We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22-to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease, which causes significant problems to aquaculture worldwide. A recent study (Soule M, Cain K, LaFrentz S, Call DR [2005] Infect Immun 73:3799-3802) identified two 16S rRNA gene sequence variants (6 base differences) within the variable stem-loop region 3 for F. psychrophilum strains ATCC 49418 and CSF 259-93. That study also hypothesized that F. psychrophilum is composed of at least 2 distinct genetic lineages (I and II) described by a microarray-based comparative genomics study. In the present study, we augmented an existing 16S rDNA microarray to detect both 16S rRNA sequence variants from F. psychrophilum. Subsequent microarray experiments showed that CSF 259-93 hybridized as expected, but ATCC 49418 was positive for both sequence variants. We then developed a PCR-restriction fragment length polymorphism (RFLP) assay (MnlI and MaeIII) to distinguish between the 2 sequences. Gel isolation of PCR-RFLP products, cloning, and sequencing confirmed that ATCC 49418 harbors both 16S rRNA sequences. Microarray experiments showed that 11 of 14 strains from genetic Lineage I harbor both the CSF 259-93 and ATCC 49418 16S rRNA sequence variants, whereas all 15 Lineage II strains were only positive for the CSF 259-93 sequence (p < 0.0001). Elastin hydrolysis and tetracycline resistance were most closely associated with the latter strains (p < 0.0001 and p = 0.024, respectively). These data support the hypothesis that F. psychrophilum is composed of at least 2 distinct genetic lineages that are closely associated with host origin. KEY WORDS: 16S rRNA · Flavobacterium psychrophilum · Bacterial coldwater disease · Elastin hydrolysis · Microarray hybridization · Tetracycline resistance Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [209][210][211][212][213][214][215][216] 2005 In general, Flavobacterium psychrophilum strains are biochemically homogeneous, with the exception of some variation in the presence or absence of caseinases, gelatinases, and elastases (Bertolini et al. 1994, Lorenzen et al. 1997, Madetoja et al. 2001. Not all strains of F. psychrophilum are capable of causing the same degree of disease (Holt 1987, Madetoja et al. 2002, LaFrentz et al. 2003 and they can be antigenically diverse (Evensen & Lorenzen 1996, Crump et al. 2001, Madetoja et al. 2002. Immunization with killed cells of one strain does not necessarily convey protective immunity from infection by other strains (Holt et al. 1989, Obach & Laurencin 1991.Genetic variation has been assessed between strains using ribotyping, plasmid profiles, randomly amplified polymorphic DNA (RAPD), and PCR-RFLP of specific gene fragments (Chakroun et al. 1997, Chakroun et al. 1998, Madetoja et al. 2001, Izumi et al. 2003. Studies of outbreak strains from Danish trout farms suggest dominance by a single ribotype , Madetoja et al. 2002, but other studies show greater genetic variation across geographic scales (Chakroun et a...

Reciprocal subtractive libraries were prepared for two strains of Flavobacterium psychrophilum, one virulent and the other avirulent in a trout challenge model. Unique clones were sequenced and their distribution assessed among 34 strains. The analysis showed that F. psychrophilum is composed of two genetic lineages, possibly reflecting host specificity.Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease and rainbow trout fry syndrome, both of which affect salmonid fish and impact commercial aquaculture and resource enhancement hatcheries worldwide (12,19,21). Coho salmon (Oncorhynchus kisutch [Walbaum]) and rainbow trout (O. mykiss) are particularly susceptible, although F. psychrophilum also infects other fish species (21). F. psychrophilum strains vary greatly in the ability to establish disease (virulence). For example, one well-studied strain (ATCC 49418 [4,6]) is unable to cause significant mortality (9) while strain CSF 259-93 causes high mortality (16) in a trout challenge model. There are no commercial vaccines available for bacterial coldwater disease, although several research groups have active programs in this area (11,14,20). From these studies we know that not all strains elicit an antibody response that is effective against other strains and consequently there may be considerable genetic variation between strains.The goals of this project were (i) to examine genetic differences between two strains of F. psychrophilum and ATCC 49418 [16]) and (ii) to assess the extent and distribution of genetic variation between other strains relative to geographical source and host species. Strains for the latter assessment were chosen based on availability at the time of this study. All strains were stored at Ϫ80°C and were cultured at 16 to 17°C in tryptone yeast extract salts medium (TYES; 0.4% tryptone, 0.04% yeast extract, 0.05% calcium chloride, 0.05% magnesium sulfate, pH 7.2). Genomic DNA (gDNA) was extracted using the DNeasy tissue kit (QIAGEN, Valencia, CA).We prepared reciprocal suppression subtractive hybridization libraries for the two strains by using the PCR-Select Bacterial Genomic Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturer's protocol, except that tester and driver gDNAs were restriction enzyme digested with RsaI (supplied with the kit) and DraI (New England Biolabs, Inc,, Beverly, MA) and the hybridization step was at 59°C. Resulting DNA fragments were cloned in pCR2.1 (Invitrogen, Carlsbad, CA), and 576 randomly chosen recombinant clones were used to make a microarray as previously described (8, 10). gDNA (0.5 g) was nick translated for 2 h in the presence of biotindATP (BioNick Labeling System; Invitrogen) and hybridized to the microarray (data not shown). Microarray slides were processed, imaged, and analyzed as described previously (23). Using stringent selection criteria, where the median probe intensity was Ն95% of the maximum pixel intensity in one strain and Յ5% of the maximum pixel intensity in the other strain, we identified...

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BackgroundThe ecology of columnaris disease, caused by Flavobacterium columnare, is poorly understood despite the economic losses that this disease inflicts on aquaculture farms worldwide. Currently, the natural reservoir for this pathogen is unknown but limited data have shown its ability to survive in water for extended periods of time. The objective of this study was to describe the ultrastructural changes that F. columnare cells undergo under starvation conditions. Four genetically distinct strains of this pathogen were monitored for 14 days in media without nutrients. Culturability and cell viability was assessed throughout the study. In addition, cell morphology and ultrastructure was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Revival of starved cells under different nutrient conditions and the virulence potential of the starved cells were also investigated.ResultsStarvation induced unique and consistent morphological changes in all strains studied. Cells maintained their length and did not transition into a shortened, coccus shape as observed in many other Gram negative bacteria. Flavobacterium columnare cells modified their shape by morphing into coiled forms that comprised more than 80% of all the cells after 2 weeks of starvation. Coiled cells remained culturable as determined by using a dilution to extinction strategy. Statistically significant differences in cell viability were found between strains although all were able to survive in absence of nutrients for at least 14 days. In later stages of starvation, an extracellular matrix was observed covering the coiled cells. A difference in growth curves between fresh and starved cultures was evident when cultures were 3-months old but not when cultures were starved for only 1 month. Revival of starved cultures under different nutrients revealed that cells return back to their original elongated rod shape upon encountering nutrients. Challenge experiments shown that starved cells were avirulent for a fish host model.ConclusionsSpecific morphological and ultrastructural changes allowed F. columnare cells to remain viable under adverse conditions. Those changes were reversed by the addition of nutrients. This bacterium can survive in water without nutrients for extended periods of time although long-term starvation appears to decrease cell fitness and resulted in loss of virulence.