Draft Genome Sequences of Flavobacterium columnare Strains ARS1 and BGFS27, Isolated from Channel Catfish (Ictalurus punctatus).

Flavobacterium columnare strain BGFS27 was isolated from an apparently healthy wild channel catfish (Ictalurus punctatus) collected from the Mobile River in 2005. F. columnare strain ARS1 was isolated from a channel catfish suffering from columnaris disease in a commercial farm in 1996. BGFS27 belongs to genomovar II (genetic group 2), while ARS1 belongs to genomovar III (genetic group 3). Here, we report the draft genome sequences of F. columnare BGFS27 and ARS1, obtained by PacBio sequencing.

Biofilm and Sediment are Major Reservoirs of Virulent Aeromonas hydrophila (vAh) in Catfish Production Ponds.

The genus Aeromonas comprises more than 60 recognized species that include many important fish pathogens such as the causative agents of furunculosis and motile Aeromonas septicemia (MAS). Although MAS is typically considered a secondary infection, a new virulent A. hydrophila (vAh) strain has been causing devastating losses to the catfish industry in Alabama since 2009. The objective of this study was to characterize the spatiotemporal distribution of Aeromonas sp. and, specifically, vAh in a commercial catfish farm in western Alabama. We sampled biofilm, sediment, and water from three ponds during four consecutive months during the growing season. Total aerobic counts were between 8.8 × 105 and 1.5 × 106  CFU/mL but were significantly higher in biofilm and sediment than in water throughout the sampling period. Total Aeromonas counts in water samples significantly increased in all three ponds after the month of August and ranged from 7.8 × 103 to 4.9 × 104  CFU/mL. A similar trend was observed in biofilm and sediment samples for which total Aeromonas counts increased in samples taken in late summer to early fall. Over time, the concentration of Aeromonas in water samples decreased by one order of magnitude, while there was a significant increase in sediments as temperature dropped. The virulent vAh was detected in 35.4% of biofilm samples and 22.9% of sediment samples, suggesting that both environments serve as the major reservoir for this pathogen. Future monitoring efforts should focus on targeting sediment and biofilms since samples of these appear to naturally enrich for the presence of vAh and other Aeromonas species.

Fish intestinal microbiome: diversity and symbiosis unravelled by metagenomics.

The gut microbiome of vertebrates plays an integral role in host health by stimulating development of the immune system, aiding in nutrient acquisition and outcompeting opportunistic pathogens. Development of next-generation sequencing technologies allows researchers to survey complex communities of microorganisms within the microbiome at great depth with minimal costs, resulting in a surge of studies investigating bacterial diversity of fishes. Many of these studies have focused on the microbial structure of economically significant aquaculture species with the goal of manipulating the microbes to increase feed efficiency and decrease disease susceptibility. The unravelling of intricate host-microbe symbioses and identification of core microbiome functions is essential to our ability to use the benefits of a healthy microbiome to our advantage in fish culture, as well as gain deeper understanding of bacterial roles in vertebrate health. This review aims to summarize the available knowledge on fish gastrointestinal communities obtained from metagenomics, including biases from sample processing, factors influencing assemblage structure, intestinal microbiology of important aquaculture species and description of the teleostean core microbiome.

Protective efficacy of Nigella sativa seeds and oil against columnaris disease in fishes.

Columnaris disease, caused by the bacterium Flavobacterium columnare, is currently the most frequently reported bacterial disease affecting farm-raised channel catfish in the USA. Common treatments against the disease include the use of medicated feed that has led to emergent antibiotic resistant strains of F. columnare. Nigella sativa (Black cumin) is a medicinal herb commonly used by many cultures as a natural remedy for numerous disorders. Recently, we have discovered the antibacterial activity of N. sativa and its oil extract against F. columnare. In this study, we showed N. sativa oil (NSO) strongly inhibited the growth of all of the strains of F. columnare tested and yielded significantly larger zones of inhibition than those produced by oxytetracyclin. We tested the protective effect against columnaris disease in vivo by incorporating NSO (5%) or N. sativa seeds (NSS) (5%) into fish feeds. Fishes (Ictalurus punctatus and Danio rerio) fed amended diets displayed significantly lower mortality than those fed control diets. Per cent mortalities in control groups ranged from 77% to 44% and from 70% to 18% in zebrafish and channel catfish, respectively. A dose study using different NSS concentrations showed that 5% NSS offered the most protection against columnaris disease in channel catfish.

