wide variety of physiologically important processes and potential significance in protein unfolded states. Recent work by several authors has shown that residues besides proline can adopt this structure. A scale of relative P(II)-helix-forming propensities has been generated but only for single guest residues in a proline-based host system. Here, we present multiple guest residues in a proline-based host system. Using circular dichroism spectroscopy, we have shown that not only single residues, but also short sequences of non-proline residues can adopt the P(II) conformation.     

Impact of PCB on resistance to Flavobacterium psychrophilum after experimental infection of rainbow trout Oncorhynchus mykiss eggs by nanoinjection

The effects of sublethal exposure of a commercial blend of polychlorinated biphenyls (PCB), i.e. Clophen A50, on disease resistance to the aetiological agent of rainbow trout fry syndrome, Flavobacterium psychrophilum, were investigated. Newly fertilised rainbow trout Oncorhynchus mykiss eggs were nanoinjected with 2 doses of Clophen A50 (0.4 or 2 microg egg(-1)) and/or 100 colony forming units of F. psychrophilum. The mean cumulative mortality in control groups, and groups exposed to the lower dose of Clophen A50 (0.4 microg egg(-1)) was below 5.0%. The mean cumulative mortality in groups exposed to the higher dose of Clophen A50 (2.0 microg egg(-1)) was 5.8%, which was not significantly different from the control groups. In all groups infected with F. psychrophilum, with or without exposure to Clophen A50, significantly higher cumulative mortalities compared with control groups were recorded. No differences in mortality were recorded between groups exposed to bacteria alone or bacteria in combination with the higher dose of Clophen A50 (21.6 and 20.4%, respectively). Decreased disease resistance was recorded in groups exposed to F. psychrophilum and the lower dose of Clophen A50, with a mean cumulative mortality of 56.0%. These results could be due to non dose-dependent effects on the immune system, or toxic effects of PCB or their metabolites on the bacteria in groups exposed to the higher dose of Clophen A50. The present study indicates that maternal transfer of PCB might affect disease resistance to vertically transmitted F. psychrophilum.     

Conversions of the left-handed form and the protonated form of DNA back to the bound right-handed form by sanguinarine and ethidium: a comparative study

The interaction of sanguinarine and ethidium with right-handed (B-form), left-handed (Z-form) and left-handed protonated (designated as H(L)-form) structures of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) was investigated by measuring the circular dichroism and UV absorption spectral analysis. Both sanguinarine and ethidium bind strongly to the B-form DNA and convert the Z-form and the H(L)-form back to the bound right-handed form. Circular dichroic data also show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation either in Z-form or in H(L)-form. Both the rate and extent of B-form to Z-form transition were decreased by sanguinarine and ethidium under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. The rate of decrease is faster in the case of ethidium as compared to that of sanguinarine. Scatchard analysis of the spectrophotometric data shows that sanguinarine binds strongly to both the polynucleotides in a non-cooperative manner under B-form conditions, in sharp contrast to the highly-cooperative binding under Z-form and H(L)-form conditions. Correlation of binding isotherms with circular dichroism data indicates that the cooperative binding of sanguinarine under the Z-form and the H(L)-form conditions is associated with a sequential conversion of the polymer from a left-handed to a bound right-handed conformation. Determination of bound alkaloid concentration by spectroscopic titration technique and the measurement of circular dichroic spectra have enabled us to calculate the number of base pairs of Z-form and H(L)-form that adopt a right-handed conformation for each bound alkaloid. Analysis reveals that 2-3 base pairs (bp) of Z-form of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) switch to the right-handed form for each bound sanguinarine, while approximately same number of base pairs switch to the bound right-handed form in complexes with H(L)-form of these polynucleotides. Comparative binding analysis shows that ethidium also converts approximately 2 bp of Z-form or H(L)-form to bound right-handed form under same experimental conditions. Since sanguinarine binds preferentially to alternating GC sequences, which are capable of undergoing the B to Z or B to H(L) transition, these effects may be an important part in understanding its extensive biological activities.     

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

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.