Temperature effect on high salinity depuration of Vibrio vulnificus and V. parahaemolyticus from the Eastern oyster (Crassostrea virginica).

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are opportunistic human pathogens naturally associated with the Eastern oyster Crassostrea virginica. The abundances of both pathogens in oysters are positively correlated with temperature, thus ingestion of raw oysters during the warm summer months is a risk factor for contracting illness from these bacteria. Current post-harvest processing (PHP) methods for elimination of these pathogens are expensive and kill the oyster, changing their organoleptic properties and making them less appealing to some consumers. High salinity has proven effective in reducing Vv numbers in the wild and our research aims at developing an indoor recirculating system to reduce pathogenic Vibrios while maintaining the taste and texture of live oysters. The goal of this study was to determine the influence of temperature on the efficacy of high salinity depuration. Vv was enumerated as most probable number (MPN) per gram of oyster tissue using the FDA-approved modified cellobiose polymyxin colistin (mCPC) protocol and with an alternative Vibrio specific media CHROMagar™ Vibrio (CaV). CaV was also used to quantify Vp. Oysters were held at 35 psu for 10 days at three temperatures: low (20°C), mid (22.5°C) and high (25°C). There was no difference in MPN/g of Vv between media; however more Vv isolates were obtained from mCPC than CaV. There was no significant effect of temperature on reduction of Vv or Vp throughout depuration but there was a tendency for low temperatures to be less effective than the higher ones. High salinity resulted in a significant decrease in Vv by day 3 and again by day 10, and a decrease in Vp by day 3. Oyster condition indices were maintained throughout depuration and mortality was low (4% across three trials). Overall these results support the use of mCPC for Vv enumeration and demonstrate the promise of high salinity depuration for PHP of the Eastern oyster. The trend for lower temperatures to be less effective is surprising and indicates a potential interaction between salinity and temperature that should be further investigated.

Characterization of the gut microbiota of three commercially valuable warmwater fish species.

AIMS: Due to the strong influence of the gut microbiota on fish health, dominant bacterial species in the gut are strong candidates for probiotics. This study aimed to characterize the gut microbiota of channel catfish Ictalurus punctatus, largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus to provide a baseline for future probiotic studies. METHODS AND RESULTS: The gut microbiota of five pooled individuals from each fish species was identified using 16S rRNA pyrosequencing. Microbiota differed significantly between fish species in terms of bacterial species evenness. However, all gut communities analysed were dominated by the phylum Fusobacteria, specifically the species Cetobacterium somerae. Relatively high abundances of the human pathogens Plesiomonas shigelloides and Fusobacterium mortiferum, as well as members of the genus Aeromonas, suggest these species are normal inhabitants of the gut. CONCLUSIONS: The overwhelming dominance of the genus Cetobacterium in all species warrants further investigation into its role in the fish gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first characterization of the gut microbiota of three economically significant fishes and establishes a baseline for future probiotic trials.

Distribution and survival of Vibrio vulnificus genotypes in postharvest Gulf Coast (USA) oysters under refrigeration.

AIMS: The effect of refrigeration on the seafood-borne pathogen Vibrio vulnificus was investigated in terms of genotype selection and persistence in refrigerated oysters. METHODS AND RESULTS: Naturally occurring numbers of V. vulnificus in oysters from two different locations were compared during a 2-week period under refrigeration conditions. At different time points, V. vulnificus isolates were recovered from oysters and ascribed to 16S rRNA gene type A, B or AB using restriction fragment length polymorphism. Initial V. vulnificus numbers were higher than 10(4) most probable number (MPN) g(-1) and remained unchanged throughout the duration of the study. 16S rRNA gene type B isolates accounted for 53% of the isolates recovered. Amplified fragment length polymorphism analysis confirmed the high genetic variability previously observed within this species but revealed the presence of two main genetic groups within the species that matched 16S rRNA gene ascription. CONCLUSIONS: Vibrio vulnificus numbers in oysters did not significantly declined over the shelf life of the product and refrigeration did not select for specific V. vulnificus types. SIGNIFICANCE AND IMPACT OF THE STUDY: The prevalence of V. vulnificus 16S rRNA gene type B in oysters was higher than previously reported from the same geographic area and was not significantly reduced during the storage period. Vibrio vulnificus is divided into two clear genotypes, regardless of the genetic marker used.

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare.

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac-Col(®) . The strain of F. columnare used to generate the avirulent rifampicin-resistant mutant used in Aquavac-Col(®) belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin-resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin-resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.

Comparison of soil fungal community structure in different peanut rotation sequences using ribosomal intergenic spacer analysis in relation to aflatoxin-producing fungi.

The present study focuses on determining soil fungal community structure in different peanut-cropping sequences by using a high-resolution DNA fingerprinting technique: ribosomal intergenic spacer analysis (RISA). This study was initiated to determine fungal community profiles in four peanut-cropping sequences (continuous peanut, 4 years of continuous bahiagrass followed by peanut, peanut-corn-cotton, and peanut-cotton rotations), with a special focus to evaluate whether the profiles under investigation may have also indicated microbial differences that could affect Aspergillus flavus populations. Results indicated 75% similarities among fungal communities from the same cropping sequences as well as with similar times of sampling. Polymerase chain reaction (PCR)-based detection of A. flavus directly from these soils was carried out using A. flavus-specific primers (FLA1 and FLA2) and also through quantitative estimation on A. flavus and A. parasiticus agar medium. Population levels of A. flavus in soil samples ranged from zero to 1.2 × 10(3) CFU g(-1) of soil (based on culturable methods); however, the fungus was not detected with A. flavus-specific primers. The minimum threshold limit at which these aflatoxin-producing fungi could be detected from the total soil genomic DNA was determined through artificial inoculation of samples with 10-fold increases in concentrations. The results indicated that a minimum population density of 2.6 × 10(6) CFU g(-1) of soil is required for PCR detection in our conditions. These results are useful in further determining the relative population levels of these fungi in peanut soils with other soil fungi. This is a new approach to understanding soil fungal communities and how they might change over time and under different rotation systems.

In vivo gene expression of cold shock and other stress-related genes in Vibrio vulnificus during shellstock temperature control conditions in oysters.

AIMS: To determine Vibrio vulnificus response to shellstock refrigeration conditions while the bacterium was embedded in oysters (in vivo). METHODS AND RESULTS: Depurated oysters were artificially inoculated with V. vulnificus. Several cold-shock conditions were examined according to the National Sanitation Shellfish Program guidelines. Culturability of cells along the refrigeration period was measured using specific colony dot-blot. Gene expression of putative cold-shock genes (csp1, csp3, csp4 and csp5) as well as three stress-related genes (rpoS, oxyR and katG) was determined by reverse transcriptase polymerase chain reaction. Vibrio vulnificus exhibited a decline in cell viability after cold shock but a cold adaptation response was observed when oysters were kept at suboptimal (15 degrees C) temperatures. 16SrRNA, and rpoS genes were constitutively expressed while expression of csp genes varied among strains and time points. CONCLUSIONS: Vibrio vulnificus culturability was reduced after oysters were subjected to shellstock refrigeration conditions. When V. vulnificus was allowed to acclimate to cold temperatures, its survival after cold shock was higher. None of the cold shock genes analysed behaved as csp type I genes. SIGNIFICANCE AND IMPACT OF THE STUDY: A model for artificially inoculated specific strains of V. vulnificus into oysters has been established. For the first time, V. vulnficus gene expression was assessed with the pathogen embedded in oysters.

Use of suppressive subtractive hybridization to identify Flavobacterium columnare DNA sequences not shared with Flavobacterium johnsoniae.

AIMS: To identify specific sequences in the fish pathogen Flavobacterium columnare not shared by Flavobacterium johnsoniae. METHODS AND RESULTS: Suppressive subtractive hybridization (SSH) was used to selectively amplify and clone F. columnare-specific sequences. A highly virulent strain of F. columnare was used as tester and the type strain of F. johnsoniae was used as driver. After library construction, 192 clones were selected and sequenced. From those, 110 clones contained unique F. columnare-specific sequences that were verified using dot blot hybridization. Sequence sizes ranged from 55 to 872 bp with 45,363 bp sequenced in total. CONCLUSIONS: Specific F. columnare sequences representing all but one (motility related) functional categories were annotated. Several putative virulence factors were identified in F. columnare such as a collagenase, a chondroitinase, proteases, as well as drug resistance and iron transport-related genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Suppressive subtractive hybridization is a cost-effective method for identifying genetic differences between Flavobacterium spp. The number of sequences available from F. columnare has been doubled.

Molecular typing of Streptococcus agalactiae isolates from fish.

The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using single-stranded conformation polymorphism (SSCP) analysis of the intergenic spacer region (ISR), and amplified fragment length polymorphism (AFLP) fingerprinting. A total of 46 S. agalactiae cultures isolated from different fish species and geographic origins as well as related reference strains were included in the study. ISR-SSCP divided the S. agalactiae isolates analysed into five distinct genotypes. Genotype 1 grouped all Kuwait isolates while genotype 4 clustered the majority of non-Kuwait isolates (USA, Brazil and Honduras). AFLP analysis offered a higher resolution level by dividing the isolates into 13 different genotypes. Two different AFLP profiles were identified within the Kuwait isolates. When data from both ISR-SSCP and AFLP were combined through a multidimensional analysis (MDS), a good correlation between geographical origin and genotypes was observed. Both AFLP and ISR-SSCP revealed genetic differences between S. agalactiae isolates from fish. While AFLP offered a higher resolution, ISR-SSCP also provided valid information being a simpler and faster method.

Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus).

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.

[Behavioral factors and sensorial identificatory deficits as predictors for dementia of the Alzheimer's type]. / Factores de comportamiento y déficit sensoriales identificatorios como predictores de la demencia tipo Alzheimer.

INTRODUCTION: Several behavior factors had been studied as associated to Alzheimer, but not its prediction power. AIM: To examine whether five behavior variables (psychosocial introversion, coping deficit, apathy, demotivation and perception blockade) could predict the beginning of dementia in mild cognitive impairment initially non dementia cases, after a six-year follow-up period. SUBJECTS AND METHODS: 197 mild cognitive impairment non dementia cases (mean: 72.6 years) and an equal number age, gender, schooling and familial income matched normal cases (mean: 73.0 years) were selected to participate from an university data base of older than 60 year people initiated at 1994 for epidemiologic multifactorial studies. Behavior variables were recovery by means of a protocol, applied directly or with the help of relative caregivers, and designed under the hypothetical theory of Alzheimer as a conclusion of a progressive psychosocial capsuletion's process. RESULTS: Introversion, coping deficit, apathy and demotivation appear as frequently and progressive but not necessarily simultaneously or unchained factors, but with possibility of reversion or taking a different way. Perception blockade (three identificatory component of senses impairment at least) seems like the irreversible phase in the process, with near 100% of previous detection for the final diagnostic of Alzheimer's cases. CONCLUSION: Perception blockade, in spite of its great fluctuation and individual variations, appears as more objective, easy, non invasive and firmly as reveille and predictor of this dementia, and we suggest that it appears as the immediate cause of the wide reported histopathological disorders by way of a disintegration of neural net owing to the normal reinforcement deficit.

Factores de comportamiento y déficit sensoriales identificatorios como predictores de la demencia tipo Alzheimer / Behavioral factors and sensorial identificatory deficits as predictors for dementia of the Alzheimer´s type

Introducción. Se sabe que diversos comportamientos se hallan asociados a la demencia tipo Alzheimer, pero no se ha explorado su poder predictor. Objetivo. Determinar si en adultos mayores de 60 años inicialmente con afectaciones cognitivas o ejecutivas sin demencia, cinco factores de comportamiento (introversión psicosocial, déficit de afrontamiento de pérdidas personales, apatía, desmotivación y bloqueo perceptivo) pueden predecir dicha demencia. Sujetos y métodos. 197 personas con déficit cognitivo leve sin demencia (edad media: 72,6 años) e igual número con envejecimiento normal (edad media: 73 años), igualadas en edad, género, escolaridad e ingresos, se seleccionaron de una base de datos iniciada en 1994 para estudios epidemiológicos multifactoriales. Se utilizó un protocolo aplicado bien directamente o a través de familiares cuidadores, y diseñado especialmente bajo la teoría hipotética del Alzheimer como la culminación de un proceso progresivo de aislamiento social. Resultados. La introversión, el déficit de afrontamiento de las pérdidas personales, la apatía y la desmotivación se muestran frecuentes y los dos últimos progresivos pero no siempre simultáneos o encadenados, aunque con la posibilidad de reversión o de tomar un camino diferente al Alzheimer. El bloqueo perceptivo (déficit de al menos tres componentes identificatorios de los sentidos) aparece como la instancia irreversible del proceso, con cerca del 100% de detección previa a los casos diagnosticados como Alzheimer. Conclusiones. El bloqueo sensorial, pese a ser fluctuante y variable individualmente, es el indicador más objetivo, no invasivo, sencillo y firme como diana y predictor del curso hacia esta demencia. Se conjetura que el déficit sensorial identificatorio puede causar la desintegración de las redes neuronales por déficit de reforzamiento y conducir a las alteraciones de las funciones cerebrales típicas del Alzheimer

Introduction. Several behavior factors had been studied as associated to Alzheimer, but not its prediction power. Aim. To examine whether five behavior variables (psychosocial introversion, coping deficit, apathy, demotivation and perception blockade) could predict the beginning of dementia in mild cognitive impairment initially non dementia cases, after a six-year follow-up period. Subjects and methods. 197 mild cognitive impairment non dementia cases (mean: 72.6 years) and an equal number age, gender, schooling and familial income matched normal cases (mean: 73.0 years) were selected to participate from an university data base of older than 60 year people initiated at 1994 for epidemiologic multifactorial studies. Behavior variables were recovery by means of a protocol, applied directly or with the help of relative caregivers, and designed under the hypothetical theory of Alzheimer as a conclusion of a progressive psychosocial capsuletion’s process. Results. Introversion, coping deficit, apathy and demotivation appear as frequently and progressive but not necessarily simultaneously or unchained factors, but with possibility of reversion or taking a different way. Perception blockade (three identificatory component of senses impairment at least) seems like the irreversible phase in the process, with near 100% of previous detection for the final diagnostic of Alzheimer’s cases. Conclusion. Perception blockade, in spite of its great fluctuation and individual variations, appears as more objective, easy, non invasive and firmly as reveille and predictor of this dementia, and we suggest that it appears as the immediate cause of the wide reported histopathological disorders by way of a disintegration of neural net owing to the normal reinforcement deficit

Comparison of lipopolysaccharide and protein profiles between Flavobacterium columnare strains from different genomovars.

Lipopolysaccharide (LPS) and total protein profiles from four Flavobacterium columnare isolates were compar. These strains belonged to genetically different groups and/or presented distinct virulence properties. Flavobacterium columnare isolates ALG-00-530 and ARS-1 are highly virulent strains that belong to different genomovars while F. columnare FC-RR is an attenuated mutant used as a live vaccine against F. columnare. Strain ALG-03-063 is included in the same genomovar group as FC-RR and presents a similar genomic fingerprint. Electrophoresis of LPS showed qualitative differences among the four strains. Further analysis of LPS by immunoblotting revealed that the avirulent mutant lacks the higher molecular bands in the LPS. Total protein analysis displayed by immunoblotting showed differences between the strains analysed although common bands were present in all the isolates. FC-RR lacked two distinct common bands (34 and 33 kDa) shared by the other three isolates. Based on the difference of LPS and total protein profiles, it is possible to discriminate the attenuated mutant FC-RR from other F. columnare strains.

Combined use of 16S ribosomal DNA and automated ribosomal intergenic spacer analysis to study the bacterial community in catfish ponds.

AIMS: To apply culture-independent techniques to explore the bacterial community composition in catfish pond water. METHODS AND RESULTS: 16S rDNA libraries were constructed and sequenced from 15 pond water samples. Automated ribosomal intergenic spacer analysis (ARISA) was used to fingerprint each bacterial community. A broad diversity in bacterial species composition was found by 16S rDNA analysis. Alphaproteobacteria was the most represented class in all ponds, followed by Gammaproteobacteria and Gram-positive high G + C content bacteria. Uniqueness of bacterial communities from each individual pond was confirmed by ARISA. Catfish pathogens were detected sporadically. CONCLUSIONS: Bacterial communities in a catfish aquaculture setting can vary from pond to pond at one given point. No correlation could be made between bacteria composition and fish strain or between bacterial profile and the presence of catfish pathogens in a particular pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing the composition of bacterial communities in catfish ponds. Fish health specialists and catfish aquaculture managers should be aware of the wide differences in bacterial communities between ponds and include this variable in fish husbandry practices.

Isolation and characterization of strains of Flavobacterium columnare from Brazil.

Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare.

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish.

AIMS: To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare. METHODS AND RESULTS: Genetic variability among Fl. columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP) fingerprinting. Thirty Fl. columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study. Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II. Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates. The maximum resolution was provided by AFLP fingerprinting, as up to 22 AFLP profiles could be defined within the species. CONCLUSIONS: We confirmed the division of Fl. columnare isolates from cultured fish into different genogroups. We showed that both genomovars I and II are present in channel catfish from the US. We described a unique genetic group represented by four Fl. columnare isolates from tilapia in Brazil which appears to be related to both genomovars. We were able to further subdivide the species by analysing the ISR. Finally, the use of AFLP allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl. columnare